Search

Summary of Patients and Clinical Data.

PDF file - 86K

Supplementary Figure 1. Unsupervised Clustering on CCA samples. Supplementary Figure 2. Alterations Found in CCA Clusters. Supplementary Figure 3. MAP2K4 Homozygous Deletions and ERBB2 Amplifications. Supplementary Figure 4. Alterations Related to Structural Variations Found in CCAs. Supplementary Figure 5. FIREFLY Analysis of Pathways Systematically Dysregulated by Somatic Promoter Mutations that Alter Transcription Factor Binding. Supplementary Figure 6. Epigenetic Clusters and Mutation Signatures.

PDF file - 122K, Clinical and pathological findings of HCC patients, and the expression level of YAP and AREG detected by western blot analysis in hepatocellular carcinoma patients.

Structural Variations across 71 WGS CCAs.

Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor that is methylated in many human cancers, including hepatocellular carcinoma (HCC). RASSF1A has been shown to suppress tumors via activation of the Hippo tumor suppressor pathway, including mammalian STE20-like kinase (MST). Amphiregulin (AREG), a target gene for Yes-associated protein (YAP), is a known oncogenic component of the Hippo pathway; however, the tumor-suppressive effect of RASSF1A on AREG in regard to regulation of the Hippo pathway remains unclear in HCC. Overexpression of RASSF1A in HCC cells, which lack functional RASSF1A, significantly inhibited cell proliferation and induced apoptosis by activating the Hippo pathway. Consequently, overexpression of RASSF1A inhibited the oncogenic functions of YAP, leading to a significant reduction in AREG secretion via regulation of the Hippo pathway. In human specimens, greater expression of RASSF1A was observed in chronic hepatitis/cirrhosis than in HCC, whereas expression of YAP and AREG was higher in 81% and 86% of HCC than in corresponding chronic hepatitis/cirrhosis, respectively. Furthermore, RASSF1A protein gradually decreased as multistep hepatocarcinogenesis progressed from chronic hepatitis/cirrhosis dysplastic nodules toward HCC, whereas the protein expression of YAP and AREG gradually increased. These findings provide mechanistic insight into the regulation of YAP and AREG by RASSF1A in human multistep hepatocarcinogenesis. Mol Cancer Res; 11(7); 748–58. ©2013 AACR.

Summary of CCA Alterations.

PDF file - 2527K, Relationship between RASSF1A, YAP, and AREG histoscores.

Further details of methods, in addition to the supplementary figure and table legends.

PDF file - 1379K, Immunohistochemical analysis of nuclear and cytoplasmic YAP protein localization in human multistep hepatocarcinogenesis.

Alterations in CCA Clusters.

Coverage Statistics and List of Genes for Targeted Sequencing.

Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis to low-grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multilayered genomic, epigenomic, and transcriptomic aberrations in the stepwise hepatocarcinogenesis. Initial DNA methylation was observed in eHCC (e.g., DKK3, SALL3, and SOX1) while more extensive methylation was observed in pHCC. In addition, eHCCs showed an initial loss of DNA copy numbers of tumor suppressor genes in the 4q and 13q regions, thereby conferring survival benefits to cancer cells. Transcriptome analysis revealed that HGDNs expressed endoplasmic reticulum (ER) stress–related genes, while eHCC started to express oncogenes. Furthermore, integrative analysis indicated that expression of the serine peptidase inhibitor, Kazal type 1 (SPINK1), played a pivotal role in eHCC development. Significant demethylation of SPINK1 was observed in eHCC compared to HGDN. The study also demonstrated that ER stress may induce SPINK1 demethylation and expression in liver cancer cells. In conclusion, these results reveal the dynamics of multiomic aberrations during malignant conversion of liver cancer, thus providing novel pathobiological insights into hepatocarcinogenesis.Significance:Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.

Purpose: Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathologic parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment.Experimental Design: Microarray technologies were used to generate and analyze gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients.Results: We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the six-gene signature in an additional independent cohort.Conclusions: The risk score derived from the six-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy. Clin Cancer Res; 17(7); 1850–7. ©2011 AACR.

