Interplay of hypoxia and cancer stemness via Ephrin-A3/EphA2 axis in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most lethal cancer types with limited therapeutic options. Highly proliferative nature of solid HCC tumors frequently result in intra-tumoral hypoxic microenvironment, with regions of low oxygen concentrations. Although intra-tumoral hypoxia is known to be a key driver of disease progression in HCC, it persists to be a challenge due to the lack of mechanistic understanding of the phenomenon. Our integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia led to the identification of EFNA3 (Ephrin-A3) as a hypoxia- inducible gene. We found it is frequently upregulated in HCC cases. Ephrin-A3 is a ligand which can activate multiple receptor tyrosine kinases (RTKs) of the Eph family. Ephrins and Eph receptors are involved in various developmental processes and are found to be frequently dysregulated in multiple types of cancers. We validated the upregulation of EFNA3 in a larger cohort of our HCC samples (p<0.0001, n=97) and TCGA-LIHC database. Higher expression of EFNA3 correlated with aggressive tumor behaviour with more venous invasion and advanced tumor stage. HIF-1α directly regulated the EFNA3 expression under hypoxia. Moreover, the copy number gain of EFNA3 we observed might co-operate to further overexpress the gene. Functionally, stable knockdown of EFNA3 via short-hairpin RNA interference significantly reduced self-renewal, proliferation and migration of HCC cells. Orthotopic liver xenograft and limiting dilution assay confirmed the promoting role of EFNA3 in tumor incidence, growth and lung metastases in vivo. Mechanistically, we identified EphA2 as a key functional downstream mediator of Ephrin-A3. Knockdown of EPHA2 also resulted in slower proliferation and self-renewal of HCC cells in vitro. Subcutaneous xenotransplantation and limiting dilution assay confirmed that EPHA2 is involved in HCC tumor growth and initiation, respectively, in vivo. Recombinant Ephrin-A3 treatment caused strong induction of EphA2 tyrosine kinase activity in HCC cells and Ephrin-A3/EphA2 forward signaling axis promoted self-renewal in vitro. Limiting dilution assay suggested that this axis enhanced tumor initiation. Whole transcriptomic approach showed that this axis significantly altered the metabolic profile of HCC cells, with SREBP transcriptional activity being one of the most significantly reduced pathways in stable EFNA3/EPHA2 knockdown cells. Since SREBP transcripts regulate de novo lipogenesis and cholesterol synthesis, we validated that KD of EPHA2 significantly reduced intra-cellular lipid levels and cholesterol levels. Ephrin-A3/EphA2 axis caused the maturation of SREBP1 and increase in its target transcript levels. Both SREBP1 and ACLY, a key enzyme involved in the regulation of de novo lipogenesis and cholesterol synthesis, were

confirmed to mediate the self-renewal function of Ephrin-A3/EphA2 axis. Furthermore, ACLY was validated to be significantly associated with the expression of EFNA3, EPHA2 and hypoxia markers in two HCC cohorts. Its expression was also found to be significantly higher in sub-populations enriched with HCC stemness markers i.e. CD13, CD24 and EpCAM in human HCC samples. Taken together, we have identified Ephrin-A3/EphA2 axis as a modulator of de novo lipogenesis and a key contributor of HCC initiation and progression in hypoxic microenvironment. This axis may serve as an effective therapeutic target against HCC.