Cancerous Inhibitor Of Protein Phosphatase 2A Levels Research Articles

Cancerous inhibitor of protein phosphatase 2A enhances chemoresistance of gastric cancer cells to oxaliplatin.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a newly discovered oncogene. It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer (GC) cells. CIP2A is functionally related to chemoresistance in various types of tumors according to recent studies. The underlying mechanism, however, is unknown. Further, the primary treatment regimen for GC is oxaliplatin-based chemotherapy. Nonetheless, it often fails due to chemoresistance of GC cells to oxaliplatin. The goal of this study was to examine CIP2A expression and its association with oxaliplatin resistance in human GC cells. Immunohistochemistry was used to examine CIP2A expression in GC tissues and adjacent normal tissues. CIP2A expression in GC cell lines was reduced using small interfering RNA. After confirming the silencing efficiency, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium and flow cytometry assays were used to evaluate cell proliferation and apoptosis caused by oxaliplatin treatment. Further, the key genes and protein changes were verified using real-time quantitative reverse transcription PCR and Western blotting, respectively, before and after intervention. For bioinformatics analysis, we used the R software and Bioconductor project. For statistical analysis, we used GraphPad Prism 6.0 and the Statistical Package for the Social Sciences software version 20.0 (IBM, Armonk, United States). A high level of CIP2A expression was associated with tumor size, T stage, lymph node metastasis, Tumor Node Metastasis stage, and a poor prognosis. Further, CIP2A expression was higher in GC cells than in normal human gastric epithelial cells. Using small interfering RNA against CIP2A, we discovered that CIP2A knockdown inhibited cell proliferation and significantly increased GC cell sensitivity to oxaliplatin. Moreover, CIP2A knockdown enhanced oxaliplatin-induced apoptosis in GC cells. Hence, high CIP2A levels in GC may be a factor in chemoresistance to oxaliplatin. In human GC cells, CIP2A regulated protein kinase B phosphorylation, and chemical inhibition of the protein kinase B signaling pathway was significantly associated with increased sensitivity to oxaliplatin. Therefore, the protein kinase B signaling pathway was correlated with CIP2A-enhanced chemoresistance of human GC cells to oxaliplatin. CIP2A expression could be a novel therapeutic strategy for chemoresistance in GC.

Reciprocal regulation of CIP2A and AR expression in prostate cancer cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples (p = 0.023). Furthermore, the protein level of CIP2A was positively correlated with patients’ short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A level, associated with high protein and phosphorylation levels of AR. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR protein levels and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced protein levels of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR protein levels, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa.

OCT4 Represses Inflammation and Cell Injury During Orchitis by Regulating CIP2A Expression.

Octamer-binding transcription factor 4 (OCT4) and cancerous inhibitor of protein phosphatase 2A (CIP2A) are upregulated in testicular cancer and cell lines. However, its contribution to orchitis (testicular inflammation) is unclear and was thus, investigated herein. Cell-based experiments on a lipopolysaccharide (LPS)-induced orchitis mouse model revealed robust inflammation, apoptotic cell death, and redox disorder in the Leydig (interstitial), Sertoli (supporting), and, germ cells. Meanwhile, real-time quantitative PCR revealed low OCT4 and CIP2A levels in testicular tissue and LPS-stimulated cells. A gain-of-function study showed that OCT4 overexpression not only increased CIP2A expression but also repressed LPS-induced inflammation, apoptosis, and redox disorder in the aforementioned cells. Furthermore, the re-inhibition of CIP2A expression by TD-19 in OCT4-overexpressing cells counteracted the effects of OCT4 overexpression on inflammation, apoptosis, and redox equilibrium. In addition, our results indicated that the Keap1-Nrf2-HO-1 signaling pathway was mediated by OCT4 and CIP2A. These findings provide insights into the potential mechanism underlying OCT4- and CIP2A-mediated testicular inflammation.

Constitutive CHK1 Expression Drives a pSTAT3-CIP2A Circuit that Promotes Glioblastoma Cell Survival and Growth.

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 in vitro and in vivo 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 in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A. IMPLICATIONS: 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.