Supplementary Figures S1-S9 S1: The representative case showing CD90 expression. Morphology and character of hepatic stellate cell lines. S2: The expression change of KRT19 following S3I-201, TPA, U0126, and SCH772984. The transfection efficiency of HCC cell lines. S3: The HGF concentrations in conditioned media from hTERT-HSC, LX-2, and primary human hepatic stellate cells. Western blot analysis of hepatocellular carcinoma cell lines, showing expression of phospho-MET, MET, phsopho-ERK1/2, and KRT19. mRNA and protein expression of KRT19 in HepG2 and SNU475 cells, following transient expression of MET. S4: Morphologic changes in HepG2 and SNU423 induced by conditioned media (CM) from inducible HGF knockdown stable cell lines established from hTERT-HSC: shHGF-1 and shHGF-2. mRNA expression levels of KRT19 and morphologic changes in HepG2 and SNU423, following CM treatment combined with hepatocyte growth factor (HGF) neutralizing antibody. S5: The gene knock-down efficiencies of two pooled siRNAs targeting JUN, JUND, FOSL1, or FOSL2. The gene knock-down efficiencies of siRNAs targeting JUN or FOSL1. S6: Expression of HGF in hepatic stellate cell line of hTERT-HSC, and HCC cell lines of HepG2 and SNU423. Distribution of HGF and MET expression in HepG2 cells, at various time points after conditioned media (CM) treatment. S7: S7. Increased KRT19 expression and nuclear translocation of ERK1/2, phospho-ERK1/2, FOSL1, phospho-FOSL1 after the conditioned media treatment in HepG2. Rapid decline in phosphor-MET levels after removal of conditioned media. S8: Schematic representation of regulatory mechanism of KRT19 gene by cancer-associated fibroblast (CAF) derived HGF via MET-ERK1/2- AP1 and SP1 axis. S9: Conservation of AP1 and SP1 binding sites in KRT19 promoter.

Supplementary Table S1: Tissue specimens used for RNA-seq analysis. Supplementary Table S2: Tissues specimens used for immunohistochemical analysis. Supplementary Table S3: Gene signatures used in the study. Supplementary Table S4: Differentially methylated probes during hepatocarcinogenesis. Supplementary Table S5: Univariate and multivariate analysis for SPINK1 expression and HCC clinico-pathological features in TCGA-LIHC data.

Supplementary Figures S1-S6; Supplementary Tables S1-S2.

Supplementary Materials and Methods

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.

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.

Supplementary Figure S1: Overall study design for multi-omic data analysis. Supplementary Figure S2: The distribution of beta-values in DNA methylation data. Supplementary Figure S3: The distribution of differentially methylated probes (DMPs). Supplementary Figure S4: Differentially methylated probes in the integrated data of GSE44970 and YSHCC. Supplementary Figure S5: Distribution of CNAs during stepwise hepatocarcinogenesis. Supplementary Figure S6: DNA methylation of oncogenes and TSGs during multi-step hepatocarcinogenesis. Supplementary Figure S7: Frequencies of nonsynonymous or missense mutations in each patient. Supplementary Figure S8: Functionally enriched expression in each step of hepatocarcinogenesis. Supplementary Figure S9: Validation of the three genes including SPINK1, CAP2, and RRAGD. Supplementary Figure S10: SPINK1 overexpression increases proliferation and invasion of Huh7 cells. Supplementary Figure S11: Correlation between methylation and expression. Supplementary Figure S12: Western blot of DNMT1 expression. Supplementary Figure S13: GRP78 expression under ER stress. Supplementary Figure S14: Effects of knockdown of p65 expression in the expression of GRP78 and SPINK1.

Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis to low-grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multilayered genomic, epigenomic, and transcriptomic aberrations in the stepwise hepatocarcinogenesis. Initial DNA methylation was observed in eHCC (e.g., DKK3, SALL3, and SOX1) while more extensive methylation was observed in pHCC. In addition, eHCCs showed an initial loss of DNA copy numbers of tumor suppressor genes in the 4q and 13q regions, thereby conferring survival benefits to cancer cells. Transcriptome analysis revealed that HGDNs expressed endoplasmic reticulum (ER) stress–related genes, while eHCC started to express oncogenes. Furthermore, integrative analysis indicated that expression of the serine peptidase inhibitor, Kazal type 1 (SPINK1), played a pivotal role in eHCC development. Significant demethylation of SPINK1 was observed in eHCC compared to HGDN. The study also demonstrated that ER stress may induce SPINK1 demethylation and expression in liver cancer cells. In conclusion, these results reveal the dynamics of multiomic aberrations during malignant conversion of liver cancer, thus providing novel pathobiological insights into hepatocarcinogenesis. Significance: Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.

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.

