Plk1 Protein Research Articles

Abstract P2-10-43: The combination of immunohistochemistry for predicting TP53 mutation is useful prognostic marker in breast cancer.

Abstract The comprehensive expression analysis classified subtypes of breast cancer which is clinically applied for immunohistochemistry (IHC) with ER, HER2 and Ki67. On the other hand, recent whole genome sequencing reveals a certain quantity of TP53 mutation in breast cancer, especially with ER negative tumor. Therefore, it is important for risk evaluation that TP53 mutation is applied for IHC. However, p53 IHC is not well correlated with TP53 mutation because of false negative (deletion mutant) and false positive (stabilization of p53). This study is initiated to improve accuracy to combine the IHC of p53 and p53-regulated protein for predicting TP53 mutation. First, we explored p53-dependent down regulated proteins to rescue the false negative of p53 IHC cases. Using the microarray analysis and DNA sequencing of TP53 gene in 37 breast cancer patients, we extracted 97genes which were overexpressed in TP53 mutation breast cancer cases compared with TP53 wild cases. Furthermore, using the microarray analysis by two tet-inducible p53 cell lines, 597 genes were extracted for p53-dependent repressed genes. There were 29 common genes between both of them. In 29genes, we picked up three genes, BIRC5, BUB1 and PLK1 which were confirmed with p53-dependent repression by RT-PCR or luciferase assay. Next, the correlation of TP53 status and IHC of these genes products and p53 were evaluated. In 37 breast cancer patients, TP53 mutations were detected in 19 cases (51%). There were 11 missense mutations, 5 protein-truncating mutations and two splicing-site mutations. p53 IHC positive (>10%) were 12 cases (32.4%) which included only 9 TP53 mutations. Five protein-truncating TP53 mutations were all negative staining of p53. Among BIRC5, BUB1 and PLK1 proteins, positive staining of PLK1 is the most correlated with TP53 mutations, sensitivity 0.78, specificity 0.84 and positive predictive value 0.78 (p < 0.0001). Third, we tested well known p53-tageted gene product p21 IHC whether it is used for decreasing false positive of p53 IHC. Although strong staining of p21 is only 3 cases (8.1%), all of three were wild type TP53. Therefore IHC of p53 + and p21 – (to decrease false positive of p53) or p53- and PLK1+ (to save false negative of p53) were defined as IHC-TP53 mutation+. The IHC-TP53 mutation + were strongly correlated with TP53 mutations, sensitivity 0.89, specificity 0.95 and positive predictive value 0.94 (p < 0.0001). Furthermore, p53 IHC, TP53 mutation and IHC-TP53 mutation were evaluated as prognostic marker. In only 37 breast cancers, TP53 mutation and IHC-TP53 mutation+ were statistically significant for poor prognosis (log rank test, p = 0.005, p = 0.023, respectively) whereas p53 IHC was not. We next carried out IHC of other 157 breast cancers to validate IHC-TP53 mutation as prognostic marker. In this cases, p53 IHC had also statically significant for prognosis (p = 0.009), however IHC-TP53 mutation were more strong relation to prognosis (p = 0.0006). The combination of p53, p21 and PLK1 IHC are well predicted TP53 mutations and is a useful biological, prognostic marker such as ER or HER2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-43.

Polo-Like Kinase 1 (PLK1) Inhibition Reduces Cell Proliferation and Induces Apoptosis in Childhood Acute Lymphoblastic Leukemia

