The natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells

Background. Metastatic breast cancer (MBC) remains a major clinical challenge, particularly in estrogen receptor α (ERα)-positive patients who develop resistance to endocrine therapy (ET). While hotspot mutations such as Y537S in the ligand-binding domain (LBD) are well-characterized drivers of resistance, other ERα variants remain poorly studied. Understanding the molecular mechanisms underlying resistance in these variants is crucial for identifying novel therapeutic strategies. Here, we investigated the functional role of the L370F and E471D ERα variants, which are spatially close in the ERα structure. Methods. Stable overexpressing HEK293 cells and CRISPR/CAS9 engineered MCF-7 cells were generated and treated with 17β-estradiol (E2), fulvestrant (Ful) and all-trans retinoic acid (ATRA) to measure ERα stability, transcriptional activity and gene expression analyses using different cellular assays and RNASeq techniques. Direct in vitro measurement of ligand binding affinity to ERα were performed using the purified full-length wild type (wt) as well as L370F and Y537S ERα. In silico structural simulations were also performed to predict the structure of the mutated L370F ERα. Senescent analyses of MCF-7 and Y537S MCF-7 cells were performed using direct measurement β-galactosidase activity in vitro and in cell lines. Results The L370F variant conferred resistance to Ful in terms of in vitro ERα binding, ERα transcriptional activity, receptor degradation and cell proliferation by modifying the folding of the receptor structure. Furthermore, L370F-expressing cells exhibited a hyperactive response to low doses of E2 and basally upregulated late estrogen responsive genes. Additionally, we found that both L370F and Y537S ERα variants displayed increased RARα expression, rendering them highly sensitive to ATRA. Notably, ATRA killed L370F-expressing cells and induced senescence in Y537S-expressing cells, highlighting mutation-specific responses. Conclusions Our findings expand the understanding of ERα mutations beyond known hotspots, identifying L370F as a novel mutation contributing to ET resistance and further indicate the necessity to characterize all the less-studied ERα variants found in MBC. Furthermore, we demonstrate that ATRA selectively targets MBC cells harboring L370F and Y537S mutations, suggesting its potential as a mutation-specific therapeutic agent. These results support further investigation of ATRA in clinical settings to improve treatment strategies for ERα-mutant MBC.