The function of tumor suppressor genes in ovarian cancer: the role of LSAMP

2482 We have investigated the relationship between LSAMP (limbic system-associated membrane protein) expression and human sporadic epithelial ovarian cancer (EOC) to determine whether LSAMP is a candidate tumor suppressor. LSAMP is a member of the IgLON family of immunoglobulin domain-containing glycosylphosphatidylinositol-anchored cell adhesion molecules. The IgLONs comprise LSAMP, OPCML (opioid binding protein cell adhesion molecule-like), HNT (Neurotrimin) and NEGR1/Kilon (neuronal growth regulator 1). IgLONs may function mainly as heterodimers termed Diglons. We previously demonstrated tumor suppressor function for OPCML and also an association of altered IgLON expression, including LSAMP, in EOC. LSAMP is also a candidate tumor suppressor in clear cell renal cell carcinoma. Two alternative exons encoding LSAMP signal peptides have been identified: 1a & 1b. CpG island methylation was detected by methylation-specific PCR (MS-PCR). In ovarian tumors, methylation at exons 1a and 1b was detected in 46% (n=68) and 20% (n=60) of cases, respectively. Normal ovaries were unmethylated (n=13). Somatic methylation at exons 1a and 1b was found in 79% (n=15) and 54% (n=11) of cases, respectively. In cancer cell lines methylation associated with exons 1a and 1b was 90% and 51% (n=84), respectively. Isoform non-specific quantitative RT-PCR in cancer cell lines demonstrated low levels of LSAMP expression, with the exception of SKOV-3 and PEO16. There was no evidence of correlation between methylation status and expression level in the cell lines. In addition, azacytidine treatment of SKOV-3 cells resulted in decreased LSAMP expression, suggesting that a mechanism other than CpG island methylation may influence expression. Stable transfection of SKOV-3 with sense and antisense LSAMP constructs (exon 1b isoform) generated cell lines for functional analyses. This resulted in elevated and reduced levels of LSAMP expression, respectively. The antisense cell lines displayed suppressed cell growth in both in vitro and in vivo (sub-cutaneous and intra-peritoneal) assays. Of two sense cell lines, one displayed enhanced growth in vivo but not in vitro , whereas the other displayed suppressed growth in both assays. The LSAMP sense cell lines show decreased migration towards fibronectin compared to control cells. Our data suggests the involvement of altered LSAMP expression in ovarian carcinogenesis. We present evidence that LSAMP is inactivated in EOC and that methylation is common in ovarian tumors. LSAMP may function as a suppressor of migration towards extracellular matrix components, fibronectin in particular. The evaluation of LSAMP function is complex and may be dependent upon, and indeed may influence or be influenced by, the expression of other IgLONs, including the tumor suppressor OPCML. Further functional studies are ongoing to elucidate the precise role of LSAMP and other IgLONs in EOC.