| Summary: | 博士 === 長庚大學 === 臨床醫學研究所 === 92 === Hepatocellular carcinoma (HCC) is the most common and devastating malignant tumor in Taiwan. Transformation of hepatocytes to the malignant phenotype may be induced by chronic liver injury and regeneration, with genetic mutations. Increasing evidence suggests that additional oncogenes, tumor suppressor genes and certain growth factors (through the regeneration) were involved in the progression of hepatoma. In the present study, we set forth to explore the role of the newly identified growth factor, HDGF and tumor suppressor gene, PTEN in the pathogenesis of hepatocellular carcinoma
Hepatoma-derived growth factor (HDGF) was originally isolated from the cultured media of human hepatoma cell line, HuH-7 cells. Previous studies indicated that HDGF participates in many cellular processes including astrocytes proliferation, renal development, vascular lesion formation, and cardiovascular differentiation. To explore the role of HDGF in the carcinogenesis of hepatocellular carcinoma (HCC), in vitro functional analysis in hepatoma cell lines and immunohistochemical studies on 105 HCC specimens were performed. We found that HDGF was upregulated in malignant hepatoma cell lines. Besides, malignant hepatoma cells exhibited higher mitogenic responses to exogenous HDGF, which might be attributed to their higher capability to uptake HDGF into nucleus. Immunohistochemical studies indicated that HDGF immunostaining was detected in nucleus and cytoplasm of hepatocytes and hepatoma cells in HCC specimens. The HDGF labeling index (LI) in hepatoma tissues were significantly higher than that in the adjacent non-tumor tissues (P < 0.05). Statistical analysis revealed that elevated nuclear HDGF LI correlated with HCC dedifferentiation (P = 0.033), absence of tumor capsules (P = 0.008), and high alpha-fetoprotein (a-FP) level (P = 0.045). Besides, the nuclear HDGF LI was strongly associated with that of proliferating cell nuclear antigen (PCNA) (P < 0.001). Kaplan-Meier analyses indicated that patients with high nuclear HDGF LI had poor survival and increased recurrence (P < 0.001). By multivariate analysis, nuclear HDGF LI is an independent prognostic factor for overall and disease free survival of HCC patients.
To further explore the roles of HDGF in angiogenesis and tumorigenesis, recombinant HDGF was generated and shown to stimulate the proliferation and migration of human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Besides, treatment of HUVEC with HDGF increased the secretion of matrix metalloproteinase-9 (MMP-9) by up to ten-fold. Implantation of hydron pellets containing HDGF induced dose-dependent neovascularization in rat corneas. Further, non-malignant NIH3T3 cells were transfected with HDGF expression vector and selected for HDGF-overexpressing stable clones. HDGF transfectants proliferated at a higher rate than that of NIH3T3 cells in serum-containing or -deprived media. Moreover, HDGF transfectants were capable of forming colonies in soft agar and inducing tumor formation when injected into nude mice. Histological analysis revealed prominent angiogenic and mitogenic activities in tumors derived from HDGF transfectants.
Inactivation of tumor suppressor genes or activation of oncogens in HCC has been widely explored. The inactivation of tumor suppressor gene PTEN, located on chromosome 10q23, is a common event in advanced stage of diverse human cancers. Although the mutation rates of PTEN were low (< 5%) in HCC, there have been 20~30% allelic loss of chromosome 10q in HCC. However, the role of PTEN in HCC is not characterized yet. Analysis of PTEN expression in hepatoma cell lines revealed that loss of PTEN expression was found in one hepatoma cell line, Mahlavu cells. Restoration of PTEN expression resulted in significant reduction in colonies formation, proliferation and migration of Mahlavu cells, indicating that PTEN gene delivery suppressed tumorigenicity of hepatoma cells. Furthermore, adenoviral gene transfer of PTEN gene into Mahlavu cells also suppressed its tumor growth in nude mice. Immunohistochemical analysis of 105 HCC tissues revealed that decreased or absence of PTEN immunostaining was found in 43 (40.9%) cases. The reduced PTEN expression correlated with increased grades (P = 0.017), advanced stages (P = 0.016), and elevated serum alpha-fetoprotein (aFP) levels (P = 0.001). Kaplan-Meier analysis indicated that patients with reduced PTEN level had shorter overall survival (P = 0.001) and higher recurrence rates (P = 0.0007) than that of patients with intact PTEN expression. Examining p53 expression unveiled an inverse correlation between p53 overexpression and PTEN reduction in HCC patients (P = 0.004), implicating that PTEN inactivation frequently occurred in HCC with p53 mutation. Besides, patients with p53 overexpression had shorter overall survival comparing to that of patients without p53 overexpression (P = 0.0014). Finally, the univariate and multivariate analysis revealed that reduced PTEN expression and high nuclear HDGF are two independent prognostic factors for survival of HCC.
In summary, the present study provided in vitro and in vivo evidences supporting that upregulation of HDGF and loss of PTEN expression participates in liver carcinogenesis. Both of them may hold potential for future treatment of HCC. The relationship between the two genes in HCC needs to be further elucidated.
|
|---|