AbstractAbstract 3529Polo-like Kinase 1 (PLK1) is an essential regulator of mitosis. It is often overexpressed and a predictor of poor prognosis in different types of solid tumours and adult hematopoietic malignancies, making PLK1 an interesting therapeutic target in these cancer types. No PLK1 inhibitor has entered clinical trials in pediatric malignancies yet. This study therefore aimed to determine the potential of PLK1 as a new target in the treatment of childhood acute lymphoblastic leukemia (ALL), the most common type of childhood cancer.Reverse phase protein arrays were performed to analyze levels of PLK1 protein expression and phosphorylation at T210, the major activating phosphorylation site, in ALL patient samples (n=174) and normal bone marrow mononuclear cells (nBM) (n=11). Both PLK1 expression and T210 phosphorylation were elevated (2.6-fold and 1.8-fold, respectively) in patients compared to nBM (p<0.0001). Furthermore, PLK1 protein expression levels were 1.3-fold higher in ALL cells carrying an E2A-PBX1 translocation than in other ALL genetic subtypes (p<0.0001). Knockdown of PLK1 expression by lentivirally delivered short hairpins led to inhibition of cell proliferation and induction of apoptosis in three precursor B-ALL cell lines and one T-ALL cell line, as indicated by decreased cell numbers and increased cleavage of PARP. This suggests an essential role for PLK1 in maintenance of the leukemic cell. An MTS cell viability assay was used to test the efficacy of the PLK1-specific inhibitor NMS-P937 (NMS-1286937) in primary ALL samples (n=15). When exposed to 120nM of NMS-P937 for 96 hours, there was a significant reverse correlation between PLK1 protein expression and leukemic cell survival (rs=−0.58; p=0.024): patient cells with a high expression were more sensitive than cells with a low expression, whereas nBM cells were resistant up to concentrations in the micromolar range. Cumulative incidence of relapse and non-response to therapy (CIR) did not significantly differ between ALL patients with high and low levels of PLK1 protein (CIR=25.9% vs 15.7%; p=0.215). Patients with a high PLK1 T210 phosphorylation level, however, showed a trend towards a higher CIR than those with a low expression (CIR=27.5% vs 14.0%; p=0.078). Finally, Sanger sequencing of PLK1 mutational hot-spots led to the identification of a new non-synonymous mutation in 1 out of 38 patients. This mutation was located in exon 5 of the PLK1 gene, resulting in Ser335Arg, and did not lead to unusual levels of PLK1 protein or T210 phosphorylation, nor to NMS-P937 sensitivity.Taken together, these results show that PLK1 is overexpressed in pediatric ALL and plays a pivotal role in the proliferation and survival of pediatric ALL cells. Moreover, they underline the potency of PLK1-inhibiting drugs as a valuable addition to current ALL treatment strategies, especially for cases expressing high levels of PLK1 protein. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

BackgroundPolo-like kinase 1 (PLK1) is highly expressed in many human cancers and regulates critical steps in mitotic progression. Previously, we have reported that PLK1 was overexpressed in non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms are not well understood. By using microRNA (miR) target prediction algorithms, we identified miR-100 that might potentially bind the 3’-untranslated region of PLK1 transcripts. The purpose of this study was to investigate the roles of miR-100 and its association with PLK1 in NSCLC development.MethodsTaqman real-time quantitative RT-PCR assay was performed to detect miR-100 expression 10 NSCLC tissues and corresponding nontumor tissues. Additionally, the expression of miR-100 in 110 NSCLC tissues and its correlation with clinicopathological factors or prognosis of patients was analyzed. Finally, the effects of miR-100 expression on growth, apoptosis and cell cycle of NSCLC cells by posttranscriptionally regulating PLK1 expression were determined.ResultsMiR-100 was significantly downregulated in NSCLC tissues, and low miR-100 expression was found to be closely correlated with higher clinical stage, advanced tumor classification and lymph node metastasis of patients. The overall survival of NSCLC patients with low miR-100 was significantly lower than that of those patients with high miR-100, and univariate and multivariate analyses indicated that low miR-100 expression might be a poor prognostic factor. Also, miR-100 mimics could lead to growth inhibition, G2/M cell cycle arrest and apoptosis enhancement in NSCLC cells. Meanwhile, miR-100 mimics could significantly inhibit PLK1 mRNA and protein expression and reduce the luciferase activity of a PLK1 3’ untranslated region-based reporter construct in A549 cells. Furthermore, small interfering RNA (siRNA)-mediated PLK1 downregulation could mimic the effects of miR-100 mimics while PLK1 overexpression could partially rescue the phenotypical changes of NSCLC cells induced by miR-100 mimics.ConclusionsOur findings indicate that low miR-100 may be a poor prognostic factor for NSCLC patients and functions as a tumor suppressor by posttranscriptionally regulating PLK1 expression.

