oxadiazoles and Triple-Negative-Breast-Neoplasms

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered on single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells' response to combinatorial treatment. We here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a likely cause of antitumor activity. We propose that the inhibition of the autophagic flux on combinatorial treatment is attributed to the major cytoskeletal changes induced on ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.

Topics: Antineoplastic Agents; Autophagy; Cell Death; Cell Line, Tumor; ErbB Receptors; Female; Gefitinib; Humans; Imidazoles; Mass Spectrometry; Oxadiazoles; Protein Kinase Inhibitors; Proteomics; rho-Associated Kinases; Triple Negative Breast Neoplasms

Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC.. Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice.. In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity.. Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.

Topics: Animals; Apoptosis; Breast; Caspase 3; Cell Line, Tumor; Datasets as Topic; Female; G2 Phase Cell Cycle Checkpoints; Humans; Intravital Microscopy; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Membrane Potentials; Mice; Oxadiazoles; Patch-Clamp Techniques; Tetrazoles; Thiourea; Triple Negative Breast Neoplasms