Abstract 5152: Targeting HIF1α-mediated metabolic alterations in pancreatic cancer

Abstract

Abstract Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths in the United States, with a five-year survival rate of less than 6%. Pancreatic tumors demonstrate high intra-tumoral hypoxia and low oxygen tension, which causes the stabilization of transcription factor Hypoxia-Inducible Factor1α (HIF-1α). HIF1α, in turn, mediates the metabolic alterations that facilitate tumor progression and metastasis. The objective of our study was to investigate the individual roles of HIF factors in mediating metabolic alterations in PC. Our extensive immunohistochemical studies in human pancreatic cancer tissue samples demonstrate an up-regulation of glycolytic enzymes and HIF-1α, in comparison to the normal tissues. The gene up-regulations were even more pronounced in the tumor cells as compared to the stroma. Additionally, we screened a panel of PC cell lines and found that almost all the cell lines demonstrate a significant increase in the uptake of [3H]-2DG, a glucose analogue, under hypoxic conditions. There was also a concurrent hypoxia-induced increase in lactate secretion and in the expression of glycolytic genes, as determined by colorimetric assays and real-time PCR analyses, respectively. To investigate the therapeutic efficacy of targeting HIF factors in pancreatic cancer, we established stable knockdowns for HIF-1α and HIF-2α; in Capan-1, Capan-2, S2-013, CFPAC-1 and T3M4 PC cell lines by utilizing short hairpin RNA technology. We then performed metabolic assays in these cells to investigate the distinct roles of HIF isoforms in the glycolytic phenotype of the PC cells. Our results indicate that knocking down HIF-1α significantly reduces glucose uptake under hypoxic conditions, while HIF-2α knockdown had no effect on glucose uptake. Real-time PCR and immunoblotting analyses demonstrated that knocking down HIF-1α, but not HIF-2α, reduced the levels of GLUT-1, HK-2 and LDHA. NMR-based metabolomics studies also confirmed the effect of digoxin-mediated HIF-1α inhibition on the levels of metabolic intermediates. To confirm our findings in vivo, we performed orthotropic implantation of control and HIF knockdown PC cells in athymic nude mice. Our in vivo glucose uptake, tumor growth, and metastasis studies confirmed our in vitro findings. Furthermore by utilizing a HIF-1α inhibitor, digoxin, we observed similar reduction in glucose uptake, tumor growth and metastasis in orthotopic implantation models. Digoxin treatment also improved survival in tumor bearing mice and a combination therapy with gemcitabine further improved survival synergistically. Overall, our results confirm the therapeutic potential of targeting HIF-1α to improve survival in PC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5152. doi:1538-7445.AM2012-5152