Study Of The Biological Function And Tumorigenic Mechanisms Of The Tumor Associated Antigen OVA66

The tumor associated antigen66(OVA66), also known as CML66(GenBankAccession No. AF283301) was first identified in an ovarian carcinoma cDNA expressionlibrary in our lab and was shown to play a significant role in process of tumorigenesis. Asfar as our knowledge of the expression profile of the protein OVA66, it‘s specificallyover-expressed in either tumor tissues from cancer patients or cancer cell lines and closelycorrelated with the process of tumorigenesis. For one thing, knocking down the expressionof OVA66in HeLa cells obviously retarded the tumorigenic capacity of HeLa cells. Foranother, OVA66stably transfected normal mouse fibroblast NIH3T3cells exhibited severalmalignant characteristics indicating that OVA66was able to induce oncogenictransformation of NIH3T3cells, suggesting that OVA66function similar as otheroncogenes. These findings suggested that OVA66might be a potential target for cancerdiagnosis or even therapy. Studying its biological function and tumorigenic mechanismsmight also be of great theoretical significance in developing novel strategies for cancerdiagnosis or therapy. Based on these, our researching work is mainly focused on three parts:1). Detecting the OVA66protein level expressed in cancer cell lines and normal cell lines,establishing the stable knocking-down or over-expressed cancer cell lines and intensivelystudying the biological function of OVA66in these cancer cell lines: we detected theprotein expression of OVA66in different types of cancer cells and normal cell lines,showed it was specifically over-expressed in cancer cell lines; in vitro and in vivo resultsshowed that knocking down the expression of OVA66in such as SK-OV-3cells or overexpressing OVA66in HO8910cells markedly inhibited or promoted the cancer cellsproliferative, invasive and survival potential;2). Based on the above results and ourprevious analytic results of gene array or other experiments, we utilized thehigh-throughput protein antibody array analysis to identify the related cell signalingmolecules whose expression or activation level would be correlated with the expression ofOVA66in cancer cells, trying to find the signaling pathway that OVA66might be involvedin and elucidate the possible tumorigenic mechanisms of OVA66: we carried out ahigh-throughput protomic technique, which is the protein phosphorylation antibody array analysis (RTK and MAPK antibody array), using the control and OVA66stably knockeddown SK-OV-3cells which display obviously different phenotypes both in vitro and invivo. Results of RTK antibody array showed that knocking down the expression of OVA66in SK-OV-3cells caused a significant decrease in phosphporylation level of many signalingmolecules such as the Insulin-like growth factor1receptor (IGF-1R); MAPK signalingpathway array analysis suggested that the phosphorylation level of such as ERK1/2andHSP27molecules is also closely related to the expression level of OVA66in SK-OV-3cells. Our further analysis by Western Blot indicated that the expression level of proteinOVA66is quite crucial for the aberrant activation of IGF-1R-ERK1/2-HSP27signalingpathway in cancer cell lines. Thus, we proposed that OVA66functions as an importantoncoprotein promoting the tumorigenesis in cancer cells at least partially by regulating thephosphorylation level of IGF-1R, ERK1/2and HSP27signaling molecules;3). We used thespecific molecular inhibitor of the related signaling pathway or the specific siRNA targetedto the related molecule to inhibit the aberrant activation of the signaling pathway related tohigh level of protein OVA66, studying the difference of the biological function of OVA66in cancer cells in the case of blocking the relevant signaling pathway: We make use of thespecific inhibitor of IGF-1R signaling pathway Linsitinib or IGF-1R targeted specific smallinterfering RNAs (siRNAs) to inhibit the IGF-1R signaling, studying its effect on thebiological function of OVA66in these cancer cells in terms of cell proliferation, invasionand anti-apoptotic capacity. Several experimental results revealed that specific blockade ofIGF-1R signaling obviously retarded the OVA66promoted cell viability, invasion andsurvival, for instance the control cell lines or OVA66over-expressed cancer cells actsimilarly in cell proliferation, invasion or survival compared with OVA66knocked downcells or empty vector transfected cells.Finally, as our results suggested that OVA66also regulated the IGF-1R mediatedERK1/2-MAPK signaling pateway, and activation of ERK1/2signaling pathway relieslargely on process of the E3ligase MDM2mediated ubiquitination of IGF-1R. While ourprevious gene array findings in HeLa cells showed the mRNA level of OVA66gene istightly correlated with the mRNA level of MDM2gene, we thus performed an immunoprecipitation（IP）experiment and proved that down regulated expression level ofOVA66decreased the ligand IGF-1stimulated ubiquitination level of IGF-1R molecule inseveral types of cancer cell lines. Simultaneously, we also found that in the presence ofIGF-1factors, both the mRNA and protein level of MDM2in OVA66knocked downcancer cells are relatively lower than negative control cell lines.In summary, the above results revealed that the tumor associated antigen OVA66wasindispensible in promotion of cancer cell proliferation, invasion and survival probably viaits certain regulating role in activation of the IGF-1R-ERK1/2-HSP27signaling pathway.Meanwhile, the expression of OVA66was also positively correlated with the expression ofMDM2in various types of cancer cell lines, which both affected the ligand IGF-1stimulated the ubiquitination process of IGF-1R and activation of its downstreamERK1/2-MAPK signaling cascades, and therefore functioned as an oncoprotein enhancingthe cancer cells potential of viability, invasiveness and survival.