Large-Scale Label-Free Comparative Proteomics Analysis of Polo-Like Kinase 1 Inhibition via the Small-Molecule Inhibitor BI 6727 (Volasertib) in BRAFV600E Mutant Melanoma Cells
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†Department of Dermatology, ‡Molecular and Environmental Toxicology Center, and §School of Pharmacy, University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706, United States
∥ William S. Middleton Memorial VA Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705, United States
*Tel: (608) 263-5359. Fax: (608) 263-2919. E-mail: [email protected].
Copyright © 2014 American Chemical Society
Abstract
Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAFV600E mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC–MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1’s role in cancer cell survival.
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Introduction
ARTICLE SECTIONS
Polo-like kinase 1 (Plk1) is the most well-studied member of the polo-like family of kinases and is most commonly known for its regulatory role during mitosis, where it has been shown to be a critical component of centrosome maturation, kinetochore–microtubule attachment, bipolar spindle formation, and cytokinesis.(1-4) The overexpression of Plk1 is common in aberrant cells and has been identified in roughly 60% of reported cancers including melanoma, breast, ovarian, thyroid, colon, prostate, pancreatic, head and neck, nonsmall cell lung, and non-Hodgkin’s lymphomas.(5-13) Furthermore, the overexpression of Plk1 has been linked to poor disease prognosis and a decreased survival rate.(14, 15) In vitro studies have shown that specific inhibition of Plk1 can significantly reduce the proliferation and viability of multiple melanoma cell lines by inducing G2/M phase cell cycle arrest and mitotic catastrophe without detriment to normal adult or neonatal human epidermal melanocytes (HEMs).(9) These data have made Plk1 an attractive target for small-molecule inhibition and provoked the development of over a dozen Plk1 inhibitors, six of which have gone to clinical trials.(16-21) However, despite Plk1 inhibition having great preclinical success for cancer treatment, the clinical potential of Plk1-targeted inhibition for human cancers has yet to be realized. This emphasizes the need for a more in-depth understanding of Plk1 signaling in cancer. This study addresses that need by employing a large-scale comparative proteomics analysis to examine the downstream effects of Plk1 inhibition using the small-molecule inhibitor BI 6727.
BI 6727 (Volasertib) is a second-generation Plk1 inhibitor and is currently the most specific Plk1 ATP-competitive inhibitor commercially available with an IC50 of 0.87 nM.(22) Plk1 inhibition by BI 6727 has been shown to have potent antitumorigenic activity in multiple preclinical studies, but the downstream mechanism by which BI 6727 confers its therapeutic effect remains largely undefined. In an effort to identify novel regulatory effects of Plk1 inhibition in melanoma, we used a label-free, relative quantitation strategy to compare the proteomes of A375 melanoma cells cultured in the presence or absence of BI 6727. The A375 melanoma cell line has been reported to express high levels of Plk1 when compared with HEMs and is sensitive to the targeted depletion of Plk1.(9) Furthermore, A375 cells harbor the BRAFV600E mutation, a mutation present in roughly 50% of melanoma cases, thus making it an ideal candidate for our proteomics analysis.(23)
Our comparative proteomics strategy was conducted by employing data-dependent nano-LC–MS/MS analysis on a Q-Exactive with the resultant data being searched against the human proteome using the Sequest search engine and further analyzed with the SIEVE software package to reveal proteins with altered expression. This analysis resulted in the positive identification of 1819 proteins (≤1% false discovery rate (FDR)), 343 of which had significantly altered expression. Using ingenuity pathway analysis (IPA) to filter the data under more stringent conditions, we identified a subset of 23 proteins that were significantly altered due to BI 6727 inhibition of Plk1, most of which have no previously reported associations with Plk1. Importantly, we have identified proteins involved in cellular metabolism, proteasomal degradation, and p53 translation, thus providing novel insight into Plk1’s role in cancer cell survival.
Materials and Methods
ARTICLE SECTIONS
Cell Culture
A375 human melanoma cells (ATCC, VA) were cultured in HyClone Dulbecco’s modified Eagle’s medium (DMEM, Thermo, CA) supplemented with 10% fetal bovine serum (FBS, Thermo, CA). Cells were maintained in a humidified incubator with 5% CO2 at 37 °C.
BI 6727 Treatment
1 × 106 A375 human melanoma cells (ATCC, VA) were seeded in 100 mm tissue culture plates (TPP, MO) containing 10 mL of supplemented DMEM (Thermo, CA) and were allowed to recover in untreated medium for 24 h under standard cell culture conditions. Following recovery, medium was aspirated, and cells were treated with 25 nM BI 6727 (Chemietek, IN) or vehicle control (DMSO) in supplemented DMEM. After 24 h, cells were collected by trypsin digestion and centrifugation at 300g for 3 min at 4 °C. Supernatant was removed and cell pellets were washed three times with PBS. The treatments were performed using identical procedures on two varying A375 cell passages three times each for a total of six experimental replicates per treatment group. Cell pellets were stored at −80 °C prior to cell lysate preparation.
Cell Lysate Preparation for Proteomics Analysis
Cell pellets were lysed mechanically with a needle in the absence of protease inhibitors or lysis buffer according to the following protocol. 0.3 mL of ice cold PBS was added to frozen cell pellets, and the resulting mixture was lysed by passing through a 23 gauge needle 15 times. The cytosolic protein fraction was isolated by centrifugation at 10 000g for 10 min at 4 °C to remove cellular debris. Protein concentration of the extracts was measured by MicroBCA assay (Thermo Fisher Scientific, IL). A total of 20 μg of protein from each of the six replicates (control and treated) was digested with 1 μg of sequencing grade trypsin (Promega, Fitchburg, WI). Following an overnight digestion at 37 °C samples were acidified with 10% formic acid and prepared for LC–MS/MS by C18 Zip-Tip purification according to the manufacturers protocol (Millipore, Billerica, MA). Peptide samples were resuspended in water with 0.1% formic acid (v/v) and analyzed by nano-LC–MS/MS.
MS/MS Protein Identification and Quantification
For label-free, relative, quantitative analysis, six replicates of each sample were analyzed by nano-LC–MS/MS. For each run, 1 μg of the digest was injected on a 100 μm × 100 mm, reverse-phase C18 BEH column with 1.7 um particles and a 300 Å pore size (Waters, Milford, MA) using a Waters nanoAcquity system. Chromatography solvents were water (A) and acetonitrile (B), both with 0.1% formic acid. Peptides were eluted from the column with the following gradient 3 to 35% B (130 min). At 140 min, the gradient increased to 95% B and was held there for 10 min. At 160 min, the gradient returned to 3% to re-equilibrate the column for the next injection. A short 50 min linear gradient blank was run between samples to prevent sample carryover. Peptides eluting from the column were analyzed by data-dependent MS/MS on a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, MA). A top-15 method was used to acquire data. In brief, the instrument settings were as follows: resolution was set to 70 000 for MS scans and 17 500 for the data-dependent MS/MS scans to increase speed. The MS AGC target was set to 106 counts, while MS/MS AGC target was set to 105. The MS scan range was from 300 to 2000 m/z. MS scans were recorded in profile mode, while the MS/MS was recorded in centroid mode, to reduce data file size. Dynamic exclusion was set to a repeat count of 1 with a 25 s duration.
Data Processing
Following LC–MS/MS acquisition, the data were searched using Sequest HT Proteome Discoverer 1.4 search engine (Thermo Fisher Scientific), against the Uniprot Human database (6/23/2013, 20 209 sequences) at a false discovery cut off ≤1%. Following protein identification, the LC–MS/MS data were aligned using Chromalign. Quantitation of peptides eluting between 38 and 145 min was performed on the processed data using SIEVE 2.1 (Thermo Fisher Scientific), which uses MS intensities from raw LC–MS data to find statistical proteomic differences between two samples. Prior to ratio quantitation in SIEVE, peak intensities from all 12 LC–MS/MS runs were normalized by the total ion chromatogram intensity. Statistical filters were set to assess the quality of the data. Proteins ratios calculated had to be significant with a p value lower than 0.05, and the CV raw MS intensities of the six replicates had to be within 30%. This helped minimize the effect of run-to-run variability.
Data Analysis
Identified proteins from the SIEVE processing were initially analyzed and filtered using IPA (Ingenuity Systems, CA) under a trial license. A data set containing proteins with only uniquely identified amino acid sequences (peptides) with a high level of confidence (p < 0.05) was uploaded into IPA with number of peptides identified, triggered MS/MS fragmentation scans (hits), and the corresponding ratio (treated/untreated) set as the observational parameters with Swiss-Prot accession numbers used for gene ID. A secondary data set was then generated using IPA filters set to proteins with more than two identified peptides on no fewer than four hits with a greater than two-fold change in expression. Another secondary data set was generated using the IPA connect tool to identify any Plk1-associated proteins with an expression change greater than two-fold. The two secondary data sets were combined, and their molecular function and biological processes were assessed using PANTHER (Protein ANalysis THrough Evolutionary Relationships).
Western Blot Analysis
5 × 105 A375 cells were plated and grown in a 10 cm culture dish and treated with BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described. Following 24 h treatment, cells were trypsinized, washed with ice-cold PBS, and lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Pierce, IL). Protein concentration was measured with BCA Protein Assay (Pierce, IL). For immunoblot analysis, 30 μg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEAN TGX precast gels and transferred onto nitrocellulose membrane. Blots were blocked in 5% nonfat dry milk in tris-buffered saline +0.1% tween-20 (TBST), followed by probing with desired primary antibodies: anti-LDHA, AurkB, p53, β-actin (Cell Signaling nos. 2012, 3094, 9282, 4970), PSMB1, PSMB2, or PSMB5 (Abcam nos. ab135830, ab166628, ab3330). Blots were then incubated with the appropriate HRP-conjugated antibodies followed by chemiluminescent detection (Pierce, IL).
Quantitative Real-Time PCR
5 ×105 A375 cells were plated and grown in a 10 cm culture dish and treated with BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described. Following 24 h of treatment, cells were trypsinized and washed with ice-cold PBS, followed by RNA isolation using the RNeasy plus mini kit (Qiagen, CA) and first strand cDNA created with M-MLV reverse transcriptase (Promega, WI) according to vendor’s protocol. Quantitative real-time RT-PCR was performed in triplicate in 20 μL reactions with SYBR Premix Ex Taq Perfect Real Time (Takara, WI) with 100 ng first-strand cDNA and 0.2 μg of each desired primer pair. The sequences for GPI, LDHB, and p21 (PrimerBankID 296080692c3, 291575126c2, 310832423c1) were acquired from the PrimerBank online database.(24) Samples were cycled once at 95 °C for 10 min, then 35 cycles of 95, 58, and 72 °C for 5, 15, and 20 s, respectively. Relative mRNA was calculated using the ΔΔCT method with GAPDH as an endogenous control.
Lactate Assay
For lactate assay, 8 × 103 A375 cells were plated and grown in a 96-well plate and treated with BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described. 24 h following treatments, 500 μL of spent media was removed from each group and immediately cleared of LDH proteins by centrifugation in 10 kDa molecular weight cutoff spin columns and stored at −80 °C. All samples were diluted by adding 10 μL of media to 90 μL of lactate assay buffer and subsequently used in a serial dilution containing a final concentration of either 1, 0.5, 0.25, or 0.125% media in lactate assay buffer with a final volume of 50 μL in black 96-well half area plates (Corning, MA). Lactate assay was performed per manufacturer’s protocol, and fluorescence intensity was measured on the Biotek Synergy H1 microplate reader at λex = 535/λem = 587.
NAD, NADH, and NADPH Assays
3 × 103 A375 cells were plated and grown in 96-well half-volume white-wall plates (Corning, MA) and treated with BI 6727, as previously described. Following 24 h treatments, samples were processed using the NAD(P)H-Glo detection system (Promega, WI) or NAD/NADH-Glo assay (Promega, WI) as per manufacturer’s protocol. Luminescence was detected using the Biotek Synergy H1 microplate reader.
20S Proteasome Activity Assay
For assessing 20S proteasome activity, A375 cells were treated with BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described. Following 24 h treatments, cells were lysed in 50 μL of ice-cold RIPA buffer for 10 min at room temperature while shaking. Next, 25 μL of lysate was added to a 96-well plate containing BCA reagent (Pierce, Rockford, IL) and incubated at 37 °C for 30 min, while 10 μL of lysate was added to the 20S Proteasome Activity Assay (Millipore, MA) prepared per manufacturer’s protocol and incubated at 37 °C for 2 h. Relative levels of protein were quantified using the BCA assay absorbance detected using the Biotek Synergy H1 microplate reader at 562 nm. 20S proteasome activity was quantified using fluorescence intensity on the Synergy H1 microplate reader at λex = 380/λem = 460. Proteasome activity was then normalized to relative protein concentration, and the mean of relative activity for each treatment (n = 6) was represented graphically.
Immunofluorescence Staining
For immunofluorescence staining, cells were plated and grown on BD Falcon CultureSlides (BD Biosciences, San Jose, CA) and treated with BI 6727 as previously described. The cells were fixed with a 4% paraformaldehyde in PBS (pH 7.1) for 15 min at room temperature, then blocked with 5% normal goat serum in 0.3% Triton X-100 for 1 h. After blocking, cells were incubated overnight in anti-β-tubulin primary antibody in blocking buffer (1:100, Cell Signaling no. 2128), followed by a secondary incubation with Alexa Fluor 594 antirabbit IgG antibody in blocking buffer (5 μg/mL, Invitrogen no. A-11037) for 1 h in the dark. The cells were then counter-stained with Hoecsht 33342 (Invitrogen, Grand Island, NY) for nuclear staining, and a ProLong antifade kit was applied per vendor’s protocol (Molecular Probes, Eugene, OR). Slides were examined under a Nikon Ti microscope using the respective manufacturer’s suggested filter sets.
Cell Cycle Analysis
1 × 105 A375 cells were plated and grown in a six-well tissue culture dish (TPP, CHE) and treated with BI 6727, as previously described. Following treatment, cells were trypsinized for 5 min and transferred to 5 mL Falcon tubes containing cultured media. Samples were centrifuged at 1000 rpm for 2 min, and the supernatant was aspirated prior to the cells being resuspended in ice-cold 100% ethanol added dropwise while gently vortexing. Samples were stored at −20 °C overnight prior to centrifugation and resuspension in 500 μL of propidium iodide (PI) buffer (PBS, 50 μg/mL PI, 0.1 mg/mL RNase A, 0.05% Triton-X). Samples were incubated at 37 °C for 40 min in dark and stored at 4 °C until processed by flow cytometry in the FL-2A channel. Cell cycle analysis was done using ModFit LT software (Verity Software, Topsham, ME).
Results and Discussion
ARTICLE SECTIONS
BI 6727 Treatment, Protein Identification, Quantification, and Analysis
In an effort to identify the downstream molecular mechanisms of Plk1 in melanoma, we elucidated quantitative changes in the proteome of human melanoma cells following Plk1 inhibition with BI 6727. Data acquired by nano-ESI–MS/MS on a Q-Exactive spectrometer were processed with SIEVE software to reveal up- or down-regulated proteins following Plk1 inhibition. To determine the most effective concentration of BI 6727, we assessed A375 cell viability following a defined range of BI 6727 treatments (0.1–1000 nM, data not shown). We determined 25 nM of BI 6727 as the lowest effective concentration to cause a statistically significant decrease in cell viability after 48 h. To assess the molecular functions that contribute to the reduction of A375 cell viability, we terminated treatments after 24 h to limit the necrotic and late apoptotic cell population.
Several methods for relative proteomic quantitation have been described.(25-27) Labeling methods, including chemical modification approaches (I-Traq, etc.) and stable isotope labeling, can be expensive but generally require fewer LC–MS runs to generate robust results. Label-free approaches are becoming more popular as improvements in instrumentation (resolution and mass accuracy), and improvements in data analysis software facilitate analysis of large data sets. The capabilities of our Q-Exactive spectrometer made a label-free approach attractive. The high scanning speed coupled to the resolving power and mass accuracy of the Q-Exactive Orbitrap make it particularly useful for accurate, label-free, quantitative protein analysis. This is primarily due to the higher resolution (smaller diameter) Orbitrap cell of the Q-Exactive. High-resolution and mass accuracy facilitate area-under-the-curve analysis with the SIEVE software by allowing accurate tracking of peptides through their chromatographic elution. Additionally, the improvement in resolution allows shorter transient acquisitions. This improves the duty cycle and allows for increased peptide identification at the MS/MS level. The Q Exactive has been shown to identify a higher number of compounds with better confidence in multiple comparison studies.(28-30) Thus, there is greater proteome coverage, which enhances the ability to quantitate lower abundant proteins than is achieved with spectral-counting approaches.(31-36) Indeed, the power of this combination of instrumentation and software was born out in the ability of our analysis to positively identify over 1800 proteins, at a cutoff of ≤1% FDR, with 343 of these identified proteins showing significantly altered expression (Tables S1 and S2 in the Supporting Information).
A key step in label-free quantitative proteomic analysis is the evaluation of run-to-run and sample-to-sample reproducibility/variability.(37) Prior to the full analysis, we performed a pilot study of BI 6727-treated cells and control replicates, using identical sample preparation and LC–MS parameters as previously described to assess variability. Two A375 human melanoma aliquots were seeded in 100 mm tissue culture plates as previously described and grown for 24 h under standard cell culture conditions. Both plates were lysed and digested as laid out in our methods. Two replicate injections of 1 μg of both control and treated samples were run. Resulting data were subject to SEQUEST searches against Uniprot Human at ≤2% FDR, using Proteome Discoverer. A higher FDR was used in assessing the run-to-run variability to delve deeper into the data. These four control runs had an average of 715 proteins identified, with an average of 1500 unique peptides and over 3100 total scans. Percent overlap between the two control biological replicates was 67% at the protein level and 55% at the peptide level (data not shown). Not surprisingly, percent overlap of the duplicate injections was higher with 70% overlap at the protein level and 62% at the peptide level (data not shown). Percent overlap between both injection and biological replicates is consistent with a recent review article highlighting the strengths and limitations of label-free proteomic quantification.(37) Our control runs emphasize the need for more than three experimental replicates of each sample type to accurately determine protein ratios in label-free studies. To maximize the statistical power of our analysis, we opted to analyze six replicates of control versus Plk1 treated A375 cells.
The first step in any direct comparison of treated versus control samples MS peak intensity is alignment of base peak chromatograms. SIEVE uses Chromalign, a proprietary algorithm to align the data. Chromalign evaluates the quality of alignment between samples by assigning a score. Alignments with scores above 0.75 are considered acceptable for further quantitative analysis. Our data showed average alignment scores of 0.825, with the treatment groups clustering together (Figure 1A). Because SIEVE, like many other label-free methods, allows for conserved peptides to be equally considered for all candidate proteins, we decided to take the approach of only quantitating unique peptides. This avoids inaccurate fold-change calculations due to differential regulation of proteins sharing a conserved peptide. The Q-Exactive’s speed, was key in being able to take this stringent approach to label-free proteomics. Label-free quantitation approaches do not rely on internal standards, and thus care needs to be taken to accurately filter the data to reproducibly quantitate proteins. SIEVE allows for calculated peptides ratios between samples to be filtered not only on p value using Fisher’s combined probability but also based on variation in the MS peak intensities between the experimental replicates. After data were normalized to the total ion current (TIC), we filtered all protein ratios to have a CV between the six replicates (25 nM BI 6727 or vehicle control) to be ≤30%. Peptides had to show up in all six replicates of a treatment at the MS level to be quantitated (Figure 1B,C). This stringent filter allows us to have additional confidence in our data. One of the benefits SIEVE has over spectral counting is as long as a peptide has a consistent MS peak, it only has to be positively identified once to determine its ratio. Thus, we were able to identify lower abundant proteins using our approach. Finally, peptide fold-change ratios had to be significant at a p value of ≤0.05 to be considered in our analysis.
Figure 1
Figure 1. Relative quantitation chromatograms. (A) For label-free relative quantitation, six replicates of the tryptic digests were analyzed. The chromatograms were aligned using SIEVE 2.1. An overlay of the base peak chromatograms of control (blue) and treated (red) samples shows good alignment and comparable loading in the region of peptide elution (38–145 min RT). (B) An example of a peptide that is down-regulated in response to treatment. Shown is a SIEVE-aligned, extracted-ion chromatograms for peptide 524.9481 m/z (metastasis-associated protein). Triangles indicate where MS/MS identification scans were triggered. (C) Example of a peptide that is up-regulated in response to treatment. Shown is a SIEVE-aligned, extracted-ion chromatogram for peptide 373.3691 m/z (PH-domain leucine-rich protein). Triangles indicate where MS/MS identification scans were triggered.
Our protein analysis was initially done using IPA. A data set containing proteins with only uniquely identified peptides with a high level of confidence (p < 0.05) was uploaded into IPA with the number of peptides identified (Figure 2A) and the corresponding ratio (treated/untreated) (Figure 2B) set as the observational parameters. A secondary data set was then generated using IPA filters set to proteins with more than two identified peptides, no fewer than four hits, and greater than a two-fold change in expression. Another secondary data set was generated using less stringent criteria allowing for one uniquely identified peptide and integrated the IPA connect tool to identify any Plk1-associated proteins with more than four hits and an expression change greater than two-fold. These two analyses resulted in a data set containing 23 proteins of interest (Table 1). Next, we employed PANTHER (Protein ANnalysis THrough Evolutionary Relationships) to assess the molecular function of the collective proteins and identified catalytic activity and binding as the primary protein function (Figure 2C). Interestingly, using PANTHER to identify the biological processes, cell cycle was scored at 8%, while metabolic process was scored at 57% (Figure 2D). Furthermore, when scored by protein classes, the hydrolase, nucleic acid binding and protease classes were the three highest ranks, respectively. Given the known biological function of Plk1, it would be difficult to predict that Plk1 inhibition would have considerable association with cellular metabolism, nucleic acid binding, and proteolytic activity. Therefore, we sought to further investigate these observations and substantiate our proteomics findings.
Figure 2
Figure 2. Summary of proteomics analysis data. (A) Graphical breakdown representing the number of peptides recognized in all identified proteins. (B) Graphical representation of the calculated protein ratios showing BI 6727-treated samples compared with the vehicle control. (C) Proteins of interest molecular function reported by PANTHER (Protein ANalysis THrough Evolutionary Relationships) as characterized by gene ontology. (D) Proteins of interest biological processes reported by PANTHER as characterized by gene ontology.
Table 1. Proteins Quantified with Greater than Two-Fold Changes with Unique Peptides, Frames, Hits, and Normalized Ratio Listeda
| proteins of interest | |||||||
|---|---|---|---|---|---|---|---|
| gene | Swiss-Prot ID | protein description | peptides | frames | hits | normalized ratio | fold change |
| LDHB | P07195 | lactate dehydrogenase B chain - LDHB_HUMAN | 2 | 2 | 18 | 0.058 | –17.24 |
| PPP6R2 | O75170 | serine/threonine_protein phosphatase 6 regulatory subunit 2 - PP6R2_HUMAN | 2 | 2 | 17 | 0.129 | –7.75 |
| PSMA3 | P25788 | proteasome subunit alpha type_3 - PSA3_HUMAN | 3 | 4 | 17 | 0.135 | –7.41 |
| FBL | P22087 | rRNA 2_O_methyltransferase fibrillarin - FBRL_HUMAN | 3 | 4 | 21 | 4.862 | 4.862 |
| SNRPD3 | P62318 | small nuclear ribonucleoprotein Sm D3 - SMD3_HUMAN | 3 | 3 | 19 | 0.22 | –4.55 |
| TKT | P29401 | transketolase - TKT_HUMAN | 12 | 22 | 179 | 0.238 | –4.20 |
| GOT2 | P00505 | aspartate aminotransferase_mitochondrial - AATM_HUMAN | 4 | 4 | 19 | 0.318 | –3.14 |
| HNRNPC | P07910 | heterogeneous nuclear ribonucleoproteins C1/C2 - HNRPC_HUMAN | 2 | 2 | 26 | 2.978 | 2.978 |
| LDHA | P00338 | lactate dehydrogenase A chain - LDHA_HUMAN | 7 | 11 | 145 | 0.352 | –2.84 |
| AHCY | P23526 | adenosylhomocysteinase - SAHH_HUMAN | 2 | 3 | 9 | 2.531 | 2.531 |
| RPL10A | P62906 | 60S ribosomal protein L10a - RL10A_HUMAN | 2 | 3 | 15 | 0.398 | –2.51 |
| MDH1 | P40925 | malate dehydrogenase_cytoplasmic - MDHC_HUMAN | 10 | 15 | 181 | 0.428 | –2.34 |
| GPI | P06744 | glucose_6_phosphate isomerase - G6PI_HUMAN | 2 | 2 | 7 | 0.439 | –2.28 |
| PSMB2 | P49721 | proteasome subunit beta type_2 - PSB2_HUMAN | 5 | 10 | 80 | 0.455 | –2.20 |
| JAKMIP2 | Q96AA8 | Janus kinase and microtubule_interacting protein 2 - JKIP2_HUMAN | 6 | 10 | 61 | 0.457 | –2.19 |
| HSPA9 | P38646 | stress_70 protein_ mitochondrial - GRP75_HUMAN | 2 | 1 | 12 | 2.158 | 2.158 |
| PHC1 | P78364 | polyhomeotic_like protein 1 - PHC1_HUMAN | 2 | 3 | 21 | 0.482 | –2.07 |
| polo-like kinase-1-associated genes | |||||||
|---|---|---|---|---|---|---|---|
| gene | Swiss-Prot ID | description | peptides | frames | hits | normalized ratio | fold change |
| PSMB6 | P28072 | proteasome subunit beta type_6 - PSB6_HUMAN | 1 | 1 | 7 | 0.18 | –5.56 |
| PSMA7 | O14818 | proteasome subunit alpha type_7 - PSA7_HUMAN | 1 | 3 | 8 | 0.406 | –2.46 |
| MCM6 | Q14566 | DNA replication licensing factor MCM6 - MCM6_HUMAN | 1 | 1 | 4 | 2.315 | 2.315 |
| NPM1 | P06748 | nucleophosmin - NPM_HUMAN | 1 | 3 | 78 | 2.277 | 2.277 |
| TUBB3 | Q13509 | tubulin beta_3 chain - TBB3_HUMAN | 1 | 1 | 7 | 2.118 | 2.118 |
| CLSPN | Q9HAW4 | claspin - CLSPN_HUMAN | 1 | 1 | 4 | 2.044 | 2.044 |
a
Proteins identified as having greater than a two-fold change and triggering no less than four separate MS/MS fragmentation scans (hits) of at least two uniquely identified amino acid sequences (peptides) for proteins with no known association to Plk1 or one unique peptide for proteins with a Plk1 association, as identified by ingenuity pathway analysis (IPA). Also included is the number of LC peaks within a well-defined rectangular region in the M/Z versus retention time plane (frames), where each unique peptide was identified.
