Role of the Nance Horan Syndrome protein family in breast cancer invasion

Mesenchymal cell migration is dependent on polarised polymerisation of actin in the direction of movement.Actin polymerisation preferentially occurs at the leading edge of the migrating cell resulting in the formation of broad, flat protrusions known as lamellipodia (1).Cell migration is also dependent on establishment and maintenance of a defined cellular polarity; i.e. a leading edge and a trailing edge(2).We hypothesise that this may be achieved, in part, through ligand-driven endocytosis and endosomal trafficking of receptors (e.g. Tyrosine kinase receptors, G-protein coupled receptors) thatdirect migration.In support of this, there is evidence in the literature for a role of both clathrin-dependent and clathrin-independent endocytosis pathways in migration (3-13).Fast-endophilin mediated endocytosis (FEME) is one pathway which may regulate cellular polarity during migration.FEME is a clathrin-independent mechanism of endocytosis that is triggered at the leading edge of cells in response to ligand binding(14, 15).This process involves the activity of the Endophilin A (hereby referredto as Endophilin) proteins, a subfamily of BAR domain containing proteins (14).The enriched localisation of FEME to the leading edge of the cellindicatesa role for this pathway in cell migration. In support of this, knockdown of endophilin, in both epithelial and endothelial cell lines, results in impaired migration of these cells(14, 16).Furthermore, a number of the components of the FEME priming complex, including Lpd, CIP4, FBP7 and SHIP2, have been shown to promote invasion and metastasis of breast cancer(17-20). These findings may implicate a role for FEME in breast cancercell migration and invasion.A classification of breast cancer into 10 molecular subtypes has highlighted Nance Horan Syndrome-like 2 (NHSL2) as the 5th most highly mutated gene in subtype 2 and the only gene in this subtype where mutation frequency shows a subtype-specific association(21). This finding suggests a role for NHSL2 in the pathogenesis of this subtype of breast cancer. NHSL2 is part of a protein family withtwo other proteins: Nance Horan Syndrome (NHS) and Nance Horan Syndrome-like 1 (NHSL1)(22).Work on NHS has shown that it plays a role in the regulation of actin remodelling and contains a functional WAVEhomology domain (WHD), which facilitates interaction with the Scar/WAVE complex. Scar/WAVE itself functions to promote cell migration through the activation of Arp2/3 mediated actin polymerisation (22).Recently, the Krause group has also shown that NHSL1 negatively regulates migration through the Scar/WAVE complex via two Abi binding sites (23). These binding sites and the above WHD are conserved amongst all three NHS proteins and the sole Drosophila orthologue GukH, implicating a role for this family in
5migration (22, 23). We have previously observed a vesicular localisation of both NHSL1 and NHSL2 (23, and unpublished), suggesting a role for these proteins in endosomal trafficking. NHSL2 specifically co-localises with endophilin A3 at a subset of these vesicles. Furthermore, we have found that NHSL2 co-immunoprecipitateswith all three GFP-tagged Endophilin proteins inGFP trap experiments. This interaction may point to a role of NHSL2 in FEME.

We are interested to establish whether NHSL2 regulates FEME, and whether this may serve as a mechanism by which NHSL2 regulates breast cancer cell migration and invasion.We hypothesise that NHSL2 inhibits breast cancer cell migration and invasion by counteracting polarised protrusions and directional migration through the fast endophilin-mediated endocytosis (FEME) pathway.To address this hypothesis, we have devised the following aims:1.To investigate the role of NHSL2 in fast endophilin-mediated endocytosis (FEME).2.To explore the function of NHSL2 in directed 2D migration of breast cancer cells.3.To establish whether NHSL2 regulates 3D breast cancer cell invasion.