Author Manuscript Published OnlineFirst on September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0991 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Dynamics of genomic, epigenomic, and transcriptomic aberrations during stepwise hepatocarcinogenesis Byul A Jee1,2,*, Ji-Hye Choi1,2,*, Hyungjin Rhee3,*, Sarah Yoon1,2, So Mee Kwon4, Ji Hae Nahm5, Jeong Eun Yoo5, Youngsic Jeon5,6, Gi Hong Choi7, Hyun Goo Woo1,2,†, Young Nyun Park5,6† 1Department of Physiology, Ajou University School of Medicine, Suwon, Republic of Korea; 2Department of Biomedical Science, Graduate School, Ajou University, Suwon, Republic of Korea; 3Department of Radiology, Yonsei University College of Medicine, Seoul, Republic of Korea; 4Department of Biochemistry, Ajou University School of Medicine, Suwon, Republic of Korea; 5Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea; 6BK21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; 7Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea *BAJ, JHC, and HR contributed equally to this work. Running Title Multi-layered genomic profiles during stepwise HCC Keywords Multi-omics; stepwise HCC; hepatocarcinogenesis; early HCC Financial Support This research was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (NRF-2017R1E1A1A01074733, NRF-2017M3A9B6061509, NRF-2017M3C9A6047620, NRF-2019R1A5A2026045, NRF-2017R1A2B4005871, and NRF- 2017M3A9B6061512). †Co-corresponding authors: Hyun Goo Woo, M.D., Ph.D., Department of Physiology, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Korea; Tel: 82-31-219-5045, Fax number: 82-31-219-5049, E-mail address: [email protected]. Young Nyun Park, M.D., Ph.D., Department of Pathology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, Korea; Tel: 82-2-2228-1768, Fax number: 82-2-362- 0860, E-mail address: [email protected]. 1 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0991 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Conflict of interest statement Nothing to disclose Data availability: Data from genomic profiles are available in the GEO database (http://www.ncbi.nlm.nih.gov/projects/geo) under accession number GSE99036. 2 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0991 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis (LC) to low- grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multi-layered 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. INTRODUCTION Hepatocellular carcinoma (HCC) classically develops through a multistep process involving a series of pathologic states: liver cirrhotic lesion (LC), dysplastic nodule (DN), early HCC (eHCC), and progressed HCC (pHCC). Liver injury induced by viral hepatitis or other etiologic factors produces a chronic inflammatory milieu leading to the development of LCs. LCs subsequently develop into DNs, which are further classified as low-grade or high-grade DNs (LGDNs or HGDNs, respectively) based on the presence of cytological and architectural atypia. Dysplastic lesions eventually progress to eHCC and pHCC. Molecular alterations accompanying this stepwise pathological sequence have been elucidated to determine definitive markers for eHCC. At present, key cancer-associated pathways, such as Notch, Toll-like receptor, MYC, TGF-β, WNT, and epithelial–mesenchymal-transition (EMT)- related signaling, have been demonstrated as active during hepatocarcinogenesis (1-4). Additionally, high-throughput genome-wide studies have revealed genomic aberrations that occur during stepwise HCC development, including DNA methylation (5,6), DNA copy number changes, mutations [e.g., TP53, CTNNB1, or TERT promoter] (7-9), and transcriptional deregulation (10). Recently, 3 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0991 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. multiomic studies, such as those involving The Cancer Genome Atlas (TCGA), showed have enabled an integrated and systematic view of the genomic aberrations associated with liver cancer, revealing novel biomarkers, as well as therapeutic targets (11-13). Recently, multiomic studies shifted their focus towards precancerous lesions, resulting in the creation of the Pre-Cancer Atlas (PCA), an integrated collection of molecular, structural, and functional maps that provide information regarding tumor initiation and progression (14). The PCA is expected to delineate mechanisms underlying tumor development or progression in order to enhance early detection, risk prediction, and the development of new therapeutic strategies. In this study, we generated multiomic profiling data that demonstrated a molecular landscape of the multi-layered aberrations that occur during stepwise hepatocarcinogenesis from precancerous lesions to malignant development of eHCC and pHCC. By performing integrative analyses, we identified early events that may possibly promote malignant conversion of precancerous lesions, thereby providing novel mechanistic insights into HCC development. These data may facilitate upcoming studies focused on discovering novel biomarkers or therapeutic targets for HCC. MATERIALS AND METHODS Patients and tissue specimens A total of 131 tissue specimens, including those of liver cirrhosis (LC, n = 30), high-grade dysplastic nodule (HGDN, n = 28), early HCC (eHCC, n = 30), and progressed HCC (pHCC, n = 43), resected from 76 patients was obtained (YSHCC, HCC cohorts from Yonsei University). All lesions were classified according to the criteria stipulated by the ‘‘International Consensus Group for Hepatocellular Neoplasia’’ (15). Freshly frozen specimens were obtained from the Liver Cancer Specimen Bank (part of the National Research Bank Program, Korea Science and Engineering Foundation, Ministry of Science and Technology), and subjected to RNA-seq profiling. Tissues of low- grade dysplastic nodule (LGDN) were not included in this study due to limited availability of frozen tissues. Clinico-pathological features of the 76 patients are summarized (Supplementary Table S1). This study was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (4-2014-0423), and the requirement for informed consent was waived. For validation via immunohistochemical staining, a total of 171 liver specimens from 90 patients was examined, which included LC (n = 30), LGDN (n = 30), HGDN (n = 40), eHCC (n = 40), and pHCC (n = 41) specimens. Patients were 34-76 years in age (57.7 ± 8.19, mean ± SD) and consisted of 75 males and 15 females. Most etiologies were HBV (n = 74), and the remainder included HCV (n = 5), 4 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0991 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. alcohol (n = 7), Nonalcoholic steatohepatitis (NASH, n = 1), occult HBV potential (n = 1), or of unknown etiology (n = 2). Clinico-pathological features of the 90 patients subjected to immunostaining are summarized in Supplementary Table S2. RNA-seq profiling and variant profiling RNA-seq profiling was performed in the YSHCC specimens (n = 131), including LC (n = 30), HGDN (n = 28), eHCC (n = 30), and pHCC (n = 43). Sequencing data were obtained using an Illumina HiSeq2000 for 100 bp paired end reads with a