ERα-positive Breast Cancer Cells Research Articles

A Virtual Drug Discovery Screening Illuminates Campesterol as a Potent Estrogen Receptor Alpha Inhibitor in Breast Cancer.

Breast cancer remains a global health challenge, and innovative strategies are required to target estrogen receptor α (ERα), a key player in its development. This study investigates the potential of campesterol, a natural phytosterol, as an ERα inhibitor for breast cancer. Our approach integrates in silico, in vitro, and ex vivo experiments to assess the therapeutic potential of campesterol. In silico analyses highlight campesterol as a promising ERα ligand with favorable binding affinities and dynamic properties. Structural analysis reveals conformational changes in ERα upon campesterol binding. In vitro studies confirm the selective growth inhibition of campesterol against ERα-positive breast cancer cells. This study extends to ER+ breast cancer patient-derived organoids (PDOs), showing the effectiveness of campesterol in ERα-positive breast cancer PDOs. Importantly, it emphasizes the receptor-specific nature of campesterol, providing insights into its context-dependent action. In conclusion, campesterol displays potential as an ERα inhibitor, offering new avenues for ER+ breast cancer treatment.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-03.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

Abstract 6000: A novel pathway controlling anticancer drug induced immunogenic cell death

Abstract Acting through estrogen receptor alpha (ERα), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO induces necrosis was unknown. From a genome-wide CRISPR-Cas9 screen in MCF-7 cells with negative selection against ErSO, we identified FGD3, a guanine exchange factor (GEF) of Rho GTPase Cdc42 as the top target. Consistent with the screen, through subsequent knockout and overexpression of FGD3 in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO in both 2D cell culture and a 3D organoid model. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified an actin reorganization signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its actin enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating actin reorganization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Notably, FGD3 knockout cells also show considerable resistance to necrosis or necroptosis induced by other anticancer drugs. Here, we describe a therapy that induces immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We also show that, as cancer cells respond to agents that induce immunogenic cell death through membrane rupture, an actin regulating pathway plays a critical role in life-death decisions. Citation Format: Junyao Zhu, Santanu Ghosh, Darjan Duraki, Matthew Boudreau, Musarrat Jabeen, Chengjian Mao, Ben H. Park, Georgina Cheng, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. A novel pathway controlling anticancer drug induced immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6000.

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

Spinosyn A exerts anti-tumorigenic effects on progesterone-sensitive ERα-positive breast cancer cells by modulating multiple signaling pathways

Breast cancer is one of the most common and deadly cancers in women worldwide. Current treatments for breast cancer have limitations, such as toxicity, resistance, and side effects. Therefore, there is a need to develop new and effective anti-cancer agents from natural sources. Spinosyn A (SPA) is a natural product derived from soil bacteria. SPA has been reported to have anti-parasitic, insecticidal, and anti-bacterial activities. However, its anti-cancer effects and mechanisms are not well understood. In this study, we investigated the effects of SPA on T47-D, estrogen receptor-positive breast cancer cells. We found that SPA inhibited cell proliferation and migration and induced apoptosis and cell cycle arrest. Flow cytometry and holographic imaging microscopy revealed that SPA activated MAPK and PI3K signaling pathways and altered cellular morphology. Finally, RNA-Seq analysis revealed that SPA treatment altered the expression of 1380 genes in T47-D cells, which were enriched in various biological processes and signaling pathways related to cell proliferation, cholesterol metabolism, growth factor activity, amino acid transport activity, extracellular matrix, PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, and PPAR signaling pathway. Our results suggest that SPA exerts multiple anti-cancer effects on T47-D cells by modulating multiple pathways and cellular processes involved in cell growth, survival, and motility. Our findings provide new insights into the molecular mechanisms of SPA action on breast cancer cells and its potential applications as a novel anti-cancer agent.

The effect of DNA methyltransferase 3A suppression in progression of the resistance phenotype in breast cancer cells

Introduction. Rearrangement of molecular pathways and activation of bypass signaling determine the progression of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential for breast and other cancer beneficial treatments.Materials and methods. The present work was performed on estrogen receptor α ERα-positive (ERα – estrogen receptor α) McF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERα-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing.Results. We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression of cell resistance.Conclusion. Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.

RL2 Enhances the Elimination of Breast Cancer Cells by Doxorubicin.

RL2 (recombinant lactaptin 2), a recombinant analogon of the human milk protein Κ-Casein, induces mitophagy and cell death in breast carcinoma cells. Furthermore, RL2 was shown to enhance extrinsic apoptosis upon long-term treatment while inhibiting it upon short-term stimulation. However, the effects of RL2 on the action of chemotherapeutic drugs that induce the intrinsic apoptotic pathway have not been investigated to date. Here, we examined the effects of RL2 on the doxorubicin (DXR)-induced cell death in breast cancer cells with three different backgrounds. In particular, we used BT549 and MDA-MB-231 triple-negative breast cancer (TNBC) cells, T47D estrogen receptor alpha (ERα) positive cells, and SKBR3 human epidermal growth factor receptor 2 (HER2) positive cells. BT549, MDA-MB-231, and T47D cells showed a severe loss of cell viability upon RL2 treatment, accompanied by the induction of mitophagy. Furthermore, BT549, MDA-MB-231, and T47D cells could be sensitized towards DXR treatment with RL2, as evidenced by loss of cell viability. In contrast, SKBR3 cells showed almost no RL2-induced loss of cell viability when treated with RL2 alone, and RL2 did not sensitize SKBR3 cells towards DXR-mediated loss of cell viability. Bioinformatic analysis of gene expression showed an enrichment of genes controlling metabolism in SKBR3 cells compared to the other cell lines. This suggests that the metabolic status of the cells is important for their sensitivity to RL2. Taken together, we have shown that RL2 can enhance the intrinsic apoptotic pathway in TNBC and ERα-positive breast cancer cells, paving the way for the development of novel therapeutic strategies.

