Analysis of DNA methylation patterns in hepatocellular carcinoma to identify novel therapeutic targets and biomarkers that predict response to therapy

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for over 90% of cases. The treatment outcome for HCC patients is very poor and over 70% of patients present with disease that is incurable by current therapies. Furthermore, current therapies for HCC patients often have low efficacy and significant toxicities. Thus, there is a critical need for the development of novel therapeutic approaches and the optimisation of currently used therapies to improve the outcomes for HCC patients. We developed a novel bioinformatics pipeline, which integrates genome-wide DNA methylation and gene expression data, to identify genes required for the survival of cancer cells but not normal cells. Targeting these genes may induce "synthetic lethality" (SL), specifically killing cancer cells with little or no impact on healthy ones. Based on global DNA methylation patterns, five potential HCC subgroups were identified. Subgroup 2 exhibited the most unique methylation profile and was utilised for SL gene analysis. This identified two candidate SL genes, T-lymphoma invasion and metastasis 1 (TIAM1) and lactate dehydrogenase B (LDHB). Available HCC cell lines were characterised for their global methylation patterns and expression of TIAM1 and LDHB. Analysis releveled SNU182 and PLC/PRF-5 as potential HCC subgroup 2 representative cell lines (positive expression and hypomethylation of TIAM1 and LDHB) and HepG2 and Huh-7 (negative expression and hypermethylation of TIAM1 and LDHB) as non-subgroup 2 cell lines. TIAM1 belongs to a family of guanine nucleotide exchange factors (GEFs) known to activate RAC1 signalling and plays a role in cancer cell growth, adhesion, and invasion. The HCC cell lines as well as additional non-HCC liver cell lines positive for TIAM1 and LDHB SK-Hep1 (cholangiocarcinoma) and HHL5 (immortalised hepatocytes) were used to investigate the functional relevance of TIAM1 utilising siRNA silencing and a specific TIAM1/Rac1 signalling inhibitor (NSC23766). siRNA targeting TIAM1 inhibited cell proliferation in TIAM1 positive (subgroup 2) HCC cell lines but had no effect on TIAM1 negative cell lines and cell proliferation was also suppressed at significantly lower NSC23766 concentrations in the TIAM1 positive compared with the TIAM1 negative HCC lines. Thus, confirming TIAM1 as a potential SL gene for HCC subgroup 2. LDHB is a metabolic gene that encodes the B subunit of the lactate dehydrogenase enzyme, which catalyses the interconversion of pyruvate and lactate. Since the HCC subgroup 2 representative cell lines exhibit subgroup specific expression of LDHB, we tested their sensitivity to metabolic inhibitors (2-DG and metformin) and GNE-140 (LDH inhibitor). The result showed that the sensitivity of 2-DG and metformin was HCC subgroup 2 independent. In addition, the sensitivity to the LDH inhibitor GNE-140 did not correlate with LDHB expression status. Radiotherapy is a common treatment choice for cancer patients. The development of radioresistance is common in numerous cancer types including HCC, but metabolic interventions have shown promise as radiosensitisers. Hence, we evaluated radiation synergy with metabolic inhibitors (2-DG and metformin) and its potential correlation with the LDHB status of the cells. No radiosensitisation effect for either of the inhibitor individually or in combination was observed in any of the HCC cell lines tested. However, potential synergy between 2-DG and metformin was observed in some cell lines independent of their LDHB status. Overall, the study provides evidence towards exploiting TIAM1 as a potential therapeutic target for a subgroup of HCC. In addition, a combination of 2-DG and metformin should be explored further as a potential treatment strategy for HCC.