Cancerous Inhibitor Of Protein Phosphatase 2A Expression Research Articles

CIP2A interacts with AKT1 to promote the malignant biological behaviors of oral squamous cell carcinoma by upregulating the GSK‑3β/β‑catenin pathway.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide, which is associated with a poor prognosis. The present study aimed to investigate the role of cancerous inhibitor of protein phosphatase 2A (CIP2A) in OSCC and its regulatory effect on AKT1. Firstly, CIP2A and AKT1 expression in OSCC cells was detected by western blotting. After silencing CIP2A, cell viability and cell proliferation were assessed using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining. Cell apoptosis was evaluated by TUNEL staining and the expression of apoptosis-related proteins was assessed using western blotting. Wound healing, Transwell and tube formation assays were performed to evaluate CAL-27 cell migration, invasion and human umbilical vein endothelial cell (HUVEC) tube formation. The interaction between CIP2A and AKT1 was identified by co-immunoprecipitation (co-IP). In addition, AKT1 was overexpressed in CIP2A-silenced CAL-27 cells to perform rescue experiments to analyze the malignant biological functions of CAL-27 cells. Finally, the expression of proteins in the glycogen synthase kinase (GSK)-3β/β-catenin pathway was determined by western blot analysis. Markedly elevated CIP2A and AKT1 expression was observed in OSCC cells. CIP2A knockdown inhibited the viability, proliferation, migration and invasion, and promoted the apoptosis of CAL-27 cells. Concurrently, CIP2A loss-of-function attenuated tube formation. Results of Co-IP confirmed there was an interaction between CIP2A and AKT1. Rescue experiments suggested that AKT1 overexpression alleviated the inhibitory effects of CIP2A knockdown on the viability, proliferation, migration and invasion of CAL-27 cells, as well as tube formation in HUVECs . Additionally, CIP2A silencing significantly downregulated phosphorylated-GSK-3β and β-catenin expression, which was reversed by AKT1 overexpression. In conclusion, CIP2A could interact with AKT1 to promote the malignant biological behaviors of OSCC cells by upregulating the GSK-3β/β-catenin pathway. These findings may provide a targeted therapy for OSCC treatment.

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.

Ethoxysanguinarine directly targets CIP2A to inhibit proliferation and induce autophagy of SGC7901/DDP cells

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.

CIP2A deficiency promotes depression-like behaviors in mice through inhibition of dendritic arborization.

Major depressive disorder (MDD) is a severe mental illness. Decreased brain plasticity and dendritic fields have been consistently found in MDD patients and animal models; however, the underlying molecular mechanisms remain to be clarified. Here, we demonstrate that the deletion of cancerous inhibitor of PP2A (CIP2A), an endogenous inhibitor of protein phosphatase 2A (PP2A), leads to depression-like behaviors in mice. Hippocampal RNA sequencing analysis of CIP2A knockout mice shows alterations in the PI3K-AKT pathway and central nervous system development. In primary neurons, CIP2A stimulates AKT activity and promotes dendritic development. Further analysis reveals that the effect of CIP2A in promoting dendritic development is dependent on PP2A-AKT signaling. In vivo, CIP2A deficiency-induced depression-like behaviors and impaired dendritic arborization are rescued by AKT activation. Decreased CIP2A expression and impaired dendrite branching are observed in a mouse model of chronic unpredictable mild stress (CUMS). Indicative of clinical relevance to humans, CIP2A expression is found decreased in transcriptomes from MDD patients. In conclusion, we discover a novel mechanism that CIP2A deficiency promotes depression through the regulation of PP2A-AKT signaling and dendritic arborization.

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.

