The Clinical Significance And The Function Of GA6and SHP2Protein In Hepatocellular Carcinoma

Objective：This research was performed to study the clinical significance of theGA6protein expression in human hepatocellular carcinoma (HCC), and to explorethe mechanism of the tumorigenesis function of GA6in HCC.Methods：A retrospective cohort of328consecutive HCC specimens whounderwent surgical resection in the PLA general hospital from2000to2007wereincluded, both the tumor tissues (T) and self-matched adjacent non-tumor tissues(NT). By the construction of tissue microarray, immunohistochemical staining ofGA6protein was performed and the clinical and prognostic significance was explored.The GA6expression was also confirmed by the Western blot analysis of anothercohort of49fresh HCC specimens (both the T and self-matched adjacent NT tissues).By the GA6overexpression and GA6knockdown technique in HCC cell lines, weexplored the function of GA6protein in the growth and proliferation of HCC cells inthe experiments of growth curves assay, flat plate clone forming assay, soft agar cloneforming assay, cell cycle analysis and nude mice xenografts assay.Results：1. Immunohistochemical staining found that the GA6protein expressionwas significantly higher in HCC tumor tissues than the adjacent NT tissues (H-scoreT:1.39±0.63, NT:0.92±0.57,P<0.001；positive expression rate T:94.5%, NT:85.4%,P<0.001). The high expression of GA6in HCC tumor tissues was not related to theclinical features of HCC and was also not related to short overall survival byKaplan-Meier analysis (P=0.687).2. Growth curves assay, clone forming assay andnude mice xenografts assay demonstrated that GA6overexpression could promote thegrowth of HCC cells while GA6knockdown could inhibit the growth of HCC cells.Cell cycle and the correlated protein analysis revealed that GA6could promote thegrowth and proliferation of HCC cells by adjusting the expression of G2/M phasecorrelated protein. Conclusions：GA6protein can promote the growth and proliferation of HCCcells both in vitro and in vivo. By immunohistochemistry we found that theexpression of GA6in tumor tissues was significantly higher than that in NT tissues,however, no significant clinical significance was found for the higher GA6expression.Further researches are still needed to evaluate the function of GA6protein in HCC. Objective：Nonreceptor protein-tyrosine phosphatase of SHP2(Src homologyphosphotyrosine phosphatase2) has been previously well identified as aproto-oncogene in a variety of malignancies. However, recently the tumor suppressorfunction of SHP2has also been reported. In our previous research we found by themass chromatographic analysis that YAP2protein which had an importantproto-oncogene role in the pathogenesis of hepatocellular carcinoma (HCC) had aninteraction with the SHP2protein. For this reason, the present study was conducted toinvestigate the expression of SHP2and its associated clinical significance in a largecohort of HCC specimens and to explore the interaction of SHP2protein with YAP2protein by the technique of cell culture and molecular biology.Methods：HCC patients were retrieved from2000to2007in our hospital. Underthe inclusion/exclusion criteria, we included333HCC specimens and a tissuemicroarray of HCC tumor tissue and the self-matched adjacent non-tumor tissues wasconstructed. We also included31pairs of HCC fresh specimens. SHP2expressionwas determined by immunohistochemistry, Western blotting and quantitativepolymerase chain reaction. The association of SHP2expression with clinical featureswas analyzed, overall survival analysis and multivariate analysis were performed.Two plasmids with SHP2substrate trapping mutation were constructed, and theinteraction of SHP2with YAP2was investigated by the techniques of overexpression,substrate trapping co-immunoprecipitation, dual luciferase reporter gene assay andtyrosine phosphorylation.Results：Significantly decreased SHP2expression in tumor tissues compared withadjacent non-tumor tissues could be detected and the positive rate was66.1%and96.7%, respectively. Survival analysis showed low SHP2expression was significantlyassociated with short overall survival (P<0.001). Multivariate analysis showed thedecrease of SHP2expression (ΔSHP2) was an independent prognostic marker (P=0.023, HR:0.628,95%CI:0.420-0.939). By cell culture and the molecularbiology experiments, we found a weak interaction of SHP2with the YAP2protein,for example, SHP2slightly up-regulates the endogenous YAP2expression, SHP2-DM(double substrate trapping mutations) co-immunoprecipitates with YAP2andSHP2-DM up-regulates the tyrosine phosphorylation of YAP2. However, SHP2didnot have a predominant effect in the aspects of YAP2expression, YAP2downstreamactivity and YAP2tyrosine phosphorylation.Conclusion：Although human gene PTPN11was previously well interpreted as aproto-oncogene, SHP2protein is a tumor suppressor and a new prognostic marker inhepatocellular carcinoma. The weak interaction of SHP2protein with the YAP2protein may be not the major mechanism of its tumor suppressor function inhepatocellular carcinoma. For the oncogenic role of SHP2was tissue-specific, thetherapeutic target of PTPN11and its associated mechanism should be furtherresearched.