Characterization of the mechanisms of endocrine resistance in luminal A breast cancer cell lines

Background: Endocrine therapy based on Tamoxifen (Tam) is the standard treatment for luminal A breast cancer (BC). Its efficacy, however, is often compromised by acquired resistance, leading to relapse and disease progression. The fine mechanisms accounting for the onset of drug resistance are still only partially understood, which hinders the development of effective strategies to prevent or overcome it. Aims of the study: This research investigates the molecular mechanisms of Tam resistance in luminal A BC, with a specific focus on the roles of the transcription factors TFEB and PPARγ, as well as some of the most important associated metabolic alterations. Results: The transcriptomic profiling of a subclone of MCF7 cells capable of stably growing in the presence of Tam revealed a significant difference in gene expression between parental and resistant cells. In particular, in Tam-resistant cells, TFEB, a master regulator of lysosomal biogenesis and autophagy, was significantly upregulated and predominantly localized in the nucleus. Further investigations demonstrated that Tam actively promotes nuclear translocation of TFEB by triggering lysosomal Ca²⁺ release and the ensuing calcineurin-mediated dephosphorylation of the transcription factor. In keeping with the demonstration of its dysregulation, silencing TFEB in resistant cells reduced their viability and partially restored Tam sensitivity. Analysis of RNA-seq data also evidenced that Tam-resistant cells were characterized by a significant overexpression of several genes regulating lipid metabolism, including PPARγ and several of its target genes. In both parental and Tam-resistant MCF7 cells, PPARγ silencing or pharmacological inhibition with GW9662 or Imatinib reduced cell viability, impaired colony formation, and induced spheroid disaggregation and regression. Moreover, both inhibitors decreased cell viability across all Tam-resistant cell lines tested. Imaging analyses revealed that Tam exposure induces the accumulation of cytoplasmic lipid droplets (CLDs) in both parental and resistant cell lines, largely reverted by drug removal. Whole-exome sequencing of the Tam-resistant cell lines detected many somatic mutations and copy number variations in genes regulating apoptosis, senescence, and lipid metabolism, suggesting that genetic changes largely concur to the development or maintenance of drug resistance. Conclusions: Our findings highlight that multiple mechanisms contribute to the onset of endocrine resistance in luminal A BC. We provided evidence that Tam promotes TFEB activation, eventually responsible for activation of autophagy and the enhanced survival of resistant cells. PPARγ dysregulation emerges as another critical determinant of Tam-resistant cell viability and may underlie the deep modifications observed in the lipid metabolism. Interestingly, the formation and accumulation of CLDs seem to represent a dynamic process depending on the exposure to Tam, rather than a permanent characteristic of Tam-resistant cells. Furthermore, the presence of extensive genomic alterations, including both mutations and copy number variations, underscores the wide remodeling genetic background that takes place during the onset of endocrine resistance. Taken together, these results identify TFEB and PPARγ as both prominent factors contributing to the development of Tam resistance and promising targets for the development of therapeutic strategies to prevent or overcome endocrine resistance in luminal A BC