Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers.

Abstract Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study we investigated the activation of Src family kinase (SFK) and two SFK substrates—CUB-domain containing protein 1 (CDCP1) and protein kinase C delta (PKCδ)—in 56 formalin-fixed, paraffin embedded (FFPE) TNBC. Expression of SFK phosphorylated at Y416 (SFK_pY416+) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCδ (CDCP1_ pY743+ and PKCδ_pY311+) as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (cCDCP1, 70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be bi-phosphorylated (SFK_pY416+/pY527+). Bi-phosphorylated active SFK was observed more frequently in FOXA1- TNBC. In addition, in SFK_pY416- samples, FOXA1+ TNBC tended to be SFK_pY527+ (classic “inactive” SFK), and FOXA1- TNBC tended to be SFK_pY527- (SFK “poised” for activation). Strong SFK_pY416 staining was also observed in tumor infiltrating lymphocytes (TILs) in a subset of TNBC with high TIL content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/CDCP1/PKCδ pathway for TNBC treatment.