ERBB3-dependent signaling apparently plays the crucial role in regulating the motility and invasion

of HCC cells. To examine the roles of EBBB3-dependent signaling in the tumor development and growth of HCC, we examined the biological consequences of aberrant activation or suppression of ERBB3-dependent signaling in hepatocellular carcinogenesis. Although the activation of ERBB3 by treatment with recombinant NRG1 modestly enhanced cell proliferation (Fig. 7A,B), the silencing of ERBB3, HER2, or both did not suppress the proliferation of HCC cells (Fig. 7C,D). We then examined the biological effects of ERBB3-dependent signaling on the tumor formation and growth of HCC. Neither the activation of ERBB3-dependent ABT-888 concentration signaling via treatment with recombinant NRG1 nor the silencing of the expression of ERBB3, HER2, or both in Huh7 and SK-Hep1 cells significantly suppressed tumor sphere formation in soft agar assays (data Proteases inhibitor not shown). The silencing of EGFR expression did not significantly inhibit tumor sphere formation either (data not shown). On the other hand, xenograft tumor growth of Huh7 and SK-Hep1 cells in nude mice was not significantly suppressed whether the expression of ERBB3 or HER2 had been silenced or not via transduction with lentivirus-based RNA interference (Fig. 7E,F). The silencing

of ERBB3 expression in the xenografts was confirmed with immunoblotting assays (Supporting Information Fig. 4). ERBB3-dependent pathways apparently did not have significant effects on the development and growth of HCC in in vitro or in vivo assays. Microscopic vascular invasion of HCC cells is frequently found in surgically removed HCC tumors. Such microscopic vascular invasion is strongly associated with high rates of intrahepatic metastasis and early recurrence of HCC.15-17 However, the mechanisms behind

the high frequency of microscopic vascular invasion have not been well elucidated. In this study, we provide the first direct evidence that ERBB3-dependent signaling contributes to microscopic vascular invasion and intrahepatic metastasis of HCC. First, we found MCE公司 that up-regulation of ERBB3 in HCC was strongly associated with microscopic vascular invasion, early recurrence, and poor prognosis for patients with HCC. Second, we demonstrated that aberrant activation of ERBB3-dependent signaling enhanced the migration and invasion of HCC cells, whereas the suppression of ERBB3-dependent signaling significantly inhibited the migration and invasion of HCC cells. ERBB3-dependent signaling apparently plays a crucial role in the regulation of the invasion and metastasis of HCC. Our findings also suggest that ERBB3 is a marker for the prediction of microscopic vascular invasion, intrahepatic metastasis, and early recurrence.