Abstract LB_A24: Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways

Abstract The anti-tumor properties of baicalein, a flavonoid compound, have been extensively documented. However, the specific effects of baicalein on ER𝛼 positive breast cancer cell lines, namely T47-D and MCF-7, are less well-known. In this study, we conducted comprehensive in vitro experiments to elucidate the comparative impacts of baicalein on these two cell lines. Our results demonstrate that baicalein exerts robust anti-cancer effects, inducing morphological changes, reducing colony formation, and inhibiting cell proliferation in both T47-D and MCF-7 cells. These effects were more pronounced at higher concentrations. Additionally, baicalein treatment impeded the migratory capabilities of both cell lines, as shown by wound healing assays. Furthermore, our data show that baicalein triggers apoptosis, leading to an increase in early apoptotic, late apoptotic, and dead cells. This apoptotic process appears to be caspase 3/7-dependent. Interestingly, the pro-apoptotic effects were more potent in T47-D cells, accompanied by significant upregulation of pro-apoptotic genes and downregulation of anti-apoptotic markers. Baicalein treatment also induced oxidative stress and DNA damage, evidenced by an increase in reactive oxygen species (ROS) and phosphorylation of ATM and H2A.X. Importantly, baicalein inhibited the PI3K signaling pathway, a key mediator of cancer progression. Genome-wide transcriptomic analysis revealed significant changes in gene expression patterns, with baicalein treatment causing both upregulation and downregulation of several genes. Pathway analysis revealed that cell cycle and ferroptosis pathways were activated in both cell lines upon baicalein treatment. In conclusion, our findings highlight the potential of baicalein as a therapeutic agent against breast cancer, while also revealing nuanced differences in response between the T47-D and MCF-7 breast cancer cell lines. Citation Format: Besa Xhabija, Aidan J. McLaughlin, Ibiere Lovelyn Epelle, Tristan H. Nielson, Elisabeta Ujeniuc, Mariam I. Duhaini. Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A24.

Methylation of ESR1 promoter induced by SNAI2-DNMT3B complex promotes epithelial-mesenchymal transition and correlates with poor prognosis in ERα-positive breast cancers.

Estrogen receptor α (ERα) serves as an essential therapeutic predictor for breast cancer (BC) patients and is regulated by epigenetic modification. Abnormal methylation of cytosine phosphoric acid guanine islands in the estrogen receptor 1 (ESR1) gene promoter could silence or decrease ERα expression. In ERα-negative BC, we previously found snail family transcriptional repressor 2 (SNAI2), a zinc-finger transcriptional factor, recruited lysine-specific demethylase 1 to the promoter to transcriptionally suppress ERα expression by demethylating histone H3 lysine 4 dimethylation (H3K4me2). However, the role of SNAI2 in ERα-positive BC remains elusive. In this study, we observed a positive correlation between SNAI2 and ESR1 methylation, and SNAI2 promoted ESR1 methylation by recruiting DNA methyltransferase 3 beta (DNMT3B) rather than DNA methyltransferase 1 (DNMT1) in ERα-positive BC cells. Subsequent enrichment analysis illustrated that ESR1 methylation is strongly correlated with cell adhesion and junction. Knocking down DNMT3B could partially reverse SNAI2 overexpression-induced cell proliferation, migration, and invasion. Moreover, high DNMT3B expression predicted poor relapse-free survival and overall survival in ERα-positive BC patients. In conclusion, this study demonstrated the novel mechanisms of the ESR1 methylation mediated with the SNAI2/DNMT3B complex and enhanced awareness of ESR1 methylation's role in promoting epithelial-mesenchymal transition in BC.

