ERα-positive Breast Cancer Research Articles

Perfluorooctanoic Acid (in the Presence of Fetal Bovine Serum) Induces Proliferation in ERα Positive and ERα Negative Breast Cancer Cell Lines

Perfluorooctanoic acid (PFOA) is a synthetic chemical belonging to a larger group of fluorotelomers. These compounds have been used in the production of both industrial and consumer products as surfactants and due to their chemical nature are environmentally persistent pollutants. Individuals can be exposed to PFOA through ingesting PFOA‐contaminated water or food. PFOA has been shown to have a half‐life of approximately 4 years in humans. While the long‐term effects of PFOA are largely unknown, there is increasing evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates peroxisome‐proliferator‐activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα).Previous experiments in our lab demonstrated that PFOA treatment of MCF‐7 breast cancer cells (an ERα positive cell line) decreased expression of ERα mRNA and protein, and decreased cell viability by ~20% within 48h of treatment compared to DMSO controls. However, these cells were treated in the absence of fetal bovine serum (FBS). When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF‐7 cells treated with 50μM and 100μM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in this PFOA‐induced proliferation, we carried out additional experiments in MCF‐7 cells along with experiments in another ERα‐positive cell line, T47D, as well as an ERα‐negative cell line, MDA‐MB‐231. There was a tendency for increased viability in all three cell lines when treated with PFOA, although only the MDA‐MB‐231 cells and the T47D cells showed a statistically significant (~20%) increase at 48h with 50μM PFOA and 100μM PFOA, respectively. These data suggest that the PFOA‐induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF‐7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.Support or Funding InformationAugusta University Department of Biological Sciences, Augusta University Center for Undergraduate Research and Scholarship

The promyelocytic leukemia protein isoform PML1 is an oncoprotein and a direct target of the antioxidant sulforaphane (SFN)

The gene encoding promyelocytic leukemia protein (PML) generates several spliced isoforms. Ectopic expression of PML1 promotes the proliferation of ERα-positive MCF-7 breast cancer (BC) cells, while a loss of PML by knockdown or overexpression of PML4 does the opposite. PML is an essential constituent of highly dynamic particles called PML nuclear bodies (NBs). PML NBs are heterogenous multiprotein subnuclear structures that are part of cellular stress sensing machinery. The antioxidant sulforaphane (SFN) inhibits the proliferation of BC cells and causes a redistribution of the subcellular localization of PML, a disruption of disulfide-bond linkages in nuclear PML-containing complexes, and a reduction in the number and size of PML NBs. Mechanistically, SFN modifies several cysteine residues, including C204, located in the RBCC domain of PML. PML is sumoylated and contains a Sumo-interacting motif, and a significant fraction of Sumo1 and Sumo2/3 co-localizes with PML NBs. Ectopic expression of the mutant C204A selectively inhibits the biogenesis of endogenous PML NBs but not PML-less Sumo1-, Sumo2/3, or Daxx-containing nuclear speckles. Importantly, PML1 (C204A) functions as a dominant-negative mutant over endogenous PML protein and promotes anti-proliferation activity. Together, we conclude that SFN elicits its cytotoxic activity in part by inactivating PML1's pro-tumorigenic activity.

Interfering Role of ERα on Adiponectin Action in Breast Cancer.