BI 6727 Treatment Alters Expression of Multiple Metabolic Proteins
The results of our proteomics analysis have revealed that the inhibition of Plk1 activity results in the decreased expression of multiple metabolic proteins including malate dehydrogenase 1 (MDH1), glutamic-oxaloacetic transaminase 2 (GOT2), and transketolase (TKT). Additionally, we observed a significant reduction in lactate dehydrogenase A (LDHA), confirmed by Western blot (Figure 3A) as well as glucose-6-phosphate isomerase (GPI) and lactate dehydrogenase B (LDHB), which were further assessed at the mRNA level (Figure 3B). Of interest, LDHA, LDHB, and GPI each play an essential role in glycolysis, a metabolic pathway that allows tumor cells to thrive under hypoxic conditions. Glycolysis is an evolutionarily conserved reaction that allows cells to generate ATP from glucose under hypoxic conditions. However, cellular metabolism under normoxic conditions is primarily driven by a much more efficient reaction, mitochondrial oxidative phosphorylation (OXPHOS), yet a hallmark of many cancer cells is their propensity to use the inefficient glycolytic reaction for the production of energy in the presence of oxygen, a phenomenon known as the Warburg effect or aerobic glycolysis.(38) It is interesting to speculate that Plk1 overexpression may contribute to the cancer-related switch from OXPHOS to aerobic glycolysis.
Figure 3
Figure 3. Plk1 inhibition alters cellular metabolism in melanoma cells. (A) Lactate dehydrogenase A (LDHA) protein expression was significantly reduced following Plk1 inhibition in both proteomics (top) and Western blot (bottom) analyses. (B) qPCR analysis suggests a dose-dependent decrease in polo-like kinase 1 (Plk1), lactate dehydrogenase B (LDHB), and glucose-6-phosphate isomerase (GPI) transcript levels following BI 6727 treatment (25 nM, 100 nM). (C) BI 6727 treatment significantly reduces extracellular lactate levels (p < 0.001). (D) Plk1 inhibition significantly reduces reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) levels (p < 0.001). (E) NAD and NADH levels are significantly reduced in BI 6727-treated cells when compared with control (p < 0.001). (F) NAD/NADH ratio decreases in a BI 6727 dose-dependent manner (** p < 0.01, *** p < 0.001).
To assess the effect of Plk1 inhibition on aerobic glycolysis, we first measured extracellular levels of lactate, the major byproduct of glucose breakdown. Following a 24 h incubation with BI 6727 under normoxic conditions, there was a significant decrease in lactate levels in both treatment groups (25 nM, 100 nM) when compared with control (Figure 3C). Because these data suggest a disruption in metabolic activity, we next assessed the levels of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), the enzymatic cofactors produced during glucose breakdown by glycolysis and the pentose phosphate pathway, respectively. The NADH and NADPH levels were significantly reduced in BI-6727-treated cells, resulting in nearly a five-fold decrease (Figure 3D). To determine if these observations correlate to energy production, we independently measured levels of NAD, an essential coenzyme in ATP production. Interestingly, in addition to the NAD levels being significantly decreased in both the oxidized (NAD+) and reduced (NADH) forms (Figure 3E), Plk1 inhibition also significantly altered the NAD+/NADH ratio (Figure 3F). The NAD+/NADH ratio is considered to be an indicator of the metabolic state and is important in regulating the intracellular redox state, while a shift in the NAD+/NADH ratio is closely linked to physiological and pathological states.(39, 40) Our data suggest a greater decrease in NAD+ following Plk1 inhibition, and given that the pyruvate to lactate conversion is a major route of NAD+ regeneration, it is reasonable to expect that the observed decrease in LDHA is a primary factor in the ratio shift.
Although our data suggest that Plk1 inhibition decreases the metabolic activity of melanoma cells, the question of how Plk1 signaling relates to glycolysis remains to be answered. The initial Warburg hypothesis attributed its effects to dysfunctional OXPHOS; however, recent studies have indicated that OXPHOS remains functional in cancer cells and the shift toward aerobic glycolysis is driven by tumor suppressors such as p53 and PTEN or the oncogenes Ras, C-Myc, and hypoxia-inducible factor-1α (HIF-1α).(41) Plk1 has not only been shown to be involved in p53, PTEN, and C-Myc signaling pathways; recent studies suggest that Plk1 has the potential to act as a mediator between them.(42-44) For example, the loss of PTEN expression is a frequent occurrence in human malignancies and results in elevated phosphoinositide 3-kinase (PI3K) signaling.(45) Recently, Tan et al. have shown that hyperactivation of the PI3K downstream target, PI3K-dependent protein kinase-1 (PDK1), activates Plk1, which in turn stabilizes MYC through direct phosphorylation at Ser-62.(46) While this study has shown a direct connection between two key tumorigenic pathways through Plk1, further correlations can be made when considering the numerous interactions between Plk1 and p53 (discussed later). Given these data, it is interesting to hypothesize that Plk1 overexpression may contribute to the glycolytic shift described by the Warburg effect; however, further studies are needed to define the role of Plk1 in metabolic signaling.
BI 6727 Treatment Results in the Down-Regulation of Multiple 20S Proteasomal Subunits
The ubiquitin-proteasome system (UPS) plays an integral role in cellular homeostasis through a systematic process that is responsible for 80–90% of intracellular protein degradation.(47) The 26S proteasome is the primary proteolytic complex of the system and is composed of two subcomplexes, the catalytic 20S core particle (CP) and either one or two terminal 19S regulatory particles (RPs). The RPs are responsible for protein capturing by ubiquitin recognition, followed by protein unfolding and translocation to the proteolytic channel of the CP. The CP is made up of four stacked rings, each containing seven α- (α1-7, PSMA1-7) or β-subunits (β1-7, PSMB1-7) in an α–β–β–α configuration (Figure 4A). The outer α-subunits act as a physical barrier to the active β-subunits and create a docking site for the RP, while the inner β-rings are responsible for protein cleavage through caspase-like, trypsin-like, and chymotrypsin-like activities by the catalytically active subunits β1, β2, and β5, respectively.(48)
Figure 4
Figure 4. Plk1 inhibition significantly alters 20S proteasome expression and activity. (A) Basic structure of the 20S proteasome. (B) Proteomics analysis identified four 20S proteasome subunits as being down-regulated. (C) Western blot analysis of the catalytically active 20S proteasome subunits, proteasome subunits β2, β5, and β1 (PSMB2, PSMB5, and PSMB1) following Plk1 inhibition. (D) BI 6727-treated cells have significantly decreased 20S proteasome activity (p < 0.001).
In our comparative proteomics analysis, we found that BI 6727 treatment results in significant downregulation of multiple 20S subunits in human melanoma cells. Following our IPA analysis, we initially identified subunits α3 and β2 as being 2.20- and 7.41-fold downregulated, respectively. Broadening our IPA analysis to include Plk1-associated proteins with only one unique peptide but still having a greater than a two-fold change and no fewer than four hits, we further identified the 20S subunits α7 and β6 as being downregulated by 2.46 and 5.56 fold, respectively (Figure 4B). Because these data suggest that Plk1 inhibition has an effect on a wide range of subunits, we performed a Western blot analysis of the catalytically active subunits β1, β2, and β5 following inhibition of Plk1 by BI 6727 (Figure 4C). We confirmed a decrease in β2, as reported by our proteomics analysis, and additionally observed a modest decrease in β5, while there was no apparent effect on β1. To assess if the decreased subunit expression reflected on proteasome cleavage, we measured 20S proteasome activity using a fluorophore-labeled proteasome substrate. This assay demonstrated a significant reduction in 20S activity in BI 6727-treated cells when compared with control (Figure 4D). These data suggest that Plk1 activity influences proteasome cleavage and is potentially upstream of multiple proteasome subunits in a signaling pathway that has yet to be defined. However, there is evidence of Plk1 indirectly associating with the proteasome through the transcription factors forkhead box M1 (FoxM1) and p53.(49-56)
Similar to Plk1, FoxM1 is overexpressed in numerous human carcinomas and is correlated to poor disease prognosis.(55) Plk1 is involved in a positive feedback loop with FoxM1, where Plk1-dependent phosphorylation is required for efficient FoxM1 activation, which in turn is required for expression of multiple mitotic regulators, including Plk1.(52) Interestingly, proteasome inhibitors have been shown to suppress FoxM1 transcriptional activity, suggesting that proteasome-dependent degradation may confer a level of indirect regulation to Plk1 overexpression in human carcinomas.(50) In addition to FoxM1, the classical tumor suppressor p53 also intersects with both Plk1 and the proteasome. Studies have revealed that Plk1 contributes to p53 repression by directly binding to p53 in addition to phosphorylating GTSE1 and Topors, negative regulators of p53.(42, 49, 56) Furthermore, Plk1 has also been shown to stabilize the oncoprotein MDM2, an E3 ubiquitin ligase and the principal cellular antagonist of p53.(51) MDM2 mediates p53 protein turnover through constant monoubiquitination, a critical step in the polyubiquitnation of p53 and targeted degradation by the 26S proteasome.(54) Because p53 negatively regulates Plk1, a negative feedback loop exists in the Plk1-p53 axis.(53, 57) Considering the findings presented in this study and given the relationships among Plk1, FoxM1, and p53, these data suggest that increased proteasome activity would be conducive to Plk1 overexpression.
Heterogeneous Ribonucleoprotein C1/C2 Is Up-Regulated Following BI 6727 Treatment
The heterogeneous ribonucleoprotein C1/C2 (hnRNPC) is a ubiquitous RNA-binding protein that is expressed in two main isoforms, hnRNPC1 and hnRNPC2.(58) Studies have shown hnRNPC’s biological functions to include mRNA transcript packaging, splicing, nuclear retention, and mRNA stability.(59) In this study, we report a significant up-regulation of hnRNPC following Plk1 inhibition, which was confirmed at the protein level by Western blot analysis (Figure 5A). Interestingly, a recent study has shown that hnRNPC overexpression induces micronucleation through the repression of the mitotic protein aurora kinase B (AurkB) in hepatocellular carcinoma (HCC) cells.(60) Using an immunofluorescent nuclear stain, we were able to observe a similar micronucleation phenotype in melanoma cells following Plk1 inhibition (Figure 5B). Next, we wanted to determine if there was also a repression of AurkB. Despite a significant G2/M arrest (Figure 5C), we did not observe any significant changes to AurkB expression at the protein level; however, we did observe a significant reduction in AurkB mRNA (Figure 5D). With the understanding that an accumulation of cells in G2/M would result in increased mitotic proteins such as AurkB and given the consistent protein expression we observed at 24 h, it is possible that we are seeing early evidence of AurkB protein degradation and the reduced transcript levels may be a better indicator of AurkB repression. This evidence suggests that inhibition of Plk1 activity may negatively affect AurkB expression and the mitotic catastrophe associated with Plk1 inhibition may in part be mediated through hnRNPC and AurkB.(9)
Figure 5
Figure 5. hnRNPC is up-regulated following Plk1 inhibition. (A) Heterogeneous ribonucleoprotein C1/C2 (hnRNPC) protein expression was significantly increased following Plk1 inhibition in both proteomics (top) and Western blot (bottom) analyses. (B) Immunofluorescence microscopy of β-tubulin (red) and Hoecsht DNA (blue) staining, demonstrating micronucleation following Plk1 inhibition. (C) Cell cycle analysis of BI 6727-treated cells demonstrates a pronounced G2/M arrest. (D) Western blot analysis of Aurora kinase B (AurkB) does not indicate significantly altered protein expression following BI 6727 treatment (top), but qPCR analysis reveals decreased mRNA expression levels (bottom). (E) Plk1 inhibition causes a marked increase in p53 protein expression, visualized by Western blot analysis (top) and p53 activity, demonstrated by increased p21 mRNA expression (bottom).
Although we have already discussed numerous connections between Plk1 and p53, an additional association exists through hnRNPC. hnRNPC has been shown to enhance p53 translation by directly binding to a cis-element in the 5′ coding region of p53 mRNA.(61) In accordance with these findings, we did find a corresponding increase in p53 protein expression and activity as determined by Western blot and transcriptional analysis of p21, respectively (Figure 5E). These data provide compelling evidence of a novel pathway in Plk1-mediated p53 expression through regulation of hnRNPC. However, the Plk1–p53 axis is a complex signaling network, and extensive studies will be required to determine the role of hnRNPC.
Conclusions
ARTICLE SECTIONS
Inhibition of Plk1 by small-molecule inhibitors has become an extremely active area of research based on the preclinical success of Plk1 inhibition in a wide variety of cancers. While it has been shown in multiple studies that Plk1 inhibition selectively targets cancerous cells over that of their normal counterparts, the molecular mechanisms that contribute to Plk1’s selectivity remains poorly understood. Our proteomics study of Plk1 inhibition by BI 6727 in human melanoma A375 cells has revealed the altered expression of multiple proteins that have yet to be identified as part of the Plk1 signaling network. Our proteomics analysis provides evidence of a novel relationship between Plk1 and hnRNPC and that Plk1 inhibition has considerable effect on anaerobic glycolysis and proteasome activity, two pathways that are essential in cancer cell survival. Further studies are needed to determine the significance of these protein interactions, but overall these data provide a solid foundation for novel Plk1 signaling pathways and potential candidates for future targeted therapies.
ARTICLE SECTIONS
Table S1. Complete protein list identified by nonunique peptide hits having a protein ratio of a high confidence level (p < 0.05), showing Swiss-Prot ID, protein description, number of peptides, frames, hits, and normalized ratio. Table S2. Complete protein list identified by unique peptide hits having a protein ratio of a high confidence level (p < 0.05), showing Swiss-Prot ID, protein description, number of peptides, frames, hits and normalized ratio. This material is available free of charge via the Internet at http://pubs.acs.org.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
The authors declare no competing financial interest.
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Acknowledgment
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This work was partially supported by funding from the NIH (T32 ES007015-35 to B.D.C.; R01AR059130, R01CA176748 to N.A.) and the Department of Veterans Affairs (VA Merit Review Award 1I01BX001008 to N.A.). We also acknowledge support from the NIH in form of the High-end, Shared Instrumentation grant (1S10RR029531–01) to the Analytical Instrumentation Center. Additionally, we acknowledge Brooke Hatfield for her technical contributions to this work and Travis Schmit for his contributions in the review and preparation of this manuscript.
References
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This article references 61 other publications.
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- 6Knecht, R.; Elez, R.; Oechler, M.; Solbach, C.; von Ilberg, C.; Strebhardt, K. Prognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neck Cancer Res. 1999, 59 (12) 2794– 2797[ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXktVOkur8%253D&md5=75835cab5f5bd0e831de8703a9b4767fPrognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neckKnecht, Rainald; Elez, Robert; Oechler, Martin; Solbach, Christine; Von Ilberg, Christoph; Strebhardt, KlausCancer Research (1999), 59 (12), 2794-2797CODEN: CNREA8; ISSN:0008-5472. (AACR Subscription Office)Previously, we demonstrated that the mammalian polo-like kinase (PLK), which participates in the regulation of the cell cycle, is a novel marker of cellular proliferation. Because current prognostic tools for the evaluation of patients with head and neck squamous cell cancer (HNSCC) need to be improved, we analyzed 89 patients and found elevated PLK expression in most tumors. Nodal stage as a crucial prognostic factor in HNSCC also correlated to PLK transcript levels. A Kaplan-Meier anal. showed that HNSCC patients with moderate vs. high PLK expression survived significantly longer (5-yr survival rates, 43% vs. 12%). Interestingly, a combination of nodal stage and PLK expression contributed to discriminate patients with a better prognosis in the pN0/1 and pN2/3 groups, which could improve the definition of a suitable therapy.
- 7Mito, K.; Kashima, K.; Kikuchi, H.; Daa, T.; Nakayama, I.; Yokoyama, S. Expression of Polo-Like Kinase (PLK1) in non-Hodgkin’s lymphomas Leuk. Lymphoma 2005, 46 (2) 225– 231[ Crossref], [ PubMed], [ CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFSntrjF&md5=028fb6ba93ad47cca395587736c3ce5bExpression of Polo-Like Kinase (PLK1) in non-Hodgkin's lymphomasMito, Katsuhiko; Kashima, Kenji; Kikuchi, Hiroshi; Daa, Tsutomu; Nakayama, Iwao; Yokoyama, ShigeoLeukemia & Lymphoma (2005), 46 (2), 225-231CODEN: LELYEA; ISSN:1042-8194. (Taylor & Francis Ltd.)Polo-like kinases (PLKs) are protein serine/threonine kinases that play important roles in cell division. Expression of PLK1 might, moreover, play a role in the pathogenesis of human neoplasms. The expression of PLK1 mRNA is closely correlated with survival in patients with malignant tumors. We investigated the expression of PLK1 in non-Hodgkin's lymphomas (NHLs) and analyzed the relationships between expression of PLK1, histol. grade, and prognosis. We analyzed various types of NHLs from 118 patients using monoclonal antibodies against PLK1 and Ki-67. The levels of expression of PLK1 and Ki-67 were significantly lower in low-grade NHLs than in high-grade and intermediate-grade NHLs (P < 0.001). Moreover, when patients were grouped in terms of 5-yr overall survival ( > 70%, group A; 50 - 70%, group B; 30 - 49%, group C; and < 30%, group D), levels of expression of PLK1 and Ki-67 were found to be significantly higher in group D than in group A and they were also significantly higher in group C than in group A (P < 0.001). Conversely, the level of expression, of Ki-67 was significantly lower in group D than in group C (P < 0.05). The labeling indexes specific for PLK1 were generally higher than those specific for Ki-67. Once we divided all patients into two groups in terms of the expression levels, high-level expression group of PLK1 (PLK1 index of ≥ 70%) and Ki-67 (Ki-67 indexes of ≥ 60%) and low-level expression, one of these markers (PLK1 index of < 70%, Ki-67 indexes of < 60%) had a similar prognosis, an observation that can be explained by the fact that rapidly proliferating group is more drug-sensitive than the other. Our study demonstrates that expression of PLK1 might reflect the malignant potential of NHLs and that PLK1 might be more useful than Ki-67 for the detection of proliferative cells.
- 8Salvatore, G.; Nappi, T. C.; Salerno, P.; Jiang, Y.; Garbi, C.; Ugolini, C.; Miccoli, P.; Basolo, F.; Castellone, M. D.; Cirafici, A. M.; Melillo, R. M.; Fusco, A.; Bittner, M. L.; Santoro, M. A cell proliferation and chromosomal instability signature in anaplastic thyroid carcinoma Cancer Res. 2007, 67 (21) 10148– 10158[ Crossref], [ PubMed], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1erur7I&md5=5a04652b6794a83f5927d907b6b4f208A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid CarcinomaSalvatore, Giuliana; Nappi, Tito Claudio; Salerno, Paolo; Jiang, Yuan; Garbi, Corrado; Ugolini, Clara; Miccoli, Paolo; Basolo, Fulvio; Castellone, Maria Domenica; Cirafici, Anna Maria; Melillo, Rosa Marina; Fusco, Alfredo; Bittner, Michael L.; Santoro, MassimoCancer Research (2007), 67 (21), 10148-10158CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homol. region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the neg. control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest assocd. with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential mol. target for ATC treatment.
- 9Schmit, T. L.; Zhong, W.; Setaluri, V.; Spiegelman, V. S.; Ahmad, N. Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells J. Invest. Dermatol. 2009, 129 (12) 2843– 2853[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtl2jsLrE&md5=f2f3aa721395fb9db989dda974b7febeTargeted Depletion of Polo-Like Kinase (Plk) 1 Through Lentiviral shRNA or a Small-Molecule Inhibitor Causes Mitotic Catastrophe and Induction of Apoptosis in Human Melanoma CellsSchmit, Travis L.; Zhong, Wei-Xiong; Setaluri, Vijayasaradhi; Spiegelman, Vladimir S.; Ahmad, NihalJournal of Investigative Dermatology (2009), 129 (12), 2843-2853CODEN: JIDEAE; ISSN:0022-202X. (Nature Publishing Group)Melanoma, one of the most lethal forms of skin cancer, remains resistant to currently available treatments. Therefore, addnl. target-based approaches are needed for the management of this neoplasm. Polo-like kinase 1 (Plk1) has been shown to be a crucial regulator of mitotic entry, progression, and exit. Elevated Plk1 level has been assocd. with aggressiveness of several cancer types and with poor disease prognosis. However, the role of Plk1 in melanoma is not well established. Here, we show that Plk1 is overexpressed in both clin. tissue specimens and cultured human melanoma cells (WM115, A375, and HS294T) when compared with normal skin tissues and cultured normal melanocytes, resp. Furthermore, Plk1 gene knockdown through Plk1-specific shRNA or its activity inhibition by a small-mol. inhibitor resulted in a significant decrease in the viability and growth of melanoma cells without affecting normal human melanocytes. In addn., Plk1 inhibition resulted in a significant (i) decrease in clonogenic survival, (ii) multiple mitotic errors, (iii) G2/M cell-cycle arrest, and (iv) apoptosis of melanoma cells. This study suggests that Plk1 may have a functional relevance toward melanoma development and/or progression. We suggest that the targeting of Plk1 may be a viable approach for the treatment of melanoma.
- 10Takahashi, T.; Sano, B.; Nagata, T.; Kato, H.; Sugiyama, Y.; Kunieda, K.; Kimura, M.; Okano, Y.; Saji, S. Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancers Cancer Sci. 2003, 94 (2) 148– 152[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjtFGjsbo%253D&md5=01e134d298bb5c7e619ac45b3a2df5d6Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancersTakahashi, Takao; Sano, Bun; Nagata, Takayasu; Kato, Hiroki; Sugiyama, Yasuyuki; Kunieda, Katsuyuki; Kimura, Masashi; Okano, Yukio; Saji, ShigetoyoCancer Science (2003), 94 (2), 148-152CODEN: CSACCM; ISSN:1347-9032. (Japanese Cancer Association)PLK (polo-like kinase), the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae, belongs to a family of Ser/Thr kinases. It is intimately involved in spindle formation and chromosome segregation during mitosis. The purpose of this study was to det. whether PLK1 is overexpressed in primary colorectal cancer specimens as compared with normal colon mucosa and to assess its relation to other kinases as a potential new tumor marker. In the present study, immunohistochem. analyses were performed of PLK1 expression in 78 primary colorectal cancers as well as 15 normal colorectal specimens. Furthermore, the authors examd. the relationship between other kinases, Aurora-A and Aurora-C, and PLK1 expression. In normal colon mucosa, some crypt cells showed weakly pos. staining for PLK1 in 13 out of 15 cases, the remaining cases being neg. Elevated expression of PLK1 was obsd. in 57 (73.1%) of the colorectal cancers, statistically significant assocns. being evident with pT (primary tumor invasion) (P=0.0006, Mann-Whitney U test), pN (regional lymph nodes) (P=0.008, χ2 test) and the Dukes' classification (P=0.0005, Mann-Whitney U test). Mean proliferating cell nuclear antigen-labeling index was 52.3%, with a range of 24.1% to 77.3%. Values for lesions with high and low PLK1 expression were 54.7±10.3% (mean±SD) and 45.9±11.9% (P=0.002, Student's t test). PLK1 was significantly assocd. with Aurora-A, but PLK1 staining was more diffuse and extensive than for Aurora-A or Aurora-C. Interestingly, PLK1 overexpression was significantly assocd. with p53 accumulation in colorectal cancers. These results suggest overexpression of PLK1 might be of pathogenic, prognostic and proliferative importance, so that this kinase might have potential as a new tumor marker for colorectal cancers.
- 11Wolf, G.; Elez, R.; Doermer, A.; Holtrich, U.; Ackermann, H.; Stutte, H. J.; Altmannsberger, H. M.; Rubsamen-Waigmann, H.; Strebhardt, K. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer Oncogene 1997, 14 (5) 543– 549[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXht1Git7g%253D&md5=974b1041d3535545c5242cf75cbcda05Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancerWolf, Georg; Elez, robert; Doermer, Andreas; Holtrich, Uwe; Ackermann, Hanns; Stutte, Hans Jochen; Altmannsberger, Hans-Michael; Ruebsamen-Waigmann, Helga; Strebhardt, KlausOncogene (1997), 14 (5), 543-549CODEN: ONCNES; ISSN:0950-9232. (Stockton)Previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot expts. PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived significantly longer (5 yr survival rate = 51.8%) than those with high levels of PLK transcripts (24.2%). No statistically significant correlation was found between PLK mRNA expression and age, sex, TNM status, histol. type or degree of differentiation. Interestingly, the prognosis of patients in post-surgical stages I and II was correlated with PLK expression (5 yr survival rates in stage I: 69.1% (moderate PLK) - 43.5% (high PLK), or in stage II: 51.9% (moderate PLK) - 9.9% (high PLK)). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.
- 12Wolf, G.; Hildenbrand, R.; Schwar, C.; Grobholz, R.; Kaufmann, M.; Stutte, H. J.; Strebhardt, K.; Bleyl, U. Polo-like kinase: a novel marker of proliferation: correlation with estrogen-receptor expression in human breast cancer Pathol. Res. Pract. 2000, 196 (11) 753– 759[ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjtFyqtQ%253D%253D&md5=4080e32f88ac6cab14f870dbe00c74d0Polo-like kinase: a novel marker of proliferation: correlation with estrogen-receptor expression in human breast cancerWolf, Georg; Hildenbrand, Ralf; Schwar, Christian; Grobholz, Rainer; Kaufmann, Manfred; Stutte, Hans-Jochen; Strebhardt, Klaus; Bleyl, UwePathology, Research and Practice (2000), 196 (11), 753-759CODEN: PARPDS; ISSN:0344-0338. (Urban & Fischer Verlag)Previous data have shown that the mRNA-expression of the serine/threonine-kinase polo-like kinase (PLK) is closely correlated with the survival of patients suffering from a subset of malignant tumors. PLK-mRNA and protein-expression are restricted to cells in the cell cycle. PLK-mRNA-transcripts are highly abundant in proliferating cells; no gene expression is found in G0-phase cells. Here the authors investigated the mRNA- and protein-expression of PLK- and estrogen-receptor (ER) in human breast-carcinoma by northern-blotting, RT-PCR and immunohistochem. The expression of MIB-I was detd. on serial sections. Anal. of the immunohistochem. data revealed a close correlation between the ER and PLK-expression (r = 0.677). No relation between the mRNA-expression of ER and PLK was found. Furthermore, no correlation for the protein expression of PLK and MIB-I exists. The influence of estrogen (ES) is known to have proliferative potential. The expression of ER correlates with the ES-plasma-level. In addn., the hormone cycle of premenopausal women undergoes rapid vacillations with varying effects on the proliferating tumor cells, e.g., growth induction. The authors' results therefore show that ER-expression is not only of therapeutic value for the clinician, but it may also be a tool for detg. the tumor proliferation index more precisely by integrating the hormone-mediated proliferation stimulus.
