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

Víctor Mayoral-Varo,Marta García-Hernández,Jorge Martín-Pérez,Víctor M González,María Elena Martín,Annarica Calcabrini,Valerio Frezza,María Pilar Sánchez-Bailón

doi:10.3390/cancers13030462

Abstract

Simple SummaryTriple-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.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.

Highlights

The Src family of non-receptor tyrosine kinases (SFKs) is composed of nine members, and it has a modular structure, containing the SH2 and SH3 (Src homology domains 2 and 3), which are involved in protein-protein interactions with tyrosine phosphorylated proteins or with proteins containing proline rich sequences, respectively [1,2]
We have found that the induction of SrcDN (c-Src-K295M/Y527F, which is devoid of catalytic activity but with functional SH2 and SH3 domains), a functional mirror image of c-Src-W118A/R175L, significantly reduced subpopulation mationwas efficiency (SFE) in both SUM159 and MDA-MB-231
As cell migration and invasion are steps required for triple negative breast cancer (TNBC) metastasis, we evaluated the relevance of the SH2 -c-Src domain in the regulation of these events by blocking its functionality in SUM159 and MDA-MB-231

Summary

Introduction

The Src family of non-receptor tyrosine kinases (SFKs) is composed of nine members, and it has a modular structure, containing the SH2 and SH3 (Src homology domains 2 and 3), which are involved in protein-protein interactions with tyrosine phosphorylated proteins or with proteins containing proline rich sequences, respectively [1,2]. These two domains are present in many other adapter and regulatory proteins, and facilitate the formation of intracellular signaling complexes [3]. TNBCs do not have specific therapeutic targets and are considered the most aggressive mammary tumors, with a tendency to metastasize mainly into the lung, brain, and bone [13,14]

Methods

Results

Discussion

Conclusion