Obesity is characterized by an excess of adipose tissue, due to adipocyte hypertrophy and hyperplasia. Adipose tissue is an endocrine organ producing many bioactive molecules, called adipokines. During obesity, dysfunctional adipocytes alter adipokine secretion, contributing to pathophysiology of obesity-associated diseases, including metabolic syndrome, type 2-diabetes, cardiovascular diseases and many types of malignancies. Circulating adiponectin levels are inversely correlated with BMI, thus adiponectin concentrations are lower in obese than normal-weight subjects. Many clinical investigations highlight that low adiponectin levels represent a serious risk factor in breast carcinogenesis, and are associated with the development of more aggressive phenotype. A large-scale meta-analysis suggests that BMI was positively associated with breast cancer mortality in women with ERα-positive disease, regardless menopausal status. This suggests the importance of estrogen signaling contribution in breast tumorigenesis of obese patients. It has been largely demonstrated that adiponectin exerts a protective role in ERα-negative cells, promoting anti-proliferative and pro-apoptotic effects, while controversial data have been reported in ERα-positive cells. Indeed, emerging data provide evidences that adiponectin in obese patients behave as growth factor in ERα-positive breast cancer cells. This addresses how ERα signaling interference may enhance the potential inhibitory threshold of adiponectin in ERα-positive cells. Thus, we may reasonably speculate that the relatively low adiponectin concentrations could be still not adequate to elicit, in ERα-positive breast cancer cells, the same inhibitory effects observed in ERα-negative cells. In the present review we will focus on the molecular mechanisms through which adiponectin affects breast cancer cell behavior in relationship to ERα expression.

Novel insights into adiponectin action in breast cancer: Evidence of its mechanistic effects mediated by ERα expression.

This review describes the multifaceted effects of adiponectin on breast cancer cell signalling, tumour metabolism, and microenvironment. It is largely documented that low adiponectin levels are associated with an increased risk of breast cancer. However, it needs to be still clarified what are the extents of the decrease of local/intra-tumoural adiponectin concentrations, which promote breast tumour malignancy. Most of the anti-proliferative and pro-apoptotic effects induced by adiponectin have been obtained in breast cancer cells not expressing estrogen receptor alpha (ERα). Here, we will highlight recent findings demonstrating the mechanistic effects through which adiponectin is able to fuel genomic and non-genomic estrogen signalling, inhibiting LKB1/AMPK/mTOR/S6K pathway and switching energy balance. Therefore, it emerges that the reduced adiponectin levels in patients with obesity work to sustain tumour growth and progression in ERα-positive breast cancer cells. All this may contribute to remove the misleading paradigm that adiponectin univocally inhibits breast cancer cell growth and progression independently on ERα status. The latter concept, here clearly provided by pre-clinical studies, may have translational relevance adopting adiponectin as a potential therapeutic tool. Indeed, the interfering role of ERα on adiponectin action addresses how a separate assessment of adiponectin treatment needs to be considered in novel therapeutic strategies for ERα-positive and ERα-negative breast cancer.

Abstract P5-05-16: Quantitative regression of estrogen receptor alpha positive breast cancer

Abstract Treatment of advanced ERα-positive breast cancer remains a major clinical challenge, as the standard therapeutics are mostly cytostatic agents, ultimately leading to drug resistance. Resistant ERα-positive tumors typically retain ERα expression, suggesting an opportunity to develop new therapeutics that move beyond inhibition of this nuclear receptor. Through a medicinal chemistry campaign, we have discovered an estrogen receptor selective activator, the compound ErSO, that – in stark contrast to current ERα modulators – potently kills ERα-positive breast cancer cells. ErSO is effective even against breast cancer cell lines with known mutations in ERα that confer drug resistance. ErSO hyperactivates the anticipatory unfolded protein response, leading to quantitative cell death. ErSO is well-tolerated in mice, orally bioavailable, blood-brain-barrier penetrant. Using sensitive tumor imaging, ErSO often leads to quantitative tumor eradication (without recurrence) in multiple mouse models of ERα-positive breast cancer, including those utilizing breast cancer cell lines harboring mutant ERα. This potent activity against advanced primary and metastatic ERα-positive breast cancers represents a paradigm shift in leveraging of ERα for anticancer efficacy. Citation Format: Matthew W Boudreau, Paul J Hergenrother, Darjan Duraki, Lawrence Wang, Chengjian Mao, Ben H Park, Tim M Fan, Eric R Nelson, David J Shapiro, Ramon Moreno, Elizabeth Bruckheimer, Jeffrey Kiefer, Theodore M Tarasow. Quantitative regression of estrogen receptor alpha positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-16.

