Role of HBX and IL-6 in Regulating SENP1/Stemness Properties of HBV-Related HCC under Hypoxia and Its Clinical Implication

Hepatocellular carcinoma (HCC) has been the first leading cause of cancer mortality in Taiwan in the past few decades. The molecular pathogenesis of HCC is characterized by its heterogeneity with diverse etiologies and complex signaling pathway activation. Currently there is still lack of effective individual therapy targeting on HCC derived from different etiologies. Chronic HBV hepatitis is closely associated with HCC development that continues to be a great challenge in clinical practice. Niche hypoxic microenvironment and virus-induced inflammation are critical for hepatocarcinogenesis, including expression of cancer stemness. The expression of cancer stemness is associated with poor prognosis, metastasis, and drug resistance. IL-6 has been shown to induce cancer stemness but still with unclear mechanism. Unraveling the molecular mechanisms of IL-6-derived cancer stemness in HBV-related HCC (HBV-HCC) tumorigenesis will be of crucial importance in clinical therapeutic targeting on drug resistance and early recurrence. We have previously demonstrated an important role of the SUMO1 (the small ubiquitin-like modifier) peptidase, SENP1, in stabilizing OCT4 protein stability and regulating the drug resistance in human embryonic carcinoma cells (2012, Cancer Research). Our extension studies showed a high positive correlation between OCT4 and SENP1 expressions in human HCC tissues (R2=0.9, 万=119). The concurrent expression of OCT4/SENP1 /serum IL-6 was mostly confined to HBV-HCC, and the HBV-HCC patients were significantly associated with short DFS. IL-6 stimulates the expressions of OCT4 and SENP1 in HBV+Hep3B cells, and overexpression of SENP1 under hypoxia effectively increases the protein levels of HIF-2a and OCT4 in HBV-Huh7 cells. Together with these results strongly highlight the potential role of HBV virus and hypoxia-IL-6-SENP1 axis in stemness expression and early recurrence of HBV-HCC. As HBx has been hypothesized to be major factor for HCC formation, thus this proposal aims to identify the interaction of HBx and niche hypoxia/IL-6 in regulating SENP1 and OCT4 expressions in human HBV-HCC and to verify their clinical significance and prognosis. Findings in this research will not only identify the molecular mechanism for niche hypoxia/IL-6-induced expressions of cancer stemness in HBV-HCC but also facilitate clinical targeting on individual therapy against this intractable cancer. Three specific aims will be addressed: Aim 1: To examine the role HBx in hypoxia/IL-6-induced SENP1 and stemness properties (such as OCT4 protein expression) in HBV-HCC using a Tet-ON-HBx-overexpression system in Huh7 cells (comparing with Tet-OFF-HBx system in Huh7 cell)(cell line model) Aim 2: To examine the role of SENP1 in IL-6-induced stemness expression (such as OCT4) in Tet-ON-HBx-Huh7 cells in vitro and in vivo Aim 3: To verify the clinical association of serum IL-6 level, tissue OCT4 and SENP1 expression levels with early recurrence of HCC (Using HCC serum sample, tissue array, and frozen tissues)