Overexpression of cancerous inhibitor ofPP2A (CIP2A) in acute myeloid leukemia

ABSTRACT Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. Protein phosphatase 2A Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase and tumor suppressor that negatively regulates numerous signal transduction pathways. Cancerous inhibitor of PP2A (CIP2A) is an endogenous inhibitor of PP2A. CIP2A overexpression was shown to be a recurrent event in cytogenetic normal AML patients. The aim of the study is to evaluate the prognostic significance of CIP2A overexpression in patients with AML. Research design and methods The study included 174 newly diagnosed cytogenetic normal AML patients. Detection of CIP2A expression was performed using quantitative real-time PCR. Results CIP2A was overexpressed in 125/174 (71.8%) of patients. Correlation of CIP2A overexpression with other prognostic factors showed significant association with CD34 expression (p = 0.04). CIP2A overexpression was significantly associated with a lower rate of (complete remission) CR (p = 0.019) and shorter disease free survival (DFS) and overall survival (OS) (p < 0.001 and <0.001, respectively). In multivariate analysis, CIP2A overexpression was an independent adverse prognostic factor that negatively affected DFS and OS (p < 0.001, HR:2.8,95%CI:1.7–4.7 and p = 0.002, HR:1.8; 95%CI:1.2–2.65, respectively). Conclusion CIP2A overexpression is a useful prognostic marker in AML.

ChK1 activation induces reactive astrogliosis through CIP2A/PP2A/STAT3 pathway in Alzheimer's disease.

Cancerous Inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor, is upregulated and causes reactive astrogliosis, synaptic degeneration, and cognitive deficits in Alzheimer's disease (AD). However, the mechanism underlying the increased CIP2A expression in AD brains remains unclear. We here demonstrated that the DNA damage-related Checkpoint kinase 1 (ChK1) is activated in AD human brains and 3xTg-AD mice. ChK1-mediated CIP2A overexpression drives inhibition of PP2A and activates STAT3, then leads to reactive astrogliosis and neurodegeneration in vitro. Infection of mouse brain with GFAP-ChK1-AAV induced AD-like cognitive deficits and exacerbated AD pathologies in vivo. In conclusion, we showed that ChK1 activation induces reactive astrogliosis, degeneration of neurons, and exacerbation of AD through the CIP2A-PP2A-STAT3 pathway, and inhibiting ChK1may be a potential therapeutic approach for AD treatment.

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A): Could It Be a Promising Biomarker and Therapeutic Target in Parkinson\u2019s Disease?

Parkinson’s disease (PD) is an incurable neurodegenerative disease characterized by aggregation of pathological alpha-synuclein (α-syn) and loss of dopaminergic neuron in the substantia nigra. Inhibition of phosphorylation of the α-syn has been shown to mediate alleviation of PD-related pathology. Protein phosphatase 2A (PP2A), an important serine/threonine phosphatase, plays an essential role in catalyzing dephosphorylation of the α-syn. Here, we identified and validated cancerous inhibitor of PP2A (CIP2A), as a potential diagnostic biomarker for PD. Our data showed that plasma CIP2A concentrations in PD patients were significantly lower compared to age- and sex-matched controls, 1.721 (1.435–2.428) ng/ml vs 3.051(2.36–5.475) ng/ml, p < 0.0001. The area under the curve of the plasma CIP2A in distinguishing PD from the age- and sex-matched controls was 0.776. In addition, we evaluated the role of CIP2A in PD-related pathogenesis in PD cellular and MPTP-induced mouse model. The results demonstrated that CIP2A is upregulated in PD cellular and MPTP-induced mouse models. Besides, suppression of the CIP2A expression alleviates rotenone induced aggregation of the α-syn as well as phosphorylation of the α-syn in SH-SY5Y cells, which is associated with increased PP2A activity. Taken together, our data demonstrated that CIP2A plays an essential role in the mechanisms related to PD development and might be a novel PD biomarker.

Integrative assessment of CIP2A overexpression and mutational effects in human malignancies identifies possible deleterious variants

