Cancer cells adapt to hypoxia by the stabilization of hypoxia inducible factor (HIF)-a isoforms that increase the transcription of several genes. Among the genes regulated by HIF are enzymes that play a role in invasion, metastasis and metabolism. We engineered triple (estrogen receptor/progesterone receptor/HER2/neu) negative, invasive MDA-MB-231 and SUM149 human breast cancer cells to silence the expression of HIF-1a, HIF-2a or both isoforms of HIF-a. We determined the metabolic consequences of HIF silencing and the ability of HIF-a silenced cells to invade and degrade the extracellular matrix (ECM) under carefully controlled normoxic and hypoxic conditions. We found that silencing HIF-1a alone was not sufficient to attenuate invasiveness in both MDA-MB-231 and SUM149 cell lines. Significantly reduced metastatic burden was observed in single (HIF-1a or HI -2a) and double a-isoform silenced cells, with the reduction most evident when both HIF-1a and HIF 2a were silenced in MDA-MB-231 cells. HIF-2a played a major role in altering cell metabolism. Lipids and lipid droplets were significantly reduced in HIF-2a and double silenced MDA-MB-231 and SUM149 cells, implicating HIF in their regulation. In addition, lactate production and glucose consumption were reduced. These results suggest that in vivo, cells in or near hypoxic regions are likely to be more invasive. The data indicate that targeting HIF-1a alone is not sufficient to attenuate invasiveness, and that both HIF-1a and HIF-2a play a role in the metastatic cascade in these two cell lines.
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