Non-canonical pathways of HIF1-alpha regulation in ovarian cancer: implications for tumor angiogenesis and metastasis.

Abstract

The high rates of mortality associated with epithelial ovarian cancer (EOC) are a direct consequence of its metastatic nature. Activation of angiogenesis is a significant factor in generation of metastases and is contingent upon the cellular response to hypoxia within the tumor microenvironment. Hypoxia-inducible factor 1 (HIF1) is a transcription factor composed of HIF1α and HIF1β subunits and is the master regulator of the hypoxic response. Hypoxia and HIF1 are therefore critical mediators of tumor angiogenesis and metastasis. Regulation of HIF1 is primarily at the level of protein. In normoxia, the HIF1α subunit is hydroxylated via an oxygen- and iron-dependent mechanism and targeted for destruction. In hypoxia, low oxygen levels preclude hydroxylation and HIF1α is stabilized, allowing for its association with constitutively expressed HIF1β to form bioactive HIF1. We have identified two novel mechanisms of HIF1α regulation that are oxygen-responsive in EOC cells (EOCCs). The first involves dynamins, a class of proteins involved in endocytic processes such as transferrin/iron uptake. Exposing EOCCs to hypoxic conditions results in lower levels of dynamin 2. Impairment of dynamin 2 activity in normoxia causes accumulation of HIF1α protein due to a rapid decrease in intracellular iron levels and HIF1α polyubiquitination. Treatment with a form of iron that is not dependent on dynamins for endocytosis reverses this effect. Conversely, overexpression of dynamin 2 in hypoxia results in suppression of HIF1α protein levels. A second novel mechanism of HIF1α control involves microRNAs (miRNAs), ~22 nucleotide, non-coding RNA molecules that repress translation of target mRNAs by binding their 3' untranslated regions (UTRs). Using microarray and qPCR analysis, we found that exposing EOCCs to hypoxia reduced levels of miR-199a-1, a miRNA that is located in an intron within the dynamin 2 gene and is predicted in silico to target the HIF1α 3' UTR. We further demonstrated that miR-199a-1 directly targets the HIF1α 3' UTR and overexpression of miR-199a-1 suppresses HIF1α protein levels and HIF1-driven gene expression. Moreover, cells stably overexpressing miR-199a-1 exhibit marked defects in migratory ability. We corroborated these findings in vivo by overexpressing miR-199a-1 in a mouse model of metastatic EOC and found significant reductions in tumor vessel density and tumor burden. Together, these findings provide insight into non-canonical, dynamin-dependent and miRNA-based mechanisms of HIF1 regulation that may have important implications in the progression of EOC.