The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

Bis(monoacylglycero)phosphates (BMPs), a class of lipids highly enriched within endolysosomal organelles, are key components of the lysosomal intraluminal vesicles responsible for activating sphingolipid catabolic enzymes. While BMPs are understudied relative to other phospholipids, recent reports associate BMP dysregulation with a variety of pathological states including neurodegenerative diseases and lysosomal storage disorders. Since the dramatic lysosomal remodeling characteristic of cellular transformation could impact BMP abundance and function, we employed untargeted lipidomics approaches to identify and quantify BMP species in several in vitro and in vivo models of breast cancer and comparative non-transformed cells and tissues. We observed lower BMP levels within transformed cells relative to normal cells, and consistent enrichment of docosahexaenoic acid (22:6) fatty acyl chain-containing BMP species in both human- and mouse-derived mammary tumorigenesis models. Our functional analysis points to a working model whereby 22:6 BMPs serve as reactive oxygen species scavengers in tumor cells, protecting lysosomes from oxidant-induced lysosomal membrane permeabilization. Our findings suggest that breast tumor cells might divert polyunsaturated fatty acids into BMP lipids as part of an adaptive response to protect their lysosomes from elevated reactive oxygen species levels, and raise the possibility that BMP-mediated lysosomal protection is a tumor-specific vulnerability that may be exploited therapeutically.