Abstract 6328: Metformin induces cell cycle arrest and inhibits proliferation in <i>KRAS</i>-mutated colorectal cancer

Abstract Purpose: Our objective was to evaluate the potential of metformin for colorectal cancer (CRC) prevention. Methods: HT-29 (KRAS wild-type) and SW837 (KRAS G12D mutated) CRC cells were used for in vitro analyses. Cells were treated with 10mM (HT-29) or 2mM (SW837) metformin for up to 96 hours. Proliferation was assessed by raw cell counts, CellTiter 96 Aqueous Proliferation Assay, and 0.5% crystal violet staining. Propidium iodide staining was used to evaluate cell cycle and analyzed on a BD FACS Aria III. Experimental results were analyzed using Student’s t-test or Mann-Whitney U and expressed as the mean +/- standard deviation. Statistical analysis of in vitro experiments was performed in Prism 10.0 (GraphPad) and significance was set at p&amp;lt;0.05. Sequencing was completed using an Illumina NovaSeq X Plus (Novogene). Differential expression analysis was performed using DESeq2RPackage and enrichment analysis using clusterProfiler and GSEA for Go, KEGG, Reactome, DO, and DisGeNET. Statistical significance was set at an adjusted p&amp;lt;0.05. Results: We found that metformin treatment of CRC cells in vitro significantly reduced proliferation and cellular growth after 48 and 96 hours in SW837, but not HT-29 cells. Cell cycle analysis showed that SW837 metformin treatment increased the percent of cells in G1, suggesting G1 cell cycle arrest. Sequencing analysis of SW837 cells identified 2738 and 2492 genes significantly up- and downregulated with metformin treatment, respectively. The top significantly differentially regulated genes were ATF3, SNAI2, UPP1, GDF15, and SLC20A1. Gene ontology analysis of metformin-treated SW837 cells showed differentially regulated genes involved in chromosome segregation, DNA replication, and cell cycle checkpoint, supporting our findings. Independent validation of top hits and key genes in pathways of interest, including CDC42, BRIP1, EME1, and POLE2, is ongoing. Conclusions: Treatment of KRAS-mutated CRC cells with metformin significantly reduced cell proliferation and induced cell cycle arrest. Sequencing analysis showed significant regulation of cell cycle and proliferation-associated pathways, supporting our observed phenotype and providing insight into previously unidentified pathways regulated by metformin. The results from this study may have implications for the future use of metformin for CRC prevention. Citation Format: Devanshi Patel, Michelle Pham, Parthiv Medidi, Holli Ann Loomans-Kropp. Metformin induces cell cycle arrest and inhibits proliferation in KRAS-mutated colorectal cancer [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 6328.