KIAA1524 is the gene encoding the human cancerous inhibitor of PP2A (CIP2A) protein which is regarded as a novel target for cancer therapy. It is overexpressed in 65%–90% of tissues in almost all studied human cancers. CIP2A expression correlates with cancer progression, disease aggressivity in lung cancer besides poor survival and resistance to chemotherapy in breast cancer. Herein, a pan-cancer analysis of public gene expression datasets was conducted showing significant upregulation of CIP2A in cancerous and metastatic tissues. CIP2A overexpression also correlated with poor survival of cancer patients. To determine the non-coding variants associated with CIP2A overexpression, 5′UTR and 3′UTR variants were annotated and scored using RegulomeDB and Enformer deep learning model. The 5′UTR variants rs1239349555, rs1576326380, and rs1231839144 were predicted to be potential regulators of CIP2A overexpression scoring best on RegulomeDB annotations with a high “2a” rank of supporting experimental data. These variants also scored the highest on Enformer predictions.Analysis of the 3′UTR variants of CIP2A predicted rs56255137 and rs58758610 to alter binding sites of hsa-miR-500a-5 and (hsa-miR-3671, hsa-miR-5692a) respectively. Both variants were also found in linkage disequilibrium with rs11709183 and rs147863209 respectively at r2 ≥ 0.8. The aforementioned variants were found to be eQTL hits significantly associated with CIP2A overexpression. Further, analysis of rs11709183 and rs147863209 revealed a high “2b” rank on RegulomeDB annotations indicating a probable effect on DNAse transcription factors binding. The MuTarget analysis indicated that somatic mutations in TP53 are significantly associated with upregulated CIP2A in human cancers. Analysis of missense SNPs on CIP2A solved structure predicted seven deleterious effects. Four of these variants were also predicted as structurally and functionally destabilizing to CIP2A including; rs375108755, rs147942716, rs368722879, and rs367941403. Variant rs1193091427 was predicted as a potential intronic splicing mutation that might be responsible for the novel CIP2A variant (NOCIVA) in multiple myeloma. Finally, Enrichment of the Wnt/β-catenin pathway within the CIP2A regulatory gene network suggested potential of therapeutic combinations between FTY720 with Wnt/β-catenin, Plk1 and/or HDAC inhibitors to downregulate CIP2A which has been shown to be essential for the survival of different cancer cell lines.

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.

Polyphyllin I attenuates cognitive impairments and reduces AD-like pathology through CIP2A-PP2A signaling pathway in 3XTg-AD mice.

Polyphyllin I (PPI) is a natural phytochemical drug isolated from plants which can inhibit the proliferation of cancer cells. One of the PPI tumor-inhibitory effects is through downregulating the expression of Cancerous Inhibitor of PP2A (CIP2A), the latter, is found upregulated in Alzheimer's disease (AD) brains and participates in the development of AD. In this study, we explored the application of PPI in experimental AD treatment in CIP2A-overexpressed cells and 3XTg-AD mice. In CIP2A-overexpressed HEK293 cells or primary neurons, PPI effectively reduced CIP2A level, activated PP2A, and decreased the phosphorylation of tau/APP and the level of Aβ. Furthermore, synaptic protein levels were restored by PPI in primary neurons overexpressing CIP2A. Animal experiments in 3XTg-AD mice revealed that PPI treatment resulted in decreased CIP2A expression and PP2A re-activation. With the modification of CIP2A-PP2A signaling, the hyperphosphorylation of tau/APP and Aβ overproduction were prevented, and the cognitive impairments of 3XTg-AD mice were rescued. In summary, PPI ameliorated AD-like pathology and cognitive impairment through modulating CIP2A-PP2A signaling pathway. It may be a potential drug candidate for the treatment of AD.

Prognostic value of CIP2A gene expression in adult Egyptian acute myeloid leukemia patients

BackgroundProtein phosphatase 2A (PP2A) functions as a tumor suppressor in many cancers. Cancerous inhibitor of protein phosphatase 2A (CIP2A) inhibits PP2A proteolytic degradation of c-Myc and enhances cell growth and tumor formation in several tissues. CIP2A expression in acute myeloid leukemia (AML) and its effect on outcome of treatment were not reported in Egyptian patients.ResultsNo significant difference was detected in CIP2A expression between AML and control groups (P = 0.48). However, in AML patients, those with low CIP2A expression had a longer median overall survival than those with high expression (P = 0.059). CIP2A expression was not related to the clinical and laboratory variables and did not affect response to treatment.ConclusionHigh-CIP2A expression was associated with a trend of shorter overall survival in adult Egyptian AML patients. It might serve as a useful marker to predict poor prognosis. CIP2A may represent a potential target for cancer therapy.

