Tetraspanin TM4SF5-mediated self-renewal stemness of hepatocellular carcinoma cells

Abstract

EMT is involved in tumor progression at the levels of dissemination, metastasis, drug resistance, and self-renewal. As a tetraspan(in), TM4SF5 is highly expressed in diverse cancers and causes EMT, metastasis of liver cancer cells, and gefitinib resistance of NSCLC. Here, the self-renewal by TM4SF5 was mechanistically explored using hepatocellular carcinoma cells with or without TM4SF5 expression. TM4SF5-positive cells well-formed spheroids without serum components and adhesive environments, whereas mutants of N-glycosylation residues (N138A/N155Q) or treatment of anti-TM4SF5 reagent (TSAHC) that disturbs its N-glycosylation status resulted in no formation of spheroids. TM4SF5 expressing cells down-regulate CD24 and express CD44. Further, when another single pass type Ⅰ membrane protein CD44 was physically bound to TM4SF5, the cells formed spheroids

N-glycosylation mutant TM4SF5 did not bind to CD44 and not form spheroids and anti-TM4SF5 reagent had CD44 released free leading to no spheroid formation. Meanwhile, the spheroid formation capacity correlated with tumor formation in xenografts injected with 200 ~ 5000 cells per mouse and in serial xenografts. Moreover orthotopic injection of TM4SF5 expressing cells into mouse livers efficiently formed tumors and led the TM4SF5-positive cells to circulate in mouse peripheral blood even 4 weeks after the injection. TM4SF5 up-regulates twist1 through STAT3 signaling activation resulted in inducing EMT, and up-regulating bim1 expression. These observations suggest that TM4SF5 can render self-renewal stemness during HCC initiation and progression