Preliminary Study On The Contribution Of C-Kit To The Proliferation And Invasion Of Hepatocellular Carcinoma

Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumor that has been claimed as the third most lethal tumor in the world, accounting for the second death cause of china's tumor patients. There are now one million HCC patients in the globe, with an increase of 300 thousands each year, 45% of which are Chinese who lives in south eastern and coastal area of china. With recent years'prevalence of hepatitis B virus, the geographical distribution and incidence of liver cancer has significantly changed. Onset peak trends and mortality rate also goes to the young age group. At present, the treatment of hepatocellular carcinoma is mainly based on surgical resection, after which the use of chemotherapy-related drugs in hepatocellular carcinoma will be largely limited. Therefore, the research on the impact of proto-oncogene on the proliferation and invasion of HCC is of great significance for further treatment.c-Kit is a proto-oncogene encoding receptor tyrosine kinase family. The current study shows that c-Kit is closely linked with the development, and prognosis of a variety of tumors. Many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia and lung cancer were proved with the abnormal expression of c-Kit. Aimed directly at these c-Kit high expression tumors, tyrosine kinase inhibitors have been successfully used in clinical patient. At present, research on expression of c-Kit receptor in HCC was rarely reported. The detailed mechanisms of KIT/SCF signaling pathway in HCC need to be further elucidated.Our extensive literature review showed the following information: First, patients with increased c-Kit proto-oncogene expression implies poor prognosis. Second, in normal liver, c-Kit exists only in oval cells that have uneven distribution in tumor. Research indicates its changment during the process of liver cirrhosis-liver cancer. Third, the PI3K/AKT signaling pathway, a part of the down stream of KIT/SCF signaling pathway, is closely linked with Wnt pathway that is one of the most important invasion and metastasis path of HCC. On the basis of the above theories, we hypothesized that the downstream of KIT/SCF pathway develops corresponding alteration, promoting the invasion and metastasis of HCC after its activation. This study aimed to verify these assumptions and elucidate the possible underlying molecular mechanisms of c-Kit in the proliferation and invasion of HCC.Materials and methods1. 89 clinic HCC patients were selected and examined for c-Kit expression by immunohistochemistry. Results were analyzed for the correlation between c-Kit expression and clinical pathological factors. Two MHCC-97 sub cell lines with different invasion and metastasis potential, MHCC-97L and HCC-97H, were chosen and examined for c-Kit expression by Immunofluorescence, Realtime-PCR and Western-blot, and were analyzed for the correlation between c-Kit expression and invasive potential.2. An adenovirus vector system, Adeasy, was adopted for the construction of c-Kit containing vector. The vector was then transfected into cell line Hep-G2. Western-blot, invasion test and motility test were carried out for the contrast study of c-Kit's role in invasion and metastasis process of HCC. Nude mice were injected with Hep-G2 that had been infected by c-Kit gene-contained recombinant adenovirus vector and its blank control. The tumor formation potential was observed and the role of c-Kit in the process of tumor forming and invasion was studied.3. An exogenous ligand of c-Kit receptor was introduced into SMMC-7721. the impact of c-Kit/SCF signaling system on the distribution ofβ-catenin was analyzed. Meanwhile, whether this impact is conducted through the PI3K/AKT signaling system was analyzed. And the phosphorylation status of AKT and GSK-3, the key factor of PI3K/AKT signaling system, was examined.4. FAK expression in the 89 clinical HCC patients were examined by immunohistochemistry and the result was evaluated for the correlation between FAK and c-Kit with other pathological factors. Co-immunoprecipitation was adopted to examine the binding between FAK and KIT in cell line SMMC-7721 and Hep-G2. Results and Conclusions1. c-Kit gene expression was detected and the following results in clinical HCC tissue samples and HCC cell lines with varied potential of invasion and metastasis were obtained:1.1. Expression of c-Kit was significantly increased in HCC tumor cells.1.2. c-Kit expression level is positively correlated with vascular invasion, TNM classification, HBV DNA level and HBSAg, which indicates that c-Kit expression was closely correlated with HCC progression and its increased level with the HCC development.1.3. Among the sub-cell lines of MHCC-97 that with various potential of invasion and metastasis, c-Kit was significantly higher in MHCC-97H than in MHCC-97L. This result confirmed that c-Kit was up-regulated as the invasion and metastasis increased.2. Recombinant adenovirus containing c-Kit was successfully constructed and transfected into HCC cell line Hep-G2. After the expression of c-Kit was verified, it was observed that exogenous c-Kit increased the invasion and motility of Hep-G2.3. After the exogenous ligand of c-Kit receptor was introduced into SMMC-7721, PI3K/AKT signaling system was activated and its key factor AKT and GSK-3βunderwent significant phosphorylation,β-catenin showed an intranuclear redistribution. After the addition c-Kit/SCF inhibitor Imatinib,β-catenin intranuclear-redistribution was blocked.4. FAK gene expression was detected and the following results in clinical HCC tissue samples and HCC cell lines with varied potential of invasion and metastasis were obtained.4.1.FAK was significantly up-regulated in HCC cells.4.2.FAK level was significantly correlated with great vessel invasion,TNM classification, HBsAg and HBV DNA level, indicating that FAK expression is up-regulated as the tumor progressing. And FAK and c-Kit expression was closely connected in HCC cells.To sum up, the follow conclusion was obtained form this study:1. c-Kit gene expression was closely correlated with tumor progression and its up-regulation indicates poor prognosis. Exogenous c-Kit gene increases the invasion, motility and tumor forming potential of HCC. 2. As a crucial gene in HCC, c-Kit increases invasion potential of HCC through activation of PI3K/AKT signaling system, showing synergistic effect with FAK.3. Kit/SCF signaling system inhibitor Imatinib may have protective effect on HCC progression.4. KIT and FAK could bind in hepatoma cell line.