Down-regulation of microRNA-26a (miR-26a) is associated with poor prognosis of hepatocellular carcinoma (HCC), but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of miR-26a in tumor growth and metastasis of HCC and found that miR-26a was frequently down-regulated in HCC tissues. Down-regulation of miR-26a correlated with HCC recurrence and metastasis. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit in vitro cell proliferation, migration, and invasion. In addition, miR-26a induced G1 arrest and promoted apoptosis of HCC cells. Importantly, miR-26a suppressed in vivo tumor growth and metastasis in nude mice models bearing human HCC. Interleukin-6 (IL-6) was identified as a target of miR-26a. Knockdown of IL-6 induced effects on HCC cells similar to those induced by miR-26a. In contrast, IL-6 treatment abrogated the effects induced by miR-26a up-regulation. Moreover, miR-26a dramatically suppressed expression of signal transducer and activator of transcription 3 (Stat3) target genes, including Bcl-2, Mcl-1, cyclin D1, and MMP2. IL-6 treatment antagonized this effect, while knockdown of IL-6 by IL-6 short hairpin RNA (shIL-6) induced inhibitory effects on the expression of p-Stat3 and its main target genes, similar to miR-26a. The messenger RNA and protein levels of IL-6 inversely correlated with miR-26a in HCCs. Patients with high miR-26a or low IL-6 in HCC tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, IL-6, and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Conclusion: miR-26a could suppress tumor growth and metastasis of HCC through IL-6-Stat3 signaling and is a novel prognostic marker and therapeutic target for HCC. (HEPATOLOGY 2013)

Sorafenib is a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), but its clinical effects are still limited. In this study we identify Quiescin sulfhydryl oxidase 1 (QSOX1) acting as a cellular pro-oxidant, specifically in the context of sorafenib treatment of HCC. QSOX1 disrupts redox homoeostasis and sensitizes HCC cells to oxidative stress by inhibiting activation of the master antioxidant transcription factor NRF2. A negative correlation between QSOX1 and NRF2 expression was validated in tumor tissues from 151 HCC patients. Mechanistically, QSOX1 restrains EGF-induced EGFR activation by promoting ubiquitination-mediated degradation of EGFR and accelerating its intracellular endosomal trafficking, leading to suppression of NRF2 activity. Additionally, QSOX1 potentiates sorafenib-induced ferroptosis by suppressing NRF2 in vitro and in vivo. In conclusion, the data presented identify QSOX1 as a novel candidate target for sorafenib-based combination therapeutic strategies in HCC or other EGFR-dependent tumor types.

Background: Aberrant AKT activation contributes to cancer stem cell (CSC) traits in hepatocellular carcinoma (HCC). We previously reported that CD73 activated AKT signaling via the Rap1/P110β cascade. Here, we further explored the roles of CD73 in regulating CSC characteristics of HCC. Methods: CD73 expression modulations were conducted by lentiviral transfections. CD73+ fractions were purified by magnetic-based sorting, and fluorescent-activated cell sorting was used to assess differentiation potentials. A sphere-forming assay was performed to evaluate CSC traits in vitro, subcutaneous NOD/SCID mice models were generated to assess in vivo CSC features, and colony formation assays assessed drug resistance capacities. Stemness-associated gene expression was also determined, and underlying mechanisms were investigated by evaluating immunoprecipitation and ubiquitylation. Results: We found CD73 expression was positively associated with sphere-forming capacity and elevated in HCC spheroids. CD73 knockdown hindered sphere formation, Lenvatinib resistance, and stemness-associated gene expression, while CD73 overexpression achieved the opposite effects. Moreover, CD73 knockdown significantly inhibited the in vivo tumor propagation capacity. Notably, we found that CD73+ cells exhibited substantially stronger CSC traits than their CD73– counterparts. Mechanistically, CD73 exerted its pro-stemness activity through dual AKT-dependent mechanisms: activating SOX9 transcription via c-Myc, and preventing SOX9 degradation by inhibiting glycogen synthase kinase 3β. Clinically, the combined analysis of CD73 and SOX9 achieved a more accurate prediction of prognosis. Conclusions: Collectively, CD73 plays a critical role in sustaining CSCs traits by upregulating SOX9 expression and enhancing its protein stability. Targeting CD73 might be a promising strategy to eradicate CSCs and reverse Lenvatinib resistance in HCC.