Supplementary materials and methods about the generation of conditioned media and ELISA, quantitative real time PCR, western blot analysis and phospho-receptor tyrosine kinase array, plasmids, siRNA, and transfection, dual luciferase assay, oligo pull-down and ChIP, immunofluorescence staining. Supplementary tables S1-S10. S1: Clinicopathologic characteristics of hepatocellular carcinoma cohort (n=339). S2: List of antibodies used for the immunohistochemistry. S3: Criteria for qualitative immunohistochemistry analysis. S4: Criteria for semiquantitative immunohistochemistry analysis. S5: Primer sequences for PCR reaction. S6: List of antibodies. S7: shRNA/siRNA target sequence for gene knockdown. S8: Clinicopathologic characteristics of hepatocellular carcinoma with HGF or MET expression. S9: Univariate and multivariate analyses of disease-specific survival. S10: Univariate and multivariate analyses of disease-free survival.
ARTICLE ABSTRACT
Keratin (KRT) 19 is a poor prognostic marker for hepatocellular carcinoma (HCC); however, regulatory mechanisms underlying its expression remain unclear. We have previously reported the presence of fibrous tumor stroma in KRT19-positive HCC, suggesting that cross-talk between cancer-associated fibroblasts (CAF) and tumor epithelial cells could regulate KRT19 expression. This was investigated in this study using an in vitro model of paracrine interaction between HCC cell lines (HepG2, SNU423) and hepatic stellate cells (HSC), a major source of hepatic myofibroblasts. HSCs upregulated transcription and translation of KRT19 in HCC cells via paracrine interactions. Mechanistically, hepatocyte growth factor (HGF) from HSCs activated c-MET and the MEK–ERK1/2 pathway, which upregulated KRT19 expression in HCC cells. Furthermore, AP1 (JUN/FOSL1) and SP1, downstream transcriptional activators of ERK1/2, activated KRT19 expression in HCC cells. In clinical specimens of human HCC (n = 339), HGF and KRT19 protein expression correlated with CAF levels. In addition, HGF or MET protein expression was associated with FOSL1 and KRT19 expression and was found to be a poor prognostic factor. Analysis of data from The Cancer Genome Atlas also revealed KRT19 expression was closely associated with CAF and MET-mediated signaling activities. These results provide insights into the molecular background of KRT19-positive HCC that display an aggressive phenotype.Significance: These findings reveal KRT19 expression in hepatocellular carcinoma is regulated by cross-talk between cancer-associated fibroblasts and HCC cells, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(7); 1619–31. ©2018 AACR.