Dissecting the phenotypes of Plk1 inhibition in cancer cells using novel kinase inhibitory chemical CBB2001

Polo-like kinase 1 (Plk1) is a mitotic serine/threonine kinase and its kinase activity is closely interrelated to cell cycle progression, various types of cancer development and often correlates with poor prognosis. Thus, it is of prime importance to characterize the phenotypes of Plk1 inhibition in cells for drug development and clinical application. Here, we report a novel kinase inhibitory chemical, CBB2001, which specifically inhibited Plk1 kinase activity in vitro with an IC50 of 0.39 μM. In cervical carcinoma HeLa cells, we found that treatment of CBB2001 caused mitotic cell cycle arrest (EC50=0.72 μM) and induction of ‘polo' cells (EC50=0.32 μM). Interestingly, the cell cycle arrest induced by CBB2001 was associated with accumulation of Plk1 (EC50=0.61 μM) and Geminin (EC50=0.43 μM) proteins, but distinct from the phenotypes induced by Aurora kinase inhibitors. The inhibitory effects of CBB2001 were phenocopied by RNA interferences of Plk1. We also confirmed the cell cycle inhibitory effects of CBB2001 in other cancer cells. Moreover, CBB2001 inhibited the growth of HeLa cells with an IC50 of 0.85 μM in MTT assays, which is better than that of reported Plk1 inhibitory chemicals ON01910 (IC50=6.46 μM) and LFM-A13 (IC50=37.36 μM). CBB2001 also inhibited mouse xenograft tumor growth. Furthermore, CBB2001 inhibited mitotic exit and delayed degradation of APC/C substrates, Geminin, Cyclin B1 and Aurora A. These specific phenotypes may serve as specific features for Plk1 inhibition and for Plk1-based clinic trials.

Numb Regulates Stability and Localization of the Mitotic Kinase PLK1 and Is Required for Transit through Mitosis

Numb functions in progenitor cell fate determination and early development, but it is also expressed in postdevelopmental tissues and cancers where its role is unclear. In this study, we report that a targeted knockdown of Numb expression causes a G(2)-M arrest and reduced cell growth in human melanoma cells. Co-immunoprecipitation and colocalization studies showed that Numb interacts with the serine/threonine polo-like kinase Plk1 and Numb cycles in a cell-cycle-dependent fashion along with this mitotic regulator. Interestingly, Numb expression was required for Plk1 protein stability and localization to the spindle poles during mitosis. Reduction in Numb expression resulted in mislocalization of Plk1 at both metaphase and anaphase, leading to disorganized γ-tubulin recruitment in centrosomes. Together, our findings present a novel function for Numb during symmetric cell division. We suggest that dysregulation of Numb expression results in mislocalized Plk1 and poor centrosomal γ-tubulin recruitment, potentially contributing to mitotic errors, aneuploidy, and cancer development.

SmSak, the Second Polo-Like Kinase of the Helminth Parasite Schistosoma mansoni: Conserved and Unexpected Roles in Meiosis

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.

Abstract 2131: Plk1 decreases DNA double strand break (DSB) formation during replication- stalling genotoxic stress

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. The aim of the present study was to determine the ability of Plk1 to affect the DNA damage response in the face of different forms of DSB-inducing genotoxic stress. We employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], the anticancer drug and topoisomerase II inhibitor, etoposide, as well as the oxidizer, H2O2. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by the respective genotoxins under the condition of Plk1 activation by using the comet assay and γH2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased &amp;gt; 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated both Cr(VI) and etoposide-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control-transfected cells. In sharp contrast, treatment with 150 μM H2O2 induced a similar level of DNA DSBs in both the c/a T210D mutant- and vector-transfected cells. These data highlight the ability of Plk1 to decrease DNA DSBs uniquely under conditions of replication-stalling genotoxic stress. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates decreased DNA DSBs after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to abrogate DNA DSB formation after either Cr(VI) or etoposide exposure may occur at the expense of genomic stability. Supported by NIH grants CA107972 and ES017334 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2131. doi:1538-7445.AM2012-2131

High expression of polo-like kinase 1 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

