Up-regulation of ERBB3 was strongly associated with microscopic vascular invasion of HCC (P = 0.034; Table 1) and early recurrence (P = 003; Fig. 2C). We next asked whether up-regulation of ERBB3 is associated with I-BET-762 in vivo constitutive activation of ERBB3. We assayed the coexpression of other ERBB members and NRGs, the ligands of ERBB3. EGFR and HER2 were expressed
in most of the HCC cells, whereas ERBB3 and NRG1 were expressed in all of the HCC cells (Fig. 3A). ERBB4 was not detected in any of the HCC cells (Fig. 3A). In addition, both NRG1 and pERBB3 were also detected in all of the tested HCC tissues (Fig. 3B), and this suggested constitutive activation of ERBB3 in HCC, very likely via an NRG1/ERBB3 autocrine mechanism. To confirm the involvement of an NRG1/ERBB3 autocrine loop in ERBB3 activation in HCC, we determined whether HCC cells secrete bioactive NRG1 to activate ERBB3 of HCC cells. Phosphorylation of ERBB3 of starved Huh7 and HepG2 cells was induced (presumably activated) R788 ic50 by treatment with the recombinant NRG1 (Fig. 4A) or the conditioned media of most of the HCC cells (Fig. 4B). To verify the presence of bioactive
NRG1 in the conditioned media, we used antibodies against NRG1 to block its interaction with ERBB3. As shown in Fig. 4C, phosphorylation of ERBB3 was abolished because the conditioned media had been treated with antibodies against NRG1 before its administration to HCC cells.
Pretreatment of the conditioned media with antibodies against the extracellular domain of ERBB3 was used as the positive control (Fig. 4C). In parallel, MCE HeLa cells, which did not express ERBB3, were treated with the conditioned media of HCC cells to rule out the possibility of contaminants of pERBB3 in the conditioned media due to lysis of the donor HCC cells (Fig. 4D). Treatment of HeLa cells with recombinant NRG1 was used as the control. If ERBB3 of the HCC cells was activated via an autocrine loop, we expected that silencing of the expression of endogenous NRG1 would suppress phosphorylation of their own ERBB3 and abolish the bioactivity of the conditioned media to phosphorylate ERBB3 of the target cells. As shown in Fig. 4E, silencing of the expression of endogenous NRG1 by RNA interference in HCC cells suppressed their own ERBB3 phosphorylation (Fig. 4E) and eliminated the activity of their conditioned media to phosphorylate ERBB3 of the target cells (Fig. 4F). Altogether, we conclude that the constitutive activation of ERBB3 in HCC cells was achieved via an autocrine mechanism by the synthesis/secretion of bioactive NRG1 from HCC cells to activate their own ERBB3. To identify the partners for the dimerization and activation of ERBB3 upon NRG1 binding, we investigated whether EGFR or HER2 was required for ERBB3 activation in SK-Hep1, Huh7, and HepG2 cells.