- 13Weichert, W.; Denkert, C.; Schmidt, M.; Gekeler, V.; Wolf, G.; Kobel, M.; Dietel, M.; Hauptmann, S. Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma Br. J. Cancer. 2004, 90 (4) 815– 821[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXht12iurk%253D&md5=2663adc8427cfbe6be77bb65b7cd1a78Polo-like kinase isoform expression is a prognostic factor in ovarian carcinomaWeichert, W.; Denkert, C.; Schmidt, M.; Gekeler, V.; Wolf, G.; Koebel, M.; Dietel, M.; Hauptmann, S.British Journal of Cancer (2004), 90 (4), 815-821CODEN: BJCAAI; ISSN:0007-0920. (Nature Publishing Group)The Polo-like kinase (PLK) family comprises three serine/threonine kinases, functionally involved in signal transduction pathways essential for the accomplishment of mitosis in both normal and malignant cells. Moreover, certain PLKs have been functionally linked to cytoskeletal reorganization. In this study, the expression of PLK1 and PLK3 was detd. immunohistochem. in tissue specimen of normal ovaries (n=9), cystadenomas (n=17), borderline tumors (n=13) and ovarian carcinomas (n=77). PLK 1 and PLK3 expression was low in normal ovarian surface epithelium and borderline tumors, with moderately higher expression levels in cystadenomas. In ovarian carcinomas, 26% of cases were PLK1 pos. and 50.6% of cases were PLK3 pos. A pos. correlation of both PLK1 and PLK3 expression with indicators of mitotic frequency could be established. The overexpression of either isoenzyme had an impact on patient prognosis with shortened survival time for patients with tumors pos. for PLK1 (P=0.02) and PLK3 (P=0.02), but only PLK1 expression remained a prognostic factor in multivariate survival anal. (P=0.03). The results of this study, if interpreted in the context of recently published functional data, suggest that inhibition of PLKs might represent an interesting new targeted approach for chemotherapy of epithelial ovarian cancer. Furthermore, this study suggests that PLK1 is a novel independent prognostic marker in ovarian carcinomas.
- 14Strebhardt, K.; Kneisel, L.; Linhart, C.; Bernd, A.; Kaufmann, R. Prognostic value of pololike kinase expression in melanomas JAMA, J. Am. Med. Assoc. 2000, 283 (4) 479– 480[ Crossref], [ PubMed], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3c7itlaltQ%253D%253D&md5=0e0cd545d9bac745249ff24898506de0Prognostic value of pololike kinase expression in melanomasStrebhardt K; Kneisel L; Linhart C; Bernd A; Kaufmann RJAMA : the journal of the American Medical Association (2000), 283 (4), 479-80 ISSN:0098-7484.There is no expanded citation for this reference.
- 15Weichert, W.; Schmidt, M.; Gekeler, V.; Denkert, C.; Stephan, C.; Jung, K.; Loening, S.; Dietel, M.; Kristiansen, G. Polo-like kinase 1 is overexpressed in prostate cancer and linked to higher tumor grades Prostate 2004, 60 (3) 240– 245[ Crossref], [ PubMed], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntFGks7s%253D&md5=b19f96f40a54625fb9bbcbfa4868e6a7Polo-like kinase I is overexpressed in prostate cancer and linked to higher tumor gradesWeichert, Wilko; Schmidt, Mathias; Gekeler, Volker; Denkert, Carsten; Stephan, Carsten; Jung, Klaus; Loening, Stefan; Dietel, Manfred; Kristiansen, GlenProstate (New York, NY, United States) (2004), 60 (3), 240-245CODEN: PRSTDS; ISSN:0270-4137. (Wiley-Liss, Inc.)Polo-like kinase 1 (PLK1) is known to be one of the key players in the regulation of mitosis of both normal and malignant transformed cells. Moreover, several studies reported an overexpression of PLK1 in human malignancies compared to the corresponding tissue of origin. In this study, expression of PLK1 was investigated by immunohistochem. in 78 tissue specimens of prostate carcinoma and in adjacent normal prostate tissue as well as in benign prostate hyperplasia. PLK1 expression was semiquant. scored and subsequently correlated to clinicopathol. parameters and patient prognosis. No significant PLK1 expression was obsd. in normal prostate glandular epithelium and stroma. Specimens of benign prostate hyperplasia were PLK1-neg. as well. In contrast, 52.6% of all prostate carcinomas showed strong expression of PLK1. High grade intraepithelial lesions, if present, stained almost invariably in the same manner as the resp. invasive tumors. Expression of PLK1 correlated pos. with Gleason grade. No other significant correlations of PLK1 expression with either tumor stage, WHO tumor grade, preoperative PSA, age, or resection margins could be established. In an anal. for differences in PSA-relapse-free survival time, PLK1 expression was not a prognostic marker. These results demonstrate a high rate of PLK1-positivity in prostate cancer which suggests involvement of PLK1 in tumorigenesis and progression in this tumor entity. Therefore, targeted strategies focusing on PLK1 inhibition might represent a promising new chemotherapeutic approach in prostate cancer.
- 16Hikichi, Y.; Honda, K.; Hikami, K.; Miyashita, H.; Kaieda, I.; Murai, S.; Uchiyama, N.; Hasegawa, M.; Kawamoto, T.; Sato, T.; Ichikawa, T.; Cao, S.; Nie, Z.; Zhang, L.; Yang, J.; Kuida, K.; Kupperman, E. TAK-960, a novel, orally available, selective inhibitor of polo-like kinase 1, shows broad-spectrum preclinical antitumor activity in multiple dosing regimens Mol. Cancer Ther. 2012, 11 (3) 700– 709[ Crossref], [ PubMed], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlKis7g%253D&md5=9b4798370cd6f19205c6c1982890861fTAK-960, a Novel, Orally Available, Selective Inhibitor of Polo-Like Kinase 1, Shows Broad-spectrum Preclinical Antitumor Activity in Multiple Dosing RegimensHikichi, Yuichi; Honda, Kohei; Hikami, Kouki; Miyashita, Hitoshi; Kaieda, Isao; Murai, Saomi; Uchiyama, Noriko; Hasegawa, Maki; Kawamoto, Tomohiro; Sato, Takashi; Ichikawa, Takashi; Cao, Sheldon; Nie, Zhe; Zhang, Lilly; Yang, Johnny; Kuida, Keisuke; Kupperman, ErikMolecular Cancer Therapeutics (2012), 11 (3), 700-709CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been assocd. with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G2-M cells, aberrant polo mitosis morphol., and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC50 >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clin. evaluation in patients with advanced cancers.
- 17Mross, K.; Frost, A.; Steinbild, S.; Hedbom, S.; Rentschler, J.; Kaiser, R.; Rouyrre, N.; Trommeshauser, D.; Hoesl, C. E.; Munzert, G. Phase I dose escalation and pharmacokinetic study of BI 2536, a novel Polo-like kinase 1 inhibitor, in patients with advanced solid tumors J. Clin. Oncol. 2008, 26 (34) 5511– 5517[ Crossref], [ PubMed], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVSktg%253D%253D&md5=b2a7c464ca76909221e953a266757e89Phase I dose escalation and pharmacokinetic study of BI 2536, a novel polo-like kinase 1 inhibitor, in patients with advanced solid tumorsMross, Klaus; Frost, Annette; Steinbild, Simone; Hedbom, Susanne; Rentschler, Jochen; Kaiser, Rolf; Rouyrre, Nicolas; Trommeshauser, Dirk; Hoesl, Cornelia E.; Munzert, GerdJournal of Clinical Oncology (2008), 26 (34), 5511-5517CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology)BI 2536 is a novel, potent, and highly specific inhibitor of polo-like kinase 1 (Plk1), which has an essential role in the regulation of mitotic progression. The aim of this trial was to identify the max. tolerated dose (MTD) of BI 2536 and to det. the safety, pharmacokinetics, and antitumor activity in patients who had advanced solid tumors. This phase I trial followed an open label, toxicity-guided, dose-titrn. design. Single doses of BI 2536 (25 to 250 mg) were administered as a 1-h i.v. infusion; patients who experienced clin. benefit were eligible for addnl. treatment courses. Safety and pharmacokinetics were investigated. Tumor response was evaluated according to Response Evaluation Criteria in Solid Tumors Group guidelines. The MTD was defined at 200 mg in a total of 40 patients entered; reversible neutropenia constituted the dose-limiting toxicity (DLT) and the most frequent adverse event at the MTD (grade 3 to 4; 56%). Nausea (52%), fatigue (52%), and anorexia (44%) also were common and were mostly of mild to moderate intensity (Common Terminol. Criteria of Adverse Events ≤ grade 2). One patient experienced a transient partial response. At doses equal to or greater than the MTD, 23% of patients experienced disease stabilization for 3 or more months. Dose-proportional increases in the max. plasma concn. and total exposure were obsd. BI 2536 showed a high total clearance and high distribution into tissue. The MTD of BI 2536 when administered as a single-dose, 1-h infusion was 200 mg; BI 2536 was well tolerated and showed a favorable pharmacokinetic profile. Antitumor activity of BI 2536 was obsd.
- 18Jimeno, A.; Li, J.; Messersmith, W. A.; Laheru, D.; Rudek, M. A.; Maniar, M.; Hidalgo, M.; Baker, S. D.; Donehower, R. C. Phase I study of ON 01910.Na, a novel modulator of the Polo-like kinase 1 pathway, in adult patients with solid tumors J. Clin. Oncol. 2008, 26 (34) 5504– 5510[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVSksQ%253D%253D&md5=d7ef274ca89ecf6d5c1b1ce08660b54dPhase I study of ON 01910.Na, a novel modulator of the polo-like kinase 1 pathway, in adult patients with solid tumorsJimeno, Antonio; Li, Jing; Messersmith, Wells A.; Laheru, Daniel; Rudek, Michelle A.; Maniar, Manoj; Hidalgo, Manuel; Baker, Sharyn D.; Donehower, Ross C.Journal of Clinical Oncology (2008), 26 (34), 5504-5510CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology)Purpose: We conducted a first-in-man (to our knowledge) phase I study to det. the dose-limiting toxicities (DLTs), characterize the pharmacokinetic profile, and document any antitumor activity of ON 01910.Na, a new chem. entity that arrests cancer cells in G2/M by modulating mitotic regulatory pathways including polo-like kinase 1 (Plk1). Patients and Methods: Patients had solid tumors refractory to std. therapy. ON 01910.Na was administered as a 2-h infusion on days 1, 4, 8, 11, 15, and 18 in 28-day cycles. The starting dose was 80 mg, and an accelerated titrn. schedule (single-patient cohorts) was used for escalation. Pharmacokinetics were studied on days 1 and 15 of cycle 1. Results: Twenty patients (11 women and nine men; age 46 to 73 years) were enrolled onto the study. Dose levels of 80, 160, 320, 480, 800, 1,280, 2,080, and 3,120 mg were evaluated in single-patient cohorts. A DLT and addnl. grade 2 toxicities made the 4,370-mg dose (n = 6) not tolerable, and the next lower dose cohort (3,120 mg) was expanded to six assessable patients. Toxicities were skeletal, abdominal, and tumor pain; nausea; urge to defecate; and fatigue. Hematol. toxicity was infrequent and mild. ON 01910.Na pharmacokinetics were characterized by a rapid distribution phase (distribution half-life, 1 h) and a relatively slow elimination phase (elimination half-life, 27 h). A refractory ovarian cancer patient had an objective response after four cycles and remained progression free for 24 mo. Conclusion: ON 01910.Na showed a distinct but moderate toxicity pattern. The recommended phase II dose of ON 01910.Na with this schedule of administration is 3,120 mg. Single-agent activity was documented in an ovarian cancer patient.
- 19Schoffski, P.; Awada, A.; Dumez, H.; Gil, T.; Bartholomeus, S.; Wolter, P.; Taton, M.; Fritsch, H.; Glomb, P.; Munzert, G. A phase I, dose-escalation study of the novel Polo-like kinase inhibitor volasertib (BI 6727) in patients with advanced solid tumours Eur. J. Cancer 2012, 48 (2) 179– 186[ Crossref], [ PubMed], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC387gs1Oitg%253D%253D&md5=ca4316cf6705a2cb92ab243c1c151c35A phase I, dose-escalation study of the novel Polo-like kinase inhibitor volasertib (BI 6727) in patients with advanced solid tumoursSchoffski Patrick; Awada Ahmad; Dumez Herlinde; Gil Thierry; Bartholomeus Sylvie; Wolter Pascal; Taton Martine; Fritsch Holger; Glomb Patricia; Munzert GerdEuropean journal of cancer (Oxford, England : 1990) (2012), 48 (2), 179-86 ISSN:.BACKGROUND: Volasertib (BI 6727) is a potent and selective cell-cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Polo-like kinase (Plk). This phase I dose-escalation study evaluated the maximum tolerated dose (MTD) of volasertib, safety and efficacy, and pharmacokinetic (PK) parameters. METHODS: This trial followed an open-label, toxicity-guided dose-titration design. Patients with progressive advanced or metastatic solid tumours received a single 1-h infusion of volasertib every 3 weeks. A total of 65 patients were treated at doses of 12-450 mg. RESULTS: Reversible haematological toxicity was the main side-effect; thrombocytopenia, neutropenia, and febrile neutropenia constituting the main dose-limiting events. Anaemia (all grades 22%; grade 3: 8%), neutropenia (15%; grade 3/4: 14%), fatigue (15%; grade 3: 2%), and thrombocytopenia (14%; grade 3/4: 14%) were the most frequent drug-related adverse events. The MTD was 400mg; however, 300 mg was the recommended dose for further development based on overall tolerability. Three patients achieved confirmed partial response. Stable disease as best response was reported in 40% of patients. Two patients remained progression free for >1 year. PK analysis showed no indication of deviation from 'dose-linear PK' behaviour, a large volume of distribution (>4000 l), moderate clearance and a long half-life (~111 h). CONCLUSION: This first-in-man trial demonstrated a favourable PK profile of volasertib, with manageable toxicities. As expected, the most common events were haematological. Encouraging preliminary antitumour activity has been observed, supporting Plk inhibition as a therapeutic approach. Clinical development of volasertib in phase II monotherapy and combination trials is ongoing.
- 20Olmos, D.; Barker, D.; Sharma, R.; Brunetto, A. T.; Yap, T. A.; Taegtmeyer, A. B.; Barriuso, J.; Medani, H.; Degenhardt, Y. Y.; Allred, A. J.; Smith, D. A.; Murray, S. C.; Lampkin, T. A.; Dar, M. M.; Wilson, R.; de Bono, J. S.; Blagden, S. P. Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies Clin. Cancer. Res. 2011, 17 (10) 3420– 3430[ Crossref], [ PubMed], [ CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVGit7c%253D&md5=3cf3213934f3a34aaeee469dc9cb6529Phase I Study of GSK461364, a Specific and Competitive Polo-like Kinase 1 Inhibitor, in Patients with Advanced Solid MalignanciesOlmos, David; Barker, Douglas; Sharma, Rohini; Brunetto, Andre T.; Yap, Timothy A.; Taegtmeyer, Anne B.; Barriuso, Jorge; Medani, Hanine; Degenhardt, Yan Y.; Allred, Alicia J.; Smith, Deborah A.; Murray, Sharon C.; Lampkin, Thomas A.; Dar, Mohammed M.; Wilson, Richard; de Bono, Johann S.; Blagden, Sarah P.Clinical Cancer Research (2011), 17 (10), 3420-3430CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)PURPOSE: GSK461364 is an ATP-competitive inhibitor of polo-like kinase 1 (Plk1). A phase I study of two schedules of i.v. GSK461364 was conducted. Exptl. Design: GSK461364 was administered in escalating doses to patients with solid malignancies by two schedules, either on days 1, 8, and 15 of 28-day cycles (schedule A) or on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (schedule B). Assessments included pharmacokinetic and pharmacodynamic profiles, as well as marker expression studies in pretreatment tumor biopsies. RESULTS: Forty patients received GSK461364: 23 patients in schedule A and 17 in schedule B. Dose-limiting toxicities (DLT) in schedule A at 300 mg (2 of 7 patients) and 225 mg (1 of 8 patients) cohorts included grade 4 neutropenia and/or grade 3-4 thrombocytopenia. In schedule B, DLTs of grade 4 pulmonary emboli and grade 4 neutropenia occurred at 7 or more days at 100 mg dose level. Venous thrombotic emboli (VTE) and myelosuppression were the most common grade 3-4, drug-related events. Pharmacokinetic data indicated that AUC (area under the curve) and Cmax (max. concn.) were proportional across doses, with a half-life of 9 to 13 h. Pharmacodynamic studies in circulating tumor cells revealed an increase in phosphorylated histone H3 (pHH3) following drug administration. A best response of prolonged stable disease of more than 16 wk occurred in 6 (15%) patients, including 4 esophageal cancer patients. Those with prolonged stable disease had greater expression of Ki-67, pHH3, and Plk1 in archived tumor biopsies. CONCLUSIONS: The final recommended phase II dose for GSK461364 was 225 mg administered i.v. in schedule A. Because of the high incidence (20%) of VTE, for further clin. evaluation, GSK461364 should involve coadministration of prophylactic anticoagulation. Clin Cancer Res; 17(10); 3420-30.
- 21Ma, W. W.; Messersmith, W. A.; Dy, G. K.; Weekes, C. D.; Whitworth, A.; Ren, C.; Maniar, M.; Wilhelm, F.; Eckhardt, S. G.; Adjei, A. A.; Jimeno, A. Phase I study of Rigosertib, an inhibitor of the phosphatidylinositol 3-kinase and Polo-like kinase 1 pathways, combined with gemcitabine in patients with solid tumors and pancreatic cancer Clin. Cancer. Res. 2012, 18 (7) 2048– 2055[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFOlsrY%253D&md5=04ca490691ff61a39988ff9bc1008792Phase I Study of Rigosertib, an Inhibitor of the Phosphatidylinositol 3-Kinase and Polo-like Kinase 1 Pathways, Combined with Gemcitabine in Patients with Solid Tumors and Pancreatic CancerMa, Wen Wee; Messersmith, Wells A.; Dy, Grace K.; Weekes, Colin D.; Whitworth, Amy; Ren, Chen; Maniar, Manoj; Wilhelm, Francois; Eckhardt, S. Gail; Adjei, Alex A.; Jimeno, AntonioClinical Cancer Research (2012), 18 (7), 2048-2055CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)PURPOSE: Rigosertib, a dual non-ATP inhibitor of polo-like kinase 1 (Plk1) and phosphoinositide 3-kinase pathways (PI3K), and gemcitabine have synergistic antitumor activity when combined in preclin. studies. This phase I study aimed to det. the recommended phase II dose (RPTD) of the combination of rigosertib and gemcitabine in patients with cancer. Exptl. Design: Patients with solid tumors who failed std. therapy or were candidates for gemcitabine-based therapy were eligible. Gemcitabine was administered on days 1, 8, and 15 on a 28-day cycle and rigosertib on days 1, 4, 8, 11, 15, and 18. Pharmacokinetic studies were conducted during an expansion cohort of patients with advanced pancreatic ductal adenocarcinoma (PDA). RESULTS: Forty patients were treated, 19 in the dose-escalation phase and 21 in the expansion cohort. Dose levels evaluated were (gemcitabine/rigosertib mg/m2): 750/600 (n = 4), 750/1,200 (n = 3), 1,000/600 (n = 3), 1,000/1,200 (n = 3), and 1,000/1,800 (n = 6 + 21). One dose-limiting toxicity (death) occurred at the highest dose level (1,000/1,800) tested. Non-dose-limiting ≥grade II/III toxicities included neutropenia, lymphopenia, thrombocytopenia, fatigue, and nausea. Grade III/IV neutropenia, thrombocytopenia, and fatigue were seen in two, one, and two patients in the expansion cohort. Partial responses were obsd. in PDA, thymic cancer, and Hodgkin lymphoma, including gemcitabine-pretreated PDA. The pharmacokinetic profile of rigosertib was not affected by gemcitabine. CONCLUSION: The RPTD established in this study is rigosertib 1,800 mg/m2 and gemcitabine 1,000 mg/m2. This regimen is well tolerated with a toxicity profile of the combination similar to the profile of gemcitabine alone. Antitumor efficacy was obsd. in patients who previously progressed on gemcitabine-based therapy. Clin Cancer Res; 18(7); 2048-55.
- 22Rudolph, D.; Steegmaier, M.; Hoffmann, M.; Grauert, M.; Baum, A.; Quant, J.; Haslinger, C.; Garin-Chesa, P.; Adolf, G. R. BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity Clin. Cancer. Res. 2009, 15 (9) 3094– 3102[ Crossref], [ PubMed], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltFOrtbo%253D&md5=daed550be9d9b99ac6797c359039d295BI 6727, A Polo-like Kinase Inhibitor with Improved Pharmacokinetic Profile and Broad Antitumor ActivityRudolph, Dorothea; Steegmaier, Martin; Hoffmann, Matthias; Grauert, Matthias; Baum, Anke; Quant, Jens; Haslinger, Christian; Garin-Chesa, Pilar; Adolf, Guenther R.Clinical Cancer Research (2009), 15 (9), 3094-3102CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. The first compd. in this series, suitable for i.v. administration, has entered clin. development. To fully explore the potential of Polo-like kinase 1 inhibition in oncol., we have profiled addnl. compds. and now describe a novel clin. candidate. BI 6727 is a highly potent (enzyme IC50 = 0.87 nmol/L, EC50 = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high vol. of distribution and a long terminal half-life in mice (Vss = 7.6 L/kg, t1/2 = 46 h) and rats (Vss = 22 L/kg, t1/2 = 54 h). Second, BI 6727 has physicochem. and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clin. studies have been initiated.
- 23Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Mutations of the BRAF gene in human cancer Nature 2002, 417 (6892) 949– 954[ Crossref], [ PubMed], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XkvVagsLo%253D&md5=4625be53ad9a382d51b6ee753d0c17e8Mutations of the BRAF gene in human cancerDavies, Helen; Bignell, Graham R.; Cox, Charles; Stephens, Philip; Edkins, Sarah; Clegg, Sheila; Teague, Jon; Woffendin, Hayley; Garnett, Mathew J.; Bottomley, William; Davis, Neil; Dicks, Ed; Ewing, Rebecca; Floyd, Yvonne; Gray, Kristian; Hall, Sarah; Hawes, Rachel; Hughes, Jaime; Kosmidou, Vivian; Menzies, Andrew; Mould, Catherine; Parker, Adrian; Stevens, Claire; Watt, Stephen; Hooper, Steven; Wilson, Rebecca; Jayatilake, Hiran; Gusterson, Barry A.; Cooper, Colin; Shipley, Janet; Hargrave, Darren; Pritchard-Jones, Katherine; Maitland, Norman; Chenevix-Trench, Georgia; Riggins, Gregory J.; Bigner, Darell D.; Palmieri, Giuseppe; Cossu, Antonio; Flanagan, Adrienne; Nicholson, Andrew; Ho, Judy W. C.; Leung, Suet Y.; Yuen, Siu T.; Weber, Barbara L.; Seigler, Hilliard F.; Darrow, Timothy L.; Paterson, Hugh; Marais, Richard; Marshall, Christopher J.; Wooster, Richard; Stratton, Michael R.; Futreal, P. AndrewNature (London, United Kingdom) (2002), 417 (6892), 949-954CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Cancers arise owing to the accumulation of mutations in crit. genes that alter normal programs of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for anal. signalling pathways in which at least one gene is mutated in human cancer. The RAS-RAF-MEK-ERK-MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
- 24Spandidos, A.; Wang, X.; Wang, H.; Seed, B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification Nucleic Acids Res. 2010, 38 (Database issue) D792– D799[ Crossref], [ PubMed], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlOlsw%253D%253D&md5=b140f43714130491f9134a839b011f16PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantificationSpandidos, Athanasia; Wang, Xiaowei; Wang, Huajun; Seed, BrianNucleic Acids Research (2010), 38 (Database Iss), D792-D799CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)PrimerBank (http://pga.mgh.harvard.edu/primerbank/) is a public resource for the retrieval of human and mouse primer pairs for gene expression anal. by PCR and Quant. PCR (QPCR). A total of 306 800 primers covering most known human and mouse genes can be accessed from the PrimerBank database, together with information on these primers such as Tm, location on the transcript and amplicon size. For each gene, at least one primer pair has been designed and in many cases alternative primer pairs exist. Primers have been designed to work under the same PCR conditions, thus facilitating high-throughput QPCR. There are several ways to search for primers for the gene(s) of interest, such as by: GenBank accession no., NCBI protein accession no., NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). In all, 26 855 primer pairs covering most known mouse genes have been exptl. validated by QPCR, agarose gel anal., sequencing and BLAST, and all validation data can be freely accessed from the PrimerBank web site.
- 25Neilson, K. A.; Ali, N. A.; Muralidharan, S.; Mirzaei, M.; Mariani, M.; Assadourian, G.; Lee, A.; van Sluyter, S. C.; Haynes, P. A. Less label, more free: approaches in label-free quantitative mass spectrometry Proteomics 2011, 11 (4) 535– 553[ Crossref], [ PubMed], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVSnu74%253D&md5=4b993f82d81a20eaf1602869b481e032Less label, more free: Approaches in label-free quantitative mass spectrometryNeilson, Karlie A.; Ali, Naveid A.; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Garine; Lee, Albert; van Sluyter, Steven C.; Haynes, Paul A.Proteomics (2011), 11 (4), 535-553CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)In this review we examine techniques, software, and statistical analyses used in label-free quant. proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further expts. to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labeling techniques, and forward-looking applications for label-free quant. data are presented. We conclude that label-free quant. proteomics is a reliable, versatile, and cost-effective alternative to labeled quantitation.
- 26Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics Mol. Cell. Proteomics 2005, 4 (10) 1487– 1502[ Crossref], [ PubMed], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFeltbzI&md5=73387faedda13dcb475b7f5a9f54ca15Comparison of label-free methods for quantifying human proteins by shotgun proteomicsOld, William M.; Meyer-Arendt, Karen; Aveline-Wolf, Lauren; Pierce, Kevin G.; Mendoza, Alex; Sevinsky, Joel R.; Resing, Katheryn A.; Ahn, Natalie G.Molecular and Cellular Proteomics (2005), 4 (10), 1487-1502CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. The authors describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using std. proteins spiked into a complex sample. Linearity and good agreement between obsd. vs. expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio ests. Peak intensity values useful for protein quantitation ranged from 107 to 1011 counts with no obvious satn. effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (±2σ) when ≥3 peptides/protein were shared between samples. Protein ratios were detd. with high confidence from spectral counts when max. spectral counts were ≥4 spectra/protein, and replicates showed equiv. measurements well within 95% confidence limits. In further tests, complex samples were sepd. by gel exclusion chromatog., quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios detd. by spectral counting agreed well with those detd. from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate ests. of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p < 0.1 showed good correlations with parallel measurements of changes in mRNA expression.
