CDK5RAP3 is a novel repressor of p14ARF in hepatocellular carcinoma cells

CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14(ARF) tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14(ARF). Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14(ARF) promoter in vivo. Furthermore, knockdown of p14(ARF) in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14(ARF).published_or_final_versio

Impact of G 2 checkpoint defect on centromeric instability

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G 2 checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G 2 to M phase transition, impaired G 2 checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G 2 checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G 2 checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G 2 checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G 2 checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G 2 checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells. © 2011 Macmillan Publishers Limited All rights reserved.postprin

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy

BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850

Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4-d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine (BOS172722)

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4-d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4-d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate

CDK5RAP3 inhibits neuronal differentiation via downregulation of CDK5 activator

Co-organized and hosted by the RIKEN Center for Developmental BiologyConference Theme: Biology at the Interfac

Characterization of CDK5RAP3 in hepatocellular carcinoma

Poster Section 8 - Functional Identification of New Cancer Genes: abstract no. 243CDK5 regulatory subunit associated protein 3 (CDK5RAP3, also called C53 and LZAP) is a novel cyclin-dependent kinase 5 (CDK5) activator interacting protein that has been shown to regulate apoptosis induced by genotoxic stress. More recently, CDK5RAP3 has also been found to regulate p53 and NF-κB signaling. Here, we have characterized the role of CDK5RAP3 in hepatocellular carcinoma (HCC) cell lines. Using real-time quantitative RT-PCR, we found that 58% (39/67) of human HCC samples had significantly higher expression (at least 2-fold) of CDK5RAP3 transcripts as compared to their corresponding nontumorous livers. The overexpression of CDK5RAP3 was statistically correlated with clinicopathological feature of more metastatic phenotype. Using stable expression clones, we demonstrated that knockdown of CDK5RAP3 in HCC cell line inhibited anchorage-independent growth and tumorigenicity of cells while overexpression of CDK5RAP3 remarkably promoted tumorigenicity of cells. Our western blotting data indicated that knockdown of CDK5RAP3 in HCC cell line SMMC-7721 resulted in an upregulation of p14ARF. Using luciferase reporter assay, we showed that ectopic expression of CDK5RAP3 suppressed p14ARF promoter activity in a dose-dependent manner. Taken together, our data provide the first evidence that CDK5RAP3 is overexpressed in HCC and overexpression of CDK5RAP3 may contribute to the development of HCC via downregulation of p14ARF. In summary, CDK5RAP3 is a potential oncoprotein in promoting hepatocarcinogenesis, the regulation behind may attribute to its regulation on p14ARF expression.link_to_OA_fulltextThe 101st Annual Meeting of the American Association for Cancer Research (AACR 2010), Washington, DC., 17-21 April 2010

Functional characterization of CDK5RAP3 in Human Hepatocellular Carcinoma Cells

Conference Theme: New Paradigms in Cancer Prevention, Diagnostics, and TherapiesThe 16th Hong Kong International Cancer Congress & 6th Annual Meeting, Hong Kong, 4-6 November 2009

Functional characterization of C53 and its possible roles in tumorigenesis

Conference Theme: Oncogenes and Human Cancer- The next 25 year

Overexpression of a novel activator of PAK4, the CDK5 kinase-associated protein CDK5RAP3, promotes hepatocellular carcinoma metastasis

The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA-mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that overexpression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation. © 2011 American Association for Cancer Research.link_to_subscribed_fulltex