Effect of CIP2A and its mechanism of action in the malignant biological behavior of colorectal cancer

BackgroundIncreasing evidence has revealed a close correlation between cancerous inhibitor of protein phosphatase 2A (CIP2A) and cancer progression. CIP2A has been shown to participate in diverse biological processes, such as development, tumorigenic transformation and chemoresistance. However, the functions of CIP2A in colorectal cancer (CRC) and its underlying mechanisms of action are not yet completely understood. The purpose of this study was to explore its clinical significance, function and relevant pathways in CRC.MethodsQuantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), western blotting and enzyme-linked immunosorbent assay (ELISA) were used to identify the expression of CIP2A in CRC tissues, sera and CRC cell lines. The association between the expressions of CIP2A and patient survival was analyzed using the Kaplan-Meier curves. Additionally, the functional role of CIP2A in the cell lines was identified through small interfering RNA (siRNA)-mediated depletion of the protein followed by analyses of proliferation and xenograft growth in vivo using short hairpin (sh) RNAs. Effects of the C-myc inhibitor 10,058-F4 on the expressions of C-myc, and CIP2A in CRC cell lines and its potential mechanisms of action were investigated. Finally, the potential molecular pathways associated with CIP2A were screened using the phosphokinase array and identified through western blotting.ResultsCIP2A mRNA and protein levels were upregulated in CRC tissues compared to those of the corresponding normal tissues. It can be used as an independent prognostic indicator to determine overall survival (OS) and disease-free survival (DFS). Depletion of CIP2A substantially suppressed the growth of CRC cells and colony formation in vitro, and inhibited the growth of xenograft tumors in vivo. Additionally, the levels of CIP2A in the sera of patients with CRC were higher than those of the control subjects. Multivariate analyses revealed that the levels of CIP2A in the sera were not independent prognostic indicators in patients with CRC. Moreover, 10,058-F4 could effectively inhibit the growth of CRC cells in vitro, which could be correlated with an inhibition in the expressions of C-myc, CIP2A and its downstream regulatory anti-apoptotic proteins. Furthermore, the Human Phosphokinase Antibody Array was used to gain insights into the CIP2A-dependent intermediary signaling pathways. The results revealed that several signaling pathways were affected and the protein levels of p-p53 (S392), p-STAT5a (Y694), Cyclin D1, p-ERK1/2 and p-AKT (T308) had decreased in CIP2A-shRNA group based on the results of the western blot analysis.ConclusionsCIP2A could promote the development of CRC cells and predict poor prognosis in patients with CRC, suggesting that it may serve as a potential prognostic marker and therapeutic target against CRC.-HSBbqB3LRSmwufLCKjmKbVideo Graphical abstract

Cancerous inhibitor of protein phosphatase 2A (CIP2A) modifies energy metabolism via 5' AMP-activated protein kinase signalling in malignant cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an adverse biomarker across many malignancies. Using K562 cells engineered to have high or low CIP2A expression, we show that high CIP2A levels significantly bias cellular energy production towards oxidative phosphorylation (OXPHOS) rather than glycolysis. Mass spectrometric analysis of CIP2A interactors and isobaric tagging for relative and absolute protein quantitation (ITRAQ) experiments identified many associated proteins, several of which co-vary with CIP2A level. Many of these CIP2A associating and co-varying proteins are involved in energy metabolism including OXPHOS, or in 5' AMP-activated protein kinase (AMPK) signalling, and manipulating AMPK activity mimics the effects of low/high CIP2A on OXPHOS. These effects are dependent on the availability of nutrients, driven by metabolic changes caused by CIP2A. CIP2A level did not affect starvation-induced AMPK phosphorylation of Unc-51 autophagy activating kinase 1 (ULK-1) at Ser555, but autophagy activity correlated with an increase in AMPK activity, to suggest that some AMPK processes are uncoupled by CIP2A, likely via its inhibition of protein phosphatase 2A (PP2A). The data demonstrate that AMPK mediates this novel CIP2A effect on energy generation in malignant cells.

MicroRNA-218 inhibits the cell proliferation and migration in clear cell renal cell carcinoma through targeting cancerous inhibitor of protein phosphatase 2A.