- 27Zhu, W.; Smith, J. W.; Huang, C. M. Mass spectrometry-based label-free quantitative proteomics J. Biomed. Biotechnol. 2010, 2010, 840518[ Crossref], [ PubMed], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1MjlvF2gsg%253D%253D&md5=c97206c0c551ee4bf2ab4bde6aeb9c6eMass spectrometry-based label-free quantitative proteomicsZhu Wenhong; Smith Jeffrey W; Huang Chun-MingJournal of biomedicine & biotechnology (2010), 2010 (), 840518 ISSN:.In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies.
- 28Fedorova, G.; Randak, T.; Lindberg, R. H.; Grabic, R. Comparison of the quantitative performance of a Q-Exactive high-resolution mass spectrometer with that of a triple quadrupole tandem mass spectrometer for the analysis of illicit drugs in wastewater Rapid. Commun. Mass. Sp. 2013, 27 (15) 1751– 1762
- 29Jones, K. A.; Kim, P. D.; Patel, B. B.; Kelsen, S. G.; Braverman, A.; Swinton, D. J.; Gafken, P. R.; Jones, L. A.; Lane, W. S.; Neveu, J. M.; Leung, H. C.; Shaffer, S. A.; Leszyk, J. D.; Stanley, B. A.; Fox, T. E.; Stanley, A.; Hall, M. J.; Hampel, H.; South, C. D.; de la Chapelle, A.; Burt, R. W.; Jones, D. A.; Kopelovich, L.; Yeung, A. T. Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers J. Proteome Res. 2013, 12 (10) 4351– 4365[ ACS Full Text
], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtl2rtLfE&md5=0d14c16c3ea10d538b386ab7cc343f1fImmunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass SpectrometersJones, Kelly A.; Kim, Phillip D.; Patel, Bhavinkumar B.; Kelsen, Steven G.; Braverman, Alan; Swinton, Derrick J.; Gafken, Philip R.; Jones, Lisa A.; Lane, William S.; Neveu, John M.; Leung, Hon-Chiu E.; Shaffer, Scott A.; Leszyk, John D.; Stanley, Bruce A.; Fox, Todd E.; Stanley, Anne; Hall, Michael J.; Hampel, Heather; South, Christopher D.; de la Chapelle, Albert; Burt, Randall W.; Jones, David A.; Kopelovich, Levy; Yeung, Anthony T.Journal of Proteome Research (2013), 12 (10), 4351-4365CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Plasma proteomic expts. performed rapidly and economically using several of the latest high-resoln. mass spectrometers were compared. Four quant. hyperfractionated plasma proteomics expts. were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each expt. compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 mo. Several anal. algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest no. of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity. - 30Oppermann, F. S.; Klammer, M.; Bobe, C.; Cox, J.; Schaab, C.; Tebbe, A.; Daub, H. Comparison of SILAC and mTRAQ quantification for phosphoproteomics on a quadrupole orbitrap mass spectrometer J. Proteome Res. 2013, 12 (9) 4089– 4100[ ACS Full Text], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFOqurnE&md5=9ae3d83d2489e05abe91ee4360b6e2a9Comparison of SILAC and mTRAQ Quantification for Phosphoproteomics on a Quadrupole Orbitrap Mass SpectrometerOppermann, Felix S.; Klammer, Martin; Bobe, Caroline; Cox, Juergen; Schaab, Christoph; Tebbe, Andreas; Daub, HenrikJournal of Proteome Research (2013), 12 (9), 4089-4100CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Advances in mass spectrometric methodol. and instrumentation have promoted a continuous increase in anal. performance in the field of phosphoproteomics. Here, the authors employed the recently introduced quadrupole Orbitrap (Q Exactive) mass spectrometer for quant. signaling anal. to a depth of >15,000 phosphorylation sites. In parallel to the commonly used SILAC approach, the authors evaluated the nonisobaric chem. labeling reagent mTRAQ as an alternative quantification technique. Both enabled high phosphoproteome coverage in H3122 lung cancer cells. Replicate quantifications by mTRAQ identified almost as many significant phosphorylation changes upon treatment with ALK kinase inhibitor crizotinib as found by SILAC quantification. Overall, mTRAQ was slightly less precise than SILAC as evident from a somewhat higher variance of replicate phosphosite ratios. Direct comparison of SILAC- and mTRAQ-quantified phosphosites revealed that the majority of changes were detected by either quantification techniques, but also highlighted the aspect of false neg. identifications in quant. proteomics applications. Further inspection of crizotinib-regulated phosphorylation changes unveiled interference with multiple antioncogenic mechanisms downstream of ALK fusion kinase in H3122 cells. In conclusion, the authors' results demonstrate a strong anal. performance of the Q Exactive in global phosphoproteomics, and establish mTRAQ quantification as a useful alternative to metabolic isotope labeling.
- 31Gamez-Pozo, A.; Sanchez-Navarro, I.; Calvo, E.; Agullo-Ortuno, M. T.; Lopez-Vacas, R.; Diaz, E.; Camafeita, E.; Nistal, M.; Madero, R.; Espinosa, E.; Lopez, J. A.; Fresno Vara, J. A. PTRF/cavin-1 and MIF proteins are identified as non-small cell lung cancer biomarkers by label-free proteomics PLoS One 2012, 7 (3) e33752[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1Citb8%253D&md5=ca235b8ad48bee4f8ef223df049378d8PTRF/Cavin-1 and MIF proteins are identified as non-small cell lung cancer biomarkers by label-free proteomicsGamez-Pozo, Angelo; Sanchez-Navarro, Iker; Calvo, Enrique; Agullo-Ortuno, Maria Teresa; Lopez-Vacas, Rocio; Diaz, Esther; Camafeita, Emilio; Nistal, Manuel; Madero, Rosario; Espinosa, Enrique; Lopez, Juan Antonio; Vara, Juan Angel FresnoPLoS One (2012), 7 (3), e33752CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)With the completion of the human genome sequence, biomedical sciences have entered in the "omics" era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technol. advances and their application in the clin. setting. Our work is designed to build bridges between high-performance proteomics and clin. routine. Protein exts. were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontol. and interactome analyses identified signaling pathways altered on tumor tissue. We have identified 2 proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochem. The application of discovery-based proteomics analyses in clin. samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer.
- 32Guillaume, E.; Berger, B.; Affolter, M.; Kussmann, M. Label-free quantitative proteomics of two Bifidobacterium longum strains J. Proteomics 2009, 72 (5) 771– 784[ Crossref], [ PubMed], [ CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotFCgurc%253D&md5=0d9e9fd732952a7facab0507c7f70f3bLabel-free quantitative proteomics of two Bifidobacterium longum strainsGuillaume, Elisabeth; Berger, Bernard; Affolter, Michael; Kussmann, MartinJournal of Proteomics (2009), 72 (5), 771-784CODEN: JPORFQ; ISSN:1874-3919. (Elsevier Ltd.)Bifidobacteria are gram-pos. anaerobes with a high Guanine/Cytosine genome content. Specific strains are used as probiotics because of their health benefits. Probiotics are live microorganisms with a pos. influence on their host's health. They are used as nutritional supplements and their resistance to conditions of food manufg. and of the gastro-intestinal tract is studied. We report on differential proteomics of two Bifidobacterium longum strains that differ in their heat shock resistance. This was achieved by a comparative qual. survey of their proteomes and relative LC-MS/MS-based label-free protein quantification. Deploying a nano LC-ESI ion-trap mass spectrometer, 165 proteins expressed by the two probiotic strains were identified. Around 50% of these were common to both strains with the remaining half identified in either one of the strains. Using a label-free technol. based on the 3D overlay of retention times, mass-over-charge ratios and ion intensities between LC-MS runs, we found quant. differences in the relative abundance of 19 of the proteins common to both strains. The differentially expressed proteins were classified into categories involving glucose metab., protein synthesis and heat shock proteins. Six of these 19 proteins have been previously reported as being assocd. to heat stress response.
- 33Lopez, M. F.; Sarracino, D. A.; Vogelsang, M.; Sutton, J. N.; Athanas, M.; Krastins, B.; Garces, A.; Prakash, A.; Peterman, S.; Demirjian, Z.; Inglessis-Azuaje, I.; Feeney, K.; Elia, M.; McMullin, D.; Dec, G. W.; Palacios, I.; Lo, E. H.; Buonanno, F.; Ning, M. Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow J. Invest. Med. 2012, 60 (8) 1122– 1130[ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXis1Wltro%253D&md5=093b6e62df55d6420ae78c5118df3a68Heart-Brain Signaling in Patent Foramen Ovale-Related Stroke: Differential Plasma Proteomic Expression Patterns Revealed With a 2-Pass Liquid Chromatography-Tandem Mass Spectrometry Discovery WorkflowLopez, Mary F.; Sarracino, David A.; Vogelsang, Maryann; Sutton, Jennifer N.; Athanas, Michael; Krastins, Bryan; Garces, Alejandra; Prakash, Amol; Peterman, Scott; Demirjian, Zareh; Inglessis-Azuaje, Ignacio; Feeney, Kathleen; Elia, Mikaela; McMullin, David; William Dec, G.; Palacios, Igor; Lo, Eng H.; Buonanno, Ferdinand; Ning, Ming MingJournal of Investigative Medicine (2012), 60 (8), 1122-1130CODEN: JINVFI; ISSN:1081-5589. (Lippincott Williams & Wilkins)Patent foramen ovale (PFO) is highly prevalent and assocd. with more than 150,000 strokes per yr. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiol. to individualize the best clin. treatment for each patient. We initiated a study using a novel quant., 2-pass discovery workflow using high-resoln. liq. chromatog.-mass spectrometry/mass spectrometry coupled with label-free anal. to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quant. differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the mol. landscape of PFO-related physiol., our methods have yielded biol. relevant information on the synergistic and functional redundancy of various cell-signaling mols. with respect to PFO circulatory physiol. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.
- 34Qu, J.; Lesse, A. J.; Brauer, A. L.; Cao, J.; Gill, S. R.; Murphy, T. F. Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses BMC Microbiol. 2010, 10, 162[ Crossref], [ PubMed], [ CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3crht1yjtg%253D%253D&md5=c7700fe8c899a68766de42f902e99caeProteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responsesQu Jun; Lesse Alan J; Brauer Aimee L; Cao Jin; Gill Steven R; Murphy Timothy FBMC microbiology (2010), 10 (), 162 ISSN:.BACKGROUND: Nontypeable Haemophilus influenzae colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, H. influenzae, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by H. influenzae in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient. RESULTS: A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions. CONCLUSIONS: Proteomic expression profiling of H. influenzae grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that H. influenzae adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival in these conditions.
- 35Sutton, J.; Richmond, T.; Shi, X.; Athanas, M.; Ptak, C.; Gerszten, R.; Bonilla, L. Performance characteristics of an FT MS-based workflow for label-free differential MS analysis of human plasma: standards, reproducibility, targeted feature investigation, and application to a model of controlled myocardial infarction Proteomics: Clin. Appl. 2008, 2 (6) 862– 881[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotF2gu7c%253D&md5=a575ec4fe4f417491aa09afc06345562Performance characteristics of an FT MS-based workflow for label-free differential MS analysis of human plasma: standards, reproducibility, targeted feature investigation, and application to a model of controlled myocardial infarctionSutton, Jennifer; Richmond, Tori; Shi, Xu; Athanas, Michael; Ptak, Celeste; Gerszten, Robert; Bonilla, LeoProteomics: Clinical Applications (2008), 2 (6), 862-881CODEN: PCARCU; ISSN:1862-8346. (Wiley-VCH Verlag GmbH & Co. KGaA)Proteomics is undergoing a rapid transformation from a qual. global peptide sequencing discipline into a quant., reproducibility-driven practice. Nowhere is this more evident than in the rapidly expanding field of protein biomarker discovery where the general goal is to uncover statistically robust patterns of differential expression between or among subjects/samples representing distinct biol./temporal states. This report presents the anal. characterization of a label-free LC FT-ICR-MS workflow for differential proteomics anal. of human plasma. The key elements discussed include (i) methodologies for performing properly replicated expts. with highly reproducible sample prepn. and anal., including the use of internal stds. to quantify variance at different steps in the process, (ii) a new methodol. for performing sample re-anal. that uses off-line targeted robotic acquisition of complementary spectral data (e.g. ECD and/or IRMPD) to enhance the identification of differentially expressed peptides/proteins, and (iii) data processing pipelines capable of integrating the automatic statistical anal. of the label-free (LC-) MS signal, together with the intuitive and highly interactive curation and annotation of differential features using the output from std. sequence database search programs. The authors illustrate the application of the complete sample-to-annotated-differential-peptides (-proteins) workflow by describing the acquisition and anal. of a large multidimensional dataset from patients undergoing a controlled myocardial infarction resulting in an exptl. setup in which each patients serve as their own control. Furthermore, the authors discuss a couple illustrative examples of mid-level proteins obsd. in this study whose plasma concns. change consistently within and across patients, in a treatment- and time-dependent fashion.
- 36Tabata, T.; Sato, T.; Kuromitsu, J.; Oda, Y. Pseudo internal standard approach for label-free quantitative proteomics Anal. Chem. 2007, 79 (22) 8440– 8445[ ACS Full Text], [ CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFCrs7rJ&md5=c09279bd627e01348210cf945e8925c2Pseudo Internal Standard Approach for Label-Free Quantitative ProteomicsTabata, Tsuyoshi; Sato, Toshitaka; Kuromitsu, Junro; Oda, YoshiyaAnalytical Chemistry (Washington, DC, United States) (2007), 79 (22), 8440-8445CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Quant. proteome anal. has become a versatile tool to understand biol. functions. Although stable isotope labeling is the most reliable method for quant. mass spectrometry, prepn. of isotope-labeled compds. is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without stds. is not generally accepted as reliable, esp. for small mols. We have developed a novel label-free quant. proteome anal. using pseudo internal stds. (PISs). This idea was derived from northern blotting anal., in which housekeeping genes are used as stds. to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal stds. This new approach is simple and does not require addnl. stds. or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitation and may also be applicable to quant. metabolome anal.
- 37Nilsson, T.; Mann, M.; Aebersold, R.; Yates, J. R., 3rd; Bairoch, A.; Bergeron, J. J. Mass spectrometry in high-throughput proteomics: ready for the big time Nat. Methods 2010, 7 (9) 681– 685[ Crossref], [ PubMed], [ CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtV2hsb3E&md5=5a83e7edaf1a783502a8649fca8480fcMass spectrometry in high-throughput proteomics: ready for the big timeNilsson, Tommy; Mann, Matthias; Aebersold, Ruedi; Yates, John R.; Bairoch, Amos; Bergeron, John J. M.Nature Methods (2010), 7 (9), 681-685CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Mass spectrometry has evolved and matured to a level where it is able to assess the complexity of the human proteome. We discuss some of the expected challenges ahead and promising strategies for success.
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- 39Lin, S. J.; Guarente, L. Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and disease Curr. Opin. Cell Biol. 2003, 15 (2) 241– 246[ Crossref], [ PubMed], [ CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXitV2msL0%253D&md5=a3daf490bbfd30a1abdeaceb742e9235Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and diseaseLin, Su-Ju; Guarente, LeonardCurrent Opinion in Cell Biology (2003), 15 (2), 241-246CODEN: COCBE3; ISSN:0955-0674. (Elsevier Science Ltd.)A review. NAD is a ubiquitous biol. mol. that participates in many metabolic reactions. Recent studies show that NAD also plays important roles in transcriptional regulation, longevity, calorie-restriction-mediated life-span extension and age-assocd. diseases. It has been shown that NAD affects longevity and transcriptional silencing through the regulation of the Sir2p family, which are NAD-dependent deacetylases. Many human diseases are assocd. with changes in NAD level and/or the NAD : NADH ratio, raising the possibility that the Sir2p family might play a role in these diseases.
- 40Sun, F.; Dai, C.; Xie, J.; Hu, X. Biochemical issues in estimation of cytosolic free NAD/NADH ratio PLoS One 2012, 7 (5) e34525[ Crossref], [ PubMed], [ CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsVGht70%253D&md5=5c2f13a3e6c52ec8e09e2bcded4651c3Biochemical issues in estimation of cytosolic free NAD/NADH ratioSun, Feifei; Dai, Chunyan; Xie, Jiansheng; Hu, XunPLoS One (2012), 7 (5), e34525CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al., reported a method to est. this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chem. equil. Numerous studies used L/P ratio to est. the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equil. but not verifying how near it was. In addn., it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equil.; (2) that cytosolic free NAD/NADH could be accurately estd. by L/P only when the conversion is at or very close to equil. otherwise a calcn. error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estn. of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.
- 41Zheng, J. Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review) Oncol. Lett. 2012, 4 (6) 1151– 1157[ PubMed], [ CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVKgtLnO&md5=d692211905f9ecd90cf144a1c533c53eEnergy metabolism of cancer: glycolysis versus oxidative phosphorylation (review)Zheng, JieOncology Letters (2012), 4 (6), 1151-1157CODEN: OLNEB5; ISSN:1792-1074. (Spandidos Publications Ltd.)A review. Metabolic activities in normal cells rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate ATP for energy. Unlike in normal cells, glycolysis is enhanced and OXPHOS capacity is reduced in various cancer cells. It has long been believed that the glycolytic phenotype in cancer is due to a permanent impairment of mitochondrial OXPHOS, as proposed by Otto Warburg. This view is challenged by recent investigations which find that the function of mitochondrial OXPHOS in most cancers is intact. Aerobic glycolysis in many cancers is the combined result of various factors such as oncogenes, tumor suppressors, a hypoxic microenvironment, mtDNA mutations, genetic background and others. Understanding the features and complexity of the cancer energy metab. will help to develop new approaches in early diagnosis and effectively target therapy of cancer.
- 42Liu, X. S.; Li, H.; Song, B.; Liu, X. Polo-like kinase 1 phosphorylation of G2 and S-phase-expressed 1 protein is essential for p53 inactivation during G2 checkpoint recovery EMBO Rep. 2010, 11 (8) 626– 632[ Crossref], [ PubMed], [ CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvFSgtbY%253D&md5=ed86deedb26769e42edcc069273a5b27Polo-like kinase 1 phosphorylation of G2 and S-phase-expressed 1 protein is essential for p53 inactivation during G2 checkpoint recoveryLiu, X. Shawn; Li, Hongchang; Song, Bing; Liu, XiaoqiEMBO Reports (2010), 11 (8), 626-632CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)In response to G2 DNA damage, the p53 pathway is activated to lead to cell-cycle arrest, but how p53 is eliminated during the subsequent recovery process is poorly understood. It has been established that Polo-like kinase 1 (Plk1) controls G2 DNA-damage recovery. However, whether Plk1 activity contributes to p53 inactivation during this process is unknown. In this study, we show that G2 and S-phase-expressed 1 (GTSE1) protein, a neg. regulator of p53, is required for G2 checkpoint recovery and that Plk1 phosphorylation of GTSE1 at Ser 435 promotes its nuclear localization, and thus shuttles p53 out of the nucleus to lead to its degrdn. during the recovery.
- 43Liu, X. S.; Song, B.; Elzey, B. D.; Ratliff, T. L.; Konieczny, S. F.; Cheng, L.; Ahmad, N.; Liu, X. Polo-like kinase 1 facilitates loss of PTEN tumor suppressor-induced prostate cancer formation J. Biol. Chem. 2011, 286 (41) 35795– 35800[ Crossref], [ PubMed], [ CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1yhtrnI&md5=7cd13ab637f92943b832ef3d8041bf90Polo-like Kinase 1 Facilitates Loss of Pten Tumor Suppressor-induced Prostate Cancer FormationLiu, X. Shawn; Song, Bing; Elzey, Bennett D.; Ratliff, Timothy L.; Konieczny, Stephen F.; Cheng, Liang; Ahmad, Nihal; Liu, XiaoqiJournal of Biological Chemistry (2011), 286 (41), 35795-35800, S35795/1-S35795/13CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the mol. mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress and that nuclear function of Pten, but not its phosphatase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a crit. regulator of the cell cycle. We show that overexpression of Plk1 correlates with genetic inactivation of Pten during prostate neoplasia formation. Significantly, we find that elevated Plk1 is crit. for Pten-depleted cells to adapt to mitotic stress for survival and that reintroduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.
- 44Padmanabhan, A.; Li, X.; Bieberich, C. J. Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific Manner J. Biol. Chem. 2013, 288 (20) 14158– 14169[ Crossref], [ PubMed], [ CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvVGqsbs%253D&md5=3591c224c9f45f24a4abc0049b139ff6Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific MannerPadmanabhan, Achuth; Li, Xiang; Bieberich, Charles J.Journal of Biological Chemistry (2013), 288 (20), 14158-14169CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)MYC levels are tightly regulated in cells, and deregulation is assocd. with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degrdn. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate pos. feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biol. roles of the different PKA catalytic subunits.
- 45Song, M. S.; Salmena, L.; Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor Nat. Rev. Mol. Cell. Biol. 2012, 13 (5) 283– 296[ PubMed], [ CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFSrurg%253D&md5=c0d6524414a050876046c647caacb9faThe functions and regulation of the PTEN tumour suppressorSong, Min Sup; Salmena, Leonardo; Pandolfi, Pier PaoloNature Reviews Molecular Cell Biology (2012), 13 (5), 283-296CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. The importance of the physiol. function of phosphatase and tensin homolog (PTEN) is illustrated by its frequent disruption in cancer. By suppressing the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway through its lipid phosphatase activity, PTEN governs a plethora of cellular processes including survival, proliferation, energy metab. and cellular architecture. Consequently, mechanisms regulating PTEN expression and function, including transcriptional regulation, post-transcriptional regulation by non-coding RNAs, post-translational modifications and protein-protein interactions, are all altered in cancer. The repertoire of PTEN functions has recently been expanded to include phosphatase-independent activities and crucial functions within the nucleus. Our increasing knowledge of PTEN and pathologies in which its function is altered will undoubtedly inform the rational design of novel therapies.
- 46Tan, J.; Li, Z.; Lee, P. L.; Guan, P.; Aau, M. Y.; Lee, S. T.; Feng, M.; Lim, C. Z.; Lee, E. Y.; Wee, Z. N.; Lim, Y. C.; Karuturi, R. K.; Yu, Q. PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell activation, and resistance to mTOR-targeted therapy Cancer Discovery 2013, 3 (10) 1156– 1171[ Crossref], [ PubMed], [ CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1WnsrnI&md5=2cc7af3a1c063ec2f7e1181d41037f9ePDK1 Signaling Toward PLK1-MYC Activation Confers Oncogenic Transformation, Tumor-Initiating Cell Activation, and Resistance to mTOR-Targeted TherapyTan, Jing; Li, Zhimei; Lee, Puay Leng; Guan, Peiyong; Aau, Mei Yee; Lee, Shuet Theng; Feng, Min; Lim, Cheryl Zihui; Lee, Eric Yong Jing; Wee, Zhen Ning; Lim, Yaw Chyn; Karuturi, R. K. Murthy; Yu, QiangCancer Discovery (2013), 3 (10), 1156-1171CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)Although 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been predominately linked to the phosphoinositide 3-kinase (PBK)-AKT pathway, it may also evoke addnl. signaling outputs to promote tumorigenesis. Here, we report that PDK1 directly induces phosphorylation of Polo-like kinase I (PLK1), which in turn induces MYC phosphorylation and protein accumulation. We show that PDK1-PLK1-MYC signaling is crit. for cancer cell growth and survival, and small-mol. inhibition of PDK1/PLK1 provides an effective approach for therapeutic targeting of MYC dependency. Intriguingly, PDK1-PLK1-MYC signaling induces an embryonic stem cell-like gene signature assocd. with aggressive tumor behaviors and is a robust signaling axis driving cancer stem cell (CSC) self-renewal. Finally, we show that a PLK1 inhibitor synergizes with an mTOR inhibitor to induce synergistic antitumor effects in colorectal cancer by antagonizing compensatory MYC induction. These findings identify a novel pathway in human cancer and CSC activation and provide a therapeutic strategy for targeting MYC-assocd. tumorigenesis and therapeutic resistance.
- 47Shen, M.; Schmitt, S.; Buac, D.; Dou, Q. P. Targeting the ubiquitin-proteasome system for cancer therapy Expert Opin. Ther. Targets 2013, 17 (9) 1091– 1108[ Crossref], [ PubMed], [ CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1ymtb%252FF&md5=f592626af4335402281ab4cdd9e9edacTargeting the ubiquitin-proteasome system for cancer therapyShen, Min; Schmitt, Sara; Buac, Daniela; Dou, Q. PingExpert Opinion on Therapeutic Targets (2013), 17 (9), 1091-1108CODEN: EOTTAO; ISSN:1472-8222. (Informa Healthcare)A review. Introduction: The ubiquitin-proteasome system (UPS) degrades 80 - 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway. Areas covered: This review provides a broad overview of the UPS and its role in supporting cancer development and progression, esp. in aspects of p53 inactivation, p27 turnover and NF-κB activation. Also, efforts toward the development of small mol. inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed. Expert opinion: Whereas some of the targets in the UPS, such as the 20S proteasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest.
- 48Sahara, K.; Kogleck, L.; Yashiroda, H.; Murata, S. The mechanism for molecular assembly of the proteasome Adv. Biol. Regul. 2014, 54C, 51– 58
- 49Ando, K.; Ozaki, T.; Yamamoto, H.; Furuya, K.; Hosoda, M.; Hayashi, S.; Fukuzawa, M.; Nakagawara, A. Polo-like kinase 1 (Plk1) inhibits p53 function by physical interaction and phosphorylation J. Biol. Chem. 2004, 279 (24) 25549– 26661[ Crossref], [ PubMed], [ CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXksFygtr8%253D&md5=eef2ecb9875f8a7ba8937701ae08f884Polo-like Kinase 1 (Plk1) Inhibits p53 Function by Physical Interaction and PhosphorylationAndo, Kiyohiro; Ozaki, Toshinori; Yamamoto, Hideki; Furuya, Kazushige; Hosoda, Mitsuchika; Hayashi, Syunji; Fukuzawa, Masahiro; Nakagawara, AkiraJournal of Biological Chemistry (2004), 279 (24), 25549-25561CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addn. to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 phys. binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunopptn. analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the phys. interaction with Plk1. The ectopically over-expressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21WAF1, MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter anal., Plk1 had an ability to block the p53-dependent induction of the endogenous p21WAF1. In addn., Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a crit. target of Plk1, and its function is abrogated through the phys. interaction with Plk1.