Abstract P6-03-18: The accumulation of IGFBP-1 results in EGFR/MAPK activation and drives the development of tamoxifen resistance in breast cancer cells

Abstract Insulin-like growth factor (IGF) system plays a significantly role in cellular processes, including proliferation, survival, and mitogenesis. The IGF system consists of two soluble ligands, IGF-1 and IGF-2; two transmembrane receptors, IGF-1R and IGF-2R; and six high affinity IGF binding proteins, IGFBPs 1-6. Evidence shows that the IGF system is involved in breast cancer. For example, at least 50% of breast cancer tumors have elevated IGF-1R signaling; the level of circulating IGF-1 is found positively associated with the incident of estrogen receptor positive breast cancer. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. However, tamoxifen resistance also occurs, and its exact mechanism is not well understood. Recently, we discovered that the accumulation of IGFBP-1 was stimulated by tamoxifen treatment, which subsequently prevented the activation of IGF-1R and inhibited the proliferation in MCF-7 cells. As a result, we hypothesized that the accumulation of IGFBP-1 due to the long-term tamoxifen exposure would result in tamoxifen resistance. In order to mimic the accumulation of IGFBP-1 after tamoxifen treatment, we employed MCF-7 and T-47D breast cancer cells to generate stable cell lines with IGFBP-1 overexpression. We found that the expression of IGF-1R was largely reduced in both MCF7-BP1 and T47D-BP1 cells, which was also observed in tamoxifen resistant MCF-7 and T-47D cells. Furthermore, we demonstrated that both MCF7-BP1 and T47D-BP1 were not responsive to tamoxifen, and epidermal growth factor receptor (EGFR) signaling pathway was significantly upregulated, as shown by higher levels of EGFR, phospho-EGFR (pEGFR), and phospho-Erk (pErk). Also, we found that the inhibition of Erk phosphorylation by MEK1 inhibitor PD98059 sensitized the stable cells to tamoxifen, showing that elevated pErk was protective against tamoxifen. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 resulted in significantly lower level of pErk and increased sensitivity to tamoxifen, further showing the role of IGFBP1 accumulation in the establishment of tamoxifen resistance and the activation of EGFR/MAPK pathway. Citation Format: Yan Zheng. The accumulation of IGFBP-1 results in EGFR/MAPK activation and drives the development of tamoxifen resistance in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-18.

Abstract P6-04-11: LLGL2 is involved in resistance to tamoxifen in estrogen receptor-alpha positive breast cancer patients

