Activation of MMP-2 by endometrial cancer cells in vitro is enhanced by LPA

Abstract

16047 Background: Matrix metalloproteinases (MMPs) are responsible for the breakdown of extracellular matrix components, and are important in the metastatic phenotype. HEC-1A is an endometrial cancer cell line, derived from a moderately-differentiated tumor. These cells do not produce MMP-2, an important mediator of invasive behavior. We sought to: evaluate if HEC-1A cells have the capacity to activate proMMP-2 if given the proenzyme exogenously; and determine if the addition of lysophosphatidic acid (LPA), a known activator of MMPs in other cancers, would further enhance this conversion. Methods: HEC-1A cells were maintained per known protocols. Conditioned media from HT1080 chondrosarcoma cells, a source of proMMP-2, was added to HEC-1A cells. Cells were also treated with 0.1, 1.0 and 10uM LPA. Conditioned media from the cells was analyzed for the presence of MMP-2 and its proenzyme utilizing gelatin zymography. Cellular invasion through collagen matrix was quantified using a modified Boyden chamber assay. Migration was evaluated using a wound healing assay with quantification of wound closure at 48 hours. Proliferation was assessed following cell culture for 48 hours using an MTS tetrazolium salt/phenazine methosulfate assay. Results: HEC-1A cells activated exogenous proMMP-2 to MMP-2, as confirmed by gelatin zymography. This occurred in both the presence and absence of LPA. Invasion of HEC-1A cells treated with proMMP-2 was significantly increased as compared to controls (p < .001). LPA increased invasion in cells with and without exogenous proMMP-2, with the most invasive cells being those treated with both LPA and exogenous proMMP-2. The addition of both exogenous MMP-2 and LPA significantly increased cellular migration as quantified using the wound healing assay (p < .001). Cellular proliferation was significantly increased with LPA vs. controls. The addition of proMMP-2 did not lead to a significant difference in proliferation. Conclusions: HEC-1A cells can convert exogenous proMMP-2 to its active form, and in doing so exhibit a more invasive phenotype. LPA serves to further increase these behaviors. This may reflect the tumor microenvironment, with endometrial cancer cells responding to bioactive lipids and proenzymes secreted by the surrounding stroma. No significant financial relationships to disclose.