J Breast Cancer.  2011 Jun;14(2):88-95. 10.4048/jbc.2011.14.2.88.

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

• Choi JY
• Jang YS
• Min SY
• Song JY

• KMID: 2286506
• DOI: http://doi.org/10.4048/jbc.2011.14.2.88

Abstract

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)-1alpha 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-1alpha 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-1alpha and MMP-9 under hypoxic conditions. Luciferase and ChiP assays were performed to demonstrate that MMP-9 promoter activity was regulated by HIF-1alpha.
RESULTS
HIF-1alpha was stabilized under hypoxic conditions and stimulated MMP-9 expression, which affected the tumor invasiveness of breast cancer cells. HIF-1alpha 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-1alpha, and in turn upregulates MMP-9 expression. We also demonstrate that MMP-9 is upregulated concurrently with HIF-1alpha in tumor tissues from patients with breast cancer.
CONCLUSION
These results suggest that HIF-1alpha promotes cell invasion through a MMP-9-dependent mechanism and that future antitumor agents could be used to target HIF-1alpha and MMP-9.

Keyword

Angiogenesis; Breast neoplasms; Hypoxia-inducible factor 1 alpha subunit; Matrix metalloproteinases

MeSH Terms

Anoxia
Antineoplastic Agents
Basement Membrane
Breast
Breast Neoplasms
Extracellular Matrix
Fluorescent Antibody Technique
Gelatinases
GTP Phosphohydrolases
Humans
Luciferases
Matrix Metalloproteinases
Neoplasm Metastasis
Oxygen
Antineoplastic Agents
GTP Phosphohydrolases
Gelatinases
Luciferases
Matrix Metalloproteinases
Oxygen

Figure

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  Figure 1 Hypoxia enhances tumor cell invasion. (A) MDA-MB-231 cells were incubated in transwell chamber under normoxic and hypoxic conditions for 24 hours, and the cells were stained with hematoxyline. (B) Invaded cells were counted in four microscopic fields per well.

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  Figure 2 Hypoxia enhances tumor cell invasion through matrix metalloproteinases (MMPs). MDA-MB-231 cells were incubated under normoxic and hypoxic conditions for 24 hours, and then total RNA was isolated. MMP-1, MMP-2, MMP-3, MMP-9, TIMP-2, and TIMP-3 levels were analyzed using reverse transcription polymerase chain reaction. GAPDH was used as a control.

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  Figure 3 Hypoxia induces matrix metalloproteinase-9 (MMP-9) expression via hypoxia-inducible factor (HIF)-1α induction. (A) MDA-MB-231 cells and NCI-H-1299 cells were incubated in the presence or absence of CoCl2 or desferoxamine (DFO), and total proteins were extracted and analyzed by Western blotting using HIF-1α antibody. MMP-9 was detected in culture medium. The data shown are representative of three independent experiments. (B) MDA-MB-231 cells were transfected with HIF-1α wt DNA construct and incubated in the presence of protease inhibitor, MG132. HIF-1α was detected in cell lysates and MMP-9 was analyzed in culture medium by Western blotting. (C) Zymography assay was used to examine MMP-9 activity. MDA-MB-231 cells were incubated under normoxic or hypoxic conditions for 24 hours and cell culture medium samples were collected. Samples were loaded on denaturing 8% polyacrylamide-SDS-gel containing 0.1% gelatin. Gels were washed in distilled water and were incubated in assay buffer, and then were stained with 0.1% Coomassi Brilliant Blue. Clear band marked in 82 kDa shows the gelatinase activity.

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  Figure 4 Hypoxia induces matrix metalloproteinase-9 (MMP-9) expression via hypoxia-inducible factor (HIF)-1α induction. Immunofluorescence assay was performed to examine expression of HIF-1α and MMP-9 in cellular level by imaging. Each slide was stained with antibodies against HIF-1α or MMP-9, and then incubated with FITC-conjugated rabbit antibody for HIF-1α or with TRITC-conjugated mouse antibody for MMP-9.

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  Figure 5 Ras-GTP induces hypoxia-inducible factor (HIF)-1α stabilization, and consequently leads to matrix metalloproteinase-9 (MMP-9) gene expression. (A) MDA-MB-231 cells were incubated in normoxic or hypoxic conditions for 1 hour (upper panel). Ras activity was measured GTP-Ras bound to GST-Ras-PBD fusion protein (low panel). Cell lysates were prepared and Western blotting was performed using antibodies against phospho-ERK or ERK. Tubulin was used as a loading control. (B) MDA-MB-231 cells were transfected with HIF-1α wt, SiHIF-1α, Ras DN or Ras Ac DNA construct and incubated in the presence or absence of CoCl2. Total protein was prepared, and Western blotting was performed with HIF-1α antibody. MMP-9 was detected in culture medium. Tubulin indicated amount of loading.

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  Figure 6 Hypoxia-inducible factor (HIF)-1α and Ras regulates matrix metalloproteinase-9 (MMP-9) promoter activity. (A) MMP-9 luciferase construct was cotransfected with HIF-1α wt, SiHIF-1α, Ras Ac or Ras DN in MDA-MB-231 cells. After 24 hours, cells were incubated in hypoxic condition or treated with MG132. Bars represent the average luciferase activity of four independent experiments. β-galactosidase assay were used to normalize for transfection efficiencies. NCI-H-1299 cells showed the same results as MDA-MB-231 cells (data not shown). (B) MDA-MB-231 cells were incubated in the normoxic or hypoxic conditions for 6 hours, and chromatin was immunoprecipitated with Rabbit IgG, HIF-1α, p300, acetylated-H3 histone or acetylated-H4 histone antibody. Purified DNA was amplified by semiquantitative polymerase chain reaction (PCR) using MMP-9 primers. Five-fold dilutions of DNA were amplified as an internal control of the PCR.

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  Figure 7 Hypoxia-inducible factor (HIF)-1α plays a role in tumor invasion by inducing matrix metalloproteinase-9 (MMP-9) gene expression in cancer patients. Tissues from patients with breast cancer and corresponding normal tissues were prepared, and lysed. HIF-1α and MMP-9 expression were detected by Western blotting (arrows). N=normal; T=tumor tissue.

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