Abstract Background: Resistance to endocrine therapy and metastatic progression is a leading cause of death in estrogen receptor-alpha(ERα)-positive breast cancer patients. Recently, the scaffolding protein LLGL2 was reported to promote cellular proliferation in ERα-positive breast cancer cells. LLGL2 forms a trimetric complex with the leucine transport protein SLC7A5. LLGL2 and SLC7A5 were reported to be involved in resistance to tamoxifen treatment. Here, we investigated LLGL2 and SLC7A5 mRNA expression in breast cancer patients during long-term follow-up. Materials and methods: A total of 627 invasive breast cancer tissues was available for analysis of LLGL2 mRNA using a TaqMan PCR system. We sought correlations between clinicopathological factors and levels of LLGL2 expression in these samples. We also examined the mRNA expression of SLC7A5. The median follow-up period was 10.8 years. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Receiver operating characteristic (ROC) analysis was performed to determine the cut-off values of LLGL2 and SLC7A5 mRNA expression in breast cancers. Results: We found a positive correlation between high expression of LLGL2 mRNA and shorter disease-free survival (DFS) in all patients analyzed (P=0.01). We also identified positive correlations between high expression of LLGL2 mRNA and shorter DFS and overall survival (OS) in ERα-positive breast cancer patients (P=0.0006 and P=0.004, respectively), and but not in ERα-negative patients (P=0.67 and P=0.63, respectively). Positive correlations of LLGL2 mRNA expression with DFS and OS were observed in ERα-positive patients who received tamoxifen as an adjuvant therapy (P=0.02 and P=0.03, respectively). We also found positive correlations of SLC7A5 mRNA expression with DFS and OS in ERα-positive patients who received tamoxifen as an adjuvant therapy (P=0.006 and P=0.049, respectively); interestingly, this was not observed in ERα-positive patients who did not receive tamoxifen. Multivariate analysis indicated that high LLGL2 mRNA expression was an independent factor for prognosis both in breast cancer patients overall (Table 1) and ERα-positive breast cancer patients. Conclusion: LLGL2 and SLC7A5 expression levels are associated with the prognosis of ERα-positive breast cancer patients, especially those who receive tamoxifen as an adjuvant therapy. Furthermore, higher expression of LLGL2 is an independent predictive factor of poor prognosis in ERα-positive breast cancer patients. LLGL2 and SLC7A5 could be promising candidate biomarkers and therapeutic targets in ERα-positive breast cancer patients who receive tamoxifen as an adjuvant therapy. Table 1. Multivariate Cox regression analysis of factors associated with prognosis in all breast cancer patients.Variablesn (%)Multivariate (DFS)P valueHR (95% CI)Tumor size≤ 2cm241 (38)1> 2cm383 (61)0.4771.14 (0.8-1.64)Node statusNegative340 (54)1Positive253 (40)<0.00013.7 (2.61-5.25)Grade1164 (263)12 and 3445(71)0.0221.67 (1.07-2.69)ER statusPositive486 (77)1Negative131(21)0.0971.59 (0.92-2.75)LLGL2 mRNALow340 (54)1High287 (46)0.0231.46 (1.05-2.03) Citation Format: Tomoka Hisada, Naoto Kondo, Yumi Endo, Tomoko Asano, Yasuaki Uemoto, Sayaka Nishikawa, Yusuke Katagiri, Mitsuo Terada, Akiko Kato, Yu Dong, Hiroyuki Kato, Satoru Takahashi, Tatsuya Toyama. LLGL2 is involved in resistance to tamoxifen in estrogen receptor-alpha positive breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-11.

Abstract P6-04-16: Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1

Abstract Despite the availability of effective drugs that target ERα-positive breast cancer, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is a potent and clinically relevant metabolite of tamoxifen. Therefore, characterization of endoxifen resistance mechanisms may be crucial to understanding tamoxifen resistance. We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen over a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. We subjected endoxifen resistant cells to a genome-wide, CRISPR-mediated knockout screen to identify genes involved in mediating endoxifen resistance. Subsequent analyses revealed that disruption of genes regulated by the glucocorticoid receptor (GR) enhanced cells’ ability to survive and proliferate in the presence of endoxifen, suggesting that GR signaling blocks growth of resistant cells. We provide evidence that GR activation via dexamethasone significantly inhibits the proliferation rates of endoxifen resistant cells by 50-60%, with little to no inhibitory effects in endoxifen sensitive models. Follow-up studies suggested that alpha zinc-glycoprotein 1 (AZGP1), a secreted lipolytic enzyme induced by GR, plays a role in mediating this phenotype. AZGP1 RNA and protein are highly expressed in normal breast tissue, but corresponding levels are lower in ERα-positive breast malignancies. In these tumors, low AZGP1 expression correlates with worse prognosis. Additionally, high expression of AZGP1 is associated with improved outcomes in patients with prostate, pancreatic, gastric and breast cancer. AZGP1 is expressed in endocrine sensitive MCF7 and T47D cells, but is silenced in endoxifen resistant models at the mRNA and protein level. Further, treatment of resistant cells with conditioned medium containing AZGP1 resulted in significant inhibition of cell proliferation and migration, and antibody-mediated depletion of AZGP1 from conditioned medium completely reversed these effects. While there are no reports regarding the molecular functions of AZGP1 in cancer we provide evidence that AZGP1 directly inhibits oncogenic signaling in endocrine resistant breast cancer through interaction with, and inhibition of, β6 integrin. Through the analysis of endoxifen resistant breast cancer cells, we have identified novel roles for GR, AZGP1 and β6 integrin in the development and progression of endocrine resistant breast cancer. We demonstrate that activation of GR may elicit therapeutic benefit in endocrine resistant breast cancer, specifically in tumors expressing AZGP1 and β6 integrin. Further, AZGP1 and β6 integrin may represent novel prognostic and/or predictive biomarkers in endocrine resistant disease. Citation Format: Calley J. Jones, Santiago Acero Bedoya, Anna V. Mykytyn, Matthew P. Goetz, John R. Hawse. Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-16.