MiR-383 Down-Regulates the Oncogene CIP2A to Influence Glioma Proliferation and Invasion.

BackgroundRecent evidence showed cancerous inhibitor of protein phosphatase 2A (CIP2A) plays carcinogenesis roles in several types of human cancer. However, the expression and function of CIP2A in gliomas are unknown.MethodsqRT-PCR, IHC and Western blot were used to evaluate CIP2A expression in glioma tissues and cell lines. The influence of CIP2A on prognosis was analyzed by KM curve and Cox regression. CCK8, clonal formation, transwell and tumor xenograft assays were used to analyze cell proliferation and invasion. The upstream microRNA of CIP2A was verified by luciferase and RIP assays.ResultsCIP2A was overexpressed in gliomas and associated with tumor size, WHO grade and postoperative overall survival rate. Depletion of CIP2A inhibited glioma cellular proliferation, invasion and xenograft tumorigenicity. miR-383 could bind to the 3ʹ-UTR of CIP2A and inhibit CIP2A expression by forming an RNA-induced silencing complex with Ago2.ConclusionCIP2A plays a carcinogenesis role in glioma progression and is one of the potential targets of miR-383.

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

Relationship Between CIP2A and Endometrium Cancer.

To determine whether there is a relationship between endometrium cancer and the oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A). A descriptive study. Balıkesir University Medical Faculty Hospital, Balıkesir, Turkey, from January 2013 to July 2018. CIP2A was studied immunohistochemically and molecularly in paraffin blocks. The correlation between CIP2A expression and different endometrial pathologies were evaluated. In addition, the relationship between CIP2A expression in tumor tissue and clinicopathological prognostic parameters and also between Ki67, P53, HER2 with CIP2A expression were investigated. A higher expression of CIP2A was found in endometrium cancer tissues compared to normal endometrial tissues. In addition, CIP2A expression according to molecular and immunohistochemical results was associated with significant poor prognostic factors such as FIGO Stage, FIGO Grade, cervical involvement, myometrial invasion and HER2 positivity. CIP2A could be a promising and therapeutic target in endometrium cancer. Invention of the complex connections of CIP2A with other oncoproteins may lead to amazing and interesting developments in both early diagnosis and treatment. Key Words: CIP2A, Endometrium cancer, Expression, Oncoprotein.

Genistein Decreases APP/tau Phosphorylation and Ameliorates Aβ Overproduction Through Inhibiting CIP2A.

Upregulation of Cancerous Inhibitor of PP2A (CIP2A) plays an important role in disease-related phosphorylation of tau/APP and tau pathology/Aβ overproduction through inhibiting PP2A in AD brain. Genistein has been shown to potently reduce CIP2A in experimental cancer treatment research. Whether Genistein can ameliorate AD pathology through targeting CIP2A needs further investigation. The inhibitory effects of Genistein on tau/APP phosphorylation and Aβ overproduction in AD cell models have been explored. HEK293-T cells were co-transfected with CIP2A and APP plasmids, or CIP2A and tau plasmids, with Genistein incubation at 0, 30, 60 or 120 µM for 48 h, cell viability and PP2A activities were measured. HEK293-T cells with CIP2A/APP overexpression treated with Genistein at 30 µM for 48 h were collected and lyzed for Western blotting detection of CIP2A, PP2Ac, APP-T668, total APP, PS1, BACE1, sAPPα and sAPPβ. Aβ40 and Aβ42 levels in cell supernatant, soluble fraction (RIPA) and insoluble fraction (formic acid soluble) of cell lysates were measured by ELISA. HEK293-T cells with CIP2A/tau overexpression treated with Genistein at 30 µM for 48 h were collected for Western blotting detection of CIP2A, PP2Ac, tau-S396, tau-S404 and total tau. Genistein effectively reduced CIP2A expression, and restored PP2A activities both in CIP2A/APP, CIP2A/tau co-expressed cells. Genistein reduced APP phosphorylation at T668 site and inhibited Aβ production. Meantime, Genistein ameliorated tau hyperphosphorylation through repressing the inhibitory effect of CIP2A on PP2A. CIP2A is a target of Genistein in AD therapy. Genistein reduces APP/tau hyperphosphorylation and Aβ production through inhibiting the effect of CIP2A on PP2A.

