Society: AGA

Background: RNA-binding motif protein 47 (RBM47) is a component of the APOBEC-1 dependent C-to-U RNA editosome required for RNA editing. However, the range of targets and functions of RBM47 in intestine is unknown. Aims: We studied spontaneous and induced tumor susceptibility in intestine-specific Rbm47 knockout mice (Rbm47^IKO) and human colorectal cancer (CRC). Results: We observed spontaneous intestinal and colonic adenomatous polyps in 92% (11/12) of 12-month-old chow-fed Rbm47^IKO mice, while only 8% (1/13) of aged Rbm47^flox controls developed polyps. High milk fat-fed Rbm47 ^IKO mice exhibited enhanced polyposis with a 3-fold and 2-fold increase in total small intestine (5.9 vs 2) and colon (1.9 vs 0.71) polyp count respectively compared to control mice (Fig 1A). In the Apc^Min/+background, total numbers of polyps were similar by genotype, but Rbm47 ^IKO Apc^Min/+mice developed significantly larger polyps (3.04 vs 1.82 mm²) in the small intestine compared to Rbm47^f/f Apc^Min/+mice. In contrast, colonic polyp burden was decreased in Rbm47 ^IKO Apc^Min/+mice (4.3 vs 7.4), with no change in polyp size (Fig 1B). We also observed upregulation of stem cell markers (Lgr5, Lrig 1, Atoh 1), WNT signaling (Wnt 11, Wnt7b, Fzd8, Fzd3, Ccnd1) pathway genes. Publicly available single cell RNA-seq data (retrieved from PMID 29144463) revealed increased expression of Rbm47 in mature intestinal enterocytes compared to stem and transit amplifying cells (Fig 2A), suggesting that RBM47 normally suppresses Wnt activity and the stem cell program. In addition, we observed increased expression of anti-oxidative pathway genes (Fndc5, Fndc4, Gsta4, Gstm1, Gsta3, Sod1, Nqo1) in Rbm47 ^IKO intestine. In order to examine the mechanisms involved in Rbm47-dependent regulation of mRNAs we undertook polysome profiling and mRNA stability assays on candidate target RNAs. We observed Fndc5 mRNA exhibited increased stability in Rbm47 ^IKO enteroids and colonoids (Fig 2B). We further extended these findings to humans where we observed decreased expression of Rbm47 mRNA (–ΔCT -2.53±3.12 vs 0.16±3.33, P<.0001) in CRC samples (n=24) vs paired uninvolved tissue. Tumors from CRC patients with higher N-stage exhibited lower expression of Rbm47 compared to patients with lower stage (Fig 2C). TCGA analysis showed Rbm47 overexpression (third quartile vs first quartile) is associated with improved overall (OS, HR=0.4, P=.01) and progression-free survival (PFS, HR =0.44; P=.001)) in CRC. Conclusion: Rbm47^IKO mice demonstrate increased susceptibility to both spontaneous and diet induced polyposis and exhibit accelerated polyposis in the Apc^Min/+background through upregulation of stem cell markers, Wnt signaling, and anti-oxidative pathways. RBM47 also exhibits a tumor suppressive role in human CRC as evidenced by findings of reduced expression of Rbm47 and association with OS and PFS.

Figure 1. A: Representative gross pictures of small intestine from <i>Rbm47<sup>f/f</sup></i><sup> </sup>and <i>Rbm47-IKO</i> mice fed with high milk fat diet for 6 months. B: Representative gross pictures of small intestine from <i>Apc<sup>min/+</sup></i> <i>Rbm47<sup>f/f</sup> </i>and <i>Apc<sup>min/+</sup>Rbm47-IKO</i> mice

Figure 1. A: Representative gross pictures of small intestine from Rbm47^f/fand Rbm47-IKO mice fed with high milk fat diet for 6 months. B: Representative gross pictures of small intestine from Apc^min/+ Rbm47^f/f and Apc^min/+Rbm47-IKO mice

$Figure 2. A. Top: <i>Rbm47</i> mRNA expression in small intestine epithelial cells with different levels of maturation (cell count: enterocyte 1041, EP (enterocyte progenitor) 1957, stem (3679), and TA (transit amplifying) 2311) derived from single cell RNA-seq data. Bottom: Three-dimensional exhibition of single cell RNA-seq survey for <i>Rbm 47</i> mRNA expression in murine intestinal epithelial cells. B. Relative <i>Fndc5</i> mRNA abundance in successive time-points after actinomycin treatment as a fraction of baseline <i>Fndc5</i> in enteroids (top) and colonoids (bottom). C. top:<i> Rbm47</i> mRNA expression in uninvolved and paired tumor tissue samples from colorectal cancer patients. Bottom: <i>Rbm47 </i>mRNA expressions in colorectal cancer patients with low (0) and high (1-2) N stage.$

Figure 2. A. Top: Rbm47 mRNA expression in small intestine epithelial cells with different levels of maturation (cell count: enterocyte 1041, EP (enterocyte progenitor) 1957, stem (3679), and TA (transit amplifying) 2311) derived from single cell RNA-seq data. Bottom: Three-dimensional exhibition of single cell RNA-seq survey for Rbm 47 mRNA expression in murine intestinal epithelial cells. B. Relative Fndc5 mRNA abundance in successive time-points after actinomycin treatment as a fraction of baseline Fndc5 in enteroids (top) and colonoids (bottom). C. top: Rbm47 mRNA expression in uninvolved and paired tumor tissue samples from colorectal cancer patients. Bottom: Rbm47 mRNA expressions in colorectal cancer patients with low (0) and high (1-2) N stage.

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