MicroRNAs (miRs) have emerged as critical modulators of tumor initiation and progression in numerous types of human cancer, including clear cell renal cell carcinoma (ccRCC), which is the most common subtype of renal cell carcinoma. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a newly characterized oncoprotein and its overexpression has been reported to promote cellular epithelial-mesenchymal transition and the tumor progression of ccRCC. The present study examined the effects of miR-218 on CIP2A expression in ccRCC cells. The results demonstrated that the expression level of miR-218 was lower in ccRCC tissues compared with that in adjacent non-tumor renal tissues. In addition, it was identified that miR-128 could directly bind to the 3′-untranslated region of CIP2A. Furthermore, a negative correlation between the expression levels of miR-218 and CIP2A was detected in ccRCC. Additionally, the downregulation of CIP2A or overexpression of miR-218 in ccRCC cells was revealed to inhibit cell proliferation and migration. In summary, these data suggest that miR-218 serves a role in the regulation of CIP2A and elucidate its consequences on tumor progression, tumor cell proliferation and migration. These results indicate that miR-218 may exhibit potential as a molecular target for the treatment of ccRCC.

The red wine component ellagic acid induces autophagy and exhibits anti-lung cancer activity invitro and invivo.

Red wine consists of a large amount of compounds such as resveratrol, which exhibits chemopreventive and therapeutic effects against several types of cancers by targeting cancer driver molecules. In this study, we tested the anti‐lung cancer activity of 11 red wine components and reported that a natural polyphenol compound ellagic acid (EA) inhibited lung cancer cell proliferation at an efficacy approximately equal to that of resveratrol. EA markedly increased the expression of the autophagosomal marker LC3‐II as well as inactivation of the mechanistic target of rapamycin signalling pathway. EA elevated autophagy‐associated cell death by down‐regulating the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), and CIP2A overexpression attenuated EA‐induced autophagy of lung cancer cells. Treating tumour‐bearing mice with EA resulted in significant inhibition of tumour growth with suppression of CIP2A levels and increased autophagy. In addition, EA potentiated the inhibitory effects of the natural compound celastrol on lung cancer cells in vitro and in vivo by enhancing autophagy and down‐regulating CIP2A. These findings indicate that EA may be a promising chemotherapeutic agent for lung cancer, and that the combination of EA and celastrol may have applicability for the treatment of this disease.

ER stress-related ATF6 upregulates CIP2A and contributes to poor prognosis of colon cancer.

Endoplasmic reticulum (ER) stress is an adaptive response to various stress conditions and plays emerging roles in cancer. Activating transcription factor 6 (ATF6), one of the three major ER stress transducers, has been shown to contribute to chemoresistance by altering cancer cell survival. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogene, and its expression has been correlated with the prognosis of patients with cancer. In this study, we aimed to explore the relationship between ER stress‐related ATF signaling and CIP2A. We found that CIP2A expression was positively correlated with ATF6 expression by analyzing publicly available RNA sequence data of patients with colorectal cancer (The Cancer Genome Atlas, TCGA). In addition, we demonstrated that tunicamycin‐induced ER stress in vitro upregulated ATF6 and CIP2A. Mechanistically, we found that ATF6 directly bound to the CIP2A promoter and induced CIP2A gene expression, which contributed to colon cancer cell survival. Furthermore, knockdown of CIP2A reduced the viability of cells under ER stress. Most importantly, immunohistochemical analysis of a tissue microarray from a colon cancer patient cohort showed that higher expression levels of ATF6 and CIP2A were associated with a trend toward poor prognosis. Taken together, our results show that ER stress‐related ATF6 upregulates CIP2A and contributes to the prognosis of colon cancer. Targeting CIP2A may disrupt ER stress‐mediated colon cancer cell survival and thus improve the prognosis of patients with colon cancer.

