Treatment Of ERα-positive Breast Cancers Research Articles

Exploring the Spatial Landscape of the Estrogen Receptor Proximal Proteome With Antibody-Based Proximity Labeling

Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.

Assessing Estrogenic Activity of Classical Estrogen Receptor-Binding Compounds.

The classical estrogen receptor α (ERα) has been a clinical therapeutic target for decades. ERα-targeted drugs have shown great clinical success, in particular as antagonists for the treatment of ERα-positive breast cancers. However, ERα-targeted agonists have also been clinically useful (e.g., for the treatment of osteoporosis). The breast cancer field is regularly identifying novel ERα-binding compounds with the goal of identifying new potential ERα-targeted therapeutics. To determine whether such newly identified ERα-binding compounds have clinical potential, it is important to characterize the estrogenic activity (i.e., both receptor-mediated agonism and/or antagonism) of these compounds. This chapter focuses on methods that allow determination of whether an ERα-binding compound acts as an agonist or antagonist of the receptor and whether the compound induces degradation of the receptor.

ERα-targeted endocrine therapy, resistance and the role of GPER

Endocrine therapy is an effective option for the treatment of estrogen receptor alpha (ERα)-positive breast cancers. Unfortunately, a large fraction of women relapse with endocrine-resistant tumors. The presence of constitutively active ERα mutants, found in a subset of relapse tumors, is thought to be an important endocrine resistance mechanism and has prompted the search for more effective anti-hormone drugs that can effectively inhibit these mutant versions of the receptor. The G protein-coupled estrogen receptor (GPER) is also thought to contribute to the development of endocrine resistance, in part, due to its activation by clinically used selective estrogen receptor modulators and downregulators (SERMs/SERDs). Therefore, next-generation drugs should be screened for potential activity towards GPER. Here, we highlight the need for truly ERα-selective SERMs and SERDs that do not cross-react with GPER for the treatment of ERα-positive breast cancers.

Discovery of novel natural compound inhibitors targeting estrogen receptor α by an integrated virtual screening strategy.

Estrogen receptor (ER) is a nuclear hormone receptor and plays an important role in mediating the cellular effects of estrogen. ER can be classified into two receptors: estrogen receptor alpha (ERα) and beta (ERβ), and the former is expressed in 50~80% of breast tumors and has been extensively investigated in breast cancer for decades. Excessive exposure to estrogen can obviously stimulate the growth of breast cancers primarily mediated by ERα, and thus anti-estrogen therapies by small molecules are of concern to clinicians and pharmaceutical industry in the treatment of ERα-positive breast cancers. Although a series of estrogen receptor modulators have been developed, these drugs can lead to resistance and side effects. Therefore, the development of small molecule inhibitors with high target specificity has been intensified. In this pursuit, an integrated computer-aided virtual screening technique, including molecular docking and pharmacophore model screening, was used to screen traditional Chinese medicine (TCM) databases. The compounds with high docking scores and fit values were subjected to ADME (adsorption, distribution, metabolism, excretion) and toxicity prediction, and ten hits were identified as potential inhibitors targeting ERα. Molecular docking was used to investigate the binding modes between ERα and three most potent hits, and molecular dynamic simulations were chosen to explore the stability of these complexes. The rank of the predicted binding free energies evaluated by MM/GBSA is consistent with the docking score. These novel scaffolds discovered in the present study can be used as critical starting point in the drug discovery process for treating ERα-positive breast cancer. Graphical abstract .

Abstract B255: Development of hybrid small molecules that induce degradation of estrogen receptor-alpha and necrotic cell death in breast cancer cells.

