Abstract 4917: Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway
Authors: Li Zheqi , Levine Kevin M. , Arneson Spencer , Berrett Kristofer C. , Priedigkeit Nolan M. , Bahreini Amir , Chen Jian , Zhu Li , Carroll Jason S. , Tseng George C. , Lucas Peter C. , Atkinson Jennifer M. , Gertz Jason , Lee Adrian V. , Oesterreich Steffi 
Published: September 2020

Abstract
Background: Hotspot estrogen receptor-α (ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer, with Y537S and D538G as the two most frequent hotspot mutations. A mostly unanswered question is and how these mutations facilitate metastatic processes.
Methods: Frequencies of different ESR1 hotspot mutations were compared within three publicly available cohorts. Y537S and D538G genome-edited MCF7 and T47D cell models were used for in vitro characterization. Wound scratching, spheroid collective migration, chemotaxis assays were applied to examine different metastatic properties. Tail vein injection in nude mice followed by human CK19 lung section immune staining was used to characterize in vivo metastasis. Canonical Wnt pathway activity was studied using top-flash reporter assays, immunoblotting, and overexpression of dominant negative TCF4. ChIP-seq and ATAC-seq were utilized to profile ER global binding patterns and chromatin accessibility in cell models, respectively. Porcupine inhibitor-LGK974 was used to evaluate the efficiency of Wnt-targeted therapy.
Results: D538G mutations were more frequent in ER+ metastatic lesions compared to Y537S among three metastatic breast cancer cohorts with unbiased SNV detection. In line with this, T47D-D538G ESR1 mutant cells showed strongly enhanced cell migration in vitro which was not observed in Y537S cells. We also observed increased lung micro-metastasis in vivo after tail vein injection of the D538G cells. Cell line transcriptomic analysis revealed uniquely hyperactivated Wnt pathway in D538G mutant cells, which was further confirmed in vitro by top-flash reporter and immunoblot. Suppression of canonical Wnt pathways blocked T47D-D538G specific cell migration. Combination treatment of fulvestrant and LGK974 synergistically inhibited D538G specific migration. Mechanistically, multiple Wnt regulator genes were found uniquely upregulated in D538G cells. Interestingly, none of these targets gained ER binding peaks at proximal regulatory regions, suggesting potential epigenetic regulation, which was confirmed in ATAC-seq results. Knockdown of FOXA1, a well-characterized pioneer factor, decreased canonical Wnt activity and abrogated the D538G-unique cell migration in short-term.
Conclusion: T47D-D538G cells showed uniquely enhanced cell migration via Wnt hyperactivation, potentially mediated via epigenetic remodeling. These findings suggest the further study of potential combinatorial targeting of Wnt and ER signaling in D538G ESR1 mutant tumors.
Citation Format: Zheqi Li, Kevin M. Levine, Spencer Arneson, Kristofer C. Berrett, Nolan M. Priedigkeit, Amir Bahreini, Jian Chen, Li Zhu, Jason S. Carroll, George C. Tseng, Peter C. Lucas, Jennifer M. Atkinson, Jason Gertz, Adrian V. Lee, Steffi Oesterreich. Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4917.