CIP2A mediates fibronectin-induced bladder cancer cell proliferation by stabilizing \u03b2-catenin

BackgroundFibronectin (FN) is associated with tumorigenesis and progression in bladder cancer, however, the underlying mechanisms causing this remain largely unknown. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A) has been shown to play important regulatory roles in cancer proliferation. Here, we investigated whether FN regulates CIP2A expression to promote bladder cancer cell proliferation.MethodsThe correlations of stromal FN with CIP2A and proliferating cell nuclear antigen (PCNA) expression were analyzed in a cohort bladder cancer patients. The roles of FN and CIP2A in regulating bladder cancer cell proliferation were evaluated in cell and animal models. Cycloheximide treatment was used to determine the effects of CIP2A on β-catenin stabilization. The CIP2A-β-catenin interaction was confirmed by immunofluorescence staining and co-immunoprcipitation.ResultsIn this study, we found that stromal FN expression correlated positively with the levels of CIP2A and PCNA in bladder cancer tissues. Meanwhile, in human bladder cancer cell lines (T24 and J82), exogenous FN significantly promoted cell proliferation, however, CIP2A depletion inhibited this process. Furthermore, the interaction between CIP2A and β-catenin enhanced the stabilization of β-catenin, which was involved in FN-induced cell proliferation. In vivo, CIP2A depletion repressed FN-accelerated subcutaneous xenograft growth rates.ConclusionsThese data reveal that CIP2A is a crucial mediator of FN-induced bladder cancer cell proliferation via enhancing the stabilization of β-catenin. Promisingly, FN and CIP2A could serve as potential therapeutic targets for bladder cancer treatment.

FTY720-induced enhancement of autophagy protects cells from FTY720 cytotoxicity in colorectal cancer.

FTY720, also known as fingolimod, is a widely used immunomodulator in multiple sclerosis and multiple organ transplantation. It is also an important protein phosphatase 2A (PP2A) activator. Based on this, a number of studies have recently demonstrated the cytotoxic effect of FTY720 in various cancers. Yet in colorectal cancer (CRC), the underlying mechanisms of FTY720 cytotoxicity remain less clear, especially the relationship between a drug and autophagy. We demonstrate here for the first time that FTY720 promotes the appearance of autophagic hallmarks such as autophagosome formation and light chain 3 (LC3)-II accumulation, indicating the participation of autophagy in FTY720 cytotoxicity on CRC. Moreover, inhibition of autophagy using 3-methyladenine (3-MA), a specific inhibitor of autophagy, enhanced FTY720 cytotoxicity, indicating the protective role of autophagy against the drug's own cytotoxic effect. The protective autophagy was likely affected by cancerous inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor that is closely related with poor prognosis and drug resistance. Consequently, our data not only demonstrate a new mechanism underlying the cytotoxic effect of FTY720 in CRC, but also a new strategy for CRC treatment, especially in cases resistant to conventional chemotherapies because of high CIP2A levels.

High CIP2A levels correlate with an antiapoptotic phenotype that can be overcome by targeting BCL-XL in chronic myeloid leukemia.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a predictive biomarker of disease progression in many malignancies, including imatinib-treated chronic myeloid leukemia (CML). Although high CIP2A levels correlate with disease progression in CML, the underlying molecular mechanisms remain elusive. In a screen of diagnostic chronic phase samples from patients with high and low CIP2A protein levels, high CIP2A levels correlate with an antiapoptotic phenotype, characterized by downregulation of proapoptotic BCL-2 family members, including BIM, PUMA and HRK, and upregulation of the antiapoptotic protein BCL-XL. These results suggest that the poor prognosis of patients with high CIP2A levels is due to an antiapoptotic phenotype. Disrupting this antiapoptotic phenotype by inhibition of BCL-XL via RNA interference or A-1331852, a novel, potent and BCL-XL-selective inhibitor, resulted in extensive apoptosis either alone or in combination with imatinib, dasatinib or nilotinib, both in cell lines and in primary CD34+ cells from patients with high levels of CIP2A. These results demonstrate that BCL-XL is the major antiapoptotic survival protein and may be a novel therapeutic target in CML.

PP2A Inhibition By CIP2A or SETBP1 Leads to Elevated Levels of AKT S473 Which Can be Used As a Biomarker of Outcome in Acute Myeloid Leukaemia

