Cancerous Inhibitor Of Protein Phosphatase 2A Protein Research Articles

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.

CIP2A facilitates the G1/S cell cycle transition via B-Myb in human papillomavirus 16 oncoprotein E6-expressing cells.

Infection with high‐risk human papillomaviruses (HR‐HPVs, including HPV‐16, HPV‐18, HPV‐31) plays a central aetiologic role in the development of cervical carcinoma. The transforming properties of HR‐HPVs mainly reside in viral oncoproteins E6 and E7. E6 protein degrades the tumour suppressor p53 and abrogates cell cycle checkpoints. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that is involved in the carcinogenesis of many human malignancies. Our previous data showed that CIP2A was overexpressed in cervical cancer. However, the regulation of CIP2A by HPV‐16E6 remains to be elucidated. In this study, we demonstrated that HPV‐16E6 significantly up‐regulated CIP2A mRNA and protein expression in a p53‐degradation‐dependent manner. Knockdown of CIP2A by siRNA inhibited viability and DNA synthesis and caused G1 cell cycle arrest of 16E6‐expressing cells. Knockdown of CIP2A resulted in a significant reduction in the expression of cyclin‐dependent kinase 1 (Cdk1) and Cdk2. Although CIP2A has been reported to stabilize c‐Myc by inhibiting PP2A‐mediated dephosphorylation of c‐Myc, we have presented evidence that the regulation of Cdk1 and Cdk2 by CIP2A is dependent on transcription factor B‐Myb rather than c‐Myc. Taken together, our study reveals the role of CIP2A in abrogating the G1 checkpoint in HPV‐16E6‐expressing cells and helps in understanding the molecular basis of HPV‐induced oncogenesis.

Protein phosphatase 2A (PP2A) inhibitor CIP2A indicates resistance to radiotherapy in rectal cancer.

Preoperative (chemo)radiotherapy, (C)RT, is an essential part of the treatment of rectal cancer patients, but tumor response to this therapy among patients is variable. Thus far, there are no clinical biomarkers that could be used to predict response to (C)RT or to stratify patients into different preoperative treatment groups according to their prognosis. Overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) has been demonstrated in several cancers and is frequently associated with reduced survival. Recently, high CIP2A expression has also been indicated to contribute to radioresistance in head and neck squamous cell carcinoma, but few studies have examined the connection between CIP2A and radiation response regarding other malignancies. We have evaluated CIP2A protein expression levels in relation to tumor regression after preoperative (C)RT and survival of rectal adenocarcinoma patients. The effects of CIP2A knockdown by siRNA on cell survival were further investigated in colorectal cancer cells exposed to radiation. Patients with low‐CIP2A‐expressing tumors had more frequently moderate or excellent response to long‐course (C)RT than patients with high‐CIP2A‐expressing tumors. They also had higher 36‐month disease‐specific survival (DSS) rate in categorical analysis. In the multivariate analysis, low CIP2A expression level remained as an independent predictive factor for increased DSS. Suppression of CIP2A transcription by siRNA was found to sensitize colorectal cancer cells to irradiation and decrease their survival in vitro. In conclusion, these results suggest that by contributing to radiosensitivity of cancer cells, low CIP2A protein expression level associates with a favorable response to long‐course (C)RT in rectal cancer patients.

High expression of CIP2A protein is associated with tumor aggressiveness in stage I-III NSCLC and correlates with poor prognosis.

