Role of the proto-oncogene Src in metastatic breast cancer

Summary (SAF2016_75991-R). The proto-oncogene c-Src is required for cellular homeostasis; its hyper-activation/expression is associated with tumor progression and metastasis in humans (breast, pancreas, colon, ovary, lung, etc.); however, the molecular mechanisms are not completely defined. Most of c-Src studies have concentrated on its catalytic activity, and several inhibitors were designed for clinical practice. Some groups, including ours, have shown that c-Src SH2/SH3 adapter domains are necessary for its full functionality. In breast cancer, Triple Negative Tumors (estrogen and progesterone receptors, and for HER2 negative) are highly metastatic, and do not have effective therapeutic targets; in these tumors, hyper-activation of c-Src predisposes for bone, brain and lung metastases. In metastatic cells, c-Src controls proliferation, migration, and invasion. Moreover, c-Src is involved in the regulation of secretion and protein composition of the secretome (metalloproteases, etc.) and cell-derived (Cyr61, LOXL2, etc.); in this way c-Src influences the microenvironment for the primary tumor and the metastatic niche. Our working hypothesis postulates that the SH2/SH3 adapter domains are essential for c-Src's full functionality in breast cancer metastasis. We aim to study in the human metastatic triple negative cell lines MDA-MB-231 and SUM159, the functionality of the adapter and catalytic domains of c-Src. We will conditionally express (Tet-On system) c-Src variants with loss of adapter domains functions in these cell lines. These c-Src mutants (c-Src-SH2-: c-Src-T215W; c-Src-SH3-: c-Src-D99N; c-Src-SH3--SH2-: c-Src-D99N /T215W) are defective for SH2 and/or SH3 adapter functions, but they do not induce conformational changes activating the catalytic domain. In combination with other c-Src variants currently under study, we will be able to discern the functional role of each c-Src domain. We will analyze their effects on cell proliferation, migration, and invasion under culture conditions, and on the viability of the breast cancer stem cell subpopulation. We will then study the role of c-Src variants in the microenvironment by determining the protein composition and functionality of the secretome and the exosomes derived from MDA-MB-231 or SUM159 expressing the c-Src variants. These secretome and exosome preparations will be incubated with stromal cells of the primary tumor and of the metastatic niche microenvironments (e.g., normal mammary epithelial, mesenchymal, endothelial cells), and cell proliferation and differentiation will be determined. We will also study whether secreted exosomes (fluorescently labeled) from MDA-MB-231 or SUM159PT expressing different c-Src variants distribute into the typical breast metastatic organs in mice upon retro-orbital injection, and whether they play a role in preparing metastatic niches (education of the niche). If there are differences among exosomes, will study their protein composition, and characterize the function of these differentially expressed proteins. The anticipated results should allow us to define the functionality of c-Src domains on the metastatic breast cells, the microenvironments, and predisposition of the metastatic niche. They should also stimulate the design of specific modulators for the c-Src SH2 and SH3 domains, which in combination with the catalytic inhibitors would provide better treatment for breast metastasis, as well as for other tumors where c-Src is also deregulated.