Abstract PD8-08: ESR1 mutations confer novel metastatic functions in genome-edited breast cancer cell models
Authors: Oesterreich S , Li Z , Bahreini A , Wang P , Levine KM , Tasdemir N , Chu D , Park BH , Lee AV 
Published: February 2018

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). We subsequently performed an RNA-sequencing to deeply differentiate the gene expression patterns in these mutants. The growth of these pooled mutant-cells was determined in both 2D and 3D 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 transwell assays were used to monitor the cell migration and chemotaxis. Tail vein injection were performed on nude mice, and immunofluorescent staining of lung 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 exhibits multiple ligand-independent genes overlapping between either cell lines or mutants, which were further conformed by qPCR. We also found that both Y537S and D538G mutants present ligand-independent growth in 2D and 3D ultra-low 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 mutant-cells in nude mice derived more micrometatsatic spots in the lung tissues.

Conclusion: In sum, our study presents the first in-depth metastatic functional analysis of the biology of ESR1 mutations in genomic knock-in 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: Oesterreich S, Li Z, Bahreini A, Wang P, Levine KM, Tasdemir N, Chu D, Park BH, Lee AV. ESR1 mutations confer novel metastatic functions in genome-edited breast cancer cell models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-08.