Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

The formation of Gasdermin (GSDM) pores, leading to pyroptosis or other context-dependent consequences, is directly involved in multiple diseases. Gasdermin-B (GSDMB) plays complex and controversial roles in pathologies, with pyroptosis-dependent and independent functions. GSDMB is promising oncologic therapeutic target since it exhibits either antitumor function, when immunocyte-mediated Granzyme-A (GZMA) cleaves GSDMB releasing its cytotoxic N-terminal domain, or pro-tumoral activities (invasion, metastasis, and drug resistance). However, it is still unknown the precise regulatory mechanisms of GSDMB pyroptosis as well as the differential effects of the four translated GSDMB variants (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Here, we first prove that exon 6 translation (in the interdomain protein linker) is essential for pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in large series of breast cancer samples GSDMB2 expression, and not of exon6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Moreover, cellular, and biochemical analyses combined with confocal, live cell imaging, and electron microscopy studies, demonstrated that diverse GSDMB N-terminal constructs containing exon-6 induce mitochondrial damage (increased mitochondrial ROS, membrane potential loss and mitochondrial DNA release) together with pyroptotic membrane cell lysis. While exon-6 residues are not required for membrane or mitochondrial localization, we also identified other key residues for N-terminal domain cytotoxicity. Additionally, we demonstrated that all GSDMB variants share the cleavage sites for GZMA, Neutrophil Elastase (identified in this study) and caspases. Interestingly, whereas Neutrophil Elastase and caspases produce N-terminal fragments in all GSDMB isoforms with no pyroptotic activity, thus acting as a potential inhibitory mechanism, GZMA cleavage activates pyroptosis in an isoform-dependent way. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer and other pathologies and for the future design of GSDMB-targeted therapies.