Mutated CEACAMs Disrupt Transforming Growth Factor Beta Signaling and Alter the Intestinal Microbiome to Promote Colorectal Carcinogenesis

Abstract

Background & aims: We studied interactions among proteins of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which interact with microbes, and transforming growth factor beta (TGFB) signaling pathway, which is often altered in colorectal cancer cells. We investigated mechanisms by which CEACAM proteins inhibit TGFB signaling and alter the intestinal microbiome to promote colorectal carcinogenesis.

Methods: We collected data on DNA sequences, messenger RNA expression levels, and patient survival times from 456 colorectal adenocarcinoma cases, and a separate set of 594 samples of colorectal adenocarcinomas, in The Cancer Genome Atlas. We performed shotgun metagenomic sequencing analyses of feces from wild-type mice and mice with defects in TGFB signaling (Sptbn1^+/- and Smad4^+/-/Sptbn1^+/-) to identify changes in microbiota composition before development of colon tumors. CEACAM protein and its mutants were overexpressed in SW480 and HCT116 colorectal cancer cell lines, which were analyzed by immunoblotting and proliferation and colony formation assays.

Results: In colorectal adenocarcinomas, high expression levels of genes encoding CEACAM proteins, especially CEACAM5, were associated with reduced survival times of patients. There was an inverse correlation between expression of CEACAM genes and expression of TGFB pathway genes (TGFBR1, TGFBR2, and SMAD3). In colorectal adenocarcinomas, we also found an inverse correlation between expression of genes in the TGFB signaling pathway and genes that regulate stem cell features of cells. We found mutations encoding L640I and A643T in the B3 domain of human CEACAM5 in colorectal adenocarcinomas; structural studies indicated that these mutations would alter the interaction between CEACAM5 and TGFBR1. Overexpression of these mutants in SW480 and HCT116 colorectal cancer cell lines increased their anchorage-independent growth and inhibited TGFB signaling to a greater extent than overexpression of wild-type CEACAM5, indicating that they are gain-of-function mutations. Compared with feces from wild-type mice, feces from mice with defects in TGFB signaling had increased abundance of bacterial species that have been associated with the development of colon tumors, including Clostridium septicum, and decreased amounts of beneficial bacteria, such as Bacteroides vulgatus and Parabacteroides distasonis.

Conclusion: We found expression of CEACAMs and genes that regulate stem cell features of cells to be increased in colorectal adenocarcinomas and inversely correlated with expression of TGFB pathway genes. We found colorectal adenocarcinomas to express mutant forms of CEACAM5 that inhibit TGFB signaling and increase proliferation and colony formation. We propose that CEACAM proteins disrupt TGFB signaling, which alters the composition of the intestinal microbiome to promote colorectal carcinogenesis.

Keywords: Microbiome; Signal Transduction; TCGA; Tumor Suppressor.