Many cells utilise reversible phosphorylation as a mechanism of post-translational modification for activating and deactivating key regulatory molecules involved in cell signalling. Many malignancies are characterised by overactive kinases e.g. BCR-ABL or FLT-3 in myeloid malignancy, or ERK or ErbB2 in solid tumours. A major phosphatase working in opposition to kinases is protein phosphatase 2A (PP2A) but why the cellular phosphatases such as PP2A simply do not counteract the overactive kinase activity is unclear. PP2A activity is regulated by inhibitor proteins SET, (which is stabilised by a binding protein SETBP1) and cancerous inhibitor of PP2A (CIP2A). In CML, we have shown that high levels of CIP2A at diagnosis is a prospective biomarker for future progression to blast crisis. High levels of CIP2A in other malignancies have also been reported to confer a poor prognosis. In CML, high CIP2A levels lead to the stabilisation of c-Myc and elevation of E2F1, as well as PP2A inactivation. In AML there are data suggesting that high SETBP1 levels may confer a poor outcome in AML, by PP2A inhibition via increased action of SET, though little is known of CIP2A in AML. The aim of the study was to investigate if CIP2A or network related proteins could predict clinical outcome in AML patients.CIP2A and network related proteins were investigated in 119 AML patient samples collected in the UK MRC/NCRI trials AML15, 16 and 17. These were stratified into intermediate or adverse risk, based on cytogenetics as previously defined (Grimwade et al, Blood. 1998: 92; 2322-33). Protein levels were studied in diagnostic mono-nuclear cell samples by flow cytometry. The diagnostic CIP2A protein level (mean fluorescence intensity (MFI)) for all patients was calculated. This range was 0-13.69, the interquartile range was 1.47-5.11, the median was 3 and the mean was 3.5. High CIP2A patients are defined as those patients with a CIP2A level greater than or equal to 3.In all AML samples studied high CIP2A was associated with inactive PP2A as shown by high levels of PP2AY307 (p=0.05), as well as elevated levels of c-Myc p=<0.0001 and E2F1 p=<0.0001, a similar finding to what we have observed in CML. We also found that AKTS473 and STAT5 were also elevated in high CIP2A AML patients (p=0.002 and p=0.006 respectively). Unlike CML, we found in AML patients high CIP2A was associated with high levels of SET (p=<0.0001) and SETBP1 (p=0.01).Younger intermediate AML patients had significantly higher diagnostic CIP2A levels than adverse risk patients (p=0.004). In the intermediate risk group, overall survival was dominated by the survival from relapse. For younger intermediate risk patients with high and low CIP2A levels, median survival from relapse (censored at transplant) was 56 days and 303 days respectively. Multivariate analysis adjusted for FLT3-ITD showed that a high CIP2A level predicted inferior survival from relapse (p=0.04, hazard ratio 4.02). Interestingly, high diagnostic CIP2A was strongly associated with the presence of a FLT3-ITD mutation (p=0.03).In adverse risk patients SETBP1 levels were elevated but CIP2A levels were lower. SETBP1 acts via SET as an alternative inhibitor of PP2A. In younger patients with adverse cytogenetics, high SETBP1 levels predict for an inferior overall survival (p=0.02). For all 119 AML patients those with detectable SETBP1 protein levels at diagnosis had an inferior overall survival (p=0.01). High SETBP1 was also associated with secondary AML.In univariate analysis, high levels of AKTphosphorylation at seine 473 (which is inversely correlated with PP2A activity)were associated with poor survival in all patients. Multivariate analysis of known predictors of outcome (age, cytogenetics, white count, secondary leukaemia, FLT3-ITD) together with CIP2A and related proteins (PP2AY307, SET, SETBP1, c-Myc E2F1, AKTS473, STAT5, and E2F1) was used to derive a prognostic model for survival. This ranked high levels of AKTS473 as the third most important predictor of outcome after age and cytogenetics. Our data suggest that AKTS473 levels are elevated as a result of PP2A inactivation (p=>0.0001) caused by high level of PP2A inhibitors, either CIP2A (p=0.003) or SETBP1 (p=0.03).In summary, AKT phosphorylation at S473 is a biomarker of PP2A inhibition by either CIP2A (intermediate risk) or SETBP1 (adverse risk patients). The diagnostic AKTS473 level appears to be a novel biomarker for survival in AML. DisclosuresNo relevant conflicts of interest to declare.

