Abstract
The induced death signals following oncogene inhibition underlie clinical efficacy of molecular targeted therapies against human cancer, and defects of intact cell apoptosis machinery often lead to therapeutic failure. Despite potential importance, other forms of regulated cell death triggered by pharmacologic intervention have not been systematically characterized. Pyroptotic cell death was assessed by immunoblot analysis, phase-contrast imaging, scanning electron microscopy, and flow cytometry. Tumor tissues of patients with lung cancer were analyzed using IHC. Functional impact of pyroptosis on drug response was investigated in cell lines and xenograft models. We showed that diverse small-molecule inhibitors specifically targeting KRAS-, EGFR-, or ALK-driven lung cancer uniformly elicited robust pyroptotic cell death, in addition to simultaneously invoking cellular apoptosis. Upon drug treatment, the mitochondrial intrinsic apoptotic pathway was engaged and the mobilized caspase-3 protease cleaved and activated gasdermin E (GSDME, encoded by DFNA5), which permeabilized cytoplasmic membrane and executed cell-lytic pyroptosis. GSDME displayed ubiquitous expression in various lung cancer cell lines and clinical specimens, including KRAS-mutant, EGFR-altered, and ALK-rearranged adenocarcinomas. As a result, cooccurrence and interplay of apoptosis and pyroptosis were widespread in lung cancer cells, succumbing to genotype-matched regimens. We further demonstrated that pyroptotic cell death partially contributed to the drug response in a subset of cancer models. These results pinpoint GSDME-dependent pyroptosis as a previously unrecognized mechanism of action for molecular targeted agents to eradicate oncogene-addicted neoplastic cells, which may have important implications for the clinical development and optimal application of anticancer therapeutics.
Highlights
An emerging is that the intricate molecular regulation on the inherent signal-mediated death process, generally referred to asNote: Supplementary data for this article are available at Clinical Cancer Research Online.H
We showed that diverse small-molecule inhibitors targeting KRAS, EGFR, or ALK-driven lung cancer uniformly elicited robust pyroptotic cell death, in addition to simultaneously invoking cellular apoptosis
We individually combined a range of small-molecule inhibitors to determine their agonistic or antagonistic roles, and found that cell death induced by targeted therapies was appreciably prevented by the pan-caspase inhibitor Q-VD-OPh [39], whereas necrostatin-1, ferrostatin-1, niraparib, ALLN, cyclosporine A, and chloroquine did not exhibit consistent effects (Supplementary Fig. S1A)
Summary
The notion that antitumor therapeutics act most potently through stimulating apoptosis prompted us to comprehensively investigate different subroutines of regulated cell death that likely occur in response to targeted agents. Flow cytometric analysis verified the necrotic nature of treatment-induced cell death, as exemplified by Annexin V and PI double-positive staining (Fig. 1E) These data suggested that diverse molecular targeted therapies, besides triggering apoptosis, could lead to tumor cell pyroptosis. In contrast to previous observations that no or little GSDME was expressed within other tumor types [47,48,49], GSDME protein was readily detected by immunoblot analysis in most lung cancer cell lines disregarding oncogenic drivers (Fig. 2A) We subjected this large panel of carcinoma models to their alteration-specific targeted inhibition, and uncovered that. As with GSDME in cell lines, the protein was plausibly functional in patients with lung cancer, because serum LDH concentrations significantly increased during 6-month follow-up after initial chemo- or EGFR inhibitor-based treatment (Supplementary Tables S3 and S4), with the exception of squamous cell carcinomas that were known to typically resist chemotherapy (Fig. 2F). The clinical significance of pyroptotic cell death in patients with cancer receiving targeted agents warranted prospective investigations
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