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
The 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 system
Expression 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
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.