Background: Estrogen receptor alpha (ER/ESR1) mutations are found in 20-40% of endocrine resistant ER+ metastatic breast cancers, and they are associated with worse outcome. Preclinical studies have shown that they cause ligand-independent growth, resistance to endocrine therapy, and there is growing evidence for a role in metastasis. It is not known how ESR1 mutant cancer cells cause metastases and whether such a mechanism may indicate novel therapeutic options. Methods: ddPCR was used to detect hotspot ESR1 mutations. Transcriptome data were derived from cell line models and clinical samples. For in vitro phenotypic characterization, Y537S and D538G genome-edited MCF7 and T47D cell models from four different labs were used. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and microfluidic aggregation assays. Altered expression of cell-cell adhesion genes detected in RNA seq data was validated via qRT-PCR, immunoblot and immune staining. Functional contributions of desmosome and gap junctions were tested by blocking peptide and carbenoxolone respectively. Mutant ER cistromes were profiled using ChIP-sequencing. Tail vein injection was performed on nude mice to evaluate metastasis in vivo. Mouse lung micro-metastatic foci were quantified using human-specific CK19 staining. Circulating tumor cells (CTCs) clustering propensity in vivo was assessed via intracardiac injection of ESR1 WT and mutant cells into nude mice following CTC microfilter capture. CTCs enumeration from breast cancer patients’ blood samples was performed using CellSearchTM system. Results: We identified a significant enrichment of ESR1 mutations in distant (12/48) vs local (0/27) recurrences, confirming the strong association of mutant ER with metastasis. Transcriptomic analysis revealed altered cell-to-cell interaction pathways in ESR1 mutant tumors compared to ESR1 WT tumors, suggesting a previously undescribed role of ESR1 mutations in reprogramming cell-cell adhesome. ESR1 mutant cells grown in suspension culture revealed more compact multicellular spheroids compared to WT cells. This observation was confirmed under both static and microfluidic conditions, indicative of increased cell-cell interactions. The effect was more pronounced in MCF7 compared to T47D cells, and it correlated with increased expression of multiple desmosome and gap junction pathway genes, which were also significantly enriched in ESR1 mutant tumors. Pharmacological blockade of desmosome and gap junctions significantly rescued enhanced cell-cell adhesion in ESR1 mutant cells. Mechanistically, our ER ChIP-seq did not identify any gained mutant ER binding sites in proximity to cell-cell adhesion gene loci, indicating indirect regulation by mutant ER. Consistent with this, expression of Connexin 43, one of the top upregulated gap junction components, was induced by cFOS found to be highly upregulated in ESR1 mutant cells. Tail vein injection of ESR1-mutant cells derived more distant macro- (MCF7) and micro- (T47D) metastases. Given increasing evidence for role of cell-cell attachment in CTC phenotypes, we tested CTC formation for ESR1 WT and mutant cells. In vivo studies showed MCF7 Y537S ESR1 mutant cells formed larger multi-cellular CTC clusters with increased compactness compared to WT CTCs. These preclinical data translated to clinical observation, where we observed an enrichment of CTC clusters in patients with ESR1 mutant-metastatic breast cancers. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer increased cell-cell adhesion, which facilitate breast cancer metastasis via increased CTCs clustering propensity. These findings might guide approaches to test potential repurpose of drugs targeting gap junction in ER mutant tumors.

Citation Format: Zheqi Li, Yang Wu, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben H. Park, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Jennifer M. Atkinson, Dorraya El-Ashry, Massimo Cristofanilli, Adrian V. Lee, Steffi Oesterreich. Hotspot ESR1 mutations rewire cell-cell adhesome to facilitate breast cancer metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-05.