Overexpression of MMP-9 and HIF-1α in Breast Cancer Cells under Hypoxic Conditions

Purpose: Hypoxia, which is a loss of oxygen in tissues, is a common condition in solid tumors due to the tumor outgrowing existing vasculature. Under hypoxic conditions, hypoxia-inducible factor (HIF)-1 alpha rapidly accumulates and transactivates hundreds of genes, such as matrix metalloproteinases (MMPs). MMPs contribute to invasion and metastasis of tumor cells by degrading the surrounding basement membrane and extracellular matrix barriers, which enables the easy migration and spread of cancer cells. We examined whether hypoxia increases tumor cell invasion, and whether increased invasiveness was due to HIF-1 alpha and MMP-9 expression. Methods: Transwell invasion assays were performed to demonstrate whether hypoxia enhance tumor invasion by use of MDA-MB-231 breast cancer cells. An immunofluorescence assay was used to demonstrate expression of HIF-1 alpha and MMP-9 under hypoxic conditions. Luciferase and ChiP assays were performed to demonstrate that MMP-9 promoter activity was regulated by HIF-1 alpha. Results: HIF-1 alpha was stabilized under hypoxic conditions and stimulated MMP-9 expression, which affected the tumor invasiveness of breast cancer cells. HIF-1 alpha transactivated the MMP-9 promoter by forming a transcriptional unit with p300, thus increasing expression of MMP-9 transcripts. Zymography indicated that MMP-9 had more gelatinase activity under hypoxic conditions than normoxic conditions. Furthermore, the small GTPase Ras was also activated in response to hypoxia, which then aids stabilization of HIF-1 alpha, and in turn upregulates MMP-9 expression. We also demonstrate that MMP-9 is upregulated concurrently with HIF-1 alpha in tumor tissues from patients with breast cancer. Conclusion: These results suggest that HIF-1 alpha promotes cell invasion through a MMP-9-dependent mechanism and that future antitumor agents could be used to target HIF-1 alpha and MMP-9.