Telomerase reverse transcriptase (TERT) promoter somatic mutations, related to telomerase activation, have been known to frequently occur at two hot spots located at -124 and -146 bp relative to the start codon in various cancers. In the present study, we investigated the occurrence and implications of the genetic alterations of the TERT promoter in B viral hepatocarcinogenesis. TERT promoter mutations, especially -124C>T mutation, obviously enhanced TERT promoter activity in hepatocellular carcinoma (HCC) cell lines. We identified prospero homeobox protein 1 (PROX1) as a novel transcriptional activator for TERT gene through binding to the promoter regions containing two hot spots. PROX1 binding affinity was strong to the mutant TERT promoter harboring a consensus E-twenty six/ ternary complex factor (ETS/TCF) binding sequence (CCGGAA). The incidence of TERT promoter mutations gradually increased according to the progression of human B viral multistep hepaticarcinogenesis, which was found in 9.0% of low grade dysplastic nodules (LGDNs), 13.5% of high grade dysplastic nodules (HGDNs), 27.3% of early HCCs (eHCCs) and 28.4% of progressed HCCs (pHCCs). The occurrence of TERT promoter mutations correlated with lower levels of alpha-fetoprotein (AFP) (p=0.046) and a poor overall survival (p=0.012) in B viral HCC patients. On the contrary to in vitro data, TERT mRNA expression was lower in B viral HCCs with the mutant TERT promoter compared to those without. In addition, mRNA level of PROX1 was not correlated with that of TERT in B viral HCCs, in contrast that such correlation was evident in non-B viral HCCs. Interestingly, induction of stable HBx expression inhibited PROX1-mediated TERT expression in vitro study. In conclusion, our findings suggest that TERT promoter somatic mutations are early events in B viral human multistep hepatocarcinogeneis and HBx can induce a loss of PROX1 function as transcriptional activator for TERT expression. Citation Format: Young-Joo Kim, Jeong Eun Yoo, Youngsic Jeon, Jae Uk Chong, Gi Hong Choi, Dae-Geun Song, Sang Hoon Jung, Bong-Kyeong Oh, Young Nyun Park. TERT promoter mutations in B viral humanhepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2547.

Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor that is methylated in many human cancers, including hepatocellular carcinoma (HCC). RASSF1A has been shown to suppress tumors via activation of the Hippo tumor suppressor pathway, including mammalian STE20-like kinase (MST). Amphiregulin (AREG), a target gene for Yes-associated protein (YAP), is a known oncogenic component of the Hippo pathway; however, the tumor-suppressive effect of RASSF1A on AREG in regard to regulation of the Hippo pathway remains unclear in HCC. Overexpression of RASSF1A in HCC cells, which lack functional RASSF1A, significantly inhibited cell proliferation and induced apoptosis by activating the Hippo pathway. Consequently, overexpression of RASSF1A inhibited the oncogenic functions of YAP, leading to a significant reduction in AREG secretion via regulation of the Hippo pathway. In human specimens, greater expression of RASSF1A was observed in chronic hepatitis/cirrhosis than in HCC, whereas expression of YAP and AREG was higher in 81% and 86% of HCC than in corresponding chronic hepatitis/cirrhosis, respectively. Furthermore, RASSF1A protein gradually decreased as multistep hepatocarcinogenesis progressed from chronic hepatitis/cirrhosis dysplastic nodules toward HCC, whereas the protein expression of YAP and AREG gradually increased. These findings provide mechanistic insight into the regulation of YAP and AREG by RASSF1A in human multistep hepatocarcinogenesis. Mol Cancer Res; 11(7); 748–58. ©2013 AACR.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related death. The Hippo-YAP signaling pathway is gaining recognition as it plays an important role in both organ size control and tumorigenesis. Liver-specific ablation of WW45 in rodent models (one of the tumor suppressive Hippo pathway components in mammals) has been reported to increase liver size and expand hepatic stem/progenitor cells (oval cells) and to develop HCC eventually. The expression of YAP also has been reported as an independent poor prognostic marker for human HCCs. Amphiregulin is a downstream target of YAP and is a member of the EGF family, which is implicated in cancer stem cells in HCC. Cancer stem cells have the ability to self-renew, differentiate, and proliferate, and have greater tumorigenicity, chemoresistance, and resistance to hypoxia. HCCs expressing stem cell makers have actually been reported to be associated with poor prognosis and aggressive biological behavior. The aim of this study is to investigate the Hippo-YAP pathway and its related oncogenic signaling in HCCs expressing stem cell markers. The effect of amphiregulin on the regulation of the Hippo-YAP pathway was also studied to identify potential molecular therapeutic targets for HCCs expressing stem cell markers. Under hypoxic condition, the expression of cancer stem cell markers (EpCAM, CD133, and CK19) was upregulated in a time dependent manner and the Hippo-YAP pathway was oncogenically activated in four HCC cell lines (HepG2, Hep3B, Huh7, and SK-Hep1). In addition, the treatment of recombinant human amphiregulin dephosphorylated and activated YAP pathway. Therefore, up-regulation of cancer stem cell markers with activation of oncogenic YAP pathway in HCC cell lines might suggest the acquisition of survival advantage of tumor cells under hypoxia. The relationship between the regulatory mechanism of cancer stem cells and the Hippo-YAP pathway can be used for molecular target of chemotherapy for HCC. Citation Format: Ji Su Kim, Ei Yong Ahn, Young Nyun Park. Regulation of stemness by Hippo-YAP pathway under hypoxia in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C87.