Abstract 5175: Investigating the cooperative effects of the <i>BRAF</i> oncogene and inflammation in <i>BRAF</i>-mutant CRC development
Authors: Caleb Green, Catherine Andreadi, Emma Parrott, Hong Cai, Karen Brown, Abdolrahman Shams-Nateri, Alessandro Rufini
Journal: Cancer Research (2025)

Abstract Colorectal cancer (CRC) currently represents the third most commonly diagnosed cancer worldwide, with its tumorigenesis associated with multiple risk factors, including the presence of inflammation. However, the pro-tumorigenic mechanisms of inflammation remain ill-defined, with poor understanding of the roles of individual inflammatory pathways. Mutations in the BRAF oncogene drive a CRC subtype with serrated histology, typically co-occurring with high levels of microsatellite instability. Unpublished gene set enrichment analysis revealed increased inflammatory signaling in human serrated colonic lesions from publicly available human transcriptomic data. Paradoxically, in-house RNA-sequencing analysis from mice harboring an inducible oncogenic mutation in Braf showed repression of inflammatory pathways in intestinal tissue. In addition, in vivo, Braf mutant mice exhibit long latency and low frequency of tumor formation, which may be due to repressed inflammatory signaling following BrafV600E induction. Our work aims to further investigate the impact of the Braf oncogene on specific inflammatory pathways through analysis of phosphorylated (p)STAT1 and (p)STAT3 expression, biomarkers for IFN-γ and IL6/JAK/STAT pathways respectively. This will be assessed by western blot using an in vitro mouse intestinal organoid (MIO) model, and through multiplex immunofluorescence (mIF) on in vivo murine intestinal tissue slides. To elucidate whether Braf mutations employ cell autonomous mechanisms to alter inflammatory signaling, inducible Braf mutant intestinal crypts were isolated and grown in vitro to form MIOs before treatment with IFN-γ. Western blotting of MIO lysates revealed unchanged phosphorylation status of STAT1 and STAT3 irrespective of Braf mutational status in our experimental conditions. Additionally, mIF revealed the expression and localization of phosphorylated STAT1/STAT3 in intestinal tissue were comparable in wildtype control and BrafV600E mice, 3 days and 6 weeks after induction of the mutant oncogene (n=9). However, in mice expressing BrafV600E for 6 months, there was significantly restrained signaling for both pSTAT1 (p&amp;lt;0.05) and pSTAT3 (p&amp;lt;0.05) in the mutant compared to the wildtype control animals (n=4). Overall, our data indicate temporal control of inflammatory signaling by the BrafV600E oncogene in vivo. With short term Braf induction leaving IFN-γ and IL-6 signaling unperturbed, assessed through pSTAT1/3, whereas mice with prolonged Braf expression showed modulation of both IFN-γ and IL6/JAK/STAT pathways. Further investigation is required into the repression of inflammation induced by the Braf oncogene, and whether subsequent restoration of inflammatory signaling would lead to increased potency in tumor formation. Knowledge of these mechanisms could prove crucial in understanding the development of human BRAF-mutant CRCs. Citation Format: Caleb Green, Catherine Andreadi, Emma Parrott, Hong Cai, Karen Brown, Abdolrahman Shams-Nateri, Alessandro Rufini. Investigating the cooperative effects of the BRAF oncogene and inflammation in BRAF-mutant CRC development [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 5175.