- 50Bhat, U. G.; Halasi, M.; Gartel, A. L. FoxM1 is a general target for proteasome inhibitors PLoS One 2009, 4 (8) e6593
- 51Dias, S. S.; Hogan, C.; Ochocka, A. M.; Meek, D. W. Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover FEBS Lett. 2009, 583 (22) 3543– 3548[ Crossref], [ PubMed], [ CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWls7zO&md5=2e05278d074a76910b659eec5c5609f0Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnoverDias, Sylvia S.; Hogan, Carol; Ochocka, Anna-Maria; Meek, David W.FEBS Letters (2009), 583 (22), 3543-3548CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)E3 ubiquitin ligase MDM2 (murine double-minute clone 2), promotes the degrdn. of p53 protein under normal homeostatic conditions. Several Ser residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signaling pathways that mediate these phosphorylation events are not fully understood. Here, the authors show that oncogenic and cell cycle-regulatory protein kinase Plk1 (polo-like kinase-1) phosphorylates MDM2 at one of these residues, Ser-260, and stimulates MDM2-mediated turnover of p53. These data were consistent with the idea that deregulation of Plk1 during tumorigenesis may help suppress p53 function.
- 52Fu, Z.; Malureanu, L.; Huang, J.; Wang, W.; Li, H.; van Deursen, J. M.; Tindall, D. J.; Chen, J. Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression Nat. Cell Biol. 2008, 10 (9) 1076– 1082[ Crossref], [ PubMed], [ CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVGgtLnL&md5=6614ca56c9321979d4fc23f49a721f14Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progressionFu, Zheng; Malureanu, Liviu; Huang, Jun; Wang, Wei; Li, Hao; van Deursen, Jan M.; Tindall, Donald J.; Chen, JunjieNature Cell Biology (2008), 10 (9), 1076-1082CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Proper control of entry into and progression through mitosis is essential for normal cell proliferation and the maintenance of genome stability. The mammalian mitotic kinase, Polo-like kinase 1 (Plk1), is involved in multiple stages of mitosis. Here, the authors report that transcription factor, Forkhead Box M1 (FoxM1), a substrate of Plk1, controls a transcriptional program that mediates Plk1-dependent regulation of cell-cycle progression. The C-terminal domain of FoxM1 binds Plk1, and phosphorylation of 2 key residues in this domain by Cdk1 is essential for Plk1-FoxM1 interaction. The formation of the Plk1-FoxM1 complex allows for direct phosphorylation of FoxM1 by Plk1 at G2/M and the subsequent activation of FoxM1 activity, which is required for expression of key mitotic regulators, including Plk1 itself. Thus, Plk1-dependent regulation of FoxM1 activity provides a pos.-feedback loop ensuring tight regulation of transcriptional networks essential for orderly mitotic progression.
- 53McKenzie, L.; King, S.; Marcar, L.; Nicol, S.; Dias, S. S.; Schumm, K.; Robertson, P.; Bourdon, J. C.; Perkins, N.; Fuller-Pace, F.; Meek, D. W. p53-dependent repression of polo-like kinase-1 (PLK1) Cell Cycle 2010, 9 (20) 4200– 4212[ Crossref], [ PubMed], [ CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1ahu74%253D&md5=4dbd41a29399a64db6ed1ab0f5b87fcep53-dependent repression of polo-like kinase-1 (PLK1)McKenzie, Lynsey; King, Sharon; Marcar, Lynnette; Nicol, Sam; Dias, Sylvia S.; Schumm, Katie; Robertson, Pamela; Bourdon, Jean-Christophe; Perkins, Neil; Fuller-Pace, Frances; Meek, David W.Cell Cycle (2010), 9 (20), 4200-4212CODEN: CCEYAS; ISSN:1538-4101. (Landes Bioscience)PLK1 is a crit. mediator of G2/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G2/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G1/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunopptn. anal. indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-assocd. changes in the chromatin. Downregulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Addnl., wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G2/M arrest.
- 54Michael, D.; Oren, M. The p53-Mdm2 module and the ubiquitin system Semin. Cancer Biol. 2003, 13 (1) 49– 58[ Crossref], [ PubMed], [ CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpvVWksrw%253D&md5=187aa679ba07100da1e95e1891777828The p53-Mdm2 module and the ubiquitin systemMichael, Dan; Oren, MosheSeminars in Cancer Biology (2003), 13 (1), 49-58CODEN: SECBE7; ISSN:1044-579X. (Elsevier Science Ltd.)A review. The p53 tumor suppressor protein is a short-lived protein, which is stabilized in response to cellular stress. The ubiquitination and degrdn. of p53 are largely controlled by Mdm2, an oncogenic E3 ligase. Stress signals lead to p53 stabilization either by induction of covalent modifications in Mdm2 and p53, or through altered protein-protein interactions. Mdm2 also harbors a post-ubiquitination function, probably enabling efficient targeting of ubiquitinated p53 to the proteasome. p53 ubiquitination is assocd. with its export from the nucleus into the cytoplasm. However, the exact site of degrdn. of p53 is presently under debate. P53 may be targeted by other E3 ligases besides Mdm2, as well as by non-proteasomal mechanisms. Despite extensive information about p53 degrdn., many important aspects remain unresolved.
- 55Wierstra, I. FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapy Adv. Cancer Res. 2013, 119, 191– 419[ Crossref], [ PubMed], [ CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1elt7nE&md5=6d9f85cd02e81372c6f30773464f78e3FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapyWierstra, InkenAdvances in Cancer Research (2013), 119 (), 191-419CODEN: ACRSAJ; ISSN:0065-230X. (Elsevier Inc.)A review. FOXM1 (Forkhead box M1) is a typical proliferation-assocd. transcription factor and is also intimately involved in tumorigenesis. FOXM1 stimulates cell proliferation and cell cycle progression by promoting the entry into S-phase and M-phase. Addnl., FOXM1 is required for proper execution of mitosis. In accordance with its role in stimulation of cell proliferation, FOXM1 exhibits a proliferation-specific expression pattern and its expression is regulated by proliferation and anti-proliferation signals as well as by proto-oncoproteins and tumor suppressors. Since these factors are often mutated, overexpressed, or lost in human cancer, the normal control of the foxm1 expression by them provides the basis for deregulated FOXM1 expression in tumors. Accordingly, FOXM1 is overexpressed in many types of human cancer. FOXM1 is intimately involved in tumorigenesis, because it contributes to oncogenic transformation and participates in tumor initiation, growth, and progression, including pos. effects on angiogenesis, migration, invasion, epithelial-mesenchymal transition, metastasis, recruitment of tumor-assocd. macrophages, tumor-assocd. lung inflammation, self-renewal capacity of cancer cells, prevention of premature cellular senescence, and chemotherapeutic drug resistance. However, in the context of urethane-induced lung tumorigenesis, FOXM1 has an unexpected tumor suppressor role in endothelial cells because it limits pulmonary inflammation and canonical Wnt signaling in epithelial lung cells, thereby restricting carcinogenesis. Accordingly, FOXM1 plays a role in homologous recombination repair of DNA double-strand breaks and maintenance of genomic stability, i.e., prevention of polyploidy and aneuploidy. The implication of FOXM1 in tumorigenesis makes it an attractive target for anticancer therapy, and several antitumor drugs have been reported to decrease FOXM1 expression.
- 56Yang, X.; Li, H.; Zhou, Z.; Wang, W. H.; Deng, A.; Andrisani, O.; Liu, X. Plk1-mediated phosphorylation of Topors regulates p53 stability J. Biol. Chem. 2009, 284 (28) 18588– 18592[ Crossref], [ PubMed], [ CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotVKnsrk%253D&md5=2e20a9458b44003d3315fab622eaa9a2Plk1-mediated phosphorylation of topors regulates p53 stabilityYang, Xiaoming; Li, Hongchang; Zhou, Zinan; Wang, Wen-Horng; Deng, Anping; Andrisani, Ourania; Liu, XiaoqiJournal of Biological Chemistry (2009), 284 (28), 18588-18592CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo-like kinase 1 (Plk1) overexpression is assocd. with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a neg. regulator of tumor suppressor p53, but the mechanism remains to be detd. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser718 in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degrdn. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degrdn. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.
- 57Cholewa, B. D.; Liu, X.; Ahmad, N. The role of polo-like kinase 1 in carcinogenesis: cause or consequence? Cancer Res. 2013, 73 (23) 6848– 6855[ Crossref], [ PubMed], [ CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKhurfM&md5=214e9d9c7e526aa0cae71d28843ef667The Role of Polo-like Kinase 1 in Carcinogenesis: Cause or Consequence?Cholewa, Brian D.; Liu, Xiaoqi; Ahmad, NihalCancer Research (2013), 73 (23), 6848-6855CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)A review. Polo-like kinase 1 (Plk1) is a well-established mitotic regulator with a diverse range of biol. functions continually being identified throughout the cell cycle. Preclin. evidence suggests that the mol. targeting of Plk1 could be an effective therapeutic strategy in a wide range of cancers; however, that success has yet to be translated to the clin. level. The lack of clin. success has raised the question of whether there is a true oncogenic addiction to Plk1 or if its overexpression in tumors is solely an artifact of increased cellular proliferation. In this review, we address the role of Plk1 in carcinogenesis by discussing the cell cycle and DNA damage response with respect to their assocns. with classic oncogenic and tumor suppressor pathways that contribute to the transcriptional regulation of Plk1. A thorough examn. of the available literature suggests that Plk1 activity can be dysregulated through key transformative pathways, including both p53 and pRb. On the basis of the available literature, it may be somewhat premature to draw a definitive conclusion on the role of Plk1 in carcinogenesis. However, evidence supports the notion that oncogene dependence on Plk1 is not a late occurrence in carcinogenesis and it is likely that Plk1 plays an active role in carcinogenic transformation. Cancer Res; 73(23); 6848-55. ©2013 AACR.
- 58Burd, C. G.; Swanson, M. S.; Gorlach, M.; Dreyfuss, G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts Proc. Natl. Acad. Sci. U. S. A. 1989, 86 (24) 9788– 9792[ Crossref], [ PubMed], [ CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktFyltbs%253D&md5=15bf6aa44341f77b3e6e38391a594a6cPrimary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide insertsBurd, Christopher G.; Swanson, Maurice S.; Gorlach, Matthias; Dreyfuss, GideonProceedings of the National Academy of Sciences of the United States of America (1989), 86 (24), 9788-92CODEN: PNASA6; ISSN:0027-8424.Some cDNAs for the major heterogeneous nuclear ribonucleoprotein (hnRNP) A2, B1, and C2 proteins were isolated and their nucleotide and deduced amino acid sequences were detd. The A2 and B1 cDNAs are identical except for a 36-nucleotide in-frame insert in B1. Similarly, the sequence of the C2 protein cDNA is related to that of C1 in that C2 contains an extra 39 in-frame nucleotides. Therefore, the B1 amino acid sequence is identical to A2 except for the insertion of 12 amino acids near its amino terminus, and C1 and C2 are also identical to each other except for an extra 13 amino acids near the middle of C2. All 3 proteins are members of a large family of RNA-binding proteins that contain the consensus sequence-type RNA-binding domain (CS-RBD). The A2 and B1 proteins have a modular structure similar to that of the hnRNP protein A1: they contain two CS-RBDs and a glycine-rich auxiliary domain at the carboxyl terminus. The CS-RBDs of A2 and B1 have ≈80% amino acid identity with those of A1, whereas the glycine-rich auxiliary domain is considerably more divergent with <30% of the amino acids being identical. Thus, the addn. of small peptides, probably by alternative pre-mRNA splicing, generates some of the diversity apparent among hnRNP proteins.
- 59Krecic, A. M.; Swanson, M. S. hnRNP complexes: composition, structure, and function Curr. Opin. Cell Biol. 1999, 11 (3) 363– 371[ Crossref], [ PubMed], [ CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXktFWms7g%253D&md5=66f04865aad1c966462a042802e2ea8fhnRNP complexes: composition, structure, and functionKrecic, Annette M.; Swanson, Maurice S.Current Opinion in Cell Biology (1999), 11 (3), 363-371CODEN: COCBE3; ISSN:0955-0674. (Current Biology Publications)A review with 62 refs. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins function in a staggering array of cellular activities, ranging from transcription and pre-mRNA processing in the nucleus to cytoplasmic mRNA translation and turnover. Recent studies suggest that several fundamental characteristics of hnRNPs account for their involvement in multiple regulatory pathways.
- 60Sun, D. Q.; Wang, Y.; Liu, D. G. Overexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cells Oncol. Lett. 2013, 5 (4) 1243– 1249[ PubMed], [ CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1KitLk%253D&md5=82c2d1915d97ad9a89c9bd3a0d4c0f5fOverexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cellsSun, Da-Quan; Wang, Ying; Liu, Ding-GanOncology Letters (2013), 5 (4), 1243-1249CODEN: OLNEB5; ISSN:1792-1074. (Spandidos Publications Ltd.)Heterogeneous ribonuclear protein C2 (hnRNPC2), an RNA binding protein, is a component of hnRNPC which is upregulated in many tumors. Multinucleation exists in many tumors and is pos. correlated with tumor grade. To uncover the correlation between hnRNPC2 and multinucleation in hepatocellular carcinoma SMMC-7721 cells, we constructed a pEGFP-hnRNPC2 vector and transfected it into cancer cells. Our results revealed that overexpression of hnRNPC2 induced multinucleation in SMMC-7721 cells. Tracking tests indicated that the induced multinucleated cells were unable to recover to mononuclear cells and finally died as a result of defects in cell division. Furthermore, Aurora B, which was localized at the midbody and plays a role in cytokinesis, was repressed in hnRNPC2-overexpressing cells, whose knockdown by RNA interference also induced multinucleation in SMMC-7721 cells. Quant. polymerase chain reaction (qPCR) and mRNA-protein co-immunopptn. results revealed that Aurora B mRNA did not decrease in hnRNPC2-overexpressing cells, instead it bound more hnRNPC2 and less eIF4E, an mRNA cap binding protein and translational initiation factor. Moreover, hnRNPC2 bound more eIF4E in hnRNPC2-overexpressing cells. These results indicate that hnRNPC2 repressed Aurora B binding with eIF4F, which must bind with Aurora B mRNA in order to initiate its translation. This induced multinucleation in hepatocellular carcinoma cells. In addn., hnRNPC2 accelerated hepatocellular carcinoma cell proliferation. Collectively, these data suggest that hnRNPC2 may be a potential target for hepatocellular carcinoma cell diagnosis and treatment.
- 61Christian, K. J.; Lang, M. A.; Raffalli-Mathieu, F. Interaction of heterogeneous nuclear ribonucleoprotein C1/C2 with a novel cis-regulatory element within p53 mRNA as a response to cytostatic drug treatment Mol. Pharmacol. 2008, 73 (5) 1558– 1567[ Crossref], [ PubMed], [ CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlsVWgu7Y%253D&md5=5f6856542e8072fcc854bd88090d92a6Interaction of heterogeneous nuclear ribonucleoprotein C1/C2 with a novel cis-regulatory element within p53 mRNA as a response to cytostatic drug treatmentChristian, Kyle J.; Lang, Matti A.; Raffalli-Mathieu, FrancoiseMolecular Pharmacology (2008), 73 (5), 1558-1567CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)We describe a novel cis-element in the 5' coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP)C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides down-stream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is obsd. in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qual. changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addn., we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.
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Abstract
Figure 1
Figure 1. Relative quantitation chromatograms. (A) For label-free relative quantitation, six replicates of the tryptic digests were analyzed. The chromatograms were aligned using SIEVE 2.1. An overlay of the base peak chromatograms of control (blue) and treated (red) samples shows good alignment and comparable loading in the region of peptide elution (38–145 min RT). (B) An example of a peptide that is down-regulated in response to treatment. Shown is a SIEVE-aligned, extracted-ion chromatograms for peptide 524.9481 m/z (metastasis-associated protein). Triangles indicate where MS/MS identification scans were triggered. (C) Example of a peptide that is up-regulated in response to treatment. Shown is a SIEVE-aligned, extracted-ion chromatogram for peptide 373.3691 m/z (PH-domain leucine-rich protein). Triangles indicate where MS/MS identification scans were triggered.
Figure 2
Figure 2. Summary of proteomics analysis data. (A) Graphical breakdown representing the number of peptides recognized in all identified proteins. (B) Graphical representation of the calculated protein ratios showing BI 6727-treated samples compared with the vehicle control. (C) Proteins of interest molecular function reported by PANTHER (Protein ANalysis THrough Evolutionary Relationships) as characterized by gene ontology. (D) Proteins of interest biological processes reported by PANTHER as characterized by gene ontology.
Figure 3
Figure 3. Plk1 inhibition alters cellular metabolism in melanoma cells. (A) Lactate dehydrogenase A (LDHA) protein expression was significantly reduced following Plk1 inhibition in both proteomics (top) and Western blot (bottom) analyses. (B) qPCR analysis suggests a dose-dependent decrease in polo-like kinase 1 (Plk1), lactate dehydrogenase B (LDHB), and glucose-6-phosphate isomerase (GPI) transcript levels following BI 6727 treatment (25 nM, 100 nM). (C) BI 6727 treatment significantly reduces extracellular lactate levels (p < 0.001). (D) Plk1 inhibition significantly reduces reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) levels (p < 0.001). (E) NAD and NADH levels are significantly reduced in BI 6727-treated cells when compared with control (p < 0.001). (F) NAD/NADH ratio decreases in a BI 6727 dose-dependent manner (** p < 0.01, *** p < 0.001).
Figure 4
Figure 4. Plk1 inhibition significantly alters 20S proteasome expression and activity. (A) Basic structure of the 20S proteasome. (B) Proteomics analysis identified four 20S proteasome subunits as being down-regulated. (C) Western blot analysis of the catalytically active 20S proteasome subunits, proteasome subunits β2, β5, and β1 (PSMB2, PSMB5, and PSMB1) following Plk1 inhibition. (D) BI 6727-treated cells have significantly decreased 20S proteasome activity (p < 0.001).
Figure 5
Figure 5. hnRNPC is up-regulated following Plk1 inhibition. (A) Heterogeneous ribonucleoprotein C1/C2 (hnRNPC) protein expression was significantly increased following Plk1 inhibition in both proteomics (top) and Western blot (bottom) analyses. (B) Immunofluorescence microscopy of β-tubulin (red) and Hoecsht DNA (blue) staining, demonstrating micronucleation following Plk1 inhibition. (C) Cell cycle analysis of BI 6727-treated cells demonstrates a pronounced G2/M arrest. (D) Western blot analysis of Aurora kinase B (AurkB) does not indicate significantly altered protein expression following BI 6727 treatment (top), but qPCR analysis reveals decreased mRNA expression levels (bottom). (E) Plk1 inhibition causes a marked increase in p53 protein expression, visualized by Western blot analysis (top) and p53 activity, demonstrated by increased p21 mRNA expression (bottom).
References
ARTICLE SECTIONSThis article references 61 other publications.
- 1Sumara, I.; Gimenez-Abian, J. F.; Gerlich, D.; Hirota, T.; Kraft, C.; de la Torre, C.; Ellenberg, J.; Peters, J. M. Roles of polo-like kinase 1 in the assembly of functional mitotic spindles Curr. Biol. 2004, 14 (19) 1712– 1722[ Crossref], [ PubMed], [ CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotFCkur4%253D&md5=8b3172fe1f24104d3c3342241b55f5a7Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic SpindlesSumara, Izabela; Gimenez-Abian, Juan F.; Gerlich, Daniel; Hirota, Toru; Kraft, Claudine; de la Torre, Consuelo; Ellenberg, Jan; Peters, Jan-MichaelCurrent Biology (2004), 14 (19), 1712-1722CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)The stable assocn. of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not assocd. with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Expts. with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.
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- 3Seong, Y. S.; Kamijo, K.; Lee, J. S.; Fernandez, E.; Kuriyama, R.; Miki, T.; Lee, K. S. A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cells J. Biol. Chem. 2002, 277 (35) 32282– 32293[ Crossref], [ PubMed], [ CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XmslyjsLY%253D&md5=e6ed028c69d0a7548f9671235a724755A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cellsSeong, Yeon-Sun; Kamijo, Keiju; Lee, Jae-Seon; Fernandez, Ester; Kuriyama, Ryoko; Miki, Toru; Lee, Kyung S.Journal of Biological Chemistry (2002), 277 (35), 32282-32293CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo kinases play crit. roles for proper M-phase progression. They are characterized by the presence of two regions of homol. in the C-terminal non-catalytic domain, termed polo-box 1 (PB1) and polo-box 2 (PB2). Here we demonstrate that both PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1/K82M or the C-terminal plk1ΔN induced a pre-anaphase arrest with elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibited randomly oriented spindle structures, whereas metaphase cells exhibited aberrant bipolar spindles with Mad2 localization at kinetochores of misaligned chromosomes. Microtubule nucleation activity of centrosomes was not compromised. In vivo time-lapse studies revealed that expression of plk1ΔN resulted in repeated cycles of bipolar spindle formation and disruption, suggestive of a defect in spindle stability. A prolonged arrest frequently led to the generation of micronucleated cells in the absence of sister chromatid sepn. and centrosome duplication, indicating that micronucleation is not a result of accumulated cytokinesis failures. Interestingly, bypass of the mitotic arrest by dominant-neg. spindle checkpoint components led to a failure in completion of cytokinesis. We propose that, in mammalian cells, the polo-box-dependent Plk1 activity is required for proper metaphase/anaphase transition and for cytokinesis.
- 4van Vugt, M. A.; van de Weerdt, B. C.; Vader, G.; Janssen, H.; Calafat, J.; Klompmaker, R.; Wolthuis, R. M.; Medema, R. H. Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis J. Biol. Chem. 2004, 279 (35) 36841– 36854[ Crossref], [ PubMed], [ CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvFCmsbc%253D&md5=6dd4c8622a7a43351aeb7bba24146003Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of CytokinesisVan Vugt, Marcel A. T. M.; Van de Weerdt, Barbara C. M.; Vader, Gerben; Janssen, Hans; Calafat, Jero; Klompmaker, Rob; Wolthuis, Rob M. F.; Medema, Rene H.Journal of Biological Chemistry (2004), 279 (35), 36841-36854CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to sep. their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addn., co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degrdn. of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis.
- 5Gray, P. J., Jr.; Bearss, D. J.; Han, H.; Nagle, R.; Tsao, M. S.; Dean, N.; Von Hoff, D. D. Identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancer Mol. Cancer Ther. 2004, 3 (5) 641– 646[ PubMed], [ CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjvVKnur4%253D&md5=b56542c25220e6ce32a244a51ef9b252Identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancerGray, Phillip J., Jr.; Bearss, David J.; Han, Haiyong; Nagle, Raymond; Tsao, Ming-Sound; Dean, Nicholas; Von Hoff, Daniel D.Molecular Cancer Therapeutics (2004), 3 (5), 641-646CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Pancreas cancer is the fourth leading cause of cancer-related death in adults in the United States. New mol. targets for diagnosis and therapy of this disease are desperately needed. In this study, we report on the mitotic serine-threonine kinase polo-like kinase 1 (Plk1) in pancreatic cancer. Plk1 mRNA was overexpressed in 9 of 10 tested pancreatic cancer cell lines and in 4 of 4 tested human tumors. Immunohistochem. staining of a pancreatic tissue microarray showed that 26 of the 35 tumors taken directly from patients overexpressed Plk1. We also examd. the effects of depleting Plk1 in pancreatic cancer cells by the use of antisense oligonucleotides. Antisense-treated pancreatic cancer cells showed cell cycle arrest in G2-M as well as a drastic redn. in proliferation rates. These data suggest that Plk1 is a potential therapeutic target in devising a treatment for patients with pancreatic cancer.
- 6Knecht, R.; Elez, R.; Oechler, M.; Solbach, C.; von Ilberg, C.; Strebhardt, K. Prognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neck Cancer Res. 1999, 59 (12) 2794– 2797[ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXktVOkur8%253D&md5=75835cab5f5bd0e831de8703a9b4767fPrognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neckKnecht, Rainald; Elez, Robert; Oechler, Martin; Solbach, Christine; Von Ilberg, Christoph; Strebhardt, KlausCancer Research (1999), 59 (12), 2794-2797CODEN: CNREA8; ISSN:0008-5472. (AACR Subscription Office)Previously, we demonstrated that the mammalian polo-like kinase (PLK), which participates in the regulation of the cell cycle, is a novel marker of cellular proliferation. Because current prognostic tools for the evaluation of patients with head and neck squamous cell cancer (HNSCC) need to be improved, we analyzed 89 patients and found elevated PLK expression in most tumors. Nodal stage as a crucial prognostic factor in HNSCC also correlated to PLK transcript levels. A Kaplan-Meier anal. showed that HNSCC patients with moderate vs. high PLK expression survived significantly longer (5-yr survival rates, 43% vs. 12%). Interestingly, a combination of nodal stage and PLK expression contributed to discriminate patients with a better prognosis in the pN0/1 and pN2/3 groups, which could improve the definition of a suitable therapy.
- 7Mito, K.; Kashima, K.; Kikuchi, H.; Daa, T.; Nakayama, I.; Yokoyama, S. Expression of Polo-Like Kinase (PLK1) in non-Hodgkin’s lymphomas Leuk. Lymphoma 2005, 46 (2) 225– 231[ Crossref], [ PubMed], [ CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFSntrjF&md5=028fb6ba93ad47cca395587736c3ce5bExpression of Polo-Like Kinase (PLK1) in non-Hodgkin's lymphomasMito, Katsuhiko; Kashima, Kenji; Kikuchi, Hiroshi; Daa, Tsutomu; Nakayama, Iwao; Yokoyama, ShigeoLeukemia & Lymphoma (2005), 46 (2), 225-231CODEN: LELYEA; ISSN:1042-8194. (Taylor & Francis Ltd.)Polo-like kinases (PLKs) are protein serine/threonine kinases that play important roles in cell division. Expression of PLK1 might, moreover, play a role in the pathogenesis of human neoplasms. The expression of PLK1 mRNA is closely correlated with survival in patients with malignant tumors. We investigated the expression of PLK1 in non-Hodgkin's lymphomas (NHLs) and analyzed the relationships between expression of PLK1, histol. grade, and prognosis. We analyzed various types of NHLs from 118 patients using monoclonal antibodies against PLK1 and Ki-67. The levels of expression of PLK1 and Ki-67 were significantly lower in low-grade NHLs than in high-grade and intermediate-grade NHLs (P < 0.001). Moreover, when patients were grouped in terms of 5-yr overall survival ( > 70%, group A; 50 - 70%, group B; 30 - 49%, group C; and < 30%, group D), levels of expression of PLK1 and Ki-67 were found to be significantly higher in group D than in group A and they were also significantly higher in group C than in group A (P < 0.001). Conversely, the level of expression, of Ki-67 was significantly lower in group D than in group C (P < 0.05). The labeling indexes specific for PLK1 were generally higher than those specific for Ki-67. Once we divided all patients into two groups in terms of the expression levels, high-level expression group of PLK1 (PLK1 index of ≥ 70%) and Ki-67 (Ki-67 indexes of ≥ 60%) and low-level expression, one of these markers (PLK1 index of < 70%, Ki-67 indexes of < 60%) had a similar prognosis, an observation that can be explained by the fact that rapidly proliferating group is more drug-sensitive than the other. Our study demonstrates that expression of PLK1 might reflect the malignant potential of NHLs and that PLK1 might be more useful than Ki-67 for the detection of proliferative cells.
