Dual targeting of αv and α5β1 integrins in triple-negative breast cancer (TNBC).

Abstract

e15073 Background: Integrin αv has been implicated in a stem-phenotype of breast cancer (1) and is cross-regulated by fibronectin-binding integrin α5β1 in tumor cells (2). We hypothesized that these integrins are expressed in TNBC cells and dual targeting of αv and α5β1 integrins by a bispecific antibody (BsAbαv/α5β1) will be superior to monospecific integrin antibodies (MsAbs) alone or in combination. Methods: A representative panel of basal-subtype TNBC lines was assembled: MDA-MB-231 (P53 mutated, KRAS mutated), MDA-MB-468 (P53 mutated, PTEN deleted, EGFR amplified) and BT-20 (P53 and PIK3CA mutated, EGFR amplified). Integrin expression was determined by flow cytometry and Western blot in cell lines and by IHC in the residual cancer burden (RCB) of TNBC specimens following neoadjuvant chemotherapy (positive: >2+ membranous, >10% cells). The impact of MsAbs targeting αv and α5β1 integrins alone or in combination versus the BsAbαv/α5β1 was assessed in adhesion, migration, and clonogenic survival. The impact of integrin blockade on nuclear localization of basal markers including FAK, YAP, EGFR and beta-catenin, was assessed. Results: Robust integrin αv expression was present in all TNBC lines with variable α5β1 expression. Marked reduction in α5 and in αv expression following BsAbαv/α5β1 was uniquely demonstrated by Western blot and/or flow cytometry compared to MsAbs, likely due to endosomal trafficking and lysosomal degradation of target integrins as reported previously (Joshi, MCR. 2020). Combined α5 and αv neutralization with MsAbs was superior to individual single agents in blocking adhesion and migration across all TNBC lines studied. However, the BsAbα5β1/αv was superior to combinatorial MsAbs in MDA-MB-468 and BT-20 lines in chemotaxis assays. A significant (>50%) reduction in clonogenic cell survival was noted with the BsAbα5β1/αv compared to MsAbs. BsAbαv/α5β1 blocked nuclear localization of FAK, EGFR, activated beta-catenin, and YAP in MDA-MB-231 uniquely compared to MsAbs alone or in combination. RCB tumor cells were strongly positive for membranous αv [7/9, (78%)]. Diffuse expression of integrin α5 (67%) in alpha smooth-muscle actin-expressing fibroblasts with dual staining for integrin αv was found. Marked CD68+ macrophage and CD3+ infiltration was seen in tumor and stroma of all RCB tumors. Conclusions: Combinatorial α5 and αv integrin targeting with BsAbα5/αvβ1 defines a novel therapeutic strategy with a distinct mechanism of action for further investigation in TNBC. Enrichment of integrins αv and α5 in the inflamed RCB of TNBC tumors and stroma respectively point to a potential role of these integrins in epithelial-stromal interactions and therapeutic resistance. 1. Desgrosellier, Dev Cell. 2014. 2. Joshi, MCR. 2020.