This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor-initiating stem-like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)+ patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV+ HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2-8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8--10 was further evaluated in diethylnitrosamine-induced HCCs of C57Bl/6J mice. Focally localized CD44v+ cells with a membranous staining pattern were detected in human HCV+ and mouse HCCs. CD44v9+ cells of HCCs were predominantly negative for Ki67 and P-p38, indicating decrease of cell proliferation in the CD44v9+ tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2-mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV+ HCC cases revealed that positive expression was significantly associated with poor overall and recurrence-free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor-initiating stem-like cells and a prognostic marker in HCV+ HCC patients associated with Nrf2-mediated resistance to oxidative stress. The novel finding of the present study is that CD44 antigen splicing variant isoform 9 (CD44v9) is overexpressed in tumor cell populations within human and mouse hepatocellular carcinomas (HCCs). We have performed comparative proteome analysis in formalin-fixed paraffin embedded HCC sections and different immunohistochemical analyses and found that CD44v9+ cells of HCCs were predominantly negative for Ki67 and P-p38, indicating decrease of cell proliferation in the CD44v9+ tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2, DNA repair and inhibition of xenobiotic metabolism. In human HCV+ HCC cases CD44v9 positivity was correlated with poorer overall and recurrence-free survival and clinicopathological factors including younger age, a poorly differentiated invasive phenotype of HCC, thus suggesting that CD44v9 could be a novel biomarker of liver tumor initiating stem cells (TISCs) and a prognosis factor for HCV+ HCC patients associated with Nrf2-mediated resistance to oxidative stress.
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