OPCML and the IgLON family: expression, function and links with ovarian cancer

Abstract

The IgLONs are an immunoglobulin subfamily of glycosylphosphatidylinositol-anchored cell adhesion molecules, comprising four members: OPCML, HNT, LSAMP and NEGRI. They have been mainly studied in the brain of rat and chick, where they affect cell adhesion and cell-cell recognition. Both homophilic and heterophilic interactions are thought to be important in facilitating IgLON functions.Human OPCML has been proposed as a novel tumour suppressor gene (TSG) in sporadic epithelial ovarian cancer (EOC). EOC, the leading cause of death from gynaecological malignancy, arises in the monolayer of cells overlying the ovary called the ovarian surface epithelium. OPCML is normally expressed in these cells, but is epigenetically silenced in EOC. Moreover, it has functional features typical of a TSG: suppression of cell growth in vitro and suppression of tumourigenicity in vivoThe deficiency in our knowledge of the functions of OPCML beyond the locality of the brain renders the understanding of its connexion to ovarian cancer insufficient. The relevance of the other IgLON family members in this type of cancer has not been investigated, even though there is much speculation about their interactions. Addressing these two key issues has been the main objective in the work that is presented in this thesis.In order to study the functions of OPCML, two transfected cell line resources were used: an over-expression system based on SKOV-3 ovarian cancer cells and an inducible expression system based on HeLa cervical cancer cells. Upon induction with doxycycline, selected HeLa inducible clones were shown to demonstrate regulated expression of OPCML, both at the RNA and protein levels; however, low-level uninduced expression was also detected, a feature commonly associated with this type of systemExpression studies of OPCML and the other IgLON family members were undertaken in cancer cell lines, as well as normal mouse and human tissues. Expression analysis of the IgLONs in the two OPCML-transfected cell line systems identified a transcriptional effect of OPCML on two other IgLONs in SKOV-3 cells only: expression of OPCML reduces the expression of both LSAMP and NEGRI at the RNA level. In the mouse and human, the expression profile of the family was established in panels of multiple-tissue cDNAs, where similarities but also differences among different IgLONs were highlighted. Immunohistochemical studies of OPCML were used to profile expression developmentally and in adult tissues. A comparison between IgLON RNA levels in human normal ovaries and a panel of ovarian tumours has pointed to significantly reduced levels of OPCML, LSAMP and NEGRI in specific histological subtypes of ovarian cancer; HNT expression, on the other hand, was significantly elevated. This study has revealed the importance of the IgLON family as a whole in EOC.Various assays were undertaken in the two transfected cell line systems in order to suggest potential functions of OPCML. In SKOV-3 cells, OPCML was shown to significantly decrease chemotactic migration and increase adhesion to fibronectin and vitronectin. Moreover, OPCML was found to promote cell-to-cell adhesion. The growth suppression effect of OPCML in vitro was reproduced, and the underlying mechanism was investigated. This effect is not accounted for by a difference in proliferation; on the contrary, OPCML was shown to significantly increase apoptosis. Expression of OPCML in the non-ovarian HeLa inducible cells did not recapitulate the phenotypic features identified in the ovarian SKOV-3 cells, indicating the importance of context specificity.In summary, IgLON expression profiling has yielded valuable observations; most significantly, it demonstrated a link between the family as a whole and EOC. One of the family's members, OPCML, has functional features that fit the role of a TSG in SKOV-3 cells, in particular a pro-apoptotic role. In the future, a SKOV-3 inducible system will offer a refined method to further study its functions and connexion to ovarian cancer.