Abstract P1-05-02: Estrogen-regulated enhancer RNAs control enhancer assembly and function in breast cancer cells

Abstract Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor, making it an excellent target for endocrine therapy. When endogenous and exogenous ligands engage ERα, the receptor binds to regulatory regions in the genome known as enhancers, where it (1) associates with coregulatory proteins, (2) increases histone modifications associated with active enhancers, (3) promotes chromatin looping, (4) stimulates enhancer transcription and the production of enhancer RNAs (eRNAs), and (5) ultimately regulates target gene transcription. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. In ERα-positive breast cancer cells, estrogen signaling promotes the synthesis of thousands of eRNAs that originate from ERα enhancers. We are interested in understanding the functions of these estrogen-regulated eRNAs and how they contribute to the etiology of breast cancer. Recent studies on the roles of eRNAs have suggested that they may (1) recruit regulatory proteins to enhancers; (2) promote RNA polymerase II transition; (3) regulate histone modification; and (4) increase chromosome looping. One challenge facing the field is the lack of accurate eRNA annotations and cloned full-length eRNAs; eRNAs have typically been studied as signals from genomic assays on browser tracks, not as physical entities (e.g., cDNAs) - in other words, “ghosts.” We have employed precision nuclear run-on of capped RNA (PRO-cap) to determine the transcription start sites of all expressed eRNAs. In addition, we have also used ultra-deep RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to detect eRNAs and examine for the potential splicing of eRNAs. Combining these genomic data, our annotations provide critical information for the large-scale cloning of eRNAs and testing them either by (1) tethering them to their cognate ERα enhancers by using CRISPR/dCas9 technology (i.e., cis) or (2) ectopically expressing them (i.e., trans) in MCF-7 breast cancer cells. When we tether eRNAs to their cognate ERα enhancers by using dCas9 and CRISPR guide RNAs, various aspects of ERα enhancer assembly and target gene expression are enhanced. Currently we are devising strategies to screen for functional eRNAs on a genome-wide basis to determine how they contribute to ERα enhancer assembly and function on a global scale, and ultimately identify the eRNAs that drive breast cancer initiation and growth. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Citation Format: Tim Y Hou, William L Kraus. Estrogen-regulated enhancer RNAs control enhancer assembly and function in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-02.

Abstract P6-04-10: Combining direct and indirect modes of antiestrogen ligand antagonism of estrogen receptor activity in breast cancer

