ABSTRACT
Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.
ABSTRACT
INTRODUCTION: Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. METHODS: Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. RESULTS: Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, causing ~ 300% binding increase of the beads compared to negative controls. Immunocyto- and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at ~ 75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and E-selectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. CONCLUSIONS: In summary, this investigation reveals a shear-dependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.
