RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells.

This study was undertaken to reveal the mechanisms by which RLIP76 regulates endothelial cell angiogenic responses. RLIP76 is an effector of the angiogenic modulator, R-Ras. RLIP76 is overexpressed in many tumors, required for tumor angiogenesis, and blockade of RLIP76 results in tumor regression in multiple models. We report here that RLIP76 was required for expression and secretion of vascular endothelial growth factor (VEGF) in carcinoma and melanoma cells. Conditioned medium derived from RLIP76-depleted tumor cells, but not control knockdown cells, could not stimulate proliferation, migration, or Matrigel cord formation in endothelial cell cultures, which indicates that RLIP76 regulates angiogenic components of the tumor cell secretome. Recombinant VEGF added to conditioned medium from RLIP76-knockdown tumor cells restored these endothelial cell functions. Transcriptional activity of hypoxia-inducible factor 1 (HIF-1), which drives VEGF expression, was blocked in RLIP76-depleted tumor cells. RLIP76 was required for PI3-kinase activation, known to regulate HIF-1, in these cells. However, HIF-1α expression and nuclear localization were unaffected by RLIP76 knockdown, which suggests that RLIP76 regulates HIF-1 at the functional level. Thus, RLIP76 regulates tumor cell transactivation of endothelial cells via control of VEGF expression and secretion, providing a new important link in the mechanism of tumor cell induction of angiogenesis.