Abstract Resistance to endocrine therapies in breast cancer highlights the need to explore new modes for antagonizing the activity of estrogen receptor alpha (ERα) that might lead to clinically more effective antiestrogens. Most clinically approved antiestrogens, however, have conceptually similar overall structures. Typically, this includes a lipophilic ligand core with a single side chain attached to an E-ring that uses direct steric contact to displace helix-12 of the ER ligand binding domain (LBD) from the agonist conformation. Unlike conventional antiestrogens, certain structurally bridged oxabicycloheptene-sulfonamide (OBHS-N) ER ligands have two rings (E and F) onto which side chains can be attached, thereby probing two distinct regions and modes of antagonism within the ER LBD. We attached characteristic ER antagonist side chains onto each of these two rings seeking to displace helix-12 sterically (“direct antagonism”) and/or to distort the ligand binding pocket and thereby alter the conformation of the helix-11/12 loop (a distinct mode, termed “indirect antagonism”). Several of these OBHS-N compounds have full antiproliferative activity on ERα-positive breast cancer cells and reverse estrogen-regulated gene expression, and the inhibitory potency of compounds with each type of side chain shows a distinct preference for E- or F-ring of attachment. Conformational analysis of these ER-ligand complexes using a multiplexed coregulator peptide interaction assay shows that sidechain substitution on the E-ring gives interaction profiles similar to that for hydroxytamoxifen and fulvestrant, whereas F-ring substitution gives a very different pattern. X-ray crystallographic structure analysis confirms that F-ring substituted OBHS-N compounds use a combination of direct and indirect modes of antagonism that both displaces and disorders helix-12 of the ER LBD. Our findings expand design concepts by which ERα ligands can block the activity of this receptor in breast cancers and structurally how direct and indirect modes of inhibition can be combined. This could facilitate the development of more efficacious antiestrogens for breast cancer treatment. Supported by the Breast Cancer Research Foundation (BCRF 18-084 to JAK and BSK) and the National Institutes of Health (R01 CA220284 to JAK, BSK, TI, and KWN; T32 GM070421 Fellowship to VSG). Citation Format: John A Katzenellenbogen, Jian Min, Sathish Srinivasan, Erumbi S. Rangarajan, Jerome C. Nwachukwu, Valeria Sanabria Guillen, Yvonne Ziegler, Shunchao Yan, Kathryn E. Carlson, Yingwei Hou, Sung Hoon Kim, Tina Izard, Rene Houtman, Kendall W. Nettles, Benita S. Katzenellenbogen. Combining direct and indirect modes of antiestrogen ligand antagonism of estrogen receptor activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-10.

Estradiol-Induced MMP-9 Expression via PELP1-Mediated Membrane-Initiated Signaling in ERα-Positive Breast Cancer Cells.

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1) is a novel estrogen receptor (ER) coregulator, demonstrated distinctive characters from other ERα coregulators, and has been suggested to be involved in metastasis of several cancers. In ERα-positive breast cancer, PELP1 overexpression enhanced ruffles and filopodium-like structure stimulated by estradiol (E2) through extranuclear cell signaling transduction hereby increased cell motility. However, whether PELP1 is also involved in extracellular matrix remodeling of ERα-positive breast cancer cells is still unknown. In this study, we investigated the role of PELP1 in E2-induced MMP-9 expression and the underlined mechanism. The results demonstrated the following: E2-induced ERα-positive MCF-7 breast cancer cell MMP-9 mRNA and protein expression in a rapid response and concentration-dependent manner. Knocked down PELP1 significantly suppressed E2-induced MMP-9 expression. E2-bovine serum albumin (BSA), a large molecular membrane-impenetrable conjugate of E2, can also upregulate MMP-9 protein expression in MCF-7, and the action of E2-BSA can be abolished by PI3K inhibitor LY294002; treating MCF-7 simultaneously with PELP1-shRNA and LY294002 did not show synergetic inhibitory effect on E2-BSA-induced MMP-9 expression. Our results indicated that estrogen-induced MMP-9 expression in ER-positive breast cancer cells may be through PELP1-mediated PI3K/Akt signaling pathway.

MiR-200b restrains EMT and aggressiveness and regulates matrix composition depending on ER status and signaling in mammary cancer

Secreted microRNAs (miRNAs) reside in a complex regulatory network with extracellular matrix (ECM) macromolecules, which affect cell-cell communication, therefore miRNA expression highlights its significance in several aspects of human diseases, including cancer. miRNA-mediated regulation of breast cancer has received considerable attention due to evidence that shows miRNAs to mediate estrogen receptor (ER) status, metastasis, chemoresistance and epithelial-to-mesenchymal transition (EMT). miR-200b is a pluripotent miRNA, which is inversely regulated by ERα and ERβ in mammary cancer. It has been identified as tumor suppressor and EMT inhibitor serving as a critical biomarker, as its expression in breast tumor determines the disease-free survival, thus highlighting its roles in breast cancer invasion and metastasis. The main goal of this study was to investigate the role of miR-200b in modulating the behavior of breast cancer cells with different ER status. We demonstrate that estrogen signaling through ERs reduces miR-200b expression levels in ERα-positive breast cancer cells. Moreover, miR-200b upregulation reduces the aggressive phenotype of ERβ-positive breast cancer cells by inhibiting cell invasiveness and motility, followed by ECM reorganization as well as cytoskeletal and morphological changes concluded from deep inspection of cell topography. Future investigation towards the mechanistic perspective of miR-200b effects in the behavior of aggressive mammary cancer cells appears rewarding in order to expand our understanding of miR-200b as a novel mediator beyond breast cancer diagnosis and pharmaceutical targeting.

