We previously identified OPCML (OBCAM) from 11q25 as a tumor suppressor gene frequently inactivated in sporadic epithelial ovarian cancer (EOC). OPCML is a member of the IgLON family of immunoglobulin domain-containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules. OPCML transfection into SKOV-3 ovarian cancer cells resulted in phenotypes consistent with tumor suppressor and cell adhesion molecule function: growth suppression in vitro and in vivo, and enhanced in vitro cell-cell aggregation. A somatic mis-sense mutation, P95R, resulted in loss of OPCML-enhanced cell-cell aggregation function, whereas growth suppression remained unaffected. Neither the signalling pathway nor the downstream transcriptional consequences associated with OPCML function have been described. Therefore, we have used a microarray approach to determine the downstream transcriptional consequences associated with OPCML function in SKOV-3 cells. We used the Cancer Research UK Microarray Consortium 10K cDNA array to compare the gene expression profile of the OPCML-transfected cell line with the SKOV-3 clonal parent cell line, pcDNA3.1zeo-, OPCML antisense-, and OPCML P95R-transfected SKOV-3 cell lines. GeneSpring analysis identified genes with >2-fold expression changes associated with OPCML function. Only characterised genes were considered for further analysis. Quantitative RT-PCR validation of expression differences was performed using Rotorgene real-time thermal cyclers with SYBR Green incorporation. The relative quantification method was employed by extrapolation from a standard curve and calculation of expression levels as ratios to beta actin. Thirty-four genes were identified with differential expression associated with OPCML function in SKOV-3. In keeping with the phenotypes being analysed genes with roles in cell adhesion (e.g. Cadherin 6, Galectin 1) and growth suppression (e.g. Gelsolin) were identified. Thirteen genes displayed reduced expression with wild type OPCML function. Of these, twelve genes were analysed by quantitative RT-PCR and altered expression verified in six. Sixteen genes displayed increased expression associated with wild type OPCML function and altered expression was subsequently confirmed in seven genes. Three genes displayed reduced expression with P95R mutant OPCML function and altered expression verified in two genes. Two genes displayed increased expression associated with P95R mutant OPCML function and altered expression was confirmed in both genes. Confirmation of the changes in expression of selected genes at a protein level by Western blotting is currently underway. Future work will aim to determine the functional significance of expression of the genes identified.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]