Identification and characterization of tumor suppressor gene and cancer stemness gene in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with a very poor prognosis. Despite intensive studies, the molecular mechanism underlying HCC initiation and progression remains unclear. A better understanding of the genetic and epigenetic landscape during HCC development is of great significance to prognosis and treatment. In a recent study, genome-wide methylation analysis (27,578 CpG sites) was applied to identify differentially methylated genes between 71 primary HCCs and 8 nondiseased (ND) normal liver tissues. A total of 678 genes were significantly hypermethylated in HCCs. Our previous RNA-sequencing results derived from 3 pairs of HCC cases found 407 genes were downregulated in all 3 cases (GenBank accession no. GSE33294). By combining these two databases, we identified 20 genes were overlapped and Angiopoietin-like protein 1 (ANGPTL1) is one of the significantly down-regulated genes with promoter hypermethylation. ANGPTL1 was frequently downregulated in HCC, which was significantly associated with vascular invasion, tumor thrombus, metastasis and poorer prognosis. Ectopic expression of ANGPTL1 in HCC cells could effectively decrease their in vitro and in vivo tumorigenicity, cell motility and angiogenesis. Silencing ANGPTL1 expression by RNAi with shRNA had the opposite effects. Further study found that ANGPTL1 could promote apoptosis by inhibiting the STAT3/Bcl-2 mediated anti-apoptotic pathway. ANGPTL1 decreased cell migratory and invasive abilities by inhibiting EMT via downregulating transcription factors SNAIL and SLUG expression. Furthermore, ANGPTL1 could inhibit angiogenesis by inhibiting ERK and AKT signaling. Molecular study found that ANGPTL1 interacted with integrin α1β1 receptor and suppressed downstream FAK/Src-JAK-STAT3 signaling pathway, thus affects HCC cell survival, metastasis and angiogenesis. ANGPTL1 might be used as a prognostic biomarker and a novel therapeutic agent for HCC treatment. The poor prognosis of HCC patients is largely attributed to aggressive nature of this cancer, characterized by a high potential of recurrence and metastasis. The cancer stem cell (CSC) theory suggests that a tumor is organized within a hierarchy of mixed tumor cells, with the CSC at the apex. Stemness-regulated genes in embryonic stem cells are often reactivated in those CSCs. These parallels gave rise to the hypothesis that tumors may arise from undifferentiated cancer stem cells or the cancer cells may undergo dedifferentiation during cancer progression. Clinically, the expression of stemness markers and the differentiation level of cancer are closely associated with the clinical outcome of cancer patients. DPPA3 was identified in a liver developmental model, in which DPPA3 has highest expression in liver progenitor stage and dramatically decrease following differentiation. Functional assays demonstrated that DPPA3 could enhance cancer cells capabilities of self-renewal, tumor initiation, chemodrug resistance as well as metastasis. Whole-genome methylation sequencing demonstrated that DPPA3 could lead to promoter methylation level alteration, especially in genes associated with pluripotency maintenance, suggesting that DPPA3 may exerts its function via modulating genes methylation. In summary, we discovered the molecular mechanisms of two novel genes ANGPTL1 and DPPA3 during HCC pathogenesis. Identification and characterization of their mechanism may shed a light on developing novel biomarkers and targets in HCC diagnosis and treatment.