Abstract P2-01-09: ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties
Authors: Li Z , Bahreini A , Levine KM , Wang P , Tasdemir N , Montanez MA , Sundd P , Wallace CT , Watkins SC , Chu D , Park BH , Hou W , Mooring MS , Zhu L , Tseng GC , Carroll JS , Atkinson JM , Lee AV , Oesterreich S 
Published: May 2019

Background: Estrogen receptor alpha (ERα/ESR1) is mutated in 30-40% of endocrine resistant ER+ breast cancer. These mutations, primarily located in the ligand binding domain, are associated with worse outcome in patients, and preclinical studies have shown that they cause ligand independent growth. An open question is whether these mutations contribute to actual metastatic process, or merely endocrine resistance.

Methods: Using Y537S and D538G genome-edited MCF7 and T47D cells, 3D growth was assessed in ultralow attachment plates. Cell-cell adhesion was determined using calcein-labelled adhesion assay and quantitative microfluidic fluorescence microscope (qMFM). Collagen-based adhesion and spheroid invasion assays were used to test adhesive and invasive properties. Wound scratching, spheroid collective migration and Boyden chamber transwell assays were applied to monitor cell migratory phenotypes. Mutated ER cistromes were profiled using ChIP-sequencing. ESR1 mutations in clinical samples were characterized using ddPCR.

Results: Visual inspection of cells grown in suspension culture revealed more compressed multicellular spheroids in ESR1 mutant cells, indicative of increased cell-cell interactions. This observation was confirmed in both static and microfluidic conditions. This effect was more pronounced in MCF7 than T47D cells, correlating with increased expression of desmosome and gap junction genes. Pharmacological blockade of gap junctions decreased cell-cell adhesion. Decreased attachment and increased invasion to collagen were discerned in all mutant cell types. Further functional analysis identified alterations in the TIMP3-MMP axis causing these phenotypes. The cell-cell adhesion phenotypes were restricted to MCF7-Y537S/D538G and T47D-Y537S, whereas T47D-D538G cells showed significantly increased migration. A GSEA screen identified Wnt signaling as uniquely induced in this context, and combination treatment using the Wnt inhibitor LGK974 and Fulvestrant led to synergistic inhibition of migration. ChIP-seq identified mutation-specific cistromes with an overall increased ligand-independent ER binding. However, it did not reveal binding sites in any candidate metastases genes, suggesting secondary epigenetic mechanisms. The motif analysis revealed the enrichment of FOXA1 motifs in mutated ER cistromes except T47D-D538G cells. However, knockdown of FOXA1 induced significantly higher inhibition of T47D-D538G migration than Fulvestrant treatment alone, indicating a FOXA1-dominated mechanism. Collectively, these data show that ESR1 mutant cells gain metastatic properties, in addition to endocrine resistance. To prove this using clinical samples, we measured ESR1 mutations in a well-defined cohort of endocrine resistant local or distant recurrence. Significant enrichment of ESR1 mutations in distant (9/55) vs local (0/27) recurrences confirms critical role of mutant ERα in metastases.

Conclusion: Further analysis of context dependent changes in cell-cell adhesion and migration of ESR1 mutant cells might guide the design and development of drugs targeting ERα-mutant tumors, such as inhibitors of gap junction, FOXA1, MMP, and Wnt signaling pathways.

Disclosure: The authors declare no conflict of interest.

Citation Format: Li Z, Bahreini A, Levine KM, Wang P, Tasdemir N, Montanez MA, Sundd P, Wallace CT, Watkins SC, Chu D, Park BH, Hou W, Mooring MS, Zhu L, Tseng GC, Carroll JS, Atkinson JM, Lee AV, Oesterreich S. ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-09.