Genetic and Epigenetic Mechanisms Deregulate the CRL2pVHL Complex in Hepatocellular Carcinoma

Abstract

Dysregulation of ubiquitin-proteasome pathway genes through copy number alteration, promoter hypomethylation, and miRNA deregulation is involved in cancer development and progression. Further characterizing alterations in these genes may uncover novel drug targets across a range of diseases in which druggable alterations are uncommon, including hepatocellular carcinoma (HCC). We analyzed 377 HCC and 59 adjacent non-malignant liver tissue samples, focusing on alterations to component genes of the widely studied CRL2^pVHL E3 ubiquitin ligase complex. mRNA upregulation of the component genes was common, and was correlated with DNA hypomethylation and copy number increase, but many tumours displayed overexpression that was not explained by either mechanism. Interestingly, we found 66 miRNAs, including 39 previously unannotated miRNAs, that were downregulated in HCC and predicted to target one or more CRL2^pVHL components. Several miRNAs, including hsa-miR-101-3p and hsa-miR-139-5p, were negatively correlated with multiple component genes, suggesting that miRNA deregulation may contribute to CRL2^pVHL overexpression. Combining miRNA and mRNA expression, DNA copy number, and methylation status into one multidimensional survival analysis, we found a significant association between greater numbers of alterations and poorer overall survival for multiple component genes. While the intricacies of CRL2^pVHL complex gene regulation require additional research, it is evident that multiple causes for the deregulation of these genes must be considered in HCC, including non-traditional mechanisms.

Keywords: DNA copy number; DNA hypomethylation; liver cancer; novel microRNAs; ubiquitin-proteasome.

FIGURE 1

Various combinations of mRNA upregulation, copy number increase, and hypomethylation alterations are common in CRL2^pVHL component genes in HCC. (A) The bar graph indicates the prevalence of alterations to the CRL2^pVHL component genes in TCGA-LIHC tumours for which complete data were available (n = 369). CN (copy number) increase includes both genomic gain and amplification. (B) The Venn diagrams display the numbers of HCC samples that harbor overlapping and/or unique alterations in each CRL2^pVHL component gene.

FIGURE 2

Overexpression of CRL2^pVHL component genes is linked to worse overall survival. (A–E) Kaplan-Meier plots indicating the overall survival over time of TCGA-LIHC patients (n = 365), stratified by the presence of mRNA overexpression (OE) for each CRL2^pVHL component gene. An mRNA was deemed overexpressed in a given HCC sample if its expression exceeded the upper Tukey fence for that mRNA in the non-malignant samples. Plots are cut off at 2,000 days, and the 5-year mark is denoted by a dotted line.

FIGURE 3

mRNA upregulation of CRL2^pVHL component genes is correlated with copy number increase, hypomethylation, and miRNA downregulation. (A) Comparisons of mRNA expression of CRL2^pVHL component genes between non-malignant samples and HCC samples, where the latter are stratified by the presence of CN (copy number) increase. Dashed lines represent the upper Tukey fence of the non-malignant group, and were considered the threshold of overexpression; (B) Comparison of component gene methylation between non-malignant samples and HCC samples, where the latter are stratified by the presence of gene overexpression. Dashed lines represent the lower Tukey fence of the non-malignant group, and were considered the threshold of hypomethylation. **p < 0.01; ***p < 0.001; (C) Examples of negative correlations between the mRNA expression of component genes and the expression of HCC-downregulated annotated miRNAs that were predicted to target at least one component gene.

FIGURE 4

CRL2^pVHL component gene alterations impact patient outcomes. (A,B) Kaplan-Meier plots indicating the overall survival over time of TCGA-LIHC patients, stratified by the number of alterations each had affecting the specified gene (n = 359). Groups containing five or fewer patients were combined with the nearest larger group; (C) Kaplan-Meier plot indicating the overall survival over time of TCGA-LIHC patients (n = 365), divided between patients with both ELOC hypomethylation (HM) and mRNA overexpression (OE), and all other patients; (D) Kaplan-Meier plot indicating the overall survival over time of TCGA-LIHC patients (n = 365), stratified by the number of CRL2^pVHL component genes with mRNA overexpression. For all panels, plots are cut off at 2000 days and the 5-year mark is indicated by a dotted line.