Hypoxia Induces Pro-Fibrotic and Fibrosis Marker Genes in Hepatocellular Carcinoma Cells Independently of Inflammatory Stimulation and the NF-κΒ Pathway.

Hypoxia and its key mediators hypoxia inducible Factors (HIFs) are implicated in the development of liver diseases of diverse etiologies, often in interplay with inflammatory mediators. We investigated the interplay between hypoxia and proinflammatory mediators in the development of liver fibrosis, using human hepatocellular carcinoma Huh7 cells as a model. Treatment of Huh7 with DMOG or under hypoxia, induced HIF-1α protein levels and the expression of genes for pro-fibrotic (TGF-ß1, PDGFC, PAI-1) and fibrosis (LOX, P4HA1, P4HB) markers. Knockdown of HIF-1α decreased the induction of PDGFC, LOX and P4HA1, showing the involvement of HIF-1 in their regulation. Interestingly, incubation of Huh7 cells under hypoxia did not cause activation of the NF-κΒ pathway. In contrast, inflammatory mediators such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS) activated the NF-κΒ pathway, but failed to increase HIF-1α protein levels. Moreover, TNFα had a weaker effect than hypoxia on the induction or did not induce pro-fibrotic and fibrosis markers, respectively, while LPS enhanced only the hypoxic induction of P4HB. In conclusion, the above findings suggest that hypoxia and HIF-1 play an important role in the development of fibrosis in hepatocellular carcinoma, which appears to be independent of the activation of the NF-κΒ pathway.

Expression of AGPAT2, an enzyme involved in the glycerophospholipid/triacylglycerol biosynthesis pathway, is directly regulated by HIF-1 and promotes survival and etoposide resistance of cancer cells under hypoxia.

Hypoxia inducible factor-1 (HIF-1) supports survival of normal cells under low oxygen concentration and cancer cells in the hypoxic tumor microenvironment. This involves metabolic reprogramming via upregulation of glycolysis, downregulation of oxidative phosphorylation and, less well documented, effects on lipid metabolism. To investigate the latter, we examined expression of relevant enzymes in cancer cells grown under hypoxia. We show that expression of acylglycerol-3-phosphate acyltransferase 2 (AGPAT2), also known as lysophosphatidic acid acyltransferase ß (LPAATß), was upregulated under hypoxia and this was impaired by siRNA-mediated knockdown of HIF-1α. Moreover, a sequence of the AGPAT2 gene promoter region, containing 6 putative Hypoxia Response Elements (HREs), activated transcription of a reporter gene under hypoxic conditions or in normoxic cells over-expressing HIF-1α. Chromatin immunoprecipitation experiments confirmed binding of HIF-1α to one of these HREs, mutation of which abolished hypoxic activation of the AGPAT2 promoter. Knockdown of AGPAT2 by siRNA reduced lipid droplet accumulation and cell viability under hypoxia and increased cancer cell sensitivity to the chemotherapeutic etoposide. In conclusion, our findings demonstrate that AGPAT2, which is mutated in patients with congenital generalized lipodystrophy and over-expressed in different types of cancer, is a direct transcriptional target of HIF-1, suggesting that upregulation of lipid storage by HIF-1 plays an important role in adaptation and survival of cancer cells under low oxygen conditions.