The Relevance of the SH2 Domain for c-Src Functionality in Triple-Negative Breast Cancer Cells

SIMPLE SUMMARY: Triple-Negative breast cancers (TNBC) have not specific therapeutic targets and are considered the most aggressive mammary tumors. c-Src controls several cellular processes: proliferation, differentiation, survival, motility, and angiogenesis. Alteration of c-Src functionality, by increasing its expression and/or its kinase activity, is associated to progression and metastasis of tumors in mammary gland, pancreas, colon, brain, and lung. However, c-Src tyrosine kinase inhibitors alone are not fully clinically effective, suggesting that c-Src adapter SH2/SH3 domains may be important. We questioned whether the SH2-c-Src domain is relevant for tumorigenicity of TNBC SUM159 and MDA-MB-231 human cell lines. Conditional expression of SH2 and SH3 inactivating mutants in these TNBC cells, or transfection of aptamers directed to SH2, allowed us to show that this domain is required for their tumorigenesis. Therefore, the SH2-c-Src domain could be a promising therapeutic target that, combined with c-Src kinase inhibitors, may represent a novel therapeutic strategy for TNBC patients. ABSTRACT: The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.