CIP2A is associated with multidrug resistance in cervical adenocarcinoma by a P-glycoprotein pathway.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein. Here, we investigated its role in the formation of multidrug resistance (MDR) of cervical adenocarcinoma in vitro and in vivo. MTT assay showed that knockdown of CIP2A expression increased the drug sensitivity of HeLa and Dox-resistant HeLa cells (HeLa-Dox) to doxorubicin, cisplatin, and paclitaxel significantly, while overexpression of CIP2A decreased the sensitivity of HeLa cells to chemo-drugs dramatically. When treated with different chemotherapeutics, CIP2A and P-glycoprotein (P-gp) protein levels were increased in HeLa cells simultaneously. In accordance with it, knockdown or overexpression of CIP2A expression inhibited or increased the P-gp expression in the transcription level separately. The effects of CIP2A on P-gp expression was achieved partly through its regulation on the transcription factor E2F1. Moreover, the interference of CIP2A could decrease the P-gp protein activity elucidated by Rhodamine 123 (Rh123) efflux assay in HeLa and HeLa/Dox cells. In the in vivo level, confocal microscopy data demonstrated the strong co-localization of CIP2A and P-gp protein in HeLa cells, and CIP2A protein expression was significantly associated with that of P-gp in cervical adenocarcinoma tissues. Thus, CIP2A is involved in regulating multidrug resistance of cervical adenocarcinoma upon chemotherapy by enhancing P-gp expression through E2F1. CIP2A may be an attractive target in anticancer strategies to improve the effect of chemotherapy in cervical adenocarcinoma.

Cancerous inhibitor of protein phosphatase 2A contributes to human papillomavirus oncoprotein E7-induced cell proliferation via E2F1.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in many human malignant tumors including cervical cancer. Human papillomavirus (HPV) oncoprotein E7 is the key transformation factor in cervical cancer. Our previous data showed a positive association of CIP2A and HPV-16E7 protein levels; however, how CIP2A is regulated by HPV-E7 and the roles of CIP2A in HPV-E7-mediated cell proliferation are unknown. In this study, we demonstrated that HPV-16E7 protein significantly upregulating CIP2A mRNA and protein expression depended on retinoblastoma protein pRb rather than p130. CIP2A siRNA knockdown in HPV-E7-expressing cells inhibited cell proliferation, DNA synthesis and G1/S cell cycle progression. CIP2A siRNA decreased the protein levels of cyclin-dependent kinase 1 (Cdk1), Cdk2 and their partner cyclin A2, with no change in levels of Cdk4, Cdk6 and their partner cyclin D1. The downregulation of Cdk1 and Cdk2 was independent of c-Myc; instead, E2F1 was the main target of CIP2A in this process, as overexpression of E2F1 rescued the inhibitory effects of CIP2A siRNA knockdown on cell proliferation and G1 arrest of HPV-E7-expressing cells. Our studies reveal a novel function of CIP2A in HPV-16E7-mediated cell proliferation.

Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as a proto-oncogene that is overexpressed in various types of human cancers. CIP2A acts by inhibiting protein phosphatase 2A-dependent destabilization of c-Myc, resulting in increased cell proliferation. Here, we have characterized the proximal promoter region of the human CIP2A gene in cervical, endometrial and liver carcinoma cells. The 5′ flanking minimal proximal promoter of the CIP2A gene consists of putative binding sites for Ets1 and Elk1 in forward and reverse orientations. Here, we show that Ets1 and Elk1 binding is essential for CIP2A basal expression in several urogenital cancer cell lines. Interestingly, both Ets1 and Elk1 are required together for CIP2A expression, as siRNA knockdown of Ets1 and Elk1 together decreased CIP2A gene transcription, whereas knockdown of Ets1 or Elk1 alone had no effect. Moreover, ectopic expression of Ets1 and Elk1 together increased CIP2A expression. To gain physiological significance of the Ets1 and Elk1 regulation we observed, a panel of matched human cervical carcinoma samples was analyzed for the expression of CIP2A and Ets1 and/or Elk1. We found a direct correlation between the levels of CIP2A and the levels of Ets1 and Elk1. Our results suggest that the binding of Ets1 and Elk1 together to the proximal CIP2A promoter is absolutely required for CIP2A expression in cervical, endometrial and liver carcinoma cell lines. Thus, different factors regulate CIP2A expression in a cell-type specific manner. As previous work has shown a requirement for only Ets1 in prostate and gastric carcinomas, our results now indicate that CIP2A regulation is more complex than previously determined.