The aim of this work was to examine the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in non-small cell lung cancer (NSCLC) and analyze its correlation with clinical outcomes. CIP2A protein levels were detected by immunohistochemistry (IHC). One hundred and eighty-four of 209 (88.3%) primary stage I–III NSCLC specimens and 4 of 38 (10.5%) adjacent normal lung tissue specimens expressed CIP2A protein. High expression of CIP2A was detected in 38.8% (81/209) of the NSCLC specimens. Patients diagnosed histologically with late-stage NSCLC (p<0.001) and malignant nodes (p=0.001) exhibited high CIP2A expression. Univariate analysis using the log-rank test identified CIP2A expression as a prognostic predictor for overall survival (p=0.005). In multivariate analyses using the Cox regression test, CIP2A expression, T stage, N stage, histological type, and chemotherapy were identified as independent prognostic factors (p=0.007, 0.001, 0.003, <0.001, and <0.001, respectively). Furthermore, Kaplan–Meier survival curves demonstrated that high CIP2A expression indicated poor prognosis in the subgroup of patients with squamous cell carcinoma (p=0.008). Similar results were noted in the subgroup of patients with adenocarcinoma, but the results did not reach statistical significance (p=0.084). We also used univariate analysis and multivariate analysis to assess the prognostic factors for overall survival in the subgroup of patients who received postoperative chemotherapy. CIP2A expression was also an independent prognostic factor in NSCLC patients who received postoperative chemotherapy (p=0.009), along with histological type (p=0.001) and N stage (p=0.034). In conclusion, adding to the accumulating evidence, our research suggested that the CIP2A expression is associated with aggressiveness and correlates with poor prognosis in NSCLC. Our findings also indicated that CIP2A might be a potential therapeutic target against NSCLC.

SiRNA-Mediated Silencing of CIP2A Enhances Docetaxel Activity Against PC-3 Prostate Cancer Cells.

Purpose: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an identified human oncoprotein which modulates malignant cell growth. It is overexpressed in human prostate cancer and in most of the human malignancies. The aim of this study was to investigate the effects of CIP2A silencing on the sensitivity of PC-3 prostate cancer cells to docetaxel chemotherapy.Methods: PC-3 cells were transfected using CIP2A siRNA. CIP2A mRNA and protein expression were assessed after CIP2A gene silencing using q-RT PCR and Western blotting. Proliferation and apoptosis were analyzed after treatment with docetaxol using MTT assay, DAPI staining, and flow cytometry, respectively.Results: Silencing of CIP2A enhanced the sensitivity of PC-3 cells to docetaxel by strengthening docetaxel induced cell growth inhibition and apoptosis against PC-3 cells.Conclusion: Silencing of CIP2A may potentiate the cytotoxic effects of docetaxel and this might be a promising therapeutic approach in prostate cancer treatment.

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.

CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein which participates in inhibiting tumor apoptosis in pancreatic cancer cells. Using immunohistochemical staining, we investigated the expression of CIP2A protein in 72 cases of human pancreatic ductal adenocarcinoma (PDAC) tissue and 27 cases of adjacent normal pancreatic tissue. The positive rate of CIP2A protein expression in pancreatic cancer tissue was70.83 %, which was significantly higher than that in adjacent non- cancerous pancreatic tissue (11.11%). The expression of CIP2A was found to be correlated with TNM stage, but not correlated with age, gender, tumor location, smoking status, alcohol consumption, diabetes, high blood pressure, BMI, tumor size, lymph node metastasis or distant metastases. Kaplan- Meier survival analysis showed that patients with positive CIP2A protein expression had a lower overall survival rate than patients without CIP2A expression. COX regression analysis indicated that expression of CIP2A was an independent prognostic factor for pancreatic ductal adenocarcinoma. In addition, down-regulation of CIP2A inhibited cell proliferation and increased sensitivity to gemcitabine in pancreatic cancer cells by decreasing AKT signaling pathway. Our results indicated that down-regulation of CIP2A could be a novel therapeutic strategy for pancreatic cancer.

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.

Overexpression of CIP2A in clear cell renal cell carcinoma promotes cellular epithelial-mesenchymal transition and is associated with poor prognosis

Cancerous inhibitor of protein phosphatase2A (CIP2A) is a newly characterized oncoprotein involved in a variety of malignant tumors. However, its expression pattern and biological functions in clear cell renal cell carcinoma (ccRCC) remain unclear. In the present study, our findings demonstrated that expressions of CIP2A mRNA and protein in ccRCC tissues and cell lines were significantly higher than those in paired normal renal tissues or normal renal tubular epithelial cells (P<0.05). High CIP2A level was closely correlated with Tstage (P=0.001), tumor size (P=0.009), lymph node metastasis (P=0.014), vascular invasion (P=0.018) and high Snail expression (P<0.001). Additionally, ccRCC patients with high CIP2A expression had significantly shorter overall survival (OS, P<0.001) and disease-free survival (DFS, P<0.001) when compared with patients with the low expression of CIP2A. On Cox multivariate analysis, CIP2A overexpression was an independent and significant prognostic factor for OS (P=0.010) and DFS (P=0.004). Furthermore, knockdown of the CIP2A expression significantly reduced ccRCC cell invasion, with decreased Snail and Vimentin expression, and increased E-cadherin expression. Taken together, our data identified CIP2A as a critical oncoprotein involved in cell invasion and epithelial mesenchymal transition (EMT), which could serve as a therapeutic target in ccRCC.

