Materials and methods are described in Supporting Materials and Methods. Consistent with previous reports,7 we observed increased CTGF messenger RNA (mRNA) levels in HCC tissues, compared with samples from individuals with healthy or only small changes in liver function tests (Supporting Fig. 1A). Interestingly, CTGF expression was also elevated in samples of peritumoral noncirrhotic and cirrhotic liver tissues (Supporting Fig. 1A,B).

Treatment with EGFR ligands activated EGFR phosphorylation and elicited a time- and dose-dependent increase in CTGF mRNA in Hep3B cells, which was abolished by the EGFR inhibitor, PD153035 (Fig. 1A). Similar observations were made in Huh7 cells (not shown). this website CTGF expression by EGFR triggering likely involved transcriptional activation, because it was

prevented by actinomycin-D (Act-D) (Fig. Veliparib in vivo 1A). CTGF protein was increased also in cells and conditioned media (Fig. 1B). CTGF gene transcription upon EGFR triggering was further demonstrated in HCC cells transfected with a CTGF promoter-luciferase reporter construct (Fig. 1C). Previously, we described the existence of an autocrine loop in HCC involving AR release and EGFR stimulation.14, 15 Currently, we observe that AR knockdown significantly reduces CTGF expression, suggesting an important role for AR in the basal expression of CTGF (Fig. 1D). Next, we examined whether EGFR activation could lead to CTGF expression in nontransformed human liver parenchymal cells. According to previous findings in rat hepatocytes,16 we observed that TGF-β stimulated CTGF expression in human hepatocytes, and that activation of EGFR by AR and heparin-binding epidermal growth factor (HB-EGF) treatment shared this effect (Fig. 2A,B). Different transcription factors and coactivators participate in the complex regulation

of the CTGF promoter, among them antimothers against decapentaplegic homolog 2/3, activator protein 1 (AP-1), and YAP/TEAD (TEA domain), play important roles in basal and/or growth-factor–triggered CTGF expression.4, 16-19 YAP was recently identified as an oncogene overexpressed in liver cancer,12, 13, 20 and three Pyruvate dehydrogenase lipoamide kinase isozyme 1 putative YAP/TEAD-binding sites (TB1, TB2, and TB3) exist in the human CTGF promoter (Fig. 3A).18, 19 This led us to test first whether these motifs were involved in basal CTGF expression in HCC cells. Though mutation of the most 5′ TB element (TB1) did not change reporter gene activity, mutation of the two adjacent TB sites, TB2 and TB3, caused a significant reduction in basal CTGF promoter activity (Fig. 3B). Conversely, mutation of the AP-1 site, present in our CTGF promoter construct, did not affect basal activity in Hep3B cells (Fig. 3B). Similar findings were obtained in HepG2 cells (Fig.