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.

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substrates-CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ)-in 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. 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 (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 biphosphorylated (SFK_pY416/pY527). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1) TNBCs. 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 in a subset of TNBCs with high tumor-infiltrating lymphocyte 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.