Abstract
Naturally-occurring somatic mutations in the estrogen receptor gene (ESR1) have been previously implicated in the clinical development of resistance to hormonal therapies, such as Tamoxifen. For example, the somatic mutation Y537S has been specifically associated with acquired endocrine resistance. Briefly, we recombinantly-transduced MCF7 cells with a lentiviral vector encoding ESR1 (Y537S). As a first step, we confirmed that MCF7-Y537S cells are indeed functionally resistant to Tamoxifen, as compared with vector alone controls. Importantly, further phenotypic characterization of Y537S cells revealed that they show increased resistance to Tamoxifen-induced apoptosis, allowing them to form mammospheres with higher efficiency, in the presence of Tamoxifen. Similarly, Y537S cells had elevated basal levels of ALDH activity, a marker of “stemness”, which was also Tamoxifen-resistant. Metabolic flux analysis of Y537S cells revealed a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis. Finally, to understand which molecular signaling pathways that may be hyper-activated in Y537S cells, we performed unbiased label-free proteomics analysis. Our results indicate that TIGAR over-expression and the Rho-GDI/PTEN signaling pathway appear to be selectively activated by the Y537S mutation. Remarkably, this profile is nearly identical in MCF7-TAMR cells; these cells were independently-generated in vitro, suggesting a highly conserved mechanism underlying Tamoxifen-resistance. Importantly, we show that the Y537S mutation is specifically associated with the over-expression of a number of protein markers of poor clinical outcome (COL6A3, ERBB2, STAT3, AFP, TFF1, CDK4 and CD44). In summary, we have uncovered a novel metabolic mechanism leading to endocrine resistance, which may have important clinical implications for improving patient outcomes.
Highlights
In human breast cancer patients, the hormone estrogen and its main receptor (ESR1), are key drivers of tumor initiation, cancer progression and metastasis [1,2,3,4]
Our results indicate that TIGAR over‐expression and the Rho‐GDI/PTEN signaling pathway appear to be selectively activated by the estrogen receptor point mutation (Y537S) mutation
MCF7 cells were transduced with a lentiviral vector carrying the Y537S mutation of estrogen receptor alpha or estrogen receptor 1 (ESR1) and positive “pools” of cells were selected, using a puromycin resistance cassette
Summary
In human breast cancer patients, the hormone estrogen and its main receptor (ESR1), are key drivers of tumor initiation, cancer progression and metastasis [1,2,3,4]. As a consequence, targeted therapies, such as Tamoxifen, were first developed to inhibit estrogen receptor signaling, in ER(+) breast cancer cells [5]. Endocrine therapy fails in a significant number of patients on long-term anti-estrogen therapy, due to the acquired emergence of drug-resistance. The resulting treatment failure often has dire consequences for the patient, with the emergence of tumor recurrence and distant metastasis, resulting in poor clinical outcomes. If we are going to successfully prevent or reverse treatment failure, we need to know the underlying mechanism(s) by which ER(+) tumor cells can successfully escape the effects of hormonal therapy. The prominent role of somatic mutations in the estrogen receptor, in conferring resistance to endocrine therapy, has remained largely unappreciated [4, 9]
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