ObjectivesPolo-like kinase 1 (Plk1) has been widely pursued as an oncology target because it is overexpressed in several human tumor types. To investigate whether Plk1 plays a general role in bladder urothelial carcinoma, we examined the expression of Plk1 protein in bladder urothelial carcinoma and cell lines, and analyzed the relationship among Plk1 protein expression, metastasis, and recurrence of urinary bladder urothelial carcinoma. MethodsImmunohistochemistry was used to detect the expression of Plk1 in 120 bladder urothelial carcinoma. Moreover, the expression of Plk1 was analyzed by Western blot in 60 bladder urothelial carcinoma and 21 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of scytonemin (the inhibitor of Plk1). Statistical analysis was used to discuss the association between Plk1 expression and clinicopathologic parameters, tumor metastasis and recurrence, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells. ResultsThere was a significantly higher Plk1expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Plk1 expression was positively correlated with histologic grade, pT stage, recurrence, and metastasis. With the increasing concentration of scytonemin, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G2/M stage. ConclusionPlk1 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Plk1 played an important function on the bladder cancer cells' proliferation by regulating the cancer cell cycle from G1/S to G2/M and probably promoted the invasion and metastasis of bladder cancer.

Expression, adverse prognostic significance and therapeutic small molecule inhibition of Polo-like kinase 1 in multiple myeloma

The amplified myeloma centrosome has been identified as a therapeutic target. The present study explored the expression and prognostic significance of the centrosome-associated protein PLK1 in myeloma and the effect of BI 2536, a potent and selective inhibitor of PLK1, on myeloma cells. High plasma cell expression of PLK1 protein in myeloma patient bone marrow biopsies is an independent adverse prognostic factor (HR = 2.3, p = 0.003 unadjusted; HR = 1.9, p = 0.03 in multivariable model). BI 2536 inhibits myeloma cell lines at nanomolar concentrations, and is therapeutic for xenografts in NOD/SCID mice. PLK1 inhibition is a potential new strategy for the treatment of multiple myeloma.

Induction of antitumor immunity using dendritic cells electroporated with Polo-like kinase 1 (Plk1) mRNA in murine tumor models

Polo-like kinase 1 (Plk1), a serine-threonine kinase, plays a key role in the regulation of the cell cycle. Elevated Plk1 expression in various cancers is correlated with poor prognosis and poor patient survival rates. Several Plk1 inhibitors are currently being developed as potential treatments for cancer. In the present study, we investigated whether dendritic cells (DC) electroporated with mouse Plk1RNA (mPlk1RNA/DC) can induce Plk1-specific immune responses and exert antitumor effects in various murine tumor models. Overexpression of Plk1 protein was confirmed in several mouse and human tumor cell lines and various cancer tissues. Furthermore, Plk1-specific CD4(+) and CD8(+) T cells were induced by vaccination with mPlk1RNA/DC and the cytotoxic activity of the T cells was demonstrated against several Plk1-expressing tumor cell lines. Vaccination with mPlk1RNA/DC inhibited the growth of MC-38 and B16F10 tumors in C57BL/6 mice and the growth of CT26 tumors in BALB/c mice. Depletion of CD8(+) T cells reversed the inhibition of tumor growth by mPlk1RNA/DC vaccination. Homologous human Plk1RNA-electroporated DC also inhibited tumor growth in MC-38 tumor-bearing mice. In addition, Plk1-specific cytotoxic T lymphocytes from PBMC of healthy donors could be induced using autologous monocyte-derived DC electroporated with RNA encoding the whole gene of human Plk1. Taken together, the results of the present study suggest that Plk1 could be a universal tumor antigen recognized by cytotoxic T lymphocytes for cancer immunotherapy.

