Evidence for diet-gene interaction in early-onset colorectal cancer.

Abstract

182 Background: About 10 percent of all colorectal cancers are in subjects who are not yet 50 (EO-CRC) and the occurrence of early onset colorectal cancer (EO-CRC) is rising in the US. Patients with CRC are twice as likely to have diabetes or be overweight. Using targeted exome sequencing of germline DNA from EO-CRC subjects, we identified a missense mutation at Ala98Val within the DNA binding domain of Hepatic Nuclear Factor 1 alpha (HNF1A, 12q24.31, Rs1800574). HNF1A is the most frequently mutated gene in diabetic individuals whose onset of diabetes occurs typically before age 25 (MODY3 locus). Our aim was to demonstrate that the HNF1A variant provides a genomic landscape for EO-CRC to develop in the setting of a specific diet type. Methods: HNF1A was identified using targeted exon sequencing of archived leukocyte DNA from subjects with EO-CRC. Flag-tagged WT and HNF1AA98V expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts were prepared for Electrophoretic Mobility Shift Assays (EMSAs) to test binding differences. An Hnf1aA98V mutant mouse model was generated using CRISPR/Cas9. WT, Hnf1a A98V/+ and Hnf1a A98V/A98V mice were placed on 3 diets--normal chow, a high fat (~34%) diet (HFD) or a high sugar (~62% fructose) diet (HSD) after weaning at 3 weeks. The mice were followed for 12 months, weighed monthly, and observed for clinical signs of morbidity. After euthanizing, blood, colon and liver were collected for histology and qPCR. Results: HNF1AA98V was identified in 13/145 subjects with EO-CRC. An additional 3 subjects exhibited mutations elsewhere in HNF1A. Flag-tagged WT and mutant HNF1A-expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts showed reduced HNF1AA98V binding to its consensus DNA element by EMSA. Only heterozygous or homozygous Hnf1aA98V mice on the HFD showed a significant number of colon polyps (~19%). Only 1/22 heterozygous mice developed a polyp on the HSD. RNA-Seq analysis of colonic mucosa from the homozygous mutant compared to WT mice revealed an increase lipid and b-catenin regulated genes. Specifically, there was an increase in β-catenin, LEF 1 and MYC by immunohistochemistry in the polyp of HNF1AA98V, but not in the adjacent normal tissue. Liver steatosis in the Hnf1aA98V versus WT mice on the HFD was observed. Conclusions: Occurrence of the HNF1AA98V variant is increased among a cohort of EO-CRC patients and is a loss of function mutation that creates a genetic landscape for colon polyps on a HFD.