Are CCN1 (Cysteine Rich 61-Connective Tissue Growth Factor-Nephroblastoma Overexpressed) and Integrin Receptor Subunits Expressed by KGN, an Ovarian Granulosa Tumor Cell Line?

Abstract

Tumors express a variety of pro-angiogenic factors that contribute to tumor angiogenesis and cancer progression including CCN1, vascular endothelial growth factor (VEGFa), basic fibroblast growth factor (FGF2) and matrix metalloproteinases (MMPs). Interestingly, healthy tissues undergoing physiological angiogenesis, such as those comprising the female reproductive tract, also express VEGFa, FGF2 and MMPs. We have recently reported, for the first time, that the angiogenic inducer, CCN1 (CYR61), is expressed by granulosa and theca cells of normal bovine ovarian follicles. Although it has been shown that normal ovarian surface epithelial cell lines and several epithelial ovarian carcinoma cell lines express CCN1, it is not known whether or not CCN1 plays a role in the progression of human granulosa carcinomas, a malignant ovarian sex cord tumor originating from cells that surround the oocyte. These carcinomas represent between 1-5% of all ovarian cancer diagnoses. The objective of the present study was to determine the expression of CCN1, the a and β integrin subunits that form the heterodimeric receptors for CCN1 and MMP activity, in a model of human ovarian granulosa carcinoma. KGN, a steroidogenic human granulosa-like tumor cell line, was cultured to near confluence and then maintained for 24 hours with or without 10% fetal bovine serum (FBS) and in the absence or presence of testosterone (T; 10 uM). RNA and protein were extracted for analysis of CCN1 and integrin subunit expression, and conditioned medium was collected for analysis of MMPs by gelatin zymography. Our results showed baseline expression of CCN1 mRNA in KGN cells regardless of treatment while FBS increased expression of CCN1 protein. There was, however, no effect of T on either CCN1 mRNA or protein expression. A PCR array to screen for the presence of specific integrin receptor subunits was performed and revealed that KGN cells express integrin a1, a3, a5, aV, β1 and β5 mRNA. Gelatin zymography of KGN condition medium revealed an intense band (approximately 74 kD) and a weak band (approximately 92 kD), corresponding to MMP2 and MMP9 family members, respectively. A higher molecular mass doublet (approximately 128 and 123 kD), consistent with MMP9/neutrophil gelatinase-associated lipocalin (NGAL; Lipocalin 2), was also detected. In summary, we have determined that in addition to possessing aromatase activity and the ability to produce VEGFa, KGN cells also express CCN1, integrin a and β subunits and MMPs, factors that are known to contribute to tumor angiogenesis and cancer progression. Future studies are aimed at elucidating the molecular mechanisms of CCN1 action on the progression of granulosa tumor cells, including how it interacts with specific integrin receptors and other angiogenic factors such as VEGFa, to influence granulosa cell cancer progression. (Supported by NIH grant P01 CA045548 and the Stuart Weitzman Foundation to MAM, the Multistate Regional Project NE1027 to PCWT and the Department of Veterans Affairs to JSD.)