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.
ARTICLE ABSTRACT
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.
Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.