Maspin regulates hypoxia-mediated stimulation of uPA/uPAR complex in invasive breast cancer cells - PubMed (original) (raw)

Comparative Study

Maspin regulates hypoxia-mediated stimulation of uPA/uPAR complex in invasive breast cancer cells

Sumaira Amir et al. Cancer Biol Ther. 2005 Apr.

Abstract

Maspin, a unique serine proteinase inhibitor (serpin), plays a key role in mammary gland development and is silenced during breast cancer progression. Maspin has been shown to inhibit tumor cell motility and invasion in cell culture, as well as growth and metastasis in animal models. In this study, we investigated the effect of maspin on the regulation of hypoxia-induced expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), with respect to invasive potential in metastatic breast cells MDA-MB-231. We hypothesized that maspin can neutralize or mitigate hypoxia-induced expression of uPA/uPAR in metastatic breast cancer cells, resulting in suppression of their invasive potential. To test our hypothesis, we employed the highly invasive MDA-MB-231 breast cancer cells that are devoid of maspin, and transfected them with the maspin gene, and then determined the effect of hypoxia on uPA/uPAR expression. Normal mammary epithelial cells 1436N1 were used as a control. Our findings demonstrate that maspin downregulated the basal and hypoxia-induced uPA/uPAR expression and reduced the stimulatory effect of hypoxia on the in vitro invasive ability of MDA-MB-231-cells. In addition, maspin also inhibited the enzymatic activity of secreted and cell associated uPA in MDA-MB-231 cells. These results indicate that maspin inhibits hypoxia-induced invasion of metastatic breast cancer cells by blocking the uPA system, thus illuminating an important molecular pathway for therapeutic consideration.

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Figures

Figure 1

Figure 1

Effect of maspin on uPA and uPAR expression under hypoxia. MDA-MB-231 (A) and MDA-MB-231-GFP-Maspin (B) were cultured under hypoxia for (0–24 hrs). Total RNA was then isolated and analyzed by RT-PCR using uPAR, uPA and HIF-1α specific primers. 18s rRNA primers were used as control for equal loading. The signals for uPA and uPAR mRNA were quantified by densitometric analysis and represented as the ratio of uPA:18srRNA and uPAR:18s rRNA (C and D). Results shown in the lower panels are representatives of three independent experiments and were performed using Scion image analysis and Sigma plot.

Figure 2

Figure 2

Maspin inhibits the uPAR and uPA expression in MDA-MB- 231-GFP-Maspin cells. Western blot analysis of uPAR and uPA protein in MDA-MB-231 (A) MDA-MB-231-GFP-maspin (B) and normal mammary epithelial cells 1436N1 (C) cultured for (0–24 hrs) under hypoxia. Equal amounts (25 μg) of cytosolic protein was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, transblotted onto nitorcellulose membrane, and probed with anti-HIF-1α, uPAR and uPA monoclonal antibodies. Monoclonal antibody to actin was used as a control for equal loading. The data represents the densitometric analysis of results represented as the ratio of uPA: actin and uPAR:actin (C and F). Results shown in the lower panels were performed using Scion image and Sigma plot are representatives of three to five independent experiments.

Figure 3

Figure 3

Maspin inhibits hypoxia-induced in vitro invasion in human breast cancer cells MDA-MB-231. Equal amounts (50,000) cells/wells were seeded into the upper wells of the MICS chamber containing Mito+ serum-free media, following a 24 hr incubation under either hypoxia or normoxia in the absence or presence of 20 μg/ml anti-uPAR neutralizing antibody. After 24 hrs the cells in the bottom chamber were harvested and counted. The percentage of invasion was determined by counting the number of cells that migrated through a collagen 1V/laminin/gelatin-coated polycarbonate fiter in 24 hr/total number of cells seeded X 100). The Data are expressed as a percentage of normoxic cells and are representatives of 3–4 independent experiments.

Figure 4

Figure 4

Maspin inhibits the activity of cell associated and secreted uPA in MDA-MB-231 and MDA-MB-231-GFP-Maspin cells. UPA activity of (A) cell-associated and secreted (C) in breast cancer cells MDA-MB-231, MDA-MB-231 GFP-maspin and normal mammary epithelial cells 1436N1 under hypoxia at indicated times as determined by zymography. Clear bands at 50–55 kDa represent enzymatic activity of uPA due to the activation of plasminogen. Levels of uPA activity were quantified by densitometry employing scion image analysis and sigma plot (B and D). Results shown in the lower panels are representatives of three independent experiments.

Figure 5

Figure 5

Hypothetical model for the regulation of uPA system by maspin under hypoxia in breast cancer cells. Exposure of cancer cells to hypoxic environment leads to increased secreation of prouPA which then binds to its cellular receptor uPAR. This interaction between uPA-uPAR provides inducible, transient and localized cell surface proteolytic activity, required for tissue invasion, by enhancing the conversion of plasminogen to plasmin. Plasmin can either directly degrade basement ECM or activate other zymogen proteases such as procollagenase, resulting in increase invasive potential of tumor cells. In this model maspin regulates the uPA system by blocking the interaction of uPA with uPAR by binding to uPA and inactivating it. The maspin-uPA complex together with uPAR gets internalized and degraded. Thus, inhibiting migration, invasion and ultimately metastasis by the highly invasive and metastatic breast cancer cells MDA-MB-231.

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