This difference among cell lines may be attributed, in part, to the significantly higher basal levels of YAP expression found in HepG2 cells (see below), which could compensate, in part, for the reduced binding of YAP/TEAD complexes

to a mutant TB3 site. Moreover, in addition to YAP, the expression of an active mutant β-catenin in HepG2 cells may also be an important determinant for CTGF expression in this cell line, because CTGF is a Wnt/β-catenin target gene.5 Next, we examined whether these elements were required for EGFR-mediated CTGF gene expression. Though AR-mediated transactivation of the CTGF promoter was significantly attenuated when the TB2 and TB3 sites were mutated, it was not affected by AP-1 site mutation (Fig. 3C). In agreement with this, we found that AR treatment increased the recruitment of YAP to

the CTGF Neratinib concentration promoter region encompassing the YAP/TEAD-binding sites (Fig. 4A). Furthermore, YAP knockdown in Hep3B cells significantly reduced basal and AR or HB-EGF-stimulated CTGF gene expression H 89 molecular weight (Fig. 4B). In line with these observations, we found a close correlation between YAP and CTGF mRNA levels in five human HCC cell lines and primary hepatocytes (Fig. 4C,D). In accord with previous reports,20 we observed that YAP expression was increased in HCC tissues and, also, in peritumoral noncirrhotic and cirrhotic liver tissues (Supporting Fig. 1A). Moreover, YAP mRNA levels Methocarbamol significantly correlated with those of CTGF in our collection of healthy and diseased liver samples (r = 0.53, P = 0.0019). Accordingly, the expression of both proteins was detected in cirrhotic and HCC tissues (Supporting Fig. 1B). YAP gene expression is increased in over 60% of HCCs and is frequently detected in nontransformed tissues surrounding tumor nodules.20, 21 However, the mechanisms underlying the regulation of YAP gene transcription are not known. Given the important role played by YAP in basal and EGFR-triggered CTGF expression, we examined whether

EGFR activation could influence YAP expression. We found that AR or HB-EGF treatment increased YAP mRNA and protein levels (Fig. 5A,B), an effect abolished in the presence of Act-D (Fig. 5A). Stimulation of YAP expression by AR was mediated through the EGFR receptor and the downstream activation of extracellular regulated kinase kinase-1 (MEK1), because it was prevented by the PD153035 and UO126 inhibitors, but not by PI3K inactivation (Fig. 5C). The effect of AR and these inhibitors on EGFR downstream signaling is shown in Fig. 5C. Interestingly, EGFR and MEK1 inhibitors also reduced basal YAP mRNA levels (Fig. 5C). In primary human hepatocytes, EGFR activation also elevated YAP mRNA and protein levels (Fig. 6A) and consistently up-regulated integrin β2 (ITGB2) expression, a direct transcriptional target of YAP18 (Fig. 6B).