Abstract 3914: Plk1 enhances DNA double strand break (DSB) repair during acute genotoxin exposure

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression in cancer cells during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. In this study we employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], which is a well-documented genotoxin of occupational and environmental concern. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study we measured DNA DSB induction by Cr(VI) in HLFs under the condition of Plk1 activation by using the comet assay and gamma H2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We found that DNA DSBs increased &amp;gt; 2 fold 15 min after 3 μM Cr(VI) exposure, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated Cr(VI)-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control transfected cells. Since Cr(VI)-induced DNA DSBs were equally formed by 15 min in both vector- and Plk1 T210D-transfected cells, these data highlight the heretofore unreported ability of Plk1 to enhance DNA repair. Our previous report found that Plk1 expression increased survival and mutagenesis of wt S. cerevisiae treated with Cr(VI). Therefore, we also determined the ability of Plk1 to “rescue” repair deficient rad52 yeast from Cr(VI)-induced lethality. S. cerevisae transformed with Plk1-Gal-HA-EGFP plasmid grown under inducible conditions overexpress Plk1. We found that induction of Plk1 in rad52 yeast significantly protected them from Cr(VI) clonogenic lethality. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates DNA damage repair after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to provoke DNA DSB repair after Cr(VI) exposure occurs at the expense of genomic stability. Supported by NIH grants CA107972 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2011-3914

Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for 'absolute quantification') method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope-labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as (13)C, (2)H or (15)N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase.

Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos

In ascidian embryos, the posterior-localized maternal factor Posterior End Mark (PEM) is responsible for patterning embryos along the anterior-posterior axis with regard to both cleavage pattern involving unequal cell divisions and gene expression. Although PEM plays important roles in embryogenesis, its mechanism of action is still unclear because PEM has no known functional domain. In the present study, we explored the candidate of PEM partner proteins in Halocynthia roretzi using yeast two-hybrid screening. We isolated a homologue of Polo-like kinase 1 (Plk1), a key regulator of cell division and highly conserved in eukaryotes, as the first potential binding partner of PEM. We biochemically confirmed that interaction occurred between the Plk1 and PEM proteins. Immunostaining showed that Plk1 protein concentrates in the centrosome-attracting body (CAB) at the posterior pole, where PEM protein is also localized. The CAB is a subcellular structure that plays an important role in generating the posterior cleavage pattern. Plk1 localization to the CAB was dependent on the cell cycle phases during unequal cleavage. Inhibition of Plk1 with specific drugs resulted in failure of the nucleus to migrate towards the posterior pole and formation of a microtubule bundle between the CAB and a centrosome, similarly to inhibition of PEM function, suggesting that both proteins are involved in the same process of unequal cleavages. This interrupted nuclear migration was rescued by overexpression of PEM. In Plk1-inhibited embryos, the localization of PEM protein to the CAB was impaired, indicating that Plk1 is required for appropriate localization of PEM.

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.

K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Abstract 4362: Plk1 is overexpressed in retinoblastoma and its inhibition by siRNA or small-molecule inhibitor results in growth arrest and apoptosis

Abstract Retinoblastoma (RB) is a pediatric cancer of the retina where current chemotherapeutic regimens are broad spectrum with significant side effects. Molecular target-based approaches for RB therapy are absent and may be rational for this disease. We performed a siRNA library screen for growth inhibition of human Y79 RB cells. Knockdown of Plk1 or AURKA (but not other mitotic regulatory factors) lead to greater than 90% cell growth inhibition and prompted further investigation. We determined Plk1 expression in retinoblastoma and normal retina tissue by immunohistochemistry. We also determined the effects of siRNA-mediated Plk1 knockdown and small molecule-mediated Plk1 inhibition on Y79 and Weri-Rb1 cell growth using a tetrazolium dye-based assay. Effects on proliferation and cell cycle distribution were determined by flow cytometric analysis of EdU incorporation and DNA content. Activation of DNA damage response and apoptosis was determined by western blot analysis of phospho-H2AX, cleaved caspase-3, and PARP. We observed moderate and high Plk1 protein expression in several human retinoblastoma tumor samples as compared to minimal or low expression found in normal retinal tissue. Increased Plk1 expression was observed in RB cell lines as compared to normal retina cells. 10 nM Plk1 siRNA and 30 nM BI-2536 potently inhibited Y79 and Weri-Rb1 cell growth and significantly decreased cell viability. A decrease in S phase EdU incorporation and proliferation and an increase in G1 phase population was observed in flow cytometric analysis of Plk1 inhibited RB cells. A significant increase in phospho-H2AX, cleaved caspase-3, and cleaved PARP protein levels was observed in Y79 and Weri-Rb1 cells treated with Plk1 siRNA or BI-2536. These results suggest that Plk1 may be a rational target for RB therapy and encourages preclinical studies in animal models of RB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4362.

