Abstract A70: ESR1 mutations confer novel metastatic functions in genome-edited breast cancer models
Authors: Li Zheqi , Bahreini Amir , Wang Peilu , Levine Kevin M. , Tasdemir Nilgun , Chu David , Park Ben H. , Lee Adrian V. , Oesterreich Steffi 
Published: August 2018

Abstract
Background: Estrogen receptor alpha (ERα), encoded by the ESR1 gene, is expressed in approximately 70% of breast cancers. Recent studies conducted by us and others have shown that somatic mutations in ESR1 gene play a key role in conferring endocrine resistance in ER+ breast cancer. These hotspot mutations mainly occur on the ligand-binding domain of ERα, leading to poor outcomes in 25-30% of patients with ER+ metastatic breast cancer in clinic. The mechanisms behind the potential enhanced metastasis of these mutations have become an urgent issue to be addressed, but they are not well understood due to a lack of ESR1 mutant models.
Methods: We generated and characterized genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations (Y537S and D538G), using CRIPSR/Cas9 and rAAV systems, respectively. Multiple clones for each mutant were sorted and the mutation frequencies were detected using digital droplet PCR (ddPCR). Levels of total or phosphorylated ER were determined by Western blot. We subsequently performed an RNA-sequencing and ChIP-sequencing to deeply differentiate the gene expression and ER-DNA binding patterns in these mutants at transcriptome and cistrome. The growth of these pooled mutant-cells was determined in both 2D and 3D ultralow attachment plates. The cell-matrix adhesions were measured based on ECM array, and 84-ECM adhesion related genes were further tested by qPCR array. IncuCyte real-time image system and Boyden chamber trans-well assays were used to monitor the cell migration and chemotaxis. Tail vein injections were performed on nude mice. H&E and immunofluorescent staining of lung and liver tissues with human specific cytokeratin 19 were utilized to evaluate in vivo metastatic capacities of the mutant cell models.
Results: We first identified the robust mutation frequencies at both RNA and DNA levels in our cell models. The RNA-seq and ChIP-seq exhibits multiple ligand-independent genes and ER binding events overlapping between either cell lines or mutants, which were further conformed by qPCR and ChIP. We also found that both Y537S and D538G mutants present ligand-independent growth in 2D and 3D ultralow attachment plates. Using wound-scratching assay, we observed significant higher migration rate in D538G mutant of T47D cell lines on both Matrigel and type I collagen, indicating a cell-line and mutant-specific phenotype. We also detected lower attachment of both mutants on type I collagen in both cell lines, and our qPCR array revealed that alterations in the MMP pathways could be one of the major mechanism causing this phenotype. Finally, tail vein injection of T47D-D538G mutant cells in nude mice derived more micrometatsatic spots in the lung and liver tissues.
Conclusion: In sum, our study presents the first in-depth metastatic functional analysis of the biology of ESR1 mutations in genomic knockin cell models, pointing out the enhanced migration and decreased cell-matrix adhesion as a potential novel gain-of-function of the Y537S and D538G mutant cells in vitro and in vivo. These findings suggest the potential role of enhanced metastasis of these ESR1 mutations through remodeling of transcriptional profiles, shedding lights towards the development of efficient therapies of ESR1 mutant breast cancer.
Citation Format: Zheqi Li, Amir Bahreini, Peilu Wang, Kevin M. Levine, Nilgun Tasdemir, David Chu, Ben H. Park, Adrian V. Lee, Steffi Oesterreich. ESR1 mutations confer novel metastatic functions in genome-edited breast cancer models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A70.