- 8Salvatore, G.; Nappi, T. C.; Salerno, P.; Jiang, Y.; Garbi, C.; Ugolini, C.; Miccoli, P.; Basolo, F.; Castellone, M. D.; Cirafici, A. M.; Melillo, R. M.; Fusco, A.; Bittner, M. L.; Santoro, M. A cell proliferation and chromosomal instability signature in anaplastic thyroid carcinoma Cancer Res. 2007, 67 (21) 10148– 10158[ Crossref], [ PubMed], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1erur7I&md5=5a04652b6794a83f5927d907b6b4f208A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid CarcinomaSalvatore, Giuliana; Nappi, Tito Claudio; Salerno, Paolo; Jiang, Yuan; Garbi, Corrado; Ugolini, Clara; Miccoli, Paolo; Basolo, Fulvio; Castellone, Maria Domenica; Cirafici, Anna Maria; Melillo, Rosa Marina; Fusco, Alfredo; Bittner, Michael L.; Santoro, MassimoCancer Research (2007), 67 (21), 10148-10158CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homol. region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the neg. control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest assocd. with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential mol. target for ATC treatment.
- 9Schmit, T. L.; Zhong, W.; Setaluri, V.; Spiegelman, V. S.; Ahmad, N. Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells J. Invest. Dermatol. 2009, 129 (12) 2843– 2853[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtl2jsLrE&md5=f2f3aa721395fb9db989dda974b7febeTargeted Depletion of Polo-Like Kinase (Plk) 1 Through Lentiviral shRNA or a Small-Molecule Inhibitor Causes Mitotic Catastrophe and Induction of Apoptosis in Human Melanoma CellsSchmit, Travis L.; Zhong, Wei-Xiong; Setaluri, Vijayasaradhi; Spiegelman, Vladimir S.; Ahmad, NihalJournal of Investigative Dermatology (2009), 129 (12), 2843-2853CODEN: JIDEAE; ISSN:0022-202X. (Nature Publishing Group)Melanoma, one of the most lethal forms of skin cancer, remains resistant to currently available treatments. Therefore, addnl. target-based approaches are needed for the management of this neoplasm. Polo-like kinase 1 (Plk1) has been shown to be a crucial regulator of mitotic entry, progression, and exit. Elevated Plk1 level has been assocd. with aggressiveness of several cancer types and with poor disease prognosis. However, the role of Plk1 in melanoma is not well established. Here, we show that Plk1 is overexpressed in both clin. tissue specimens and cultured human melanoma cells (WM115, A375, and HS294T) when compared with normal skin tissues and cultured normal melanocytes, resp. Furthermore, Plk1 gene knockdown through Plk1-specific shRNA or its activity inhibition by a small-mol. inhibitor resulted in a significant decrease in the viability and growth of melanoma cells without affecting normal human melanocytes. In addn., Plk1 inhibition resulted in a significant (i) decrease in clonogenic survival, (ii) multiple mitotic errors, (iii) G2/M cell-cycle arrest, and (iv) apoptosis of melanoma cells. This study suggests that Plk1 may have a functional relevance toward melanoma development and/or progression. We suggest that the targeting of Plk1 may be a viable approach for the treatment of melanoma.
- 10Takahashi, T.; Sano, B.; Nagata, T.; Kato, H.; Sugiyama, Y.; Kunieda, K.; Kimura, M.; Okano, Y.; Saji, S. Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancers Cancer Sci. 2003, 94 (2) 148– 152[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjtFGjsbo%253D&md5=01e134d298bb5c7e619ac45b3a2df5d6Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancersTakahashi, Takao; Sano, Bun; Nagata, Takayasu; Kato, Hiroki; Sugiyama, Yasuyuki; Kunieda, Katsuyuki; Kimura, Masashi; Okano, Yukio; Saji, ShigetoyoCancer Science (2003), 94 (2), 148-152CODEN: CSACCM; ISSN:1347-9032. (Japanese Cancer Association)PLK (polo-like kinase), the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae, belongs to a family of Ser/Thr kinases. It is intimately involved in spindle formation and chromosome segregation during mitosis. The purpose of this study was to det. whether PLK1 is overexpressed in primary colorectal cancer specimens as compared with normal colon mucosa and to assess its relation to other kinases as a potential new tumor marker. In the present study, immunohistochem. analyses were performed of PLK1 expression in 78 primary colorectal cancers as well as 15 normal colorectal specimens. Furthermore, the authors examd. the relationship between other kinases, Aurora-A and Aurora-C, and PLK1 expression. In normal colon mucosa, some crypt cells showed weakly pos. staining for PLK1 in 13 out of 15 cases, the remaining cases being neg. Elevated expression of PLK1 was obsd. in 57 (73.1%) of the colorectal cancers, statistically significant assocns. being evident with pT (primary tumor invasion) (P=0.0006, Mann-Whitney U test), pN (regional lymph nodes) (P=0.008, χ2 test) and the Dukes' classification (P=0.0005, Mann-Whitney U test). Mean proliferating cell nuclear antigen-labeling index was 52.3%, with a range of 24.1% to 77.3%. Values for lesions with high and low PLK1 expression were 54.7±10.3% (mean±SD) and 45.9±11.9% (P=0.002, Student's t test). PLK1 was significantly assocd. with Aurora-A, but PLK1 staining was more diffuse and extensive than for Aurora-A or Aurora-C. Interestingly, PLK1 overexpression was significantly assocd. with p53 accumulation in colorectal cancers. These results suggest overexpression of PLK1 might be of pathogenic, prognostic and proliferative importance, so that this kinase might have potential as a new tumor marker for colorectal cancers.
- 11Wolf, G.; Elez, R.; Doermer, A.; Holtrich, U.; Ackermann, H.; Stutte, H. J.; Altmannsberger, H. M.; Rubsamen-Waigmann, H.; Strebhardt, K. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer Oncogene 1997, 14 (5) 543– 549[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXht1Git7g%253D&md5=974b1041d3535545c5242cf75cbcda05Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancerWolf, Georg; Elez, robert; Doermer, Andreas; Holtrich, Uwe; Ackermann, Hanns; Stutte, Hans Jochen; Altmannsberger, Hans-Michael; Ruebsamen-Waigmann, Helga; Strebhardt, KlausOncogene (1997), 14 (5), 543-549CODEN: ONCNES; ISSN:0950-9232. (Stockton)Previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot expts. PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived significantly longer (5 yr survival rate = 51.8%) than those with high levels of PLK transcripts (24.2%). No statistically significant correlation was found between PLK mRNA expression and age, sex, TNM status, histol. type or degree of differentiation. Interestingly, the prognosis of patients in post-surgical stages I and II was correlated with PLK expression (5 yr survival rates in stage I: 69.1% (moderate PLK) - 43.5% (high PLK), or in stage II: 51.9% (moderate PLK) - 9.9% (high PLK)). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.
- 12Wolf, G.; Hildenbrand, R.; Schwar, C.; Grobholz, R.; Kaufmann, M.; Stutte, H. J.; Strebhardt, K.; Bleyl, U. Polo-like kinase: a novel marker of proliferation: correlation with estrogen-receptor expression in human breast cancer Pathol. Res. Pract. 2000, 196 (11) 753– 759[ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjtFyqtQ%253D%253D&md5=4080e32f88ac6cab14f870dbe00c74d0Polo-like kinase: a novel marker of proliferation: correlation with estrogen-receptor expression in human breast cancerWolf, Georg; Hildenbrand, Ralf; Schwar, Christian; Grobholz, Rainer; Kaufmann, Manfred; Stutte, Hans-Jochen; Strebhardt, Klaus; Bleyl, UwePathology, Research and Practice (2000), 196 (11), 753-759CODEN: PARPDS; ISSN:0344-0338. (Urban & Fischer Verlag)Previous data have shown that the mRNA-expression of the serine/threonine-kinase polo-like kinase (PLK) is closely correlated with the survival of patients suffering from a subset of malignant tumors. PLK-mRNA and protein-expression are restricted to cells in the cell cycle. PLK-mRNA-transcripts are highly abundant in proliferating cells; no gene expression is found in G0-phase cells. Here the authors investigated the mRNA- and protein-expression of PLK- and estrogen-receptor (ER) in human breast-carcinoma by northern-blotting, RT-PCR and immunohistochem. The expression of MIB-I was detd. on serial sections. Anal. of the immunohistochem. data revealed a close correlation between the ER and PLK-expression (r = 0.677). No relation between the mRNA-expression of ER and PLK was found. Furthermore, no correlation for the protein expression of PLK and MIB-I exists. The influence of estrogen (ES) is known to have proliferative potential. The expression of ER correlates with the ES-plasma-level. In addn., the hormone cycle of premenopausal women undergoes rapid vacillations with varying effects on the proliferating tumor cells, e.g., growth induction. The authors' results therefore show that ER-expression is not only of therapeutic value for the clinician, but it may also be a tool for detg. the tumor proliferation index more precisely by integrating the hormone-mediated proliferation stimulus.
- 13Weichert, W.; Denkert, C.; Schmidt, M.; Gekeler, V.; Wolf, G.; Kobel, M.; Dietel, M.; Hauptmann, S. Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma Br. J. Cancer. 2004, 90 (4) 815– 821[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXht12iurk%253D&md5=2663adc8427cfbe6be77bb65b7cd1a78Polo-like kinase isoform expression is a prognostic factor in ovarian carcinomaWeichert, W.; Denkert, C.; Schmidt, M.; Gekeler, V.; Wolf, G.; Koebel, M.; Dietel, M.; Hauptmann, S.British Journal of Cancer (2004), 90 (4), 815-821CODEN: BJCAAI; ISSN:0007-0920. (Nature Publishing Group)The Polo-like kinase (PLK) family comprises three serine/threonine kinases, functionally involved in signal transduction pathways essential for the accomplishment of mitosis in both normal and malignant cells. Moreover, certain PLKs have been functionally linked to cytoskeletal reorganization. In this study, the expression of PLK1 and PLK3 was detd. immunohistochem. in tissue specimen of normal ovaries (n=9), cystadenomas (n=17), borderline tumors (n=13) and ovarian carcinomas (n=77). PLK 1 and PLK3 expression was low in normal ovarian surface epithelium and borderline tumors, with moderately higher expression levels in cystadenomas. In ovarian carcinomas, 26% of cases were PLK1 pos. and 50.6% of cases were PLK3 pos. A pos. correlation of both PLK1 and PLK3 expression with indicators of mitotic frequency could be established. The overexpression of either isoenzyme had an impact on patient prognosis with shortened survival time for patients with tumors pos. for PLK1 (P=0.02) and PLK3 (P=0.02), but only PLK1 expression remained a prognostic factor in multivariate survival anal. (P=0.03). The results of this study, if interpreted in the context of recently published functional data, suggest that inhibition of PLKs might represent an interesting new targeted approach for chemotherapy of epithelial ovarian cancer. Furthermore, this study suggests that PLK1 is a novel independent prognostic marker in ovarian carcinomas.
- 14Strebhardt, K.; Kneisel, L.; Linhart, C.; Bernd, A.; Kaufmann, R. Prognostic value of pololike kinase expression in melanomas JAMA, J. Am. Med. Assoc. 2000, 283 (4) 479– 480[ Crossref], [ PubMed], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3c7itlaltQ%253D%253D&md5=0e0cd545d9bac745249ff24898506de0Prognostic value of pololike kinase expression in melanomasStrebhardt K; Kneisel L; Linhart C; Bernd A; Kaufmann RJAMA : the journal of the American Medical Association (2000), 283 (4), 479-80 ISSN:0098-7484.There is no expanded citation for this reference.
- 15Weichert, W.; Schmidt, M.; Gekeler, V.; Denkert, C.; Stephan, C.; Jung, K.; Loening, S.; Dietel, M.; Kristiansen, G. Polo-like kinase 1 is overexpressed in prostate cancer and linked to higher tumor grades Prostate 2004, 60 (3) 240– 245[ Crossref], [ PubMed], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntFGks7s%253D&md5=b19f96f40a54625fb9bbcbfa4868e6a7Polo-like kinase I is overexpressed in prostate cancer and linked to higher tumor gradesWeichert, Wilko; Schmidt, Mathias; Gekeler, Volker; Denkert, Carsten; Stephan, Carsten; Jung, Klaus; Loening, Stefan; Dietel, Manfred; Kristiansen, GlenProstate (New York, NY, United States) (2004), 60 (3), 240-245CODEN: PRSTDS; ISSN:0270-4137. (Wiley-Liss, Inc.)Polo-like kinase 1 (PLK1) is known to be one of the key players in the regulation of mitosis of both normal and malignant transformed cells. Moreover, several studies reported an overexpression of PLK1 in human malignancies compared to the corresponding tissue of origin. In this study, expression of PLK1 was investigated by immunohistochem. in 78 tissue specimens of prostate carcinoma and in adjacent normal prostate tissue as well as in benign prostate hyperplasia. PLK1 expression was semiquant. scored and subsequently correlated to clinicopathol. parameters and patient prognosis. No significant PLK1 expression was obsd. in normal prostate glandular epithelium and stroma. Specimens of benign prostate hyperplasia were PLK1-neg. as well. In contrast, 52.6% of all prostate carcinomas showed strong expression of PLK1. High grade intraepithelial lesions, if present, stained almost invariably in the same manner as the resp. invasive tumors. Expression of PLK1 correlated pos. with Gleason grade. No other significant correlations of PLK1 expression with either tumor stage, WHO tumor grade, preoperative PSA, age, or resection margins could be established. In an anal. for differences in PSA-relapse-free survival time, PLK1 expression was not a prognostic marker. These results demonstrate a high rate of PLK1-positivity in prostate cancer which suggests involvement of PLK1 in tumorigenesis and progression in this tumor entity. Therefore, targeted strategies focusing on PLK1 inhibition might represent a promising new chemotherapeutic approach in prostate cancer.
- 16Hikichi, Y.; Honda, K.; Hikami, K.; Miyashita, H.; Kaieda, I.; Murai, S.; Uchiyama, N.; Hasegawa, M.; Kawamoto, T.; Sato, T.; Ichikawa, T.; Cao, S.; Nie, Z.; Zhang, L.; Yang, J.; Kuida, K.; Kupperman, E. TAK-960, a novel, orally available, selective inhibitor of polo-like kinase 1, shows broad-spectrum preclinical antitumor activity in multiple dosing regimens Mol. Cancer Ther. 2012, 11 (3) 700– 709[ Crossref], [ PubMed], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlKis7g%253D&md5=9b4798370cd6f19205c6c1982890861fTAK-960, a Novel, Orally Available, Selective Inhibitor of Polo-Like Kinase 1, Shows Broad-spectrum Preclinical Antitumor Activity in Multiple Dosing RegimensHikichi, Yuichi; Honda, Kohei; Hikami, Kouki; Miyashita, Hitoshi; Kaieda, Isao; Murai, Saomi; Uchiyama, Noriko; Hasegawa, Maki; Kawamoto, Tomohiro; Sato, Takashi; Ichikawa, Takashi; Cao, Sheldon; Nie, Zhe; Zhang, Lilly; Yang, Johnny; Kuida, Keisuke; Kupperman, ErikMolecular Cancer Therapeutics (2012), 11 (3), 700-709CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been assocd. with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G2-M cells, aberrant polo mitosis morphol., and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC50 >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clin. evaluation in patients with advanced cancers.
- 17Mross, K.; Frost, A.; Steinbild, S.; Hedbom, S.; Rentschler, J.; Kaiser, R.; Rouyrre, N.; Trommeshauser, D.; Hoesl, C. E.; Munzert, G. Phase I dose escalation and pharmacokinetic study of BI 2536, a novel Polo-like kinase 1 inhibitor, in patients with advanced solid tumors J. Clin. Oncol. 2008, 26 (34) 5511– 5517[ Crossref], [ PubMed], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVSktg%253D%253D&md5=b2a7c464ca76909221e953a266757e89Phase I dose escalation and pharmacokinetic study of BI 2536, a novel polo-like kinase 1 inhibitor, in patients with advanced solid tumorsMross, Klaus; Frost, Annette; Steinbild, Simone; Hedbom, Susanne; Rentschler, Jochen; Kaiser, Rolf; Rouyrre, Nicolas; Trommeshauser, Dirk; Hoesl, Cornelia E.; Munzert, GerdJournal of Clinical Oncology (2008), 26 (34), 5511-5517CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology)BI 2536 is a novel, potent, and highly specific inhibitor of polo-like kinase 1 (Plk1), which has an essential role in the regulation of mitotic progression. The aim of this trial was to identify the max. tolerated dose (MTD) of BI 2536 and to det. the safety, pharmacokinetics, and antitumor activity in patients who had advanced solid tumors. This phase I trial followed an open label, toxicity-guided, dose-titrn. design. Single doses of BI 2536 (25 to 250 mg) were administered as a 1-h i.v. infusion; patients who experienced clin. benefit were eligible for addnl. treatment courses. Safety and pharmacokinetics were investigated. Tumor response was evaluated according to Response Evaluation Criteria in Solid Tumors Group guidelines. The MTD was defined at 200 mg in a total of 40 patients entered; reversible neutropenia constituted the dose-limiting toxicity (DLT) and the most frequent adverse event at the MTD (grade 3 to 4; 56%). Nausea (52%), fatigue (52%), and anorexia (44%) also were common and were mostly of mild to moderate intensity (Common Terminol. Criteria of Adverse Events ≤ grade 2). One patient experienced a transient partial response. At doses equal to or greater than the MTD, 23% of patients experienced disease stabilization for 3 or more months. Dose-proportional increases in the max. plasma concn. and total exposure were obsd. BI 2536 showed a high total clearance and high distribution into tissue. The MTD of BI 2536 when administered as a single-dose, 1-h infusion was 200 mg; BI 2536 was well tolerated and showed a favorable pharmacokinetic profile. Antitumor activity of BI 2536 was obsd.
- 18Jimeno, A.; Li, J.; Messersmith, W. A.; Laheru, D.; Rudek, M. A.; Maniar, M.; Hidalgo, M.; Baker, S. D.; Donehower, R. C. Phase I study of ON 01910.Na, a novel modulator of the Polo-like kinase 1 pathway, in adult patients with solid tumors J. Clin. Oncol. 2008, 26 (34) 5504– 5510[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVSksQ%253D%253D&md5=d7ef274ca89ecf6d5c1b1ce08660b54dPhase I study of ON 01910.Na, a novel modulator of the polo-like kinase 1 pathway, in adult patients with solid tumorsJimeno, Antonio; Li, Jing; Messersmith, Wells A.; Laheru, Daniel; Rudek, Michelle A.; Maniar, Manoj; Hidalgo, Manuel; Baker, Sharyn D.; Donehower, Ross C.Journal of Clinical Oncology (2008), 26 (34), 5504-5510CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology)Purpose: We conducted a first-in-man (to our knowledge) phase I study to det. the dose-limiting toxicities (DLTs), characterize the pharmacokinetic profile, and document any antitumor activity of ON 01910.Na, a new chem. entity that arrests cancer cells in G2/M by modulating mitotic regulatory pathways including polo-like kinase 1 (Plk1). Patients and Methods: Patients had solid tumors refractory to std. therapy. ON 01910.Na was administered as a 2-h infusion on days 1, 4, 8, 11, 15, and 18 in 28-day cycles. The starting dose was 80 mg, and an accelerated titrn. schedule (single-patient cohorts) was used for escalation. Pharmacokinetics were studied on days 1 and 15 of cycle 1. Results: Twenty patients (11 women and nine men; age 46 to 73 years) were enrolled onto the study. Dose levels of 80, 160, 320, 480, 800, 1,280, 2,080, and 3,120 mg were evaluated in single-patient cohorts. A DLT and addnl. grade 2 toxicities made the 4,370-mg dose (n = 6) not tolerable, and the next lower dose cohort (3,120 mg) was expanded to six assessable patients. Toxicities were skeletal, abdominal, and tumor pain; nausea; urge to defecate; and fatigue. Hematol. toxicity was infrequent and mild. ON 01910.Na pharmacokinetics were characterized by a rapid distribution phase (distribution half-life, 1 h) and a relatively slow elimination phase (elimination half-life, 27 h). A refractory ovarian cancer patient had an objective response after four cycles and remained progression free for 24 mo. Conclusion: ON 01910.Na showed a distinct but moderate toxicity pattern. The recommended phase II dose of ON 01910.Na with this schedule of administration is 3,120 mg. Single-agent activity was documented in an ovarian cancer patient.
- 19Schoffski, P.; Awada, A.; Dumez, H.; Gil, T.; Bartholomeus, S.; Wolter, P.; Taton, M.; Fritsch, H.; Glomb, P.; Munzert, G. A phase I, dose-escalation study of the novel Polo-like kinase inhibitor volasertib (BI 6727) in patients with advanced solid tumours Eur. J. Cancer 2012, 48 (2) 179– 186[ Crossref], [ PubMed], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC387gs1Oitg%253D%253D&md5=ca4316cf6705a2cb92ab243c1c151c35A phase I, dose-escalation study of the novel Polo-like kinase inhibitor volasertib (BI 6727) in patients with advanced solid tumoursSchoffski Patrick; Awada Ahmad; Dumez Herlinde; Gil Thierry; Bartholomeus Sylvie; Wolter Pascal; Taton Martine; Fritsch Holger; Glomb Patricia; Munzert GerdEuropean journal of cancer (Oxford, England : 1990) (2012), 48 (2), 179-86 ISSN:.BACKGROUND: Volasertib (BI 6727) is a potent and selective cell-cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Polo-like kinase (Plk). This phase I dose-escalation study evaluated the maximum tolerated dose (MTD) of volasertib, safety and efficacy, and pharmacokinetic (PK) parameters. METHODS: This trial followed an open-label, toxicity-guided dose-titration design. Patients with progressive advanced or metastatic solid tumours received a single 1-h infusion of volasertib every 3 weeks. A total of 65 patients were treated at doses of 12-450 mg. RESULTS: Reversible haematological toxicity was the main side-effect; thrombocytopenia, neutropenia, and febrile neutropenia constituting the main dose-limiting events. Anaemia (all grades 22%; grade 3: 8%), neutropenia (15%; grade 3/4: 14%), fatigue (15%; grade 3: 2%), and thrombocytopenia (14%; grade 3/4: 14%) were the most frequent drug-related adverse events. The MTD was 400mg; however, 300 mg was the recommended dose for further development based on overall tolerability. Three patients achieved confirmed partial response. Stable disease as best response was reported in 40% of patients. Two patients remained progression free for >1 year. PK analysis showed no indication of deviation from 'dose-linear PK' behaviour, a large volume of distribution (>4000 l), moderate clearance and a long half-life (~111 h). CONCLUSION: This first-in-man trial demonstrated a favourable PK profile of volasertib, with manageable toxicities. As expected, the most common events were haematological. Encouraging preliminary antitumour activity has been observed, supporting Plk inhibition as a therapeutic approach. Clinical development of volasertib in phase II monotherapy and combination trials is ongoing.
- 20Olmos, D.; Barker, D.; Sharma, R.; Brunetto, A. T.; Yap, T. A.; Taegtmeyer, A. B.; Barriuso, J.; Medani, H.; Degenhardt, Y. Y.; Allred, A. J.; Smith, D. A.; Murray, S. C.; Lampkin, T. A.; Dar, M. M.; Wilson, R.; de Bono, J. S.; Blagden, S. P. Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies Clin. Cancer. Res. 2011, 17 (10) 3420– 3430[ Crossref], [ PubMed], [ CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVGit7c%253D&md5=3cf3213934f3a34aaeee469dc9cb6529Phase I Study of GSK461364, a Specific and Competitive Polo-like Kinase 1 Inhibitor, in Patients with Advanced Solid MalignanciesOlmos, David; Barker, Douglas; Sharma, Rohini; Brunetto, Andre T.; Yap, Timothy A.; Taegtmeyer, Anne B.; Barriuso, Jorge; Medani, Hanine; Degenhardt, Yan Y.; Allred, Alicia J.; Smith, Deborah A.; Murray, Sharon C.; Lampkin, Thomas A.; Dar, Mohammed M.; Wilson, Richard; de Bono, Johann S.; Blagden, Sarah P.Clinical Cancer Research (2011), 17 (10), 3420-3430CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)PURPOSE: GSK461364 is an ATP-competitive inhibitor of polo-like kinase 1 (Plk1). A phase I study of two schedules of i.v. GSK461364 was conducted. Exptl. Design: GSK461364 was administered in escalating doses to patients with solid malignancies by two schedules, either on days 1, 8, and 15 of 28-day cycles (schedule A) or on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (schedule B). Assessments included pharmacokinetic and pharmacodynamic profiles, as well as marker expression studies in pretreatment tumor biopsies. RESULTS: Forty patients received GSK461364: 23 patients in schedule A and 17 in schedule B. Dose-limiting toxicities (DLT) in schedule A at 300 mg (2 of 7 patients) and 225 mg (1 of 8 patients) cohorts included grade 4 neutropenia and/or grade 3-4 thrombocytopenia. In schedule B, DLTs of grade 4 pulmonary emboli and grade 4 neutropenia occurred at 7 or more days at 100 mg dose level. Venous thrombotic emboli (VTE) and myelosuppression were the most common grade 3-4, drug-related events. Pharmacokinetic data indicated that AUC (area under the curve) and Cmax (max. concn.) were proportional across doses, with a half-life of 9 to 13 h. Pharmacodynamic studies in circulating tumor cells revealed an increase in phosphorylated histone H3 (pHH3) following drug administration. A best response of prolonged stable disease of more than 16 wk occurred in 6 (15%) patients, including 4 esophageal cancer patients. Those with prolonged stable disease had greater expression of Ki-67, pHH3, and Plk1 in archived tumor biopsies. CONCLUSIONS: The final recommended phase II dose for GSK461364 was 225 mg administered i.v. in schedule A. Because of the high incidence (20%) of VTE, for further clin. evaluation, GSK461364 should involve coadministration of prophylactic anticoagulation. Clin Cancer Res; 17(10); 3420-30.
