The Transcriptomic Landscape of Mismatch Repair-Deficient Intestinal Stem Cells

Abstract

Lynch syndrome is the most common cause of hereditary colorectal cancer and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR-deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2LoxP/LoxP) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNA-seq and mass spectrometry, followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, IHC, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, premalignant lesions, and early-stage colorectal cancers from patients with Lynch syndrome and patients with familial adenomatous polyposis (FAP) as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene-set enrichment analysis indicated inhibition of replication, cell-cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of patients with Lynch syndrome from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in Lynch syndrome colorectal premalignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of Lynch syndrome carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation. Significance: The transcriptomic and proteomic profile of MMR-deficient intestinal stem cells displays a unique set of genes with potential roles as biomarkers of cancer initiation and early progression.

Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer

“…GSEA indicated activation of key pathways—namely cancer stem cell (CSC) signatures and crypt base—in sporadic MSI rhesus CRC, which corroborates a previously described signature of human MMRd CRC [ 22 ]. The pathway enrichment between MSI-L/MSS and MSI-H indicates that these advanced, late-stage lesions are transcriptomically similar, which may be driven by the late time point rather than MSI status.…”

Recurrent Frameshift Neoantigen Vaccine Elicits Protective Immunity With Reduced Tumor Burden and Improved Overall Survival in a Lynch Syndrome Mouse Model

“…Ptmap1 is a poorly characterized gene that is ubiquitously expressed, encodes an open-reading frame with 76% amino acid identity to Ptma and may be an antagonist of Ptma. Reanalysis of previously published RNA sequencing data 45,46 showed that PTMA is up-regulated in LS patient CRCs and premalignant lesions compared to normal mucosa (P ¼ .00015, Wilcoxon 2-tailed) (Supplementary Figure 6C), suggesting the PTMA/PTMAP1 axis as a potential novel candidate mechanism in LS CRC tumorigenesis. There were no significant changes in histologically normal intestinal mucosa for immune checkpoints PD1/ PDL1, CTLA4, or LAG3 upon FSP vaccination or NSAID treatment (Supplementary Table 2).…”

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps

“…We propose a new paradigm in which damage to the proximal colon, possibly from microbiota, initiates a metaplastic cascade that may eventually select for survival/proliferative pathways, such as activating BRAF mutations. Reversion to a fetal developmental identity is a feature of WNT-independent tumorigenesis found in recent mouse models ( Han et al, 2020 ), which can be triggered by MAPK activation either via Braf -activating mutations, epithelial damage response, or stress triggered by mismatch repair deficiency ( Bommi et al, 2021 ; Leach et al, 2021 ). Critically, Braf mutations in mouse models must be accompanied by a “second hit,” such as perturbation of transforming growth factor-β (TGF-β) signaling, for tumor induction ( Han et al, 2020 ; Leach et al, 2021 ; Tong et al, 2017 ).…”