Down-regulation of CD9 in Human Ovarian Carcinoma Cell Might Contribute to Peritoneal Dissemination: Morphologic Alteration and Reduced Expression of β1 Integrin Subsets

AbstractPeritoneal dissemination is one of the main causes of death in cancer patients. Pathophysiology of metastasis has been well investigated, but the mechanism of diffuse spread of tumor colonies in the peritoneal cavity is not fully understood. CD9 is a member of tetraspanin and its down-regulation is known to be involved in poor prognosis. To investigate the significance of the down-regulation of CD9, HTOA, an ovarian carcinoma cell line that highly expressed CD9, was transiently transfected with small interfering RNA (siRNA) against CD9, and CD9-negative cells (HTOACD9−) were purified. HTOACD9− showed altered adhesion patterns on Matrigel, collagen, fibronectin, and laminin compared with those of control siRNA–transfected HTOA (control-HTOA). Flow cytometry and fluorescence cytostainings revealed that the expression levels of integrins β1, α2, α3β1, α5, and α6 were lower in HTOACD9− than those of control-HTOA. HTOACD9− showed altered expression of junctional and cytoskeletal molecules. By time-lapse video microscopy, control-HTOA showed solid adhesion to extracellular matrix and formed cobblestone pattern, whereas HTOACD9− showed weaker adhesion and were distributed as diffuse spots. To examine whether the expression level of CD9 change during tumor dissemination, HTOA-P, a highly disseminative subclone of HTOA, was established. HTOA-P showed distinctive down-regulation of CD9 at mRNA and protein levels, and showed similar morphologic alteration as HTOACD9− did. These findings indicate that the down-regulation of CD9 may be an acquired event in the process of tumor dissemination. Down-regulated CD9 may attenuate the expression of several integrins and rearrange junctional and cytoskeletal molecules that might contribute to dissemination of ovarian carcinomas.