- 21Ma, W. W.; Messersmith, W. A.; Dy, G. K.; Weekes, C. D.; Whitworth, A.; Ren, C.; Maniar, M.; Wilhelm, F.; Eckhardt, S. G.; Adjei, A. A.; Jimeno, A. Phase I study of Rigosertib, an inhibitor of the phosphatidylinositol 3-kinase and Polo-like kinase 1 pathways, combined with gemcitabine in patients with solid tumors and pancreatic cancer Clin. Cancer. Res. 2012, 18 (7) 2048– 2055[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFOlsrY%253D&md5=04ca490691ff61a39988ff9bc1008792Phase I Study of Rigosertib, an Inhibitor of the Phosphatidylinositol 3-Kinase and Polo-like Kinase 1 Pathways, Combined with Gemcitabine in Patients with Solid Tumors and Pancreatic CancerMa, Wen Wee; Messersmith, Wells A.; Dy, Grace K.; Weekes, Colin D.; Whitworth, Amy; Ren, Chen; Maniar, Manoj; Wilhelm, Francois; Eckhardt, S. Gail; Adjei, Alex A.; Jimeno, AntonioClinical Cancer Research (2012), 18 (7), 2048-2055CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)PURPOSE: Rigosertib, a dual non-ATP inhibitor of polo-like kinase 1 (Plk1) and phosphoinositide 3-kinase pathways (PI3K), and gemcitabine have synergistic antitumor activity when combined in preclin. studies. This phase I study aimed to det. the recommended phase II dose (RPTD) of the combination of rigosertib and gemcitabine in patients with cancer. Exptl. Design: Patients with solid tumors who failed std. therapy or were candidates for gemcitabine-based therapy were eligible. Gemcitabine was administered on days 1, 8, and 15 on a 28-day cycle and rigosertib on days 1, 4, 8, 11, 15, and 18. Pharmacokinetic studies were conducted during an expansion cohort of patients with advanced pancreatic ductal adenocarcinoma (PDA). RESULTS: Forty patients were treated, 19 in the dose-escalation phase and 21 in the expansion cohort. Dose levels evaluated were (gemcitabine/rigosertib mg/m2): 750/600 (n = 4), 750/1,200 (n = 3), 1,000/600 (n = 3), 1,000/1,200 (n = 3), and 1,000/1,800 (n = 6 + 21). One dose-limiting toxicity (death) occurred at the highest dose level (1,000/1,800) tested. Non-dose-limiting ≥grade II/III toxicities included neutropenia, lymphopenia, thrombocytopenia, fatigue, and nausea. Grade III/IV neutropenia, thrombocytopenia, and fatigue were seen in two, one, and two patients in the expansion cohort. Partial responses were obsd. in PDA, thymic cancer, and Hodgkin lymphoma, including gemcitabine-pretreated PDA. The pharmacokinetic profile of rigosertib was not affected by gemcitabine. CONCLUSION: The RPTD established in this study is rigosertib 1,800 mg/m2 and gemcitabine 1,000 mg/m2. This regimen is well tolerated with a toxicity profile of the combination similar to the profile of gemcitabine alone. Antitumor efficacy was obsd. in patients who previously progressed on gemcitabine-based therapy. Clin Cancer Res; 18(7); 2048-55.
- 22Rudolph, D.; Steegmaier, M.; Hoffmann, M.; Grauert, M.; Baum, A.; Quant, J.; Haslinger, C.; Garin-Chesa, P.; Adolf, G. R. BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity Clin. Cancer. Res. 2009, 15 (9) 3094– 3102[ Crossref], [ PubMed], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltFOrtbo%253D&md5=daed550be9d9b99ac6797c359039d295BI 6727, A Polo-like Kinase Inhibitor with Improved Pharmacokinetic Profile and Broad Antitumor ActivityRudolph, Dorothea; Steegmaier, Martin; Hoffmann, Matthias; Grauert, Matthias; Baum, Anke; Quant, Jens; Haslinger, Christian; Garin-Chesa, Pilar; Adolf, Guenther R.Clinical Cancer Research (2009), 15 (9), 3094-3102CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. The first compd. in this series, suitable for i.v. administration, has entered clin. development. To fully explore the potential of Polo-like kinase 1 inhibition in oncol., we have profiled addnl. compds. and now describe a novel clin. candidate. BI 6727 is a highly potent (enzyme IC50 = 0.87 nmol/L, EC50 = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high vol. of distribution and a long terminal half-life in mice (Vss = 7.6 L/kg, t1/2 = 46 h) and rats (Vss = 22 L/kg, t1/2 = 54 h). Second, BI 6727 has physicochem. and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clin. studies have been initiated.
- 23Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Mutations of the BRAF gene in human cancer Nature 2002, 417 (6892) 949– 954[ Crossref], [ PubMed], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XkvVagsLo%253D&md5=4625be53ad9a382d51b6ee753d0c17e8Mutations of the BRAF gene in human cancerDavies, Helen; Bignell, Graham R.; Cox, Charles; Stephens, Philip; Edkins, Sarah; Clegg, Sheila; Teague, Jon; Woffendin, Hayley; Garnett, Mathew J.; Bottomley, William; Davis, Neil; Dicks, Ed; Ewing, Rebecca; Floyd, Yvonne; Gray, Kristian; Hall, Sarah; Hawes, Rachel; Hughes, Jaime; Kosmidou, Vivian; Menzies, Andrew; Mould, Catherine; Parker, Adrian; Stevens, Claire; Watt, Stephen; Hooper, Steven; Wilson, Rebecca; Jayatilake, Hiran; Gusterson, Barry A.; Cooper, Colin; Shipley, Janet; Hargrave, Darren; Pritchard-Jones, Katherine; Maitland, Norman; Chenevix-Trench, Georgia; Riggins, Gregory J.; Bigner, Darell D.; Palmieri, Giuseppe; Cossu, Antonio; Flanagan, Adrienne; Nicholson, Andrew; Ho, Judy W. C.; Leung, Suet Y.; Yuen, Siu T.; Weber, Barbara L.; Seigler, Hilliard F.; Darrow, Timothy L.; Paterson, Hugh; Marais, Richard; Marshall, Christopher J.; Wooster, Richard; Stratton, Michael R.; Futreal, P. AndrewNature (London, United Kingdom) (2002), 417 (6892), 949-954CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Cancers arise owing to the accumulation of mutations in crit. genes that alter normal programs of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for anal. signalling pathways in which at least one gene is mutated in human cancer. The RAS-RAF-MEK-ERK-MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
- 24Spandidos, A.; Wang, X.; Wang, H.; Seed, B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification Nucleic Acids Res. 2010, 38 (Database issue) D792– D799[ Crossref], [ PubMed], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlOlsw%253D%253D&md5=b140f43714130491f9134a839b011f16PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantificationSpandidos, Athanasia; Wang, Xiaowei; Wang, Huajun; Seed, BrianNucleic Acids Research (2010), 38 (Database Iss), D792-D799CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)PrimerBank (http://pga.mgh.harvard.edu/primerbank/) is a public resource for the retrieval of human and mouse primer pairs for gene expression anal. by PCR and Quant. PCR (QPCR). A total of 306 800 primers covering most known human and mouse genes can be accessed from the PrimerBank database, together with information on these primers such as Tm, location on the transcript and amplicon size. For each gene, at least one primer pair has been designed and in many cases alternative primer pairs exist. Primers have been designed to work under the same PCR conditions, thus facilitating high-throughput QPCR. There are several ways to search for primers for the gene(s) of interest, such as by: GenBank accession no., NCBI protein accession no., NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). In all, 26 855 primer pairs covering most known mouse genes have been exptl. validated by QPCR, agarose gel anal., sequencing and BLAST, and all validation data can be freely accessed from the PrimerBank web site.
- 25Neilson, K. A.; Ali, N. A.; Muralidharan, S.; Mirzaei, M.; Mariani, M.; Assadourian, G.; Lee, A.; van Sluyter, S. C.; Haynes, P. A. Less label, more free: approaches in label-free quantitative mass spectrometry Proteomics 2011, 11 (4) 535– 553[ Crossref], [ PubMed], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVSnu74%253D&md5=4b993f82d81a20eaf1602869b481e032Less label, more free: Approaches in label-free quantitative mass spectrometryNeilson, Karlie A.; Ali, Naveid A.; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Garine; Lee, Albert; van Sluyter, Steven C.; Haynes, Paul A.Proteomics (2011), 11 (4), 535-553CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)In this review we examine techniques, software, and statistical analyses used in label-free quant. proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further expts. to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labeling techniques, and forward-looking applications for label-free quant. data are presented. We conclude that label-free quant. proteomics is a reliable, versatile, and cost-effective alternative to labeled quantitation.
- 26Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics Mol. Cell. Proteomics 2005, 4 (10) 1487– 1502[ Crossref], [ PubMed], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFeltbzI&md5=73387faedda13dcb475b7f5a9f54ca15Comparison of label-free methods for quantifying human proteins by shotgun proteomicsOld, William M.; Meyer-Arendt, Karen; Aveline-Wolf, Lauren; Pierce, Kevin G.; Mendoza, Alex; Sevinsky, Joel R.; Resing, Katheryn A.; Ahn, Natalie G.Molecular and Cellular Proteomics (2005), 4 (10), 1487-1502CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. The authors describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using std. proteins spiked into a complex sample. Linearity and good agreement between obsd. vs. expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio ests. Peak intensity values useful for protein quantitation ranged from 107 to 1011 counts with no obvious satn. effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (±2σ) when ≥3 peptides/protein were shared between samples. Protein ratios were detd. with high confidence from spectral counts when max. spectral counts were ≥4 spectra/protein, and replicates showed equiv. measurements well within 95% confidence limits. In further tests, complex samples were sepd. by gel exclusion chromatog., quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios detd. by spectral counting agreed well with those detd. from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate ests. of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p < 0.1 showed good correlations with parallel measurements of changes in mRNA expression.
- 27Zhu, W.; Smith, J. W.; Huang, C. M. Mass spectrometry-based label-free quantitative proteomics J. Biomed. Biotechnol. 2010, 2010, 840518[ Crossref], [ PubMed], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1MjlvF2gsg%253D%253D&md5=c97206c0c551ee4bf2ab4bde6aeb9c6eMass spectrometry-based label-free quantitative proteomicsZhu Wenhong; Smith Jeffrey W; Huang Chun-MingJournal of biomedicine & biotechnology (2010), 2010 (), 840518 ISSN:.In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies.
- 28Fedorova, G.; Randak, T.; Lindberg, R. H.; Grabic, R. Comparison of the quantitative performance of a Q-Exactive high-resolution mass spectrometer with that of a triple quadrupole tandem mass spectrometer for the analysis of illicit drugs in wastewater Rapid. Commun. Mass. Sp. 2013, 27 (15) 1751– 1762
- 29Jones, K. A.; Kim, P. D.; Patel, B. B.; Kelsen, S. G.; Braverman, A.; Swinton, D. J.; Gafken, P. R.; Jones, L. A.; Lane, W. S.; Neveu, J. M.; Leung, H. C.; Shaffer, S. A.; Leszyk, J. D.; Stanley, B. A.; Fox, T. E.; Stanley, A.; Hall, M. J.; Hampel, H.; South, C. D.; de la Chapelle, A.; Burt, R. W.; Jones, D. A.; Kopelovich, L.; Yeung, A. T. Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers J. Proteome Res. 2013, 12 (10) 4351– 4365[ ACS Full Text], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtl2rtLfE&md5=0d14c16c3ea10d538b386ab7cc343f1fImmunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass SpectrometersJones, Kelly A.; Kim, Phillip D.; Patel, Bhavinkumar B.; Kelsen, Steven G.; Braverman, Alan; Swinton, Derrick J.; Gafken, Philip R.; Jones, Lisa A.; Lane, William S.; Neveu, John M.; Leung, Hon-Chiu E.; Shaffer, Scott A.; Leszyk, John D.; Stanley, Bruce A.; Fox, Todd E.; Stanley, Anne; Hall, Michael J.; Hampel, Heather; South, Christopher D.; de la Chapelle, Albert; Burt, Randall W.; Jones, David A.; Kopelovich, Levy; Yeung, Anthony T.Journal of Proteome Research (2013), 12 (10), 4351-4365CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Plasma proteomic expts. performed rapidly and economically using several of the latest high-resoln. mass spectrometers were compared. Four quant. hyperfractionated plasma proteomics expts. were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each expt. compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 mo. Several anal. algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest no. of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
- 30Oppermann, F. S.; Klammer, M.; Bobe, C.; Cox, J.; Schaab, C.; Tebbe, A.; Daub, H. Comparison of SILAC and mTRAQ quantification for phosphoproteomics on a quadrupole orbitrap mass spectrometer J. Proteome Res. 2013, 12 (9) 4089– 4100[ ACS Full Text], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFOqurnE&md5=9ae3d83d2489e05abe91ee4360b6e2a9Comparison of SILAC and mTRAQ Quantification for Phosphoproteomics on a Quadrupole Orbitrap Mass SpectrometerOppermann, Felix S.; Klammer, Martin; Bobe, Caroline; Cox, Juergen; Schaab, Christoph; Tebbe, Andreas; Daub, HenrikJournal of Proteome Research (2013), 12 (9), 4089-4100CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Advances in mass spectrometric methodol. and instrumentation have promoted a continuous increase in anal. performance in the field of phosphoproteomics. Here, the authors employed the recently introduced quadrupole Orbitrap (Q Exactive) mass spectrometer for quant. signaling anal. to a depth of >15,000 phosphorylation sites. In parallel to the commonly used SILAC approach, the authors evaluated the nonisobaric chem. labeling reagent mTRAQ as an alternative quantification technique. Both enabled high phosphoproteome coverage in H3122 lung cancer cells. Replicate quantifications by mTRAQ identified almost as many significant phosphorylation changes upon treatment with ALK kinase inhibitor crizotinib as found by SILAC quantification. Overall, mTRAQ was slightly less precise than SILAC as evident from a somewhat higher variance of replicate phosphosite ratios. Direct comparison of SILAC- and mTRAQ-quantified phosphosites revealed that the majority of changes were detected by either quantification techniques, but also highlighted the aspect of false neg. identifications in quant. proteomics applications. Further inspection of crizotinib-regulated phosphorylation changes unveiled interference with multiple antioncogenic mechanisms downstream of ALK fusion kinase in H3122 cells. In conclusion, the authors' results demonstrate a strong anal. performance of the Q Exactive in global phosphoproteomics, and establish mTRAQ quantification as a useful alternative to metabolic isotope labeling.
- 31Gamez-Pozo, A.; Sanchez-Navarro, I.; Calvo, E.; Agullo-Ortuno, M. T.; Lopez-Vacas, R.; Diaz, E.; Camafeita, E.; Nistal, M.; Madero, R.; Espinosa, E.; Lopez, J. A.; Fresno Vara, J. A. PTRF/cavin-1 and MIF proteins are identified as non-small cell lung cancer biomarkers by label-free proteomics PLoS One 2012, 7 (3) e33752[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1Citb8%253D&md5=ca235b8ad48bee4f8ef223df049378d8PTRF/Cavin-1 and MIF proteins are identified as non-small cell lung cancer biomarkers by label-free proteomicsGamez-Pozo, Angelo; Sanchez-Navarro, Iker; Calvo, Enrique; Agullo-Ortuno, Maria Teresa; Lopez-Vacas, Rocio; Diaz, Esther; Camafeita, Emilio; Nistal, Manuel; Madero, Rosario; Espinosa, Enrique; Lopez, Juan Antonio; Vara, Juan Angel FresnoPLoS One (2012), 7 (3), e33752CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)With the completion of the human genome sequence, biomedical sciences have entered in the "omics" era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technol. advances and their application in the clin. setting. Our work is designed to build bridges between high-performance proteomics and clin. routine. Protein exts. were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontol. and interactome analyses identified signaling pathways altered on tumor tissue. We have identified 2 proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochem. The application of discovery-based proteomics analyses in clin. samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer.
- 32Guillaume, E.; Berger, B.; Affolter, M.; Kussmann, M. Label-free quantitative proteomics of two Bifidobacterium longum strains J. Proteomics 2009, 72 (5) 771– 784[ Crossref], [ PubMed], [ CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotFCgurc%253D&md5=0d9e9fd732952a7facab0507c7f70f3bLabel-free quantitative proteomics of two Bifidobacterium longum strainsGuillaume, Elisabeth; Berger, Bernard; Affolter, Michael; Kussmann, MartinJournal of Proteomics (2009), 72 (5), 771-784CODEN: JPORFQ; ISSN:1874-3919. (Elsevier Ltd.)Bifidobacteria are gram-pos. anaerobes with a high Guanine/Cytosine genome content. Specific strains are used as probiotics because of their health benefits. Probiotics are live microorganisms with a pos. influence on their host's health. They are used as nutritional supplements and their resistance to conditions of food manufg. and of the gastro-intestinal tract is studied. We report on differential proteomics of two Bifidobacterium longum strains that differ in their heat shock resistance. This was achieved by a comparative qual. survey of their proteomes and relative LC-MS/MS-based label-free protein quantification. Deploying a nano LC-ESI ion-trap mass spectrometer, 165 proteins expressed by the two probiotic strains were identified. Around 50% of these were common to both strains with the remaining half identified in either one of the strains. Using a label-free technol. based on the 3D overlay of retention times, mass-over-charge ratios and ion intensities between LC-MS runs, we found quant. differences in the relative abundance of 19 of the proteins common to both strains. The differentially expressed proteins were classified into categories involving glucose metab., protein synthesis and heat shock proteins. Six of these 19 proteins have been previously reported as being assocd. to heat stress response.
- 33Lopez, M. F.; Sarracino, D. A.; Vogelsang, M.; Sutton, J. N.; Athanas, M.; Krastins, B.; Garces, A.; Prakash, A.; Peterman, S.; Demirjian, Z.; Inglessis-Azuaje, I.; Feeney, K.; Elia, M.; McMullin, D.; Dec, G. W.; Palacios, I.; Lo, E. H.; Buonanno, F.; Ning, M. Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow J. Invest. Med. 2012, 60 (8) 1122– 1130[ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXis1Wltro%253D&md5=093b6e62df55d6420ae78c5118df3a68Heart-Brain Signaling in Patent Foramen Ovale-Related Stroke: Differential Plasma Proteomic Expression Patterns Revealed With a 2-Pass Liquid Chromatography-Tandem Mass Spectrometry Discovery WorkflowLopez, Mary F.; Sarracino, David A.; Vogelsang, Maryann; Sutton, Jennifer N.; Athanas, Michael; Krastins, Bryan; Garces, Alejandra; Prakash, Amol; Peterman, Scott; Demirjian, Zareh; Inglessis-Azuaje, Ignacio; Feeney, Kathleen; Elia, Mikaela; McMullin, David; William Dec, G.; Palacios, Igor; Lo, Eng H.; Buonanno, Ferdinand; Ning, Ming MingJournal of Investigative Medicine (2012), 60 (8), 1122-1130CODEN: JINVFI; ISSN:1081-5589. (Lippincott Williams & Wilkins)Patent foramen ovale (PFO) is highly prevalent and assocd. with more than 150,000 strokes per yr. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiol. to individualize the best clin. treatment for each patient. We initiated a study using a novel quant., 2-pass discovery workflow using high-resoln. liq. chromatog.-mass spectrometry/mass spectrometry coupled with label-free anal. to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quant. differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the mol. landscape of PFO-related physiol., our methods have yielded biol. relevant information on the synergistic and functional redundancy of various cell-signaling mols. with respect to PFO circulatory physiol. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.
- 34Qu, J.; Lesse, A. J.; Brauer, A. L.; Cao, J.; Gill, S. R.; Murphy, T. F. Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses BMC Microbiol. 2010, 10, 162[ Crossref], [ PubMed], [ CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3crht1yjtg%253D%253D&md5=c7700fe8c899a68766de42f902e99caeProteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responsesQu Jun; Lesse Alan J; Brauer Aimee L; Cao Jin; Gill Steven R; Murphy Timothy FBMC microbiology (2010), 10 (), 162 ISSN:.BACKGROUND: Nontypeable Haemophilus influenzae colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, H. influenzae, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by H. influenzae in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient. RESULTS: A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions. CONCLUSIONS: Proteomic expression profiling of H. influenzae grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that H. influenzae adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival in these conditions.
- 35Sutton, J.; Richmond, T.; Shi, X.; Athanas, M.; Ptak, C.; Gerszten, R.; Bonilla, L. Performance characteristics of an FT MS-based workflow for label-free differential MS analysis of human plasma: standards, reproducibility, targeted feature investigation, and application to a model of controlled myocardial infarction Proteomics: Clin. Appl. 2008, 2 (6) 862– 881[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotF2gu7c%253D&md5=a575ec4fe4f417491aa09afc06345562Performance characteristics of an FT MS-based workflow for label-free differential MS analysis of human plasma: standards, reproducibility, targeted feature investigation, and application to a model of controlled myocardial infarctionSutton, Jennifer; Richmond, Tori; Shi, Xu; Athanas, Michael; Ptak, Celeste; Gerszten, Robert; Bonilla, LeoProteomics: Clinical Applications (2008), 2 (6), 862-881CODEN: PCARCU; ISSN:1862-8346. (Wiley-VCH Verlag GmbH & Co. KGaA)Proteomics is undergoing a rapid transformation from a qual. global peptide sequencing discipline into a quant., reproducibility-driven practice. Nowhere is this more evident than in the rapidly expanding field of protein biomarker discovery where the general goal is to uncover statistically robust patterns of differential expression between or among subjects/samples representing distinct biol./temporal states. This report presents the anal. characterization of a label-free LC FT-ICR-MS workflow for differential proteomics anal. of human plasma. The key elements discussed include (i) methodologies for performing properly replicated expts. with highly reproducible sample prepn. and anal., including the use of internal stds. to quantify variance at different steps in the process, (ii) a new methodol. for performing sample re-anal. that uses off-line targeted robotic acquisition of complementary spectral data (e.g. ECD and/or IRMPD) to enhance the identification of differentially expressed peptides/proteins, and (iii) data processing pipelines capable of integrating the automatic statistical anal. of the label-free (LC-) MS signal, together with the intuitive and highly interactive curation and annotation of differential features using the output from std. sequence database search programs. The authors illustrate the application of the complete sample-to-annotated-differential-peptides (-proteins) workflow by describing the acquisition and anal. of a large multidimensional dataset from patients undergoing a controlled myocardial infarction resulting in an exptl. setup in which each patients serve as their own control. Furthermore, the authors discuss a couple illustrative examples of mid-level proteins obsd. in this study whose plasma concns. change consistently within and across patients, in a treatment- and time-dependent fashion.
- 36Tabata, T.; Sato, T.; Kuromitsu, J.; Oda, Y. Pseudo internal standard approach for label-free quantitative proteomics Anal. Chem. 2007, 79 (22) 8440– 8445[ ACS Full Text], [ CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFCrs7rJ&md5=c09279bd627e01348210cf945e8925c2Pseudo Internal Standard Approach for Label-Free Quantitative ProteomicsTabata, Tsuyoshi; Sato, Toshitaka; Kuromitsu, Junro; Oda, YoshiyaAnalytical Chemistry (Washington, DC, United States) (2007), 79 (22), 8440-8445CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Quant. proteome anal. has become a versatile tool to understand biol. functions. Although stable isotope labeling is the most reliable method for quant. mass spectrometry, prepn. of isotope-labeled compds. is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without stds. is not generally accepted as reliable, esp. for small mols. We have developed a novel label-free quant. proteome anal. using pseudo internal stds. (PISs). This idea was derived from northern blotting anal., in which housekeeping genes are used as stds. to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal stds. This new approach is simple and does not require addnl. stds. or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitation and may also be applicable to quant. metabolome anal.
- 37Nilsson, T.; Mann, M.; Aebersold, R.; Yates, J. R., 3rd; Bairoch, A.; Bergeron, J. J. Mass spectrometry in high-throughput proteomics: ready for the big time Nat. Methods 2010, 7 (9) 681– 685[ Crossref], [ PubMed], [ CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtV2hsb3E&md5=5a83e7edaf1a783502a8649fca8480fcMass spectrometry in high-throughput proteomics: ready for the big timeNilsson, Tommy; Mann, Matthias; Aebersold, Ruedi; Yates, John R.; Bairoch, Amos; Bergeron, John J. M.Nature Methods (2010), 7 (9), 681-685CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Mass spectrometry has evolved and matured to a level where it is able to assess the complexity of the human proteome. We discuss some of the expected challenges ahead and promising strategies for success.
- 38Koppenol, W. H.; Bounds, P. L.; Dang, C. V. Otto Warburg’s contributions to current concepts of cancer metabolism Nat. Rev. Cancer 2011, 11 (5) 325– 337[ Crossref], [ PubMed], [ CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXks1ags70%253D&md5=72988f428422daecd23b21d242aec18aOtto Warburg's contributions to current concepts of cancer metabolismKoppenol, Willem H.; Bounds, Patricia L.; Dang, Chi V.Nature Reviews Cancer (2011), 11 (5), 325-337CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. Otto Warburg pioneered quant. investigations of cancer cell metab., as well as photosynthesis and respiration. Warburg and co-workers showed in the 1920s that, under aerobic conditions, tumor tissues metabolize approx. tenfold more glucose to lactate in a given time than normal tissues, a phenomenon known as the Warburg effect. However, this increase in aerobic glycolysis in cancer cells is often erroneously thought to occur instead of mitochondrial respiration and has been misinterpreted as evidence for damage to respiration instead of damage to the regulation of glycolysis. In fact, many cancers exhibit the Warburg effect while retaining mitochondrial respiration. We re-examine Warburg's observations in relation to the current concepts of cancer metab. as being intimately linked to alterations of mitochondrial DNA, oncogenes and tumor suppressors, and thus readily exploitable for cancer therapy.
- 39Lin, S. J.; Guarente, L. Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and disease Curr. Opin. Cell Biol. 2003, 15 (2) 241– 246[ Crossref], [ PubMed], [ CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXitV2msL0%253D&md5=a3daf490bbfd30a1abdeaceb742e9235Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and diseaseLin, Su-Ju; Guarente, LeonardCurrent Opinion in Cell Biology (2003), 15 (2), 241-246CODEN: COCBE3; ISSN:0955-0674. (Elsevier Science Ltd.)A review. NAD is a ubiquitous biol. mol. that participates in many metabolic reactions. Recent studies show that NAD also plays important roles in transcriptional regulation, longevity, calorie-restriction-mediated life-span extension and age-assocd. diseases. It has been shown that NAD affects longevity and transcriptional silencing through the regulation of the Sir2p family, which are NAD-dependent deacetylases. Many human diseases are assocd. with changes in NAD level and/or the NAD : NADH ratio, raising the possibility that the Sir2p family might play a role in these diseases.