OR16-06 A Novel Pathway Controlling Anticancer Drug Induced Immunogenic Necrosis In ERα Positive Cancer Cells

Abstract Disclosure: J. Zhu: None. S. Ghosh: None. D. Duraki: None. M. Boudreau: Other; Self; Patent on ErSO. M. Jabeen: None. C. Mao: None. B. Park: None. G. Cheng: None. E.R. Nelson: None. P.J. Hergenrother: Advisory Board Member; Self; System Oncology. Other; Self; Patent on ErSO. D.J. Shapiro: Other; Self; Patent on ErSO. Acting through estrogen receptor alpha (ER), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through ERα dependent sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture and necrotic cell death. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO-induced necrosis was elusive. From a genome-wide CRISPR-Cas9 screen in MCF7 cells with negative selection against ErSO, we identified a guanine exchange factor (GEF) protein as a top target. Consistent with the screen, through subsequent knockout and overexpression of this protein in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified a cytoskeleton-modifying signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its cytoskeleton enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating cytoskeleton organization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. Unlike wild type and overexpressing cells, the knockout cells and persister cells display the persister cell phenotype - rapid re-growth of the cancer cells once ErSO is removed. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Here, we describe a therapy that induces necrosis and immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We show that, as cancer cells respond to this unique therapeutic approach, a cytoskeleton regulating pathway plays a critical role in life-death decisions. Presentation: Friday, June 16, 2023

Molecular characterization of breast cancer cell pools with normal or reduced ability to respond to progesterone: a study based on RNA-seq

BackgroundAbout one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood. MethodsWe disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses. ResultsThe overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells. ConclusionsThe present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis.

Proliferative response of ERα-positive breast cancer cells to 10 μM enterolactone, and the associated alteration in the transcriptomic landscape

Enterolactone (EL) is a product of gut-microbial metabolism of dietary plant lignans. Studies linking EL with breast cancer risk have bolstered investigations into its effects on the mammary epithelial cells, and the mechanisms thereof. While it binds to the estrogen receptor α (ERα), its effect on the proliferation of mammary tumor cell lines is reportedly ambivalent; depending on its concentration. The genomic correlates of EL actions also remain unexplored. Here we have elaborately studied the effect of EL on proliferation of ERα-positive, and ERα-negative cell lines. 10 µM EL significantly enhanced the growth of the ERα-positive MCF-7 or T47D breast cancer cells, but not the ERα-negative MDA-MB-231 or MDA-MB-453 cells. In MCF-7 cells, it significantly increased the expression of TFF1 mRNA, an estrogen-induced transcript. The binding of ERα to the estrogen response element within the TFF1 locus further demonstrated the pro-estrogenic effect of 10 µM EL. We further explored the genome-wide transcriptomic effect of 10 µM EL using the next generation sequencing technology (RNA-seq). Analysis of RNA-seq data obtained from vehicle (0.1% DMSO)- or 10 µM EL-treated MCF-7 cells revealed modulation of expression of diverse sets of functionally related genes, which reflected cell cycle progression. The manner in which 10 µM EL regulated the hallmark G2/M checkpoint, and estrogen-response-late genes correlated with proliferation inducing, and estrogen-like effects of EL on MCF-7 cells.

ERα/LKB1 complex upregulates E-cadherin expression and stimulates breast cancer growth and progression upon adiponectin exposure.

Adiponectin is the major adipocytes-secreted protein involved in obesity-related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα-positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα-coactivator. Here, we showed that adiponectin-mediated ERα transactivation enhances E-cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E-cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E-cadherin expression in ERα-positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E-cadherin gene promoter by ERα/LKB1-complex. The impact of E-cadherin on ERα-positive breast cancer cell proliferation comes from the evidence that in the presence of E-cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E-cadherin connects cell polarity and growth, we investigated if the adiponectin-enhanced E-cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin-treated MCF-7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF-7 cells revealed an enhanced E-cadherin-mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF-7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin-treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E-cadherin expression, alters cell polarity and stimulates ERα-positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.

ERα prevents tumorigenesis of both liver and breast cancer cells through CCN5

Estrogen receptor alpha (ERα)-mediated estrogen signaling has also shown to prevent hepatic tumorigenesis in mice. Consistent with this, hormone replacement therapy with estrogen supplementation dramatically reduced the risk of hepatocellular carcinoma. Silencing of ERα is also a key event for the transformation of ERα-positive breast cancer cells into malignant triple-negative breast cancer cells. However, the mechanisms underlying ERα-mediated prevention of both hepatic and mammary tumorigenesis in humans are still unclear. Here, we present a functional genomics study of ERα targeting by comparing human liver cancer cells with human breast cancer cells using “loss or gain of function” genetic assays of ERα in vitro and in vivo. We discover that cellular communication network factor 5 (CCN5) is a direct downstream target of ERα; ERα suppresses growth and prevents tumorigenesis and malignant transformation of both liver and breast cancer cells through CCN5 in humans. The ERα-CCN5 regulatory axis functions as suppressors for both hepatic and mammary tumors, which is a common mechanism of preventing tumorigenesis for both liver cancer and breast cancer in humans.

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17β-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17β-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

CircPVT1 promotes ER-positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS.

The molecular mechanisms underlying estrogen receptor (ER)-positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα-positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα-positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR-181a-2-3p, promoting the expression of ESR1 and downstream ERα-target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI-MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti-tumor immunity. Anti-sense oligonucleotide (ASO)-targeting circPVT1 inhibits ERα-positive breast cancer cell and tumor growth, re-sensitizing tamoxifen-resistant ERα-positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα-positive breast cancer in the clinic.

Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer.

Breast cancer is a major cause of cancer-related morbidity and mortality in women. Estrogen receptor-positive breast cancer accounts for roughly 70%-80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα-positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48-specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post-translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα-positive breast cancer.