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Bsacground: Lynch Syndrome (LS) is an inherited cancer syndrome associated with an increased lifetime risk of colorectal cancer (CRC) and characterised by germline mutations in DNA mismatch repair (MMR) genes. Abnormalities in the function of MMR genes lead to errors during DNA replication, including microsatellite instability. Despite regular colonoscopies, up to 15% of LS patients still develop CRC. Gut microbiome, bowel inflammation, the environment and host genetic and epigenetic factors are involved in CRC development. In this sense, the concomitant analysis of stool host microRNA (miRNA) profiles and gut microbiome composition may arise to the identification of specific fecal markers in LS explaining the molecular basis for the development of cancer in these patients. PURPOSE: In the present study, the fecal miRNome and microbiome were characterized in a time progression in an in vivo model using a mismatch repair deficient mice model that recapitulates human LS. The main aim was to evaluate the changes and crosstalk of miRNAs released in the stool by the host and the microbial population residing in the gut during the carcinogenesis process induced in animals. Findings in mice were compared and correlated to data obtained from a cohort of human subjects affected by LS. Methods: We used an in vivo model for LS based on a conditional knockout mouse with the tissue-specific inactivation of Msh2 (Msh2LoxP) in the intestinal mucosa, combining Msh2LoxP allele with the Villin-Cre transgene (VCMsh2LoxP). Stool from mice were collected at different time points (6, 9 and 12 months of age). Small RNA-sequencing (sRNA-seq) and shotgun metagenomics analyses were performed in stool from all samples. In concomitance, we collected 78 stool samples of clinically diagnosed LS subjects, at different follow up time. At the sampling, 36 resulted negative for colorectal adenomas/lesions, 12 were negative but with a previous history of lesions, 23 presented a neoplasia and 6 developed a lesion in the follow-up. Twenty-two stool samples from healthy subjects were used as controls. Results: In mice, preliminary results from sRNA-seq and metagenomics analyses showed several dysregulated miRNAs and differential microbial relative abundances, either between VCMsh2loxP and controls or at different time points. In humans, we identified 41 and 17 miRNAs respectively up- and downregulated in LS subjects who presented a lesion compared to those not having any lesion at follow-up. In the same comparison the metagenomic data showed significantly different microbial abundances, including those of Eubacterium ramulus. Conclusions: This is the first study characterizing the concomitant alterations in microbial composition and host miRNome overtime in relation to the onset of cancerous/precancerous lesions due to LS. Citation Format: Giulia Francescato, Giulio Ferrero, Marc Beltrà, Sonia Tarallo, Giulia Piaggeschi, Carla Di Battista, Antonio Francavilla, Carlijn Bruggeling, Fabio Penna, Barbara Pardini, Paola Costelli, Annemarie Boleij, Alessio Naccarati. Fecal miRNA profiles and gut metagenome composition in Lynch syndrome: results from a mouse model study and human subjects. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3797.

The 8q24 locus is enriched in cancer-associated polymorphisms and, despite containing relatively few protein-coding genes, it hosts the MYC oncogene and other genetic elements connected to tumorigenesis, including microRNAs (miRNAs). Research on miRNAs may provide insights into the transcriptomic regulation of this multiple cancer-associated region.We profiled all miRNAs located in the 8q24 region in 120 colorectal cancer (CRC) patients and 80 controls. miRNA profiling was performed on cancer/non-malignant adjacent mucosa, stool, and plasma extracellular vesicles (EVs), and the results validated with The Cancer Genome Atlas (TCGA) data. To verify if the 8q24-annotated miRNAs altered in CRC were dysregulated in other cancers and biofluids, we evaluated their levels in bladder cancer (BC) cases from the TCGA dataset and in urine and plasma EVs from a set of BC cases and healthy controls.Among the detected mature miRNAs in the region, 12 were altered between CRC and adjacent mucosa (adj. p 0.05). Five and four miRNAs were confirmed as dysregulated in the CRC and BC TCGA dataset, respectively. A co-expression analysis of tumor/adjacent tissue data from the CRC group revealed a correlation between the dysregulated miRNAs and CRC-related genes (PVT1 and MYC) annotated in 8q24 region. miR-30d-5p and miR-151a-3p, altered in CRC tissue, were also dysregulated in stool of CRC patients and urine of BC cases, respectively. Functional enrichment of dysregulated miRNA target genes highlighted terms related to TP53-mediated cell cycle control.Altered expression of 8q24-annotated miRNAs may be relevant for the initiation and/or progression of cancer.