Involvement of ROS species in amiloride derivative-induced lysosome-dependent cell death in triple-negative breast cancer cells

Drug resistance leading to cancer recurrence poses a particularly challenging barrier to clinical disease management. Since tumor cells commonly activate anti-apoptotic pathways that cause caspase-dependent pathways to malfunction, cellular resistance to apoptosis is perhaps the most critical factor contributing to the therapeutic failure of conventional and targeted therapeutic agents. Consequently, subpopulations of apoptosis-resistant cells, such as cancer stem cells (CSCs), persist after therapy to seed primary tumor recurrence and metastatic lesions, even after complete remission. The overarching goal of this project is to develop novel drugs that exploit the process of lysosome-dependent cell death, a programmed necrotic cell death mechanism, in suppressing CSC-mediated triple-negative breast cancer. We have previously observed that hexamethylene amiloride (HMA), a derivative of the FDA-approved diuretic amiloride, is cytotoxic in vitro and ex vivo toward cultured cells from a variety of tumor types but not non-transformed cells. HMA also suppresses primary and metastatic tumor outgrowth in vivo. HMA acts on breast tumor cells regardless of subtype, proliferative status, or species of origin. It engages a potent caspase- and autophagy-independent programmed necrotic death mechanism in tumor cells that alters lysosome structure, dysregulates lipid synthesis, leads to lysosomal membrane permeabilization, and acts efficiently toward therapy-resistant CSC-related subpopulations. Although some specifics of the mechanism remain unknown, we examine how the formation of reactive oxygen species is crucial to the induction of necrosis and the potency of HMA and other amiloride derivatives.