Studies of the ovarian tumour suppressor protein OPCML with a view to determining its structure and understanding its role in modulating RTK signalling

• Main Author: Zanini, Elisa
• Other Authors: Chayen, Naomi ; Mckie, Arthur
• Published: Imperial College London 2013
• Subjects: 616.99
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.693956

The opioid-binding cell adhesion molecule (OPCML) is a glycosylphosphatidylinositol (GPI) anchored protein that was found to be epigenetically inactivated in 83% of ovarian cancers and demonstrated to act as a tumour suppressor gene (TSG), inhibiting tumour cell growth in vitro and severely attenuating tumourigenesis in vivo (Sellar et al., 2003). We subsequently showed that OPCML exerted its suppressor functions by negatively regulating a spectrum of receptor tyrosine kinases (RTKs), such as ErbB2/HER2, FGFR1 and EphA2, attenuating their downstream signalling (McKie et al., 2012). Initial in vitro interaction studies revealed that OPCML physically interacted with this defined group of RTKs as a prerequisite for their downregulation. The aims of this PhD project were to define key interacting regions between OPCML and the extracellular domains of the target RTKs, to work towards elucidating the structure of OPCML and to investigate the therapeutic potential of a recombinant form of OPCML. Here it is described how a comprehensive panel of OPCML constructs was created and how these constructs were utilised for GST- and His-tagged OPCML protein production in E.coli (BL21(DE3)) and insect cells (Sf21). Selected His-tag constructs were used for expression and purification of the protein to attempt initial crystallisation studies. Tagged OPCML proteins were utilised to investigate RTK interacting partners of OPCML from members of the EGFR and FGFR families by pull-down assays initially. We then developed in-house mammalian 2-hybrid expertise in order to investigate in detail the interacting regions between OPCML and members of the EGFR, FGFR and Eph families, identifying regions of the receptors critical for the interaction with OPCML upon creating deletion constructs of their extracellular domain. We identified the importance of the distal Ig domain of OPCML to RTK interaction by virtue of our analysis of a previously identified somatic missense mutation in a primary ovarian tumour, a C-G transversion at nucleotide position 334 resulting in amino acid substitution (P95R) within the first Ig domain (Sellar et al 2003). Expression of the OPCMLP95R elicits clear disruption of cell adhesion phenotypes, in addition to disrupting the interaction of OPCML with EphA2. The FGFR1 interaction with OPCML has appeared more robust compared to the other RTK examples studied and a more precise deletion of specific FGFR1 amino acid motifs is required for loss of interaction to be observed. A His-tagged OPCML was used for the treatment of cancer cells in vitro and of two intraperitoneal models in vivo, demonstrating that exogenous administration of the recombinant protein caused profound anti-tumorigenic effects in several ovarian cancer cell lines as well as in the intraperitoneal models of EOC in mice.