Activation of the PKC Pathway Stimulates Ovarian Cancer cell Proliferation, Migration, and Expression of MMP7 and MMP10.

Abstract

Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as LH. Gonadotropins activate the PKA and PKC pathways which are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of hCG, a LH mimic, and PMA (Phorbol-12-Myristate 13-Acetate), an activator of the PKC pathway, on ovarian cancer cell proliferation, apoptosis and cell cycle kinetics in Ovcar3 cells. Cells were serum starved for 24 hrs, treated with or without PMA (20 nM) or hCG (1 IU) for varying times, and then cells were collected. Our flow cytometry data shows that PMA increased the number of cells in the S phase of the cell cycle and decreased cells in the G0/G1 phase after 24 hrs. In order to determine whether the changes in cell cycle seen in Ovcar3 cells at 24 hr were translated into changes in cell proliferation, MTS and BrdU assays were performed. When Ovcar3 cells were treated with increasing concentrations of PMA (0, 0.2, 2 or 20 nM) or hCG (1 IU) for 24 hr, no changes in proliferation were observed. To explore the possibility that the changes in cell cycle at 24 hr were manifest in cell proliferation after 24 hr, cells were treated for 48 hrs. Administration of PMA (20 nM) for 48 hr resulted in an increase in Ovcar3 cell proliferation when analyzed by both the MTS and BrdU assays. To investigate the effects of PMA and hCG on ovarian cancer cell apoptosis, we utilized FACS analysis with an annexin V assay. There was an increase in apoptotic cells after 4 hr of treatment with PMA. However, after 8 hr, PMA treatment led to the presence of less apoptotic cells whereas no changes in apoptosis were observed after 12 hr. The PKC pathway is known to differentially regulate the expression of matrix metalloproteinases (MMPs) which have an active role in cancer metastasis. PMA treatment of Ovcar3 cells increased MMP7 and MMP10 mRNA levels after 8 hr of treatment and expression remained high after 12 hr before decreasing at 24 hr The expression of MMP2, MMP9, TIMP1 and TIMP3 was not altered. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of MMP induction on ovarian cancer cell migration using transwell assays. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells after 24 hrs. The MMP2/9 inhibitor in contrast, had no effect on ovarian cancer migration. Treatment of ovarian cancer cells with GM6001 did not cause a decrease in proliferation of ovarian cancer cells indicating that the inhibitory effect of GM6001 on migration is not due to a decrease in cell proliferation. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics, proliferation and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration. Supported by NIH P20 RR15592, NIH HD057446.