Identification and characterization of stemness-related genes (RALYL and S100A10) in the development and progression of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancer which ranks the second leading cause of cancer-related deaths worldwide. During the multistep process of cancer development, cancer cells originate from a population of cancer stem cells (CSCs) which maintain the ability of self-renewal, expansion and tumor formation. Growing evidence suggested that cancer stem cells show many molecular properties and phenotype similarities to their ancestral progenitor cells. Poorly differentiated tumors which usually preserve phenotypes of stem cells and/or tissue stem cells, are commonly associated with high recurrence and poor prognosis. In the present study, we identified two genes, RALY RNA binding protein-like (RALYL) and S100A calcium binding protein A10 (S100A10), which specifically expressed in liver progenitor and hepatoblast-like cells induced from embryonic stem cells in vitro hepatic differentiation model. RALYL was rarely expressed in mature liver and higher RALYL expression was observed in CD133+ HCC cells. Interestingly, the expression of RALYL was associated with poor differentiation, metastasis, and poor prognosis in HCC clinical samples. Functional studies showed that RALYL could promote tumor cell proliferation, foci formation, colony formation, sphere formation, chemoresistance and tumorigenicity in nude mice. RALYL overexpression could increase the population of CD133+ cells. Conversely, RALYL silencing with short hairpin RNA (shRNA) decreased the percentage of CD133+ cells. RALYL overexpression also up-regulate other stemness related genes including fetal liver alpha fetoprotein (AFP), NANOG, SOX2 and c-MYC. Further studies showed that RALYL could upregulate TGF-β2 mRNA stability via decreasing N6-methyladenosine (m6A) modification. Up-regulated by RALYL, TGF-β signaling and its downstream pathways including PI3K/AKT and STAT3 contributed to enhance HCC stemness and maintain undifferentiated state of CSCs. Collectively, RALYL is a hepatic progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. S100A10 was up-regulated in HCC compared to non-tumor tissue. Upregulation of S100A10 was associated with poor prognosis clinically. Overexpression of S100A10 in HCC cells promoted tumor cell proliferation, foci formation, colony formation, sphere formation, chemoresistance and tumorigenicity in nude mice. In addition, S100A10 silencing suppressed HCC tumorigenicity, motility and chemo-resistant ability. S100A10 expression was higher in CD133+ Huh7 cells compared to CD133- cells. In vivo functional study also showed that S100A10 overexpression could promote HCC tumor formation and metastasis. In summary, S100A10 promote tumor self-renewal capacity, drug resistance, tumor formation and metastasis. Taken together, two stemness-related genes, RALYL and S100A10, were identified, which help us unveil the underlying molecular mechanism of cancer stem cells. These findings could inspire new precise therapeutic strategies targeting liver cancer stem cells