Abstract Breast cancer is the leading cause of cancer death in women. Most of the primary breast cancers express estrogen receptor α (ERα) and they show estrogen-dependent proliferation. Since tamoxifen antagonizes estrogen in breast cancers, it is widely used to treat the primary breast cancers. However, tamoxifen also shows a partial agonistic activity in different tissues and cellular context, which sometimes causes a detrimental effect. An alternative strategy to kill the estrogen signaling is to downregulate ERα protein. We have developed a protein knockdown system to induce degradation of target proteins via the ubiquitin-proteasome system (UPS) in cells. The molecules for protein knockdown, which we named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser), are composed of two different ligands connected by a linker; one is a ligand for cellular inhibitor of apoptosis protein 1 (cIAP1) and the other is a ligand for a target protein. Accordingly, SNIPER is expected to crosslink a ubiquitin ligase cIAP1 and the target protein in the cells, thereby inducing the cIAP1-mediated ubiquitylation and proteasomal degradation of the target protein. By using tamoxifen as a ligand for ERα, we developed novel SNIPER to knockdown ERα protein. SNIPER(ER) induced degradation of ERα and inhibited estrogen-dependent expression of pS2 gene in an estrogen-dependent breast cancer MCF-7 cells. Intriguingly, after the ERα degradation, the SNIPER(ER)-treated MCF-7 cells undergo rapid cell death. Detailed analysis indicated that SNIPER(ER) caused necrotic cell death accompanied by a release of HMGB1, and reactive oxygen species (ROS) mediate the necrotic cell death in the SNIPER(ER)-treated MCF-7 cells. These results indicate that SNIPER(ER) induces ERα degradation, ROS production and necrotic cell death, implying a therapeutic potential of SNIPER(ER) as a lead for the treatment of ERα-positive breast cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B255. Citation Format: Keiichiro Okuhira, Yosuke Demizu, Takayuki Hattori, Nobumichi Ohoka, Norihito Shibata, Tomoko Nishimaki-Mogami, Haruhiro Okuda, Masaaki Kurihara, Mikihiko Naito. Development of hybrid small molecules that induce degradation of estrogen receptor-alpha and necrotic cell death in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B255.

Development of hybrid small molecules that induce degradation of estrogen receptor‐alpha and necrotic cell death in breast cancer cells

Manipulation of protein stability with small molecules has a great potential for both basic research and clinical therapy. Recently, we have developed a series of hybrid small molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser) that induces degradation of target proteins via ubiquitin-proteasome system. Here we report the activities of SNIPER(ER) that targets estrogen receptor alpha (ERα) for degradation. SNIPER(ER) induced degradation of ERα and inhibited estrogen-dependent expression of pS2 gene in an estrogen-dependent breast cancer cell line MCF-7. A proteasome inhibitor MG132 and siRNA-mediated downregulation of cIAP1 abrogated the SNIPER(ER)-induced ERα degradation, suggesting that the ERα is degraded by proteasome subsequent to cIAP1-mediated ubiquitylation. Intriguingly, after the ERα degradation, the SNIPER(ER)-treated MCF-7 cells undergo rapid cell death. Detailed analysis indicated that SNIPER(ER) caused necrotic cell death accompanied by a release of HMGB1, a marker of necrosis, from the cells. Following the ERα degradation, reactive oxygen species (ROS) was produced in the SNIPER(ER)-treated MCF-7 cells, and an anti-oxidant N-acetylcysteine inhibited the necrotic cell death. These results indicate that SNIPER(ER) induces ERα degradation, ROS production and necrotic cell death, implying a therapeutic potential of SNIPER(ER) as a lead for the treatment of ERα-positive breast cancers.

Biological reprogramming in acquired resistance to endocrine therapy of breast cancer