- 40Sun, F.; Dai, C.; Xie, J.; Hu, X. Biochemical issues in estimation of cytosolic free NAD/NADH ratio PLoS One 2012, 7 (5) e34525[ Crossref], [ PubMed], [ CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsVGht70%253D&md5=5c2f13a3e6c52ec8e09e2bcded4651c3Biochemical issues in estimation of cytosolic free NAD/NADH ratioSun, Feifei; Dai, Chunyan; Xie, Jiansheng; Hu, XunPLoS One (2012), 7 (5), e34525CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al., reported a method to est. this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chem. equil. Numerous studies used L/P ratio to est. the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equil. but not verifying how near it was. In addn., it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equil.; (2) that cytosolic free NAD/NADH could be accurately estd. by L/P only when the conversion is at or very close to equil. otherwise a calcn. error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estn. of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.
- 41Zheng, J. Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review) Oncol. Lett. 2012, 4 (6) 1151– 1157[ PubMed], [ CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVKgtLnO&md5=d692211905f9ecd90cf144a1c533c53eEnergy metabolism of cancer: glycolysis versus oxidative phosphorylation (review)Zheng, JieOncology Letters (2012), 4 (6), 1151-1157CODEN: OLNEB5; ISSN:1792-1074. (Spandidos Publications Ltd.)A review. Metabolic activities in normal cells rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate ATP for energy. Unlike in normal cells, glycolysis is enhanced and OXPHOS capacity is reduced in various cancer cells. It has long been believed that the glycolytic phenotype in cancer is due to a permanent impairment of mitochondrial OXPHOS, as proposed by Otto Warburg. This view is challenged by recent investigations which find that the function of mitochondrial OXPHOS in most cancers is intact. Aerobic glycolysis in many cancers is the combined result of various factors such as oncogenes, tumor suppressors, a hypoxic microenvironment, mtDNA mutations, genetic background and others. Understanding the features and complexity of the cancer energy metab. will help to develop new approaches in early diagnosis and effectively target therapy of cancer.
- 42Liu, X. S.; Li, H.; Song, B.; Liu, X. Polo-like kinase 1 phosphorylation of G2 and S-phase-expressed 1 protein is essential for p53 inactivation during G2 checkpoint recovery EMBO Rep. 2010, 11 (8) 626– 632[ Crossref], [ PubMed], [ CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvFSgtbY%253D&md5=ed86deedb26769e42edcc069273a5b27Polo-like kinase 1 phosphorylation of G2 and S-phase-expressed 1 protein is essential for p53 inactivation during G2 checkpoint recoveryLiu, X. Shawn; Li, Hongchang; Song, Bing; Liu, XiaoqiEMBO Reports (2010), 11 (8), 626-632CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)In response to G2 DNA damage, the p53 pathway is activated to lead to cell-cycle arrest, but how p53 is eliminated during the subsequent recovery process is poorly understood. It has been established that Polo-like kinase 1 (Plk1) controls G2 DNA-damage recovery. However, whether Plk1 activity contributes to p53 inactivation during this process is unknown. In this study, we show that G2 and S-phase-expressed 1 (GTSE1) protein, a neg. regulator of p53, is required for G2 checkpoint recovery and that Plk1 phosphorylation of GTSE1 at Ser 435 promotes its nuclear localization, and thus shuttles p53 out of the nucleus to lead to its degrdn. during the recovery.
- 43Liu, X. S.; Song, B.; Elzey, B. D.; Ratliff, T. L.; Konieczny, S. F.; Cheng, L.; Ahmad, N.; Liu, X. Polo-like kinase 1 facilitates loss of PTEN tumor suppressor-induced prostate cancer formation J. Biol. Chem. 2011, 286 (41) 35795– 35800[ Crossref], [ PubMed], [ CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1yhtrnI&md5=7cd13ab637f92943b832ef3d8041bf90Polo-like Kinase 1 Facilitates Loss of Pten Tumor Suppressor-induced Prostate Cancer FormationLiu, X. Shawn; Song, Bing; Elzey, Bennett D.; Ratliff, Timothy L.; Konieczny, Stephen F.; Cheng, Liang; Ahmad, Nihal; Liu, XiaoqiJournal of Biological Chemistry (2011), 286 (41), 35795-35800, S35795/1-S35795/13CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the mol. mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress and that nuclear function of Pten, but not its phosphatase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a crit. regulator of the cell cycle. We show that overexpression of Plk1 correlates with genetic inactivation of Pten during prostate neoplasia formation. Significantly, we find that elevated Plk1 is crit. for Pten-depleted cells to adapt to mitotic stress for survival and that reintroduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.
- 44Padmanabhan, A.; Li, X.; Bieberich, C. J. Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific Manner J. Biol. Chem. 2013, 288 (20) 14158– 14169[ Crossref], [ PubMed], [ CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvVGqsbs%253D&md5=3591c224c9f45f24a4abc0049b139ff6Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific MannerPadmanabhan, Achuth; Li, Xiang; Bieberich, Charles J.Journal of Biological Chemistry (2013), 288 (20), 14158-14169CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)MYC levels are tightly regulated in cells, and deregulation is assocd. with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degrdn. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate pos. feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biol. roles of the different PKA catalytic subunits.
- 45Song, M. S.; Salmena, L.; Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor Nat. Rev. Mol. Cell. Biol. 2012, 13 (5) 283– 296[ PubMed], [ CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFSrurg%253D&md5=c0d6524414a050876046c647caacb9faThe functions and regulation of the PTEN tumour suppressorSong, Min Sup; Salmena, Leonardo; Pandolfi, Pier PaoloNature Reviews Molecular Cell Biology (2012), 13 (5), 283-296CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. The importance of the physiol. function of phosphatase and tensin homolog (PTEN) is illustrated by its frequent disruption in cancer. By suppressing the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway through its lipid phosphatase activity, PTEN governs a plethora of cellular processes including survival, proliferation, energy metab. and cellular architecture. Consequently, mechanisms regulating PTEN expression and function, including transcriptional regulation, post-transcriptional regulation by non-coding RNAs, post-translational modifications and protein-protein interactions, are all altered in cancer. The repertoire of PTEN functions has recently been expanded to include phosphatase-independent activities and crucial functions within the nucleus. Our increasing knowledge of PTEN and pathologies in which its function is altered will undoubtedly inform the rational design of novel therapies.
- 46Tan, J.; Li, Z.; Lee, P. L.; Guan, P.; Aau, M. Y.; Lee, S. T.; Feng, M.; Lim, C. Z.; Lee, E. Y.; Wee, Z. N.; Lim, Y. C.; Karuturi, R. K.; Yu, Q. PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell activation, and resistance to mTOR-targeted therapy Cancer Discovery 2013, 3 (10) 1156– 1171[ Crossref], [ PubMed], [ CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1WnsrnI&md5=2cc7af3a1c063ec2f7e1181d41037f9ePDK1 Signaling Toward PLK1-MYC Activation Confers Oncogenic Transformation, Tumor-Initiating Cell Activation, and Resistance to mTOR-Targeted TherapyTan, Jing; Li, Zhimei; Lee, Puay Leng; Guan, Peiyong; Aau, Mei Yee; Lee, Shuet Theng; Feng, Min; Lim, Cheryl Zihui; Lee, Eric Yong Jing; Wee, Zhen Ning; Lim, Yaw Chyn; Karuturi, R. K. Murthy; Yu, QiangCancer Discovery (2013), 3 (10), 1156-1171CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)Although 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been predominately linked to the phosphoinositide 3-kinase (PBK)-AKT pathway, it may also evoke addnl. signaling outputs to promote tumorigenesis. Here, we report that PDK1 directly induces phosphorylation of Polo-like kinase I (PLK1), which in turn induces MYC phosphorylation and protein accumulation. We show that PDK1-PLK1-MYC signaling is crit. for cancer cell growth and survival, and small-mol. inhibition of PDK1/PLK1 provides an effective approach for therapeutic targeting of MYC dependency. Intriguingly, PDK1-PLK1-MYC signaling induces an embryonic stem cell-like gene signature assocd. with aggressive tumor behaviors and is a robust signaling axis driving cancer stem cell (CSC) self-renewal. Finally, we show that a PLK1 inhibitor synergizes with an mTOR inhibitor to induce synergistic antitumor effects in colorectal cancer by antagonizing compensatory MYC induction. These findings identify a novel pathway in human cancer and CSC activation and provide a therapeutic strategy for targeting MYC-assocd. tumorigenesis and therapeutic resistance.
- 47Shen, M.; Schmitt, S.; Buac, D.; Dou, Q. P. Targeting the ubiquitin-proteasome system for cancer therapy Expert Opin. Ther. Targets 2013, 17 (9) 1091– 1108[ Crossref], [ PubMed], [ CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1ymtb%252FF&md5=f592626af4335402281ab4cdd9e9edacTargeting the ubiquitin-proteasome system for cancer therapyShen, Min; Schmitt, Sara; Buac, Daniela; Dou, Q. PingExpert Opinion on Therapeutic Targets (2013), 17 (9), 1091-1108CODEN: EOTTAO; ISSN:1472-8222. (Informa Healthcare)A review. Introduction: The ubiquitin-proteasome system (UPS) degrades 80 - 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway. Areas covered: This review provides a broad overview of the UPS and its role in supporting cancer development and progression, esp. in aspects of p53 inactivation, p27 turnover and NF-κB activation. Also, efforts toward the development of small mol. inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed. Expert opinion: Whereas some of the targets in the UPS, such as the 20S proteasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest.
- 48Sahara, K.; Kogleck, L.; Yashiroda, H.; Murata, S. The mechanism for molecular assembly of the proteasome Adv. Biol. Regul. 2014, 54C, 51– 58
- 49Ando, K.; Ozaki, T.; Yamamoto, H.; Furuya, K.; Hosoda, M.; Hayashi, S.; Fukuzawa, M.; Nakagawara, A. Polo-like kinase 1 (Plk1) inhibits p53 function by physical interaction and phosphorylation J. Biol. Chem. 2004, 279 (24) 25549– 26661[ Crossref], [ PubMed], [ CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXksFygtr8%253D&md5=eef2ecb9875f8a7ba8937701ae08f884Polo-like Kinase 1 (Plk1) Inhibits p53 Function by Physical Interaction and PhosphorylationAndo, Kiyohiro; Ozaki, Toshinori; Yamamoto, Hideki; Furuya, Kazushige; Hosoda, Mitsuchika; Hayashi, Syunji; Fukuzawa, Masahiro; Nakagawara, AkiraJournal of Biological Chemistry (2004), 279 (24), 25549-25561CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addn. to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 phys. binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunopptn. analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the phys. interaction with Plk1. The ectopically over-expressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21WAF1, MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter anal., Plk1 had an ability to block the p53-dependent induction of the endogenous p21WAF1. In addn., Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a crit. target of Plk1, and its function is abrogated through the phys. interaction with Plk1.
- 50Bhat, U. G.; Halasi, M.; Gartel, A. L. FoxM1 is a general target for proteasome inhibitors PLoS One 2009, 4 (8) e6593
- 51Dias, S. S.; Hogan, C.; Ochocka, A. M.; Meek, D. W. Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover FEBS Lett. 2009, 583 (22) 3543– 3548[ Crossref], [ PubMed], [ CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWls7zO&md5=2e05278d074a76910b659eec5c5609f0Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnoverDias, Sylvia S.; Hogan, Carol; Ochocka, Anna-Maria; Meek, David W.FEBS Letters (2009), 583 (22), 3543-3548CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)E3 ubiquitin ligase MDM2 (murine double-minute clone 2), promotes the degrdn. of p53 protein under normal homeostatic conditions. Several Ser residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signaling pathways that mediate these phosphorylation events are not fully understood. Here, the authors show that oncogenic and cell cycle-regulatory protein kinase Plk1 (polo-like kinase-1) phosphorylates MDM2 at one of these residues, Ser-260, and stimulates MDM2-mediated turnover of p53. These data were consistent with the idea that deregulation of Plk1 during tumorigenesis may help suppress p53 function.
- 52Fu, Z.; Malureanu, L.; Huang, J.; Wang, W.; Li, H.; van Deursen, J. M.; Tindall, D. J.; Chen, J. Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression Nat. Cell Biol. 2008, 10 (9) 1076– 1082[ Crossref], [ PubMed], [ CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVGgtLnL&md5=6614ca56c9321979d4fc23f49a721f14Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progressionFu, Zheng; Malureanu, Liviu; Huang, Jun; Wang, Wei; Li, Hao; van Deursen, Jan M.; Tindall, Donald J.; Chen, JunjieNature Cell Biology (2008), 10 (9), 1076-1082CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Proper control of entry into and progression through mitosis is essential for normal cell proliferation and the maintenance of genome stability. The mammalian mitotic kinase, Polo-like kinase 1 (Plk1), is involved in multiple stages of mitosis. Here, the authors report that transcription factor, Forkhead Box M1 (FoxM1), a substrate of Plk1, controls a transcriptional program that mediates Plk1-dependent regulation of cell-cycle progression. The C-terminal domain of FoxM1 binds Plk1, and phosphorylation of 2 key residues in this domain by Cdk1 is essential for Plk1-FoxM1 interaction. The formation of the Plk1-FoxM1 complex allows for direct phosphorylation of FoxM1 by Plk1 at G2/M and the subsequent activation of FoxM1 activity, which is required for expression of key mitotic regulators, including Plk1 itself. Thus, Plk1-dependent regulation of FoxM1 activity provides a pos.-feedback loop ensuring tight regulation of transcriptional networks essential for orderly mitotic progression.
- 53McKenzie, L.; King, S.; Marcar, L.; Nicol, S.; Dias, S. S.; Schumm, K.; Robertson, P.; Bourdon, J. C.; Perkins, N.; Fuller-Pace, F.; Meek, D. W. p53-dependent repression of polo-like kinase-1 (PLK1) Cell Cycle 2010, 9 (20) 4200– 4212[ Crossref], [ PubMed], [ CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1ahu74%253D&md5=4dbd41a29399a64db6ed1ab0f5b87fcep53-dependent repression of polo-like kinase-1 (PLK1)McKenzie, Lynsey; King, Sharon; Marcar, Lynnette; Nicol, Sam; Dias, Sylvia S.; Schumm, Katie; Robertson, Pamela; Bourdon, Jean-Christophe; Perkins, Neil; Fuller-Pace, Frances; Meek, David W.Cell Cycle (2010), 9 (20), 4200-4212CODEN: CCEYAS; ISSN:1538-4101. (Landes Bioscience)PLK1 is a crit. mediator of G2/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G2/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G1/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunopptn. anal. indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-assocd. changes in the chromatin. Downregulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Addnl., wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G2/M arrest.
- 54Michael, D.; Oren, M. The p53-Mdm2 module and the ubiquitin system Semin. Cancer Biol. 2003, 13 (1) 49– 58[ Crossref], [ PubMed], [ CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpvVWksrw%253D&md5=187aa679ba07100da1e95e1891777828The p53-Mdm2 module and the ubiquitin systemMichael, Dan; Oren, MosheSeminars in Cancer Biology (2003), 13 (1), 49-58CODEN: SECBE7; ISSN:1044-579X. (Elsevier Science Ltd.)A review. The p53 tumor suppressor protein is a short-lived protein, which is stabilized in response to cellular stress. The ubiquitination and degrdn. of p53 are largely controlled by Mdm2, an oncogenic E3 ligase. Stress signals lead to p53 stabilization either by induction of covalent modifications in Mdm2 and p53, or through altered protein-protein interactions. Mdm2 also harbors a post-ubiquitination function, probably enabling efficient targeting of ubiquitinated p53 to the proteasome. p53 ubiquitination is assocd. with its export from the nucleus into the cytoplasm. However, the exact site of degrdn. of p53 is presently under debate. P53 may be targeted by other E3 ligases besides Mdm2, as well as by non-proteasomal mechanisms. Despite extensive information about p53 degrdn., many important aspects remain unresolved.
- 55Wierstra, I. FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapy Adv. Cancer Res. 2013, 119, 191– 419[ Crossref], [ PubMed], [ CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1elt7nE&md5=6d9f85cd02e81372c6f30773464f78e3FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapyWierstra, InkenAdvances in Cancer Research (2013), 119 (), 191-419CODEN: ACRSAJ; ISSN:0065-230X. (Elsevier Inc.)A review. FOXM1 (Forkhead box M1) is a typical proliferation-assocd. transcription factor and is also intimately involved in tumorigenesis. FOXM1 stimulates cell proliferation and cell cycle progression by promoting the entry into S-phase and M-phase. Addnl., FOXM1 is required for proper execution of mitosis. In accordance with its role in stimulation of cell proliferation, FOXM1 exhibits a proliferation-specific expression pattern and its expression is regulated by proliferation and anti-proliferation signals as well as by proto-oncoproteins and tumor suppressors. Since these factors are often mutated, overexpressed, or lost in human cancer, the normal control of the foxm1 expression by them provides the basis for deregulated FOXM1 expression in tumors. Accordingly, FOXM1 is overexpressed in many types of human cancer. FOXM1 is intimately involved in tumorigenesis, because it contributes to oncogenic transformation and participates in tumor initiation, growth, and progression, including pos. effects on angiogenesis, migration, invasion, epithelial-mesenchymal transition, metastasis, recruitment of tumor-assocd. macrophages, tumor-assocd. lung inflammation, self-renewal capacity of cancer cells, prevention of premature cellular senescence, and chemotherapeutic drug resistance. However, in the context of urethane-induced lung tumorigenesis, FOXM1 has an unexpected tumor suppressor role in endothelial cells because it limits pulmonary inflammation and canonical Wnt signaling in epithelial lung cells, thereby restricting carcinogenesis. Accordingly, FOXM1 plays a role in homologous recombination repair of DNA double-strand breaks and maintenance of genomic stability, i.e., prevention of polyploidy and aneuploidy. The implication of FOXM1 in tumorigenesis makes it an attractive target for anticancer therapy, and several antitumor drugs have been reported to decrease FOXM1 expression.
- 56Yang, X.; Li, H.; Zhou, Z.; Wang, W. H.; Deng, A.; Andrisani, O.; Liu, X. Plk1-mediated phosphorylation of Topors regulates p53 stability J. Biol. Chem. 2009, 284 (28) 18588– 18592[ Crossref], [ PubMed], [ CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotVKnsrk%253D&md5=2e20a9458b44003d3315fab622eaa9a2Plk1-mediated phosphorylation of topors regulates p53 stabilityYang, Xiaoming; Li, Hongchang; Zhou, Zinan; Wang, Wen-Horng; Deng, Anping; Andrisani, Ourania; Liu, XiaoqiJournal of Biological Chemistry (2009), 284 (28), 18588-18592CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Polo-like kinase 1 (Plk1) overexpression is assocd. with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a neg. regulator of tumor suppressor p53, but the mechanism remains to be detd. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser718 in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degrdn. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degrdn. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.
- 57Cholewa, B. D.; Liu, X.; Ahmad, N. The role of polo-like kinase 1 in carcinogenesis: cause or consequence? Cancer Res. 2013, 73 (23) 6848– 6855[ Crossref], [ PubMed], [ CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKhurfM&md5=214e9d9c7e526aa0cae71d28843ef667The Role of Polo-like Kinase 1 in Carcinogenesis: Cause or Consequence?Cholewa, Brian D.; Liu, Xiaoqi; Ahmad, NihalCancer Research (2013), 73 (23), 6848-6855CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)A review. Polo-like kinase 1 (Plk1) is a well-established mitotic regulator with a diverse range of biol. functions continually being identified throughout the cell cycle. Preclin. evidence suggests that the mol. targeting of Plk1 could be an effective therapeutic strategy in a wide range of cancers; however, that success has yet to be translated to the clin. level. The lack of clin. success has raised the question of whether there is a true oncogenic addiction to Plk1 or if its overexpression in tumors is solely an artifact of increased cellular proliferation. In this review, we address the role of Plk1 in carcinogenesis by discussing the cell cycle and DNA damage response with respect to their assocns. with classic oncogenic and tumor suppressor pathways that contribute to the transcriptional regulation of Plk1. A thorough examn. of the available literature suggests that Plk1 activity can be dysregulated through key transformative pathways, including both p53 and pRb. On the basis of the available literature, it may be somewhat premature to draw a definitive conclusion on the role of Plk1 in carcinogenesis. However, evidence supports the notion that oncogene dependence on Plk1 is not a late occurrence in carcinogenesis and it is likely that Plk1 plays an active role in carcinogenic transformation. Cancer Res; 73(23); 6848-55. ©2013 AACR.
- 58Burd, C. G.; Swanson, M. S.; Gorlach, M.; Dreyfuss, G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts Proc. Natl. Acad. Sci. U. S. A. 1989, 86 (24) 9788– 9792[ Crossref], [ PubMed], [ CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktFyltbs%253D&md5=15bf6aa44341f77b3e6e38391a594a6cPrimary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide insertsBurd, Christopher G.; Swanson, Maurice S.; Gorlach, Matthias; Dreyfuss, GideonProceedings of the National Academy of Sciences of the United States of America (1989), 86 (24), 9788-92CODEN: PNASA6; ISSN:0027-8424.Some cDNAs for the major heterogeneous nuclear ribonucleoprotein (hnRNP) A2, B1, and C2 proteins were isolated and their nucleotide and deduced amino acid sequences were detd. The A2 and B1 cDNAs are identical except for a 36-nucleotide in-frame insert in B1. Similarly, the sequence of the C2 protein cDNA is related to that of C1 in that C2 contains an extra 39 in-frame nucleotides. Therefore, the B1 amino acid sequence is identical to A2 except for the insertion of 12 amino acids near its amino terminus, and C1 and C2 are also identical to each other except for an extra 13 amino acids near the middle of C2. All 3 proteins are members of a large family of RNA-binding proteins that contain the consensus sequence-type RNA-binding domain (CS-RBD). The A2 and B1 proteins have a modular structure similar to that of the hnRNP protein A1: they contain two CS-RBDs and a glycine-rich auxiliary domain at the carboxyl terminus. The CS-RBDs of A2 and B1 have ≈80% amino acid identity with those of A1, whereas the glycine-rich auxiliary domain is considerably more divergent with <30% of the amino acids being identical. Thus, the addn. of small peptides, probably by alternative pre-mRNA splicing, generates some of the diversity apparent among hnRNP proteins.
- 59Krecic, A. M.; Swanson, M. S. hnRNP complexes: composition, structure, and function Curr. Opin. Cell Biol. 1999, 11 (3) 363– 371[ Crossref], [ PubMed], [ CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXktFWms7g%253D&md5=66f04865aad1c966462a042802e2ea8fhnRNP complexes: composition, structure, and functionKrecic, Annette M.; Swanson, Maurice S.Current Opinion in Cell Biology (1999), 11 (3), 363-371CODEN: COCBE3; ISSN:0955-0674. (Current Biology Publications)A review with 62 refs. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins function in a staggering array of cellular activities, ranging from transcription and pre-mRNA processing in the nucleus to cytoplasmic mRNA translation and turnover. Recent studies suggest that several fundamental characteristics of hnRNPs account for their involvement in multiple regulatory pathways.
- 60Sun, D. Q.; Wang, Y.; Liu, D. G. Overexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cells Oncol. Lett. 2013, 5 (4) 1243– 1249[ PubMed], [ CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1KitLk%253D&md5=82c2d1915d97ad9a89c9bd3a0d4c0f5fOverexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cellsSun, Da-Quan; Wang, Ying; Liu, Ding-GanOncology Letters (2013), 5 (4), 1243-1249CODEN: OLNEB5; ISSN:1792-1074. (Spandidos Publications Ltd.)Heterogeneous ribonuclear protein C2 (hnRNPC2), an RNA binding protein, is a component of hnRNPC which is upregulated in many tumors. Multinucleation exists in many tumors and is pos. correlated with tumor grade. To uncover the correlation between hnRNPC2 and multinucleation in hepatocellular carcinoma SMMC-7721 cells, we constructed a pEGFP-hnRNPC2 vector and transfected it into cancer cells. Our results revealed that overexpression of hnRNPC2 induced multinucleation in SMMC-7721 cells. Tracking tests indicated that the induced multinucleated cells were unable to recover to mononuclear cells and finally died as a result of defects in cell division. Furthermore, Aurora B, which was localized at the midbody and plays a role in cytokinesis, was repressed in hnRNPC2-overexpressing cells, whose knockdown by RNA interference also induced multinucleation in SMMC-7721 cells. Quant. polymerase chain reaction (qPCR) and mRNA-protein co-immunopptn. results revealed that Aurora B mRNA did not decrease in hnRNPC2-overexpressing cells, instead it bound more hnRNPC2 and less eIF4E, an mRNA cap binding protein and translational initiation factor. Moreover, hnRNPC2 bound more eIF4E in hnRNPC2-overexpressing cells. These results indicate that hnRNPC2 repressed Aurora B binding with eIF4F, which must bind with Aurora B mRNA in order to initiate its translation. This induced multinucleation in hepatocellular carcinoma cells. In addn., hnRNPC2 accelerated hepatocellular carcinoma cell proliferation. Collectively, these data suggest that hnRNPC2 may be a potential target for hepatocellular carcinoma cell diagnosis and treatment.
- 61Christian, K. J.; Lang, M. A.; Raffalli-Mathieu, F. Interaction of heterogeneous nuclear ribonucleoprotein C1/C2 with a novel cis-regulatory element within p53 mRNA as a response to cytostatic drug treatment Mol. Pharmacol. 2008, 73 (5) 1558– 1567[ Crossref], [ PubMed], [ CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlsVWgu7Y%253D&md5=5f6856542e8072fcc854bd88090d92a6Interaction of heterogeneous nuclear ribonucleoprotein C1/C2 with a novel cis-regulatory element within p53 mRNA as a response to cytostatic drug treatmentChristian, Kyle J.; Lang, Matti A.; Raffalli-Mathieu, FrancoiseMolecular Pharmacology (2008), 73 (5), 1558-1567CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)We describe a novel cis-element in the 5' coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP)C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides down-stream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is obsd. in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qual. changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addn., we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.
Supporting Information
ARTICLE SECTIONSTable S1. Complete protein list identified by nonunique peptide hits having a protein ratio of a high confidence level (p < 0.05), showing Swiss-Prot ID, protein description, number of peptides, frames, hits, and normalized ratio. Table S2. Complete protein list identified by unique peptide hits having a protein ratio of a high confidence level (p < 0.05), showing Swiss-Prot ID, protein description, number of peptides, frames, hits and normalized ratio. This material is available free of charge via the Internet at http://pubs.acs.org.
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