Amentoflavone Promotes Apoptosis in Non-Small-Cell Lung Cancer by Modulating Cancerous Inhibitor of PP2A.

Non-small-cell lung cancer (NSCLC) is one of the most common human malignancies. Amentoflavone (AF) is one of bioflavonoid compounds isolated from Selaginella tamariscina Spring. This study was designed to examine the effect of AF on NSCLC. Our results indicated that AF decreased cell viability of both H1299 and H358 cells. Colony formation assay also showed that AF was able to suppress the anchorage-independent growth of NSCLC cells. AF also triggered cell cycle arrest by downregulating cyclin D1, CDK4, and CDK6. The pro-apoptotic activity of AF was confirmed by Hoechst staining and flow cytometry. The effect of AF on activation of caspase-3, upregulation of Bax, and downregulation of Bcl-2 was examined by western blot. The anti-growth and pro-apoptotic activities of AF were further validated in xenograft murine model. iTRAQ assay showed that cancerous inhibitor of PP2A (CIP2A) expression was markedly downregulated by AF treatment in H1299 cells. In addition, qRT-PCR and western blot also showed that AF was able to dose-dependently inhibit CIP2A expression. Meanwhile, the activity of protein phosphatase 2A (PP2A) was enhanced by AF treatment. The mRNA and protein expression of CIP2A as well as PP2A activity in xenograft tumor tissue were examined, which indicated that the in vivo anticancer activity of AF was associated with downregulation of CIP2A and reactivation of PP2A. Moreover, our results showed that the anti-growth and pro-apoptotic activities of AF were augmented by CIP2A knockdown and attenuated by ectopic CIP2A expression. Our results indicated that AF exhibited anticancer activity in NSCLC by targeting CIP2A. Anat Rec, 302:2201-2210, 2019. © 2019 American Association for Anatomy.

Aberrant expression of vasculogenic mimicry, PRRX1, and CIP2A in clear cell renal cell carcinoma and its clinicopathological significance

Vasculogenic mimicry (VM) involves a tubular structure with a basement membrane that is similar to and communicates with vessels but functions independent of blood vessels to nourish tumor cells, promote tumor progression, invasion, and metastasis, with reduced 5-year survival rates. Tumor cell proliferation, invasion, and metastasis are promoted by the epithelial-mesenchymal transition (EMT). Paired-related homeobox 1 (PRRX1), a newly discovered EMT inducer, has been shown to correlate with metastasis and prognosis in diverse cancer types. Cancerous inhibitor of protein phosphatase 2A (CIP2A) was initially recognized as an oncoprotein. In this study, we aimed to investigate the expression and clinical significance of the EMT markers PRRX1, CIP2A and VM in clear cell renal cell carcinoma (CCRCC) and their respective associations with clinicopathological parameters and survival.Expression of PRRX1, CIP2A and VM in whole CCRCC tissues from 110 patients was analyzed by immunohistochemical and histochemical staining. Fisher's exact test or the chi square test was used to assess associations with positive or negative staining of these markers and clinicopathological characteristics.Positive expression of CIP2A and VM presence was significantly higher and that of PRRX1 was significantly lower in CCRCC tissues than in corresponding normal tissues. Furthermore, positive expression of CIP2A and VM was significantly associated with tumor grade, size, lymph node metastasis (LNM) stage, and tumor node metastasis (TNM) stage and inversely associated with overall survival time (OST). Moreover, levels of PRRX1 were negatively associated with tumor grade, size, LNM stage, and TNM stage. The PRRX1 subgroup had a significantly longer OST time than did the PRRX1 subgroup. In multivariate analysis, high VM and CIP2A, tumor grade, LNM stage, TNM stage, and low PRRX1 levels were identified as potential independent prognostic factors for OST in CCRCC patients.VM and expression of CIP2A and PRRX1 represent promising biomarkers for metastasis and prognosis and potential therapeutic targets in CCRCC.

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.