defactinib and Triple-Negative-Breast-Neoplasms

Stemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC.. Two independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model.. Based on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

Topics: Azepines; Bcl-2-Like Protein 11; Benzamides; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Cell Survival; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Integrins; Proto-Oncogene Proteins c-myc; Pyrazines; RNA, Messenger; Signal Transduction; Sulfonamides; Transcription Factors; Triazoles; Triple Negative Breast Neoplasms

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

Topics: AC133 Antigen; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Benzamides; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Knock-In Techniques; Hepatocyte Growth Factor; Humans; Indicators and Reagents; Lung Neoplasms; Mice, Transgenic; Neoplastic Stem Cells; Phenotype; Pyrazines; Sulfonamides; Triple Negative Breast Neoplasms; Tumor Microenvironment; Xenograft Model Antitumor Assays