Germline mutations in oncogene-induced senescence pathways are associated with multiple sessile serrated adenomas

Abstract

Background & aims: Little is known about the genetic factors that contribute to the development of sessile serrated adenomas (SSAs). SSAs contain somatic mutations in BRAF or KRAS early in development. However, evidence from humans and mouse models indicates that these mutations result in oncogene-induced senescence (OIS) of intestinal crypt cells. Progression to serrated neoplasia requires cells to escape OIS via inactivation of tumor suppressor pathways. We investigated whether subjects with multiple SSAs carry germline loss-of function mutations (nonsense and splice site) in genes that regulate OIS: the p16-Rb and ATM-ATR DNA damage response pathways.

Methods: Through a bioinformatic analysis of the literature, we identified a set of genes that function at the main nodes of the p16-Rb and ATM-ATR DNA damage response pathways. We performed whole-exome sequencing of 20 unrelated subjects with multiple SSAs; most had features of serrated polyposis. We compared sequences with those from 4300 subjects matched for ethnicity (controls). We also used an integrative genomics approach to identify additional genes involved in senescence mechanisms.

Results: We identified mutations in genes that regulate senescence (ATM, PIF1, TELO2,XAF1, and RBL1) in 5 of 20 subjects with multiple SSAs (odds ratio, 3.0; 95% confidence interval, 0.9–8.9; P =.04). In 2 subjects,we found nonsense mutations in RNF43, indicating that it is also associated with multiple serrated polyps (odds ratio, 460; 95% confidence interval, 23.1–16,384; P = 6.8 x 10(-5)). In knockdown experiments with pancreatic duct cells exposed to UV light, RNF43 appeared to function as a regulator of ATMATRDNA damage response.

Conclusions: We associated germline loss-of-function variants in genes that regulate senescence pathways with the development of multiple SSAs.We identified RNF43 as a regulator of the DNA damage response and associated nonsense variants in this gene with a high risk of developing SSAs.

Figure 1. Strong Loss-of-Function Mutations Among Study Participants

(A) Gene structure diagrams for all official isoforms of mutated genes. Vertical structures along the line demonstrate exons. Downward pointing arrows indicate the site of mutations. Isoforms are labeled with RefSeq IDs. (B) Pedigrees of study participants with identifiable mutations in the gene set and family histories of colon cancer. Ages are current as of time of publication. Study participants #5 and #13 did not have family histories of colon or extracolonic cancers and are not displayed.

Figure 2. Strong Loss-of-Function Mutations Among Controls

(A) Direct protein-protein interaction network of gene set mutations discovered in controls. Red halos designate genes also found mutated in cases. (B) Cumulative MAFs of each mutated gene in order of decreasing frequency. Arrows designate genes found in cases. The skewness was 8.8 and kurtosis was 92.1.

Figure 3. RNF43 Modulates the ATM-ATR-mediated DNA Damage Response Pathway

(A) Gene-structure diagram of the germline RNF43 mutation identified from the study participants. Arrow demonstrates site of nonsense mutation. (B) Pedigrees of study participants with germline RNF43 mutations. (C) Western blots of protein isolated from KRAS mutated pancreatic duct cells. Cells were stably transfected with short hairpin constructs targeting either GFP or RNF43, and exposed to UV irradiation as indicated. (D) Efficacy of RNF43 silencing in stably transfected pancreatic duct cells. Error bars designate standard error of the mean.