DOP111 Lack of epithelial RhoA in the gut, a new colitis-associated cancer experimental model
Authors: Muccioli S E , Hidalgo-García L , Erkert L , Becker L S , Martínez-Sánchez L , González-Acera M , Vieth M , Atreya R , Becker C , Wirtz S , Brakebusch C H , Atreya I , Neurath M F , López-Posadas R 
Published: January 2025

Background

The small GTPase RhoA controls cytoskeleton dynamics in various cell types, including intestinal epithelial cells (IECs). Accordingly, we have shown that lack of RhoA in IECs in mice increases intestinal permeability and inflammation1. However, its role in cancer is controversial. While classically recognized as a potential colorectal cancer (CRC) biomarker2, RhoA inactivation was found to increase Wnt signalling and tumor risk in mouse small intestinal epithelium3. Additionally, proteins in the RhoA/Rac1 pathway were identified as highly mutated in human colitis-associated cancer (CAC)4. We hypothesize that a finely tuned RhoA function in IECs determines CRC risk and that inflammation might modulate RhoA´s pro- or anti-tumor effects in the gut.

Methods

RhoAΔIEC mice generated using the loxPCre system were our main model1. We monitored disease progression, analysed the transcriptome in colon and tumoral tissue (bulk RNA-sequencing), assessed microbiota composition (16S rRNA gene sequencing), and conducted in vitro colon organoids studies. Moreover, Rho/Rac-associated proteins were analysed across colorectal cancer (CRC) and colitis associated cancer (CAC) mouse models.

Results

RhoAΔIEC mice developed chronic intestinal inflammation and spontaneous colon tumorigenesis. Bulk RNA sequencing and Gene Set Enrichment Analysis (GSEA) highlighted four regulated pathways: inflammation, microbiota, proliferation, and epithelial-mesenchymal transition (EMT). Transcriptomic changes showed similarities with the azoxymethane/dextran sulphate sodium (AOM/DSS) and APCmin models. Microbiota analysis showed altered β-diversity, with higher abundance of Mycoplasma and Helicobacter genus. Increased Ki67 expression and non-phospho β-catenin indicated Wnt pathway activation, while YAP was not upregulated. RhoA deficient cells showed signs of EMT, as decreased E cadherin expression. Moreover, tumors in RhoAΔIEC mice depicted increased expression of CXCR2-axis chemokines (Cxcl1, 2, 5) and neutrophil infiltration. Among candidate Rho/Rac related proteins, qPCR showed DOCK2 upregulation in AOM and AOM/DSS while Prex2 increased exclusively in AOM/DSS model; DOCK3 and Radil were not regulated.

Conclusion

Lack of RhoA in IECs drives sustained intestinal inflammation and spontaneous tumorigenesis through Wnt-dependent proliferation, EMT, neutrophil activation, and dysbiosis. RhoAΔIEC mice represent a novel model of inflammation-associated tumorigenesis in the gut. Translationally, the Rac/Rho-associated PREX2 emerges as a candidate CAC-specific biomarker. We hypothesize that PREX2 increases to compensate for RhoA during inflammation.

References

1.Lopez-Posadas R, Becker C, Gunther C, et al. Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation. The Journal of Clinical Investigation. Feb 2016;126(2):611-26. doi:10.1172/JCI80997

2.Fritz G, Just I, Kaina B. Rho GTPases are over-expressed in human tumors International Journal of Cancer. 1999;81:682–687. doi:10.1002/(sici)1097-0215(19990531)81:5&lt;682::aid-ijc2&gt;3.0.co;2-b

3.Dopeso H, Rodrigues P, Cartón-García F, et al. RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis. iScience. 2024;Volume 27(Issue 4)doi:10.1016/j.isci.2024.109400

4.Robles AI, Traverso G, Zhang M, et al. Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers. Gastroenterology. Apr 2016;150(4):931-43. doi:10.1053/j.gastro.2015.12.036