167 BIOLOGICAL CHARACTERISTICS IN BLADDER CANCER DEPEND ON THE TYPE OF GENETIC INSTABILITY - CLOSE ASSOCIATION OF CENTROSOME AMPLIFICATION WITH DISEASE PROGRESSION

You have accessJournal of UrologyUrothelial Cancer: Natural History & Pathophysiology/Markers1 Apr 2010167 BIOLOGICAL CHARACTERISTICS IN BLADDER CANCER DEPEND ON THE TYPE OF GENETIC INSTABILITY - CLOSE ASSOCIATION OF CENTROSOME AMPLIFICATION WITH DISEASE PROGRESSION Yoshiaki Yamamoto, Taku Misumi, Kohsuke Sasaki, and Hideyasu Matsuyama Yoshiaki YamamotoYoshiaki Yamamoto More articles by this author , Taku MisumiTaku Misumi More articles by this author , Kohsuke SasakiKohsuke Sasaki More articles by this author , and Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.221AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A, p53, PLK1 and BUBR1 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. METHODS Expressions of the MLH1, MSH2, Aurora-A, p53, PLK1 and BUBR1 proteins, and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17 and 20q13 spots were evaluated by fluorescence in situ hybridization (FISH), and DNA ploidy was assessed by laser scanning cytometry. RESULTS The expression levels of the MMR related-proteins decreased in 9 out of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A, PLK1, and BUBR1 overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A, PLK1, and BUBR1 overexpression, increased p53 immunoreactivity and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. Of the CIN-related molecular markers investigated in this study, centrosome amplification was the strongest predictor for disease progression. CONCLUSIONS The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability, and that CIN may be dominant pathway relevant for malignant phenotype in bladder cancer. Ube, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e67-e68 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshiaki Yamamoto More articles by this author Taku Misumi More articles by this author Kohsuke Sasaki More articles by this author Hideyasu Matsuyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Polo-like Kinase 1 (Plk1) as a Novel Drug Target in Chronic Myeloid Leukemia: Overriding Imatinib Resistance with the Plk1 Inhibitor BI 2536

In most patients with chronic myeloid leukemia (CML), the disease can be kept under control using the BCR/ABL kinase inhibitor imatinib. Nevertheless, resistance or intolerance to imatinib and other BCR/ABL inhibitors may occur during therapy. Therefore, CML research is focusing on novel targets and targeted drugs. Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an essential role in mitosis. In this study, we examined the expression of Plk1 in CML cells and its potential role as a therapeutic target. Plk1 was found to be expressed in phosphorylated form in the CML cell line K562 as well as in primary CML cells in all patients tested. Inhibition of BCR/ABL by imatinib or nilotinib (AMN107) led to decreased expression of the Plk1 protein in CML cells, suggesting that BCR/ABL promotes Plk1 generation. Silencing of Plk1 in CML cells by a small interfering RNA approach was followed by cell cycle arrest and apoptosis. Furthermore, the Plk1-targeting drug BI 2536 was found to inhibit proliferation of imatinib-sensitive and imatinib-resistant CML cells, including leukemic cells, carrying the T315 mutation of BCR/ABL with reasonable IC(50) values (1-50 nmol/L). The growth-inhibitory effects of BI 2536 on CML cells were found to be associated with cell cycle arrest and apoptosis. Moreover, BI 2536 was found to synergize with imatinib and nilotinib in producing growth inhibition in CML cells. In conclusion, Plk1 is expressed in CML cells and may represent a novel, interesting target in imatinib-sensitive and imatinib-resistant CML.

Significance of Plk1 regulation by miR‐100 in human nasopharyngeal cancer

Polo-like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666-1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double-strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666-1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA-100 (miR-100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666-1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3'-UTR of Plk1 sequence, we document that miR-100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR-100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks

The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.