Abstract 2076: RNF43 is a frequent mutational target in serrated colorectal neoplasia
Authors: Whitehall Vicki L. , Bond Catherine , McKeone Diane , Ellis Jonathan , Bettington Mark , Pearson Sally , Leggett Barbara 
Published: August 2015

Abstract
BACKGROUND. The Wnt signalling pathway is altered in the majority of colorectal cancers by APC mutation. Cancers arising via the serrated neoplasia pathway are hallmarked by BRAF mutation and microsatellite instability (MSI), but do not commonly mutate APC. RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that are localised to intestinal stem cells where they cooperate to suppress Wnt signaling. Mutation of these genes may be an alternate mechanism to bypass the Wnt signal. RNF43 / ZNRF3 double knockout mice develop intestinal neoplasia and germline mutation of RNF43 has been reported in serrated polyposis. We therefore hypothesised that somatic mutation of RNF43 and/or ZNRF3 would be important in the development of serrated colorectal neoplasia.
METHODS. RNF43 and ZNRF3 mutations were assessed in a pilot series of 24 BRAF mutant cancers representing the serrated neoplasia pathway and 10 control BRAF wild type cancers using Agilent V4+UTR whole exome capture followed by sequencing on an Illumina HiSeq 2000. An extended cohort of 80 BRAF mutant cancers (48 MSI, 32 MSS) and 67 BRAF wild type cancers was examined by Sanger sequencing. The frequency of a common hotspot mutation in RNF43 was validated in a second series of 42 BRAF mutant and 22 BRAF wild type colorectal cancers. Transcript expression for RNF43 and ZNRF3 was assayed using Illumina HT-12v4 BeadChip expression arrays in 29 BRAF mutant cancers, 187 BRAF wild type cancers and a subset of 32 matched normal mucosa samples.
RESULTS. RNF43 mutations were detected in whole exome sequencing data in 11/24 BRAF mutant but no BRAF wild type cancers (P&lt;0.01). Three of these 11 cancers also had a ZNRF3 mutation. RNF43 and ZNRF3 mutations were also frequently concordant in a larger series of cancers. RNF43 mutations were associated with MSI (P&lt;0.0001), being identified in 43/48 BRAF mutant / MSI cancers (90%), 6/32 (19%) BRAF mutant / MSS cancers and 6/44 BRAF wild type / MSS cancers (14%). The high frequency of a G7 frameshift mutation at position 659 of RNF43 was similarly present in 25/33 (76%) BRAF mutant MSI cancers but only 1/34 MSS cancers from a second, validation cohort. Transcript levels of RNF43 and ZNRF3 were positively correlated (r2 0.47, P&lt;0.0001) and were significantly lower in BRAF mutant compared to BRAF wild type cancers (P&lt;0.0001).
CONCLUSIONS. RNF43 is a frequent mutational target in serrated neoplasia. The high frequency of both substitution and frameshift mutations support the functional relevance of these alterations. We hypothesise that RNF43 is mutated as an alternative to APC in serrated lesions as a mechanism of bypassing control of the Wnt signal. These data support our current model of serrated neoplasia whereby BRAF is mutated early in serrated polyp development and RNF43 is subsequently mutated to remove constraints on the Wnt signal and allow progression to cancer.
Citation Format: Vicki L. Whitehall, Catherine Bond, Diane McKeone, Jonathan Ellis, Mark Bettington, Sally Pearson, Barbara Leggett. RNF43 is a frequent mutational target in serrated colorectal neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2015-2076