The Role Of CIP2A In Cervical Carcinogenesis And The Regulation Of CIP2A Expression In Cervical Cancer Cells

Cervical carcinoma (CC) is one of the most common cancers and a leading cause of death among women worldwide. The number of patients who died from this disease each year was estimated at 200,000. An important percentage of cases occur in the developing world, where in some countries the mortality of CC is next only to that of breast cancer. In our country, there are 130,000 to 150,000 new cases of CC each year among which about 20,000 women die from this disease.At present it has been well established that the infection of Human papillomavirus (HPV) is the main etiology of cervical cancer. HPV is a kind of double-strand DNA virus. HPV can be classified into two groups on the basis of their ability of causing malignant disease:High-risk HPV and Low-risk HPV. Low-risk HPVs produce localized benign warts that do not undergo malignant progression even if left untreated, whereas persistant infection of High-risk HPVs is associated with cervical cancer. Especially HPV 16 and HPV 18 are almost detected in up to 90% of cervical cancers. Till now, the mechanism of by which HPV infection induces cervical cancer is not clarified. It is well-known that persistant infection of HPV give the virus chances to integrate viral DNA into the genome of the host cells. The integration of viral DNA leads to uncontrolled expression of HPV oncoprotein E6 and E7 which is essential for cell transformation. E6, E7 of HPV16 and HPV18 can inhibit the function of p53 and pRb respectively. The inactivation of p53 and pRb result in differential protein expression profile in host cells and disturb the cell signaling pathway. The apoptosis and senescence are repressed, finally the cell transformation happen.Though the skeleton model of HPV infection of HPV inducing cervical cancer has been established, the expression changes of protein in host cells as a result of high levels of E6 and E7 expression after the viral DNA integration are not completely clear. Therefore, new targets of E6 and E7 protein have been continuously identified over the past decade.Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a new-identified oncoprotein which inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in several human malignancies, such as gastric cancer, colon cancer, breast cancer, non-small cell lung cancer, prostatic cancer, esophageal cancer and ovarian cancer. CIP2A is associated with prognosis of breast cancer, lung cancer and ovarian cancer. Whether CIP2A is overexpressed in cervical cancer? Whether its expression is associated with HPV E6 and E7 protein? Is there any cell signaling pathway involved? These questions are still unclear. This study focused on these questions and abtained some preliminary data.Ⅰ. Overexpression of CIP2A in cervical caner We used 23 tissue samples (15 cervical cancer and 8 normal cervical samples) obtained from surgical patients at Shandong Cancer Hospital for mRNA extraction and consequently RT-PCR.We obtained 157 paraffin-embedded tissue blocks (72 cervical cancer,21 CINⅠ,25 CINⅡ,24 CINⅢand 15 normal cervical tissues) from Qilu Hospital for immunohistochemistry staining.CIP2A mRNA expression was found in 11 of 15 (73.3%) cervical cancer samples but none of 8 normal cervical tissues. CIP2A protein was absent in normal, CIN I and CIN II tissues but was present in 3 of 24 (12.5%) CIN III samples and was present in 38 of 72 (52.8%) cervical cancer samples. Meanwhile, we analyzed the correlation of CIP2A and some clinical characteristics. CIP2A protein expression was not associated with age, tumor size, differentiation status, node metastasis or clinical stage.The data of this part showed that CIP2A expression significantly differed among normal, CINⅠ, CINⅡ, CINⅢand cervical cancer samples. The overexpression of CIP2A in cervical cancer indicates that CIP2A may play an important role in driving cells from the precancerous to invasive cancer stage.II. CIP2A promotes c-Myc expression and growth of cervical cancer cells To determine whether CIP2A contributes to the proliferation of cervical cancer cells, we introduced CIP2A-specific siRNA to knock down CIP2A expression in HeLa, SiHa and Caski cells. Efficient depletion of CIP2A protein expressionwas verified by western blot analysis. In agreement with recent findings, CIP2A depletion in these cervical-cancer. CIP2A depletion with siRNA transfection downregulates the expression of c-Myc protein and impairs cell proliferation and cell growth (analyzed by CCK-8 kit) of HeLa, SiHa and Caski cells. Further more, as compared with control siRNA treatment, CIP2A siRNA treatment significantly decreased the formation of foci and colony size as well as the growth in soft agar of these cervical cancer cell lines.For clarifying why CIP2A siRNA treatment impaired the growth of cervical cancer cells, we usedβ-Galactosidase Staining kit to detect cell senescence, flow cytometry propidium iodide stain to analyzed cell cycle, flow cytometry PI/Annexin V stain to analyzed cell apoptosis. To our surprise, as compared with control siRNA treatment, there was no change in cell cycle and no more noticeable senescence and apoptosis in the CIP2A siRNA treatment cells.The data of this part showed that the growth and malignant phenotype of cervical cancer are significantly decreased after depletion of CIP2A, which indicates that CIP2A promote the growth and proliferation of cervical cancer cells while such an promotion is not achieved by changing cell cycle or repressing apoptosis.Ⅲ. CIP2A was upregulated by HPV16 E7 and inhibition of CIP2A activated Wnt signaling pathwayRecent report showed that the main oncoprotein of Helicobactor pylori Cag A upregulated CIP2A in gastric cancer cell lines. Cervical cancer is similar to gastric cancer which is associated with pathogen infection. Further more, HPV also have two important oncoprotein E6 and E7. To determine whether HPV oncoproteins have the ability of regulating CIP2A, we analyzed HPV16 E7 in cervical cancer tissues by using immunohistochemistry. Interestingly, CIP2A and HPV 16 E7 immunoreactivity showed a significantly positive correlation. CIP2A and HPV 16 E7 immunoreactivity was uniform both in location and immunointensity on the same specimens, which suggested an association of HPV16 E7 and CIP2A expression.To investigate the possible association of HPV16 E7 and CIP2A expression, we introduced HPV16 E7-specific siRNA into SiHa cells.we found both mRNA and protein levels of CIP2A significantly decreased after depletion of HPV16 E7. In addition we compared the CIP2A expression of primary human keratinocytes (PHK) and HPV16 E7 stable-transfected PHK (PHK 16E7). Compared to PHK, CIP2A showed higher expression both in mRNA and protein levels.The role of Wnt/β-catenin signaling pathway in cervical cancer attracts many reseachers'attention. In this study, we detected beta-catenin, the key protein of Wnt/β-catenin signaling, in total, cytoplasmic and nuclear protein using western blot assay. We found that beta-catenin increased in all the three protein extract after depletion of CIP2A, which indicated the activation of Wnt/β-catenin signaling.The data in this part showed that HPV16 E7 upregulates the expression of CIP2A. This finding was first reported in our study. Therefore, CIP2A is a newfound target of HPV16 E7. Furthermore, CIP2A may play a role in the regulation of Wnt/β-catenin signaling.In conclusion, we show that CIP2A is overexpressed in cervical cancer, which might result from the regulatory role of HPV16 E7 on CIP2A expression. Moreover, CIP2A plays an important role in the carcinogenesis of cervical cancer by promoting cell proliferation and cell growth. These findings help to understand the regulatory mechanism of CIP2A and untangle the novel function of HPV16 E7. The investigation of CIP2A expression in clinical tissues provides information on its possible use as a candidate biomarker and therapeutic target of cervical cancer.