Abstract P3-07-02: Inhibition of sushi domain containing 2 (SUSD2) cleavage prevents surface presentation of galectin-1 on breast cancer cells

Abstract

Abstract Routinely used therapies are not adequate to treat the heterogeneity of breast cancer (BCa), and consequently, more therapeutic targets are desperately needed. Sushi Domain Containing 2 (SUSD2) is a type I transmembrane protein composed of functional domains inherent to adhesion molecules. SUSD2 is highly expressed in BCa but has minimal expression in other normal tissues. Using an in vivo syngeneic mouse model, the Egland lab demonstrated that mice with Susd2-expressing tumors showed increased tumor growth and decreased survival compared to vector controls. Interestingly, Susd2-expressing tumors contained 50% fewer infiltrating CD4 T cells when compared to vector control tumors. In vitro co-culture assays of MDA-MB-231 and Jurkat cells showed that MDA-MB-231-SUSD2 cells increased T cell death compared to MDA-MB-231-vector cells. Our preliminary experiments suggest that SUSD2 acts through the protein Galectin-1 (Gal-1) to achieve T cell killing. Gal-1 has been implicated as a major contributor to cancer immune evasion and angiogenesis. When Gal-1 is targeted by an inhibitory molecule, cancer progression is reduced. Surface presentation of Gal-1 by cancer cells was shown to be required for T cell killing. We have previously shown that localization of Gal-1 on the surface of cells is dependent on the presence of SUSD2; therefore, disrupting the SUSD2-Gal-1 interaction offers a potential new therapeutic strategy for BCa. Western immunoblot analysis using an anti-SUSD2 antibody against the C-terminus showed that SUSD2 runs as two bands, a 110kDa full-length band and a 60kDa fragment, suggesting that SUSD2 is cleaved. When antibodies against both the C-terminus and N-terminus of SUSD2 were used, three bands were observed: a full-length 110kDa band and fragment bands of 60kDa and 50kDa. The cleavage site and significance of this cleavage with respect to SUSD2 and Gal-1 interactions previously has not been investigated. Edman degradation was used to sequence the C-terminal SUSD2 fragment and determined that the cleavage site is within the Von Willebrand type D domain, between the aspartic acid and proline of the GDPH sequence. Deletion of the GDPH sequence showed a complete inhibition of SUSD2 cleavage. Furthermore, non-cleaved SUSD2 was unable to localize to the plasma membrane indicating that SUSD2 cleavage is important for cell surface presentation. Consistently, Gal-1 was not localized to the cell surface in MDA-MB-231 cells expressing the non-cleavable SUSD2 mutant. Our results indicate that SUSD2 cleavage is a processing step required for Gal-1 surface localization and inhibiting SUSD2 processing may be a viable targeting strategy. We are treating SUSD2-expressing BCa cells with protease inhibitors to identify the protease responsible for SUSD2 cleavage. The identification of this protease would provide a potential target for decreasing BCa tumorigenesis and increasing survival of BCa patients. Citation Format: Patrick ME, Egland KA. Inhibition of sushi domain containing 2 (SUSD2) cleavage prevents surface presentation of galectin-1 on breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-02.