Abstract 3052: Butyrate reduces NLRP3 RNA expression in oral squamous cell carcinoma

Abstract Background: Oral squamous cell carcinoma (OSCC) is a type of oropharyngeal cancer associated with elevated levels of IL-1β, which can be secreted with the activation of the inflammasome NLRP3 and caspase 1 signaling pathway. High levels of IL-1β in OSCC leads to cancer progression, invasiveness, autocrine signaling which promotes metastasis, and prolonged inflammation that contributes to tumorigenesis. Inflammasomes are proteins essential for rapid responses in the innate immunity and they work with caspases to promote inflammation signaling, but persistent signaling can induce malignant transformation of cells. Additionally, OSCC is exposed to microbial products, short chain fatty acids (SCFA) which can influence transcription of inflammasomes and caspases; yet it has not been evaluated in OSCC. We have observed that butyrate treatment reduces cell viability, migration, and proliferation in OSCC cell model (OECM-1). Therefore, we aimed to investigate the SCFAs effect in regulating genes NLRP3, NLRP6, Caspase 1 and Caspase 4 in OECM-1. Methods: We extracted RNA from OECM-1 untreated and treated either with butyrate, acetate, or propionate at 5mM for 24 hours. We quantified RNA expression NLRP3, NLRP6, caspase 1, and caspase 4 by probe-based qRT-PCR using the gene RPP30 as the endogenous control. We calculated the fold change using comparative Ct method (ΔΔCt) and t-test to determine significance difference in the levels of inflammasome and caspase after SCFA treatment vs untreated cells. Results: Levels of NLRP3 significantly decreased (fold change: -2.43, p=0.022) and levels of caspase 1 significantly increased (fold change: 1.51, p<0.001) after 24 hours exposure of butyrate. There were no significant changes in gene expression levels of NLRP6 or caspase 4. Treatment with propionate resulted in the increase of NLRP3 (fold change: 1.94, p=0.086), NLRP6 (fold change: 2.22, p=0.058), caspase 1 (fold change: 2.84, p=0.026), caspase 4 (fold change: 1.87, p=0.057). No significant changes in levels of the inflammasomes or caspases were observed with acetate treatment. Conclusion: This study demonstrates that butyrate reduces NLRP3 inflammasome expression on OSCC, promotes caspase 1 expression and does not alter NLRP6 and caspase 4 expression. Conversely, our data shows that propionate increases NLRP3 and caspase 1 transcription, which increases IL-1β levels; and increments NLRP6 and caspase 4 transcription, both associated with inflammation. This proposes butyrate’s ability to reduce IL-1β levels in OSCC and potentially be promoting alternate pathways with caspase 1. Modifying the microbiome to induce greater butyrate secretion or direct inhibition of NLRP3 inflammasome through medications, can represent a novel treatment strategy for OSCC. Citation Format: Oscar Antonio Loperena Gonzalez, Angel J. Amaral Maisonet, Jurelis Torres Reyes, Jeannette L. Salgado Montilla, Gabriel Borges Velez, Josue Perez Santiago. Butyrate reduces NLRP3 RNA expression in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3052.