Study Of The Biological Function And Tumorigenic Mechanisms Of The Novel Tumor Antigen OVA12

To achieve a better understanding of mechanisms that underlie carcinogenesis and to identify novel target molecules for diagnosis and therapy of carcinoma, we previously identified 24 distinct gene clones by immunoscreening of a c DNA library derived from an ovarian cancer patient through SEREX analysis. Among these genes we focused on a novel gene terms OVA12 and which putatively encodes a 114-amino-acid protein. The gene OVA12 is located on human chromosome 9q34.3 with the ORF of 345 bp. Our previous study demonstrated that OVA12 gene was highly expressed in some tumor tissues and tumor cell lines. For one thing, downregulation of OVA12 significantly inhibited the tumorigenic capacity of Hela cells. For another, overexpression of OVA12 in SMMC-7721 cells promoted the cancer cell growth in vitro and in vivo. These findings suggested that OVA12 might be a new potential target for tumor diagnosis and therapy development. Exploring its biological function and tumorigenic mechanisms might be of great theoretical significance in developing novel strategies for tumor diagnosis and therapy. Based on these, our researching work is mainly focused on three parts: 1) Detecting the OVA12 m RNA and protein levels expressed in tumor cell lines and normal cell lines, preparing the virus vectors and establishing the stable knocking-down or over-expressed cancer cell lines; studying the biological function of OVA12 in OVA12 knocking-down and over-expressed cancer cells: we detected the m RNA and protein expression of OVA12 in different types of cancer cells and normal cell lines. The results showed OVA12 was specifically over-expressed in a panel of tumor cell lines but had lower expression levels in normal ones. Immunohistochemistry analysis showed strong expression of OVA12 in tumor regions compared to nontumor regions. By setting up an ELISA assay, we determined the Ig G levels against OVA12 in the sera of healthy donors and tumor patients. The result showed that the anti-OVA12 Ig G levels in tumor patients were significantly higher than that in healthy donors. Meanwhile, In vitro and in vivo results showed that knocking down the expression of OVA12 or overexpressing OVA12 significantly inhibited or promoted the cancer cells proliferative potential. More importantly, OVA12 could inhibit cell apoptosis induced by 5-FU through upregulation of Mcl-1 and surviving. 2) To investigate the molecular mechanism by which OVA12 ptomoted tumor growth, we tested several signaling transduction pathways that have been previously demonstrated to be critical in tumorigenesis. We found that OVA12 knockdown resulted in an increase of p53 and p21 protein levels. In contrast, p53 and p21 protein levels decreased when OVA12 was overexpressed. Additionally, we found no change in m RNA levels of p53 in OVA12 knock-down and over-expressed tumor cells, suggested a post-transcriptional regulation of p53 by OVA12. We further examined the p53 transcriptional activity using luciferase reporter system.The result showed that OVA12 knockdown significantly increased the luciferase activity, whereas overexpression of OVA12 led to attenuated intensity of the p53 luciferase reporter. In agreement with our hypothesis, we observed by co-immunoprecipitation experiments that the ectopic expression of OVA12 dramatically increased the polyubiquitination of p53. In contrast, knockdown of OVA12 led to a decrease in the polyubiquitination of p53. Furthermore, we used serial dilutions of cisplatin, a commonly cytotoxic drug that induces apoptosis in a p53 dependent manner to treat tumor cell lines. The results showed that Siha-OVA12-sh RNA cells were significantly more sensitive to cisplatin than control cells. In contrast, Caski-OVA12 cells were more significantly more resistant to cisplatin than control counterparts; 3) To address the biological significance of p53 in the tumor-promoting function of OVA12, we investigated the influence of p53 knockdown on tumorigenicity of OVA12 sh RNA-transfected cells: The depletion of OVA12 suppressed the cancer cell proliferation, while p53 knockdown restored the cell growth rate resulting from the loss of OVA12. Cell cycle analysis by flow cytometry showed that knockdown of OVA12 caused G1 arrest in Siha cells, while silencing of OVA12 and p53 together showed a marked reversal in G1 arrest. Furthermore, tumor xenograft experiments also showed that the reduction of tumor volumes/weights by OVA12 downregulation was dependent on p53 function, as knocking down both OVA12 and p53 expression reversed tumor growth to the level of the control group. These results strongly indicated OVA12 promotes tumor cell growth at least partially through the p53 signaling pathway. To further elucidate the mechanism by which OVA12 negetively regulates the p53 pathway, we performed an immunoprecipitation(IP)experiment and found that there is no interaction between OVA12 and p53. Simutaneously, we investigated the protein levels of MDM2, p-JNK and p14 ARF and found that downregulation of OVA12 elevated the expression of p14 ARF, whereas overexpressin of OVA12 inhibited the expression of p14 ARF. Overexpression of OVA12 promoted export of MDM2 from the nucleus to the cytoplasm; Co-immunoprecipitation analysis of protein interaction between MDM2 and p53 showed that overexpression of OVA12 elevaed the interaction between MDM2 and p53.In summary, OVA12 was highly expressed in tumors and played a notable role in accelerating tumor development. OVA12 could inhibit 5-FU induced apoptosis through upregulation of Mcl-1 and surviving. Furthermore, OVA12 promoted p53 protein polyubiquitination and proteasome-dependent degradation and negatively regulated the p53 pathway. OVA12 promoted tumorigenesis at least partially through the p53 signaling pathway. OVA12 might suppress the action of p14 ARF, promote export of MDM2 from the nucleus to the cytoplasm and enhance the degradation of p53.