Purpose We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2–) and, as controls, 115 ER negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer.
Most breast cancers are estrogen receptor α (ER)-positive (+) and are treated with endocrine therapies targeting ER activity. Despite efforts, the mechanisms of the frequent clinical resistance to these therapies remain largely unknown. Several recent parallel studies unveiled gain-of-function recurrent ESR1 mutations in up to 20% of patients with metastatic ER+ disease who all received endocrine therapies, which for more cases included an aromatase inhibitor. These mutations, clustered in a hotspot within the ligand-binding domain (LBD), lead to ligand independent ER activity and tumor growth, partial resistance to tamoxifen and fulvestrant, and potentially increased metastatic capacity. Together, these findings suggest that the ESR1 LBD mutations account for acquired endocrine resistance in a substantial fraction of patients with metastatic disease. The absence of detectable ESR1 mutations in treatment-naïve disease and the correlation with the number of endocrine treatments indicate a clonal expansion of rare mutant clones, selected under the pressure of treatment. New technologies to detect low/ultra rare ESR1 mutations together with tissue and liquid biopsies are required to fully expose their clinical relevance in prognosis and treatment. Pre-clinical and clinical development of rationale-based novel therapeutic strategies to inhibit these mutants has the potential to substantially improve treatment outcomes.
Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.
The ternary complex factors (TCFs) Net, Elk-1 and Sap-1 regulate immediate early genes through serum response elements (SREs) in vitro, but, surprisingly, their in vivo roles are unknown. Net is a repressor that is expressed in sites of vasculogenesis during mouse development. We have made gene-targeted mice that express a hypomorphic mutant of Net, Netd, which lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and up-regulate an immediate early gene implicated in vascular disease, egr-1. They die after birth due to respiratory failure, resulting from the accumulation of chyle in the thoracic cage (chylothorax). The mice have dilated lymphatic vessels (lymphangiectasis) as early as E16.5. Interestingly, they express more egr-1 in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds speci®-cally to SRE-5. Egr-1 has been associated with pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly be associated with obstructions that ultimately affect the lymphatics. These results show that Net is involved in vascular biology and egr-1 regulation in vivo. Keywords: egr-1/Elk-3/ERP/Net/Sap-2 IntroductionThe ternary complex factors (TCFs) form a subfamily of Ets-domain transcription factors. The three TCFs, Elk-1, Sap-1 and Net/Sap-2/Erp/Elk-3 (Price et al., 1996;Wasylyk et al., 1998), have four conserved domains, A±D. A is the Ets DNA-binding domain (DBD). The B-box interacts with the serum response factor (SRF). C is a transcriptional activation domain that is stimulated by mitogen-activated protein (MAP) kinase phosphorylation. The D-domain is a MAP kinase-binding site and a nuclear localization signal. The TCFs are nuclear mediators of cellular responses to the activation of MAP kinase pathways. Net differs from the other TCFs in that in basal conditions, in which MAP kinases are not activated, it strongly inhibits transcription. Repression is mediated by two domains, the NID (Maira et al., 1996) and the CID (Criqui-Filipe et al., 1999). The TCFs form ternary complexes with SRF on serum response elements (SREs) of immediate early gene promoters, such as c-fos, egr-1 and jun-B. The SRE is constitutively occupied by factors, and extracellular signals are thought to lead to both phosphorylation of the complex and changes in its composition due to the exchange of TCFs.The in vivo role of the TCFs is poorly understood. They may regulate the expression of immediate early genes in response to various inductive stimuli. The TCFs are expressed in many cell types and tissues (Giovane et al., 1994;Lopez et al., 1994;Magnaghi-Jaulin et al., 1996;Nozaki et al., 1996;Sgambato et al., 1998), but their precise in vivo expression patterns are not well known. Net is expressed during mouse development at E7.5±8.5 in developing vascular primordia, including the yolk sac blood islands, allantoic vessels, heart endocardium and dorsal aortae (Ayadi et al., 2001). Vascular endothelial cell expression persists ...
Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA’s selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.
The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2-ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.
Summary Breast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival, and is required in models of endocrine-resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for breast cancer patients treated with endocrine therapy. Significance ER is the defining transcription factor of luminal breast tumors, and endocrine agents that target ER are well-established standards of care in breast cancer. However, intrinsic and acquired resistance limits the success of this therapeutic strategy, highlighting the need to identify additional pathways critical for luminal tumor growth and recurrence. Our findings provide evidence that prostate derived ETS factor (PDEF) can drive luminal differentiation of basal mammary epithelial cells, regulate the survival of luminal tumor cells, and contribute to endocrine resistance. These findings suggest that increased PDEF expression may play a role in tumor recurrence following endocrine therapy and may be a clinically useful target for the treatment of patients with luminal breast cancer.
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