A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis.

While protein arginine methyltransferases (PRMTs) and PRMT-catalyzed protein methylation have been well-known to be involved in a myriad of biological processes, their functions and the underlying molecular mechanisms in cancers, particularly in estrogen receptor alpha (ERα)-positive breast cancers, remain incompletely understood. Here we focused on investigating PRMT4 (also called coactivator associated arginine methyltransferase 1, CARM1) in ERα-positive breast cancers due to its high expression and the associated poor prognosis.Methods: ChIP-seq and RNA-seq were employed to identify the chromatin-binding landscape and transcriptional targets of CARM1, respectively, in the presence of estrogen in ERα-positive MCF7 breast cancer cells. High-resolution mass spectrometry analysis of enriched peptides from anti-monomethyl- and anti-asymmetric dimethyl-arginine antibodies in SILAC labeled wild-type and CARM1 knockout cells were performed to globally map CARM1 methylation substrates. Cell viability was measured by MTS and colony formation assay, and cell cycle was measured by FACS analysis. Cell migration and invasion capacities were examined by wound-healing and trans-well assay, respectively. Xenograft assay was used to analyze tumor growth in vivo.Results: CARM1 was found to be predominantly and specifically recruited to ERα-bound active enhancers and essential for the transcriptional activation of cognate estrogen-induced genes in response to estrogen treatment. Global mapping of CARM1 substrates revealed that CARM1 methylated a large cohort of proteins with diverse biological functions, including regulation of intracellular estrogen receptor-mediated signaling, chromatin organization and chromatin remodeling. A large number of CARM1 substrates were found to be exclusively hypermethylated by CARM1 on a cluster of arginine residues. Exemplified by MED12, hypermethylation of these proteins by CARM1 served as a molecular beacon for recruiting coactivator protein, tudor-domain-containing protein 3 (TDRD3), to CARM1-bound active enhancers to activate estrogen/ERα-target genes. In consistent with its critical role in estrogen/ERα-induced gene transcriptional activation, CARM1 was found to promote cell proliferation of ERα-positive breast cancer cells in vitro and tumor growth in mice.Conclusions: our study uncovered a “hypermethylation” strategy utilized by enhancer-bound CARM1 in gene transcriptional regulation, and suggested that CARM1 can server as a therapeutic target for breast cancer treatment.

The deubiquitinating enzyme USP1 modulates ERα and modulates breast cancer progression.

Breast cancer is one of the most common malignancies worldwide, while the luminal types (ERα positive) accounts for two third of all breast cancer cases. Although ERα positive breast cancer could be effective controlled by endocrine therapy, most of the patients will develop endocrine resistance, which becomes a headache clinical issue for breast cancer field. Endocrine resistance could be caused by multiple pathway disorders, the dys-regulation of ERα signaling might be a critical factor, which makes it urgent and important to reveal the potential molecular mechanism of ERα signaling. In our current study, we identified a new deubiquitination enzyme USP1 through screening the whole DUB (Deubiquitinases) siRNA library. The expression of USP1 is elevated in human breast cancer compared with normal mammary tissues. Importantly, USP1 expression levels are specially correlated with poor survival in ERα positive patients. USP1 depletion inhibited breast cancer cell progression and ERα signaling activity. Immuno-precipitation assays indicate that USP1 associates with ERα and promotes its stability possibly via inhibiting ERα K48-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by USP1 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

Downregulation of 15-hydroxyprostaglandin dehydrogenase during acquired tamoxifen resistance and