Exploring the heterogeneity of hypoxia response and investigating the role of synonymous mutations in hepatocellular carcinoma : a multi-omics analysis

Abstract

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, arises from a complex interaction of multiple factors including hypoxia, genomic mutations, chronic viral hepatitis, long-standing liver diseases, excessive alcohol consumption, immune suppression, and among others. Notably, these risk factors often act synergistically, and individuals with multiple risk factors are significantly more likely to develop HCC.

Hypoxia, a condition characterized by a non-physiological low level of oxygen resulting from rapid cell proliferation outpacing the growth of blood vessels supplying oxygen and nutrients, triggers the activation of various cellular responses. These responses are primarily regulated by Hypoxia-Inducible Factors (HIFs). Hypoxia stabilizes HIFs, which act as transcription factors, inducing gene expression by binding to the hypoxia-responsive element (HRE) sequence. Additionally, hypoxia can promote genetic instability, fostering the selection of more aggressive tumor clones, thereby contributing to HCC progression and poor prognosis. Intriguingly, we discovered that hypoxia induces different subsets of genes across various HCC cell lines. Nonetheless, the causes and consequences of this heterogeneous response to hypoxia remain unclear.

Through integrated-omics studies, we demonstrated that diverse hypoxia responses may be shaped by the epigenetic landscape, particularly DNA methylation, which modulates the binding of HIFs to the promoter HRE sequence. Utilizing bulk RNA-Seq data in TCGA and single-cell RNA-Seq data, we unveiled the intertumoral and intratumoral heterogeneity of hypoxia response in human liver cancer. Moreover, we discovered that the heterogeneity of hypoxia response might affect the immune microenvironment. A subset of immune regulators was differentially induced by hypoxia in different tumors, and their expressions were inversely correlated with tumor CD8 T cell infiltration. This suggests a possible impact of the heterogeneity of hypoxia response on the tumor immune microenvironment.

Meanwhile, in the intricate landscape of tumorigenesis, genomic variants play a crucial role in tumor development, progression, and therapeutic response. In HCC, the impact of missense mutations in cancer-related genes such as TP53 and CTNNB1 has been extensively researched. However, the influence of synonymous mutations remains largely unexplored. Our research systematically identified the synonymous mutations in HCC patients and found that the biological processes influenced by these mutations significantly differ from those affected by other types of mutations in tumors. These processes were implicated in nucleosome and chromatin assembly and organization. Moreover, we investigated the characteristics of synonymous mutations and their changes at base and codon levels. Coupled with tRNA expression and codon usage bias, the synonymous mutations of cgG/cgT for arginine promoted tumor progression by regulating protein translation efficiency through ECM-receptor interaction and TGF-beta signaling pathways.

In summary, our findings reveal the heterogeneity of hypoxia response in HCC and its effect on the tumor immune microenvironment, and elucidate the roles of synonymous mutations in HCC. These studies enhance our understanding of the effects of heterogeneous hypoxia response and synonymous mutations on tumor development in HCC, offering potential as a biomarker to guide immunotherapy, and as drug targets for developing novel and more effective therapeutic strategies.