Identification and role of migration stimulating factor isoforms in breast carcinomas

Migration stimulating factor (MSF) is a 70 kDa truncated isoform of fibronectin containing a unique intron-derived 10 amino acid C-terminal sequence not present in any previously described full-length (250 to 280 kDa) fibronectin isoform. Unlike fibronectin, MSF is not an extracellular matrix component, but a soluble factor showing potent bioactivities relevant to cancer development, such as stimulation of cell migration, matrix remodelling and angiogenesis. MSF motogenic activity is mediated by its constituent IGD (isoleucine–glycine–aspartate) motifs. Two isoforms of MSF have been cloned. These differ by a 15 amino acid deletion and are referred to as MSF+aa (AJ535086) and MSF-aa (AJ276395). The term MSF will be used to denote both isoforms. We have identified an inhibitor of MSF+aa (MSFI) that is present in serum and in approximately 50% to 65% of breast carcinomas. MSF+aa and MSF-aa differ in their functional interaction with this inhibitor: MSF+aa is inhibited by MSFI, whereas MSF-aa is not. Both isoforms contain the same MSF-specific sequence and IGD functional domains; consequently, they are equally active in the absence of MSFI [1–4] (K. Kankova, S.J. Jones, I.R. Ellis, M.M. Florence, S.L. Schor, A.M. Schor, unpublished data, 2008).

To examine the expression of MSF isoforms and their possible role in breast tumours.

Three types of monoclonal and polyclonal antibodies were raised and characterised: identification antibodies (VSI) that recognise the MSF-unique sequence; function-neutralising antibodies (pepQ) that recognise the IGD functional domain; and antibodies (TYN) that recognise MSF-aa but not MSF+aa. MSF expression in paraffin-embedded archival specimens of breast tumours was examined by immunohistochemistry using VSI and TYN antibodies. The expression of MSF mRNA and protein by four breast carcinoma cell lines (MCF-7, T47D, MDA-MB435 and MDA-MB-231) and their response to rhMSF was determined by molecular, biochemical and functional assays.

Immunostaining with VSI and TYN antibodies indicated that approximately 80% of breast carcinomas (n = 85) overexpressed total MSF and MSF-aa. High expression was associated with poor prognosis. One of the breast carcinoma cell lines (MCF-7) did not produce MSF. The remaining three lines secreted bioactive MSF into their conditioned medium. Analysis of the type of MSF produced indicates that T47D, MDA-MB435 and MDA-MB-231 cells produce both MSF+aa and MSF-aa; the latter representing approximately 50% to 60% of total MSF. rhMSF stimulated the invasion of tumour cells through three-dimensional gels of type I collagen or through collagen-coated membranes. Conversely, cell invasion by MSF-producing tumour cells was effectively abolished by MSF-function-neutralising antibodies (pepQ).

MSF isoforms are present in most breast tumours and are secreted by breast tumour cell lines. MSF stimulates tumour cell invasion in an autocrine and paracrine manner, modulated by the type of isoform expressed and by the presence of MSFI.