Abstract 2222: Identify and evaluate novel biomarker CIP2A for cervical cancer diagnostics

Abstract Early detection of cervical cancer is critical for a favorable prognosis. HPV infection is not a reliable marker for predicting the malignancy potential of cervical lesions. Standard cytological detection methods such as Pap smear are highly subjective. As a result, there remains a very high demand for a diagnostic assay capable of determining which early stage cervical lesions will progress to cervical cancer. We sought to identify cervical cancer biomarkers and to test the hypothesis that the small percentage of HPV positive patients who progress to cervical cancer have aberrant expression of the cell signaling component, c-myc and its regulators, including protein phosphatase 2A (PP2A) and cancerous inhibitor of PP2A (CIP2A). In this study, Bio-Plex liquid microarray analysis was used to determine the HPV status cervical specimens (NNon-tumor=28, NTumor=30). qRT-PCR, Western blotting, flow cytometry, and ELISA analysis were used to access expression levels of CIP2A, c-myc, PP2A, Ki67, TOP2A, MCM2, MCM5, p14ARF, and p16INK4a. Our studies indicate CIP2A was abundantly expressed in cervical cancer cell lines and it was not expressed in normal epithelial cells. CIP2A mRNA levels were higher in cervical tissues from stage I, II, and III cervical cancer patients in comparison to the level of CIP2A mRNA in normal tissue. CIP2A protein was specifically expressed in cervical cancer tissues at different cancer stages, and not in non-cancer or adjacent non-tumor cervical tissue. Our data showed that CIP2A mRNA detection had a sensitivity of 80% and specificity of 91% and CIP2A protein expression detection had a sensitivity of 80% and specificity of 100%, similar to that of p16INK4a biomarker. Although CIP2A/PP2A/c-myc pathway may work together in the development of cervical cancer, our data strongly indicate that only CIP2A (but not PP2A or c-myc) is a reliable biomarker for detection of cervical cancer. In this study, we also examined the combined use of at least two biomarkers as a tool to detect cervical cancer. Our studies also suggest there was no strong correlation of CIP2A expression with HPV subtype, age, ethnical background, or other patient characteristics. In conclusion, our clinical studies suggest that CIP2A is a reliable and sensitive biomarker for detection of cervical cancer. CIP2A mRNA or protein can both serve as reliable markers. A combined use of CIP2A with at least on additional marker selected from the group consisting of Ki67, TOP2A, MCM2, MCM5, and p16 will improve the assay sensitivity and specificity. CIP2A is a novel biomarker for early detection of cervical cancer in humans. 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 2222. doi:10.1158/1538-7445.AM2011-2222

CIP2A Is Overexpressed in Gastric Cancer and Its Depletion Leads to Impaired Clonogenicity, Senescence, or Differentiation of Tumor Cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor stabilizing c-MYC protein and driving cellular transformation. We determine whether CIP2A expression can serve as marker for gastric cancer and investigate the mechanism underlying CIP2A-mediated transformation and cell proliferation. Normal and malignant gastric tissues derived from 37 patients with gastric cancer were analyzed for CIP2A expression using reverse transcription-PCR and immunohistochemical staining. Gastric and other cell lines with different p53 and pRB backgrounds were used to inhibit CIP2A expression using small interfering RNA and then examined for clonogenic potentials, senescence, or differentiation. CIP2A mRNA was present in 34 of 37 (90%) of tumor specimens but absent in 27 of 37 (73%) of matched normal gastric mucosa. In 10 adjacent normal tissues with detectable CIP2A mRNA, 6 of them exhibited much weaker levels of CIP2A compared with their corresponding tumors. Thus, a total of 32 (87%) gastric cancer samples overexpressed CIP2A. CIP2A protein expression was readily detectable in the tumor tissues but absent in normal gastric mucosa. Depleting CIP2A expression substantially inhibited growth and clonogenic capabilities of tumor cell lines independently of p53 and pRB pathways. Gastric cancer-derived AGS cells underwent senescence following the inhibition of CIP2A expression. Moreover, CIP2A depletion triggered partial differentiation of leukemic HL60 cells. CIP2A in tumor cells is required for sustained proliferation by preventing cell growth arrest, senescence, or differentiation and its expression is significantly (P < 0.001) discriminatory between normal and cancerous gastric tissue.