Acetalax (Oxyphenisatin acetate, NSC 59687) and Bisacodyl Cause Oncosis in Triple Negative Breast Cancer by Poisoning the Ion Exchange Membrane Protein TRPM4

Triple negative breast cancer (TNBC) is generally unresponsive to current therapies, and the development of new anticancer agents is needed. Oxyphenisatin acetate, called Acetalax, which was used as a laxative in the 1950’s, has recently been reported to have anticancer activity in murine models. In this study we demonstrate that Acetalax and its diphenolic laxative structural analog bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines. We show that both cause oncosis, a non-apoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing that leads to mitochondrial dysfunction and ATP depletion. Acetalax was further shown to enhance immune and inflammatory responses and inhibit protein synthesis. Mechanistically, we provide evidence that TRPM4 (Transient Receptor Potential Melastatin member 4) is poisoned by Acetalax and bisacodyl. By blocking TRPM4, a monovalent cation channel, Acetalax and Bisacodyl cause oncosis, and TNBC cells without endogenous TRPM4 expression as well as TRPM 4 knockout TNBC cells were found to be Acetalax- and Bisacodyl resistant. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover TRPM4 is rapidly degraded by the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and Bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and Bisacodyl efficacy, and could be used for the clinical development of these drugs as anticancer agents. We created Acetalax resistant cells by chronically exposing MDAMB468 and BT549, which were originally sensitive. We named them MDAMB468-CE(chronic exposure) and BT549-CE. Cells were seeded in a 10 cm dish, and when the dish was almost full confluent in 1–2 weeks, the cells were passaged with double the concentration Acetalax. Initially, the Acetalax concentration was started at 0.2 μM. When the Acetalax concentration was increased to 51.2 μM, the cells stopped growing. Therefore, cells chronically exposed to 40 μM Acetalax were designated as Acetalax chronic exposure cells and are Acetalax resistant clones. CE cells were used in the experiment after a 2-week Acetalax-free period. The RNA sequencing was done for MDAMB468 and BT549 parental cells and CE cells. The expressed RNAs of these 8 samples were compared to determine what RNAs were altered in the parental and CE cells and what RNAs were altered in the no-treatment and treatment groups. RNA was extracted from a control group of these cells and from cells treated with Acetalax 1 uM for 6 hours. RNA was extracted as per protocol using PureLink® RNA Mini Kit (Cat:12183018A. Invitrogen). The extracted RNA was measured for RNA Integrity Number to confirm that there were no problems with RNA quality. We used 800 ng of total RNA samples to generate the library (PolyA selected). The kits used for the library prep were NEBNext PolyA mRNA Magnetic Isolation Module (NEB #E7490), NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB, E6440) as described in the manufacturer’s manual. Sequencing was carried out in the Illumina NextSeq 2000 instrument with the NextSeq 2000 P2 kit (200 Cycles) with 101x101 pair end configuration.