Abstract Checkpoint kinase1 (CHK1) is a DNA damage kinase, which has been observed to be constitutively active in human cancers. Unfortunately a number of new generation CHK1 inhibitors, currently in clinical trials, have shown deleterious effects on normal cells, thus limiting their clinical utility. Therefore, identification and targeting of cancer specific effectors of CHK1 signaling has emerged as an attractive therapeutic alternative. Analysis of the REMBRANDT and TCGA datasets revealed a strong positive correlation for CHK1 and CIP2A expression in 422/522 and 356/454 glioma patients respectively. By contrast, there was negligible correlation observed in normal samples. Additionally, high mRNA expression of both CHK1 and CIP2A was associated with reduced overall survival in glioma patients and marked a more aggressive form of the disease. Notably, CIP2A amplification found in 14.72% cases in the Rembrandt study was associated with worse overall survival in GBM patients. Mechanistically, we identify STAT3 as a transcriptional mediator for CHK-dependent CIP2A expression in GBM cells. We demonstrate that CHK1 positively regulates CIP2A transcription by promoting phosphorylation of STAT3 at Tyrosine 705 both in vitro and in vivo. Further, depletion of STAT3 by siRNA or chemical inhibitors resulted in decreased CIP2A expression in GBM cells. Functionally, both CHK1 and CIP2A promoted viability, clonogenicity and anchorage-independent growth of GBM cells. Importantly, inhibition of cancer cell viability and clonogenicity by Chk1 inhibition can be partially rescued by exogenous overexpression of CIP2A. Higher CIP2A, p-S345-CHK1 and p-Ty705-STAT3 levels were observed in de novo and patient-derived GBM, and U251MG cells relative to levels in normal human astrocytes (NHA). Analogously, CHK1 and CIP2A mRNA expression is increased 10 to 15 fold in genetically engineered mouse(GEM) models of human GBM (Pdgf-Cre-driven Ptenf/f and Pdgf-Cre-driven Ptenf/f;/p53f/f double mutant mice compared with wild-type mice). Finally, using xenograft mouse models we show that both PF477736 (a small molecule inhibitor of CHK1) and CIP2A depletion inhibits tumor growth. These results highlight CHK1 and CIP2A expression as potential diagnostic and prognostic markers in human GBMs. Further, these findings identify CIP2A as a cancer specific therapeutic target downstream of constitutively active CHK1 and STAT3 signaling in GBM. Together, these data serves as a platform for using CIP2A expression in GBM, to either directly target CIP2A or to stratify patients for CHK1 and/or STAT3 inhibition. Note: This abstract was not presented at the meeting. Citation Format: Anchit Khanna, Brett Stringer, Bryan Day, Kathleen Ensbey, Han Shen, Andrew Boyd, Kerrie McDonald, John E. Pimanda. Keeping glioblastoma (GBM)in check by targeting the CHK1-STAT3-CIP2A axis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2014-1600
<div>Abstract<p>High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both <i>in vitro</i> and <i>in vivo</i> models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth <i>in vitro</i> and <i>in vivo</i>. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A.</p>Implications:<p>High expression of CIP2A in gliomas is maintained by a CHK1-dependent pSTAT3–CIP2A recursive loop; interrupting CIP2A induces cell senescence and slows GBM growth adding impetus to the development of CIP2A as an anticancer drug target.</p></div>
<p>PDF file - 213K, Figure S5. Regulation of MYC and cell cycle by Chk1-CIP2A pathway and effect of DNA-PK on CIP2A and pS345-Chk1 levels independent of ATM/ATR.</p>
// Anchit Khanna 1,2 , Jayant K. Rane 3 , Kati K. Kivinummi 1,4 , Alfonso Urbanucci 1,5,6 , Merja A. Helenius 1 , Teemu T. Tolonen 1 , Outi R. Saramäki 1 , Leena Latonen 1 , Visa Manni 1 , John E. Pimanda 2 , Norman J. Maitland 3 , Jukka Westermarck 7,8 and Tapio Visakorpi 1 1 Prostate Cancer Research Center (PCRC), Institute of Biosciences and Medical Technology (BioMediTech), University of Tampere and Tampere University Hospital, Tampere, Finland 2 Adult Cancer Program, The Prince of Wales Clinical School, Lowy Cancer Research Centre, UNSW Medicine, University of New South Wales, Sydney, Australia 3 YCR Cancer Research Unit, Department of Biology, The University of York, Heslington, United Kingdom 4 Department of Signal Processing, Tampere University of Technology, Tampere, Finland 5 Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway 6 Department of Cancer Prevention, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway 7 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland 8 Department of Pathology, University of Turku, Turku, Finland Correspondence to: Tapio Visakorpi, email: // Anchit Khanna, email: // Keywords : CIP2A, androgen receptor, castration-resistant prostate cancer, cancer stem-like cells Received : December 30, 2014 Accepted : April 03, 2015 Published : April 19, 2015 Abstract Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach. Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired. These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.
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