Abstract
Cancer cells adapt to hypoxia by the stabilization of hypoxia inducible factor (HIF)-α 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-1α, HIF-2α or both isoforms of HIF-α. We determined the metabolic consequences of HIF silencing and the ability of HIF-α silenced cells to invade and degrade the extracellular matrix (ECM) under carefully controlled normoxic and hypoxic conditions. We found that silencing HIF-1α 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-1α or HIF-2α) and double α-isoform silenced cells, with the reduction most evident when both HIF-1α and HIF-2α were silenced in MDA-MB-231 cells. HIF-2α played a major role in altering cell metabolism. Lipids and lipid droplets were significantly reduced in HIF-2α 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-1α alone is not sufficient to attenuate invasiveness, and that both HIF-1α and HIF-2α play a role in the metastatic cascade in these two cell lines.
Highlights
Hypoxia arises as a result of the chaotic vasculature that forms in tumors [1, 2]
Whole cell protein extracts from genetically modified MDA-MB-231 and SUM-149 cells maintained under normoxia, hypoxia or treated with the hypoxia mimetic CoCl2 for 4–5 h were resolved on 7% SDS gel and immunoblotted against hypoxiainducible factor (HIF)-1α or HIF-2α specific antibody
Both HIF-1α and HIF-2α proteins were stabilized under hypoxia in control MDA-MB-231 and SUM149 cells
Summary
Hypoxia arises as a result of the chaotic vasculature that forms in tumors [1, 2]. The adaptive response of cells to low oxygen tension is mediated through the hypoxiainducible factor (HIF) [3]. HIF-α subunits may be stabilized in the absence of hypoxia by growth factor signaling pathways [6] such as activation of RAS and Src oncogenes, mutation in the tumor suppressor PTEN gene resulting in activation of HIF that is mediated through PI3K, and loss or inactivation of the tumor suppressor gene VHL [7]. The expression of both HIF-1α and HIF-2α are increased in www.impactjournals.com/oncotarget several human tumors, including bladder, breast, colon, glial, hepatocellular, ovarian, pancreatic, prostate, and renal tumors [8]. HIF-1α activation has been associated with resistance to endocrine and chemotherapies [12]
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