Endocrine therapies targeting the proliferative effect of 17β-estradiol through estrogen receptor α (ERα) are the most effective systemic treatment of ERα-positive breast cancer. However, most breast tumors initially responsive to these therapies develop resistance through molecular mechanisms that are not yet fully understood. The long-term estrogen-deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibitors in postmenopausal women. To elucidate this resistance, genomic, transcriptomic and molecular data were integrated into the time course of MCF7-LTED adaptation. Dynamic and widespread genomic changes were observed, including amplification of the ESR1 locus consequently linked to an increase in ERα. Dynamic transcriptomic profiles were also observed that correlated significantly with genomic changes and were predicted to be influenced by transcription factors known to be involved in acquired resistance or cell proliferation (for example, interferon regulatory transcription factor 1 and E2F1, respectively) but, notably, not by canonical ERα transcriptional function. Consistently, at the molecular level, activation of growth factor signaling pathways by EGFR/ERBB/AKT and a switch from phospho-Ser118 (pS118)- to pS167-ERα were observed during MCF7-LTED adaptation. Evaluation of relevant clinical settings identified significant associations between MCF7-LTED and breast tumor transcriptome profiles that characterize ERα-negative status, early response to letrozole and tamoxifen, and recurrence after tamoxifen treatment. In accordance with these profiles, MCF7-LTED cells showed increased sensitivity to inhibition of FGFR-mediated signaling with PD173074. This study provides mechanistic insight into acquired resistance to endocrine therapies of breast cancer and highlights a potential therapeutic strategy.

Abstract A34: A mechanism for acquired resistance to estrogen deprivation in breast cancer cells

Abstract Endocrine therapies that target the proliferative effect of 17β-estradiol (17βE2) through estrogen receptor α (ERα) are the most effective form of systemic treatment of ERα-positive breast cancer. Nevertheless, through mechanisms that are not fully understood, most tumors that initially respond to therapy will recur. The long-term estrogen deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibition (AI) in postmenopausal women. To comprehensively elucidate this resistance, genomic, transcriptomic and molecular analyses were integrated into the time course of MCF7-LTED adaptation. Extensive and dynamic genomic changes were observed, including amplification of the ESR1 locus linked to an increase in ERα and its non-genomic role. Dynamic transcriptomic profiles were also observed that significantly correlated with genomic changes and that were influenced by transcription factors known to be involved in acquired resistance (IRF-1) and cell proliferation (E2F1) but not by canonical ERα genomic function. Evaluation of MCF7-LTED profiles association with pathological and clinical data shown consistent correlations with ERα-negative tumor status, letrozole effects and recurrence after tamoxifen treatment. This study describes the coordinated genomic, transcriptomic and molecular changes that mediate acquired resistance to endocrine therapies, suggesting a key role for the non-genomic function of ERα. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A34

Changes in subcellular localisation of MI-ER1α, a novel oestrogen receptor-α interacting protein, is associated with breast cancer progression

The oestrogen receptor-α (ERα) plays a key role in breast development and tumorigenesis and inhibiting its activity remains a prime strategy in the treatment of ERα-positive breast cancers. Thus, elucidation of the molecular mechanisms responsible for regulating ERα activity may facilitate the design of new, more effective breast cancer therapies. The MI-ER1α is a novel transcriptional repressor that contains an LXXLL motif for interaction with nuclear hormone receptors. We investigated the ability of MI-ER1α to bind to ERα in HEK293 and MCF-7 breast carcinoma cells, using co-immunoprecipitation assays. In both cell lines, MI-ER1α interacted with ERα in the presence and absence of oestrogen, but the interaction was stronger in the absence of ligand. Functional analysis revealed that overexpression of MI-ER1α in T47D breast carcinoma cells results in inhibition of oestrogen-stimulated anchorage-independent growth, suggesting that MI-ER1α may play a role in regulating breast carcinoma cell proliferation in vivo. To explore this further, we performed an immunohistochemical analysis of normal breast tissue and breast carcinoma; a total of 110 cases were examined in whole tissue sections and 771 cases were analysed in tissue microarrays. No consistent difference in the MI-ER1α expression level between normal breast tissue and breast carcinoma was discernible. However, there was a dramatic shift in the subcellular localisation: nuclear MI-ER1α was detectable in 75% of normal breast samples and in 77% of hyperplasia, but in breast carcinoma, only 51% of DCIS, 25% of ILC and 4% of IDC contained nuclear staining. This shift from nuclear to cytoplasmic localisation of MI-ER1α during breast cancer progression suggests that loss of nuclear MI-ER1α might contribute to the development of invasive breast carcinoma.