Abstract 3557: Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib

Abstract Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3557. doi:10.1158/1538-7445.AM2015-3557

Leucine-rich repeat-containing protein 59 mediates nuclear import of cancerous inhibitor of PP2A in prostate cancer cells.

Using yeast two-hybrid analysis, we identified several novel protein interactions for the oncoprotein Cancerous Inhibitor of PP2A (CIP2A) and confirmed a subset of these interactions in human cancer cell lines. Analysis of the interaction in prostate carcinoma cells between CIP2A and leucine-rich repeat-containing protein 59 (LRRC59) suggests that CIP2A is translocated into the nucleus at G2/M through its association with LRRC59. Recent work by others has demonstrated that nuclear CIP2A disrupts mitotic checkpoints, which promotes deregulation of the cell cycle and increases cancerous phenotypes. Thus, we provide a novel therapeutic mechanism for inhibiting CIP2A function in cancerous cells via targeting the CIP2A-LRRC59 interaction.

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.

Expression and correlation of CIP2A and OPN in bladder cancer

To explore the expressions of cancerous inhibitor of protein phosphatase 2A (CIP2A) and osteopontin (OPN) and evaluate their roles in bladder cancer. RNA was isolated from 38 cases of patients with bladder cancer and 12 cases of normal bladder tissue by TRIzol method from May 2010 to December 2012. And reverse transcription (RT)-PCR was used to detect the expressions of CIP2A and OPN. The expression levels of CIP2A and OPN in 99 cases of patients with bladder cancer and 12 cases of normal tissue were detected by immunohistochemical staining. The positive expression rate of CIP2A mRNA and OPN mRNA were 76.32% (29/38) and 92.11% (35/38) in bladder cancer while there was no expression in normal tissue (both P < 0.05). The positive rates of CIP2A and OPN protein were 63.64% (63/99)and 84.85% (84/99)in cases of bladder cancer tissues while CIP2A was not detected in normal tissues. The positive expression rate of OPN in normal tissues was 2/12 (both P < 0.05). The CIP2A and OPN proteins were both expressed in 58/99 cases of bladder cancer tissues while neither of them was expressed in 13 cases. In 8 cases, CIP2A was expressed while OPN was not. In another 20 cases, OPN was expressed while CIP2A was not (r = 0.300, P < 0.05). The expression levels of CIP2A and OPN in tissue of bladder cancer are higher than those of normal controls. And CIP2A and OPN may be used as indicators of biological behaviors and serve as new molecular diagnostic markers for bladder cancer.

Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells.

IntroductionTamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism.MethodsIn total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice.ResultsTamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors.ConclusionsInhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel “off-target“ mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0431-9) contains supplementary material, which is available to authorized users.

Clinical significance of CIP2A expression in bladder cancer

To explore the expression of cancerous inhibitor of PP2A (CIP2A) and evaluate its role in bladder cancer. RT-PCR was used to detect the expression of CIP2A mRNA from 38 cases of patients with bladder cancer and 12 cases of normal bladder tissue. The CIP2A protein expression levels in 99 cases of patients with bladder cancer and 12 cases of normal tissue was detected by immunohistochemical staining . And the serum contents of CIP2A protein of 38 patients with bladder cancer and 40 normal controls were detected by ELISA. The expression of CIP2A mRNA was detected in 29/38 cases (76.32%) of bladder cancer. And there was no expression in normal tissue (P < 0.05). The positive rate of CIP2A protein was 63.64% in 99 cases of bladder cancer tissues and no expression detected in normal tissues(P < 0.05). ELISA results showed that the serum content of CIP2A in patients with bladder cancer was significantly higher than that in normal controls (median:0.015 2 vs 0.001 8 ng/L, P < 0.05). The tissue and serum expressions of CIP2A in patients with bladder cancer are higher than those in normal controls. And CIP2A may be used as an indicator of the biological behavior of bladder cancer.

Ethoxysanguinarine Induces Inhibitory Effects and Downregulates CIP2A in Lung Cancer Cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that is able to stabilize c-Myc oncogenic transcription factor and promote proliferation and transformation of cells. CIP2A is overexpressed in many primary tumors, and pharmacological inactivation of CIP2A is an emerging concept for the development of novel anticancer agents. In this study, we demonstrate that overexpression of CIP2A predicts poor prognosis in lung cancer, and a natural compound, ethoxysanguinarine (ESG), effectively downregulates CIP2A protein and its downstream signaling molecules, c-Myc and pAkt, and induces protein phosphatase 2A (PP2A) activity. ESG inhibits proliferation and induces apoptosis of lung cancer cells, and enhances the effects of cisplatin on malignant cells. Taken together, our findings demonstrate that CIP2A is inversely associated with the clinical outcome of lung cancer, and ESG can serve as a lead compound for the development of CIP2A inhibitor for cancer therapies.

Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells

Background: Protein phosphatase 2A inhibition is one of the pre-requisites for human cell transformation. Previously, we have identified an endogenous inhibitor of PP2A, CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) in human fibrosarcoma cells (HT1080) using tandem affinity purification. CIP2A over expression has been demonstrated in almost every tumour type studied so far. However, our understanding on the mechanisms regulating CIP2A expression in human cancers, especially in sarcomas, is still emerging. Methods: Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Results: In line with previous observations, small molecule inhibitor for MEK pathway (PD98059) decreased CIP2A mRNA and protein expression. Interestingly, small molecule inhibitor for the JNK pathway, SP600125 decreased mRNA and protein levels of CIP2A oncoprotein with negligible effect of SB203580 (p38 kinase) inhibitor on CIP2A expression in HT1080 cells. However, siRNAs specific to either JNK1 or JNK2 kinases did not result in decrease in CIP2A expression. Contrarily, two different CIP2A siRNAs, which were used as positive controls, decreased JNK2 expression in HT1080 cells. Conclusion: Although it is well established that SP600125 inhibits JNK kinases, it has also been shown to inhibit a spectra of other kinases. SP600125 inhibits CIP2A protein expression both in time and concentration dependent manner. However, depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with small molecular inhibitors of kinases by independent approaches like two different target specific siRNAs. Finally, fortuitously we identify JNK2 as a CIP2A downstream target in HT1080 cells.

Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells

Background: Protein phosphatase 2A inhibition is one of the pre-requisites for human cell transformation. Previously, we have identified an endogenous inhibitor of PP2A, CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) in human fibrosarcoma cells (HT1080) using tandem affinity purification. CIP2A over expression has been demonstrated in almost every tumour type studied so far. However, our understanding on the mechanisms regulating CIP2A expression in human cancers, especially in sarcomas, is still emerging. Methods: Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Results: In line with previous observations, small molecule inhibitor for MEK pathway (PD98059) decreased CIP2A mRNA and protein expression. Interestingly, small molecule inhibitor for the JNK pathway, SP600125 decreased mRNA and protein levels of CIP2A oncoprotein with negligible effect of SB203580 (p38 kinase) inhibitor on CIP2A expression in HT1080 cells. However, siRNAs specific to either JNK1 or JNK2 kinases did not result in decrease in CIP2A expression. Contrarily, two different CIP2A siRNAs, which were used as positive controls, decreased JNK2 expression in HT1080 cells. Conclusion: Although it is well established that SP600125 inhibits JNK kinases, it has also been shown to inhibit a spectra of other kinases. SP600125 inhibits CIP2A protein expression both in time and concentration dependent manner. However, depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with small molecular inhibitors of kinases by independent approaches like two different target specific siRNAs. Finally, fortuitously we identify JNK2 as a CIP2A downstream target in HT1080 cells.

CIP2A protein expression in high‐grade, high‐stage bladder cancer

Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer.