Abstract PR04: Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia
Authors: Whitehall Vicki , Borowsky Jennifer , Bond Catherine , Bettington Mark , Pearson Sally-Ann , Kalimutho Murugan , Liu John , Dumenil Troy , McKeone Diane , Jamieson Saara , Fernando Winnie , Fennell Lochlan , Clouston Andrew , Rosty Christophe , Brown Ian , Walker Neal , Leggett Barbara 
Published: March 2017

Abstract
The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia.
Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901.
Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) samples. This did not significantly differ when samples were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P&lt;0.0001). We further demonstrated the importance of the WNT pathway in progression of serrated neoplasia by administering azoxymethane to mice with BRAF mutant-induced intestinal hyperplasia. This accelerated the serrated phenotype from 0.6 polyps per mouse to 5.9 per mouse (P&lt;0.001). These all showed typical murine serrated adenoma morphology. To investigate potential therapeutic implications we treated colorectal cancer cell lines with the porcupine inhibitor LGK974 and showed a 50% reduction in growth only in RNF43 and/or ZNRF3 mutant cell lines. In the BRAF / RNF43 / ZNRF3 mutant cell line RKO, LGK974 synergized with MEK inhibition to dramatically reduce growth by approximately 95%.
Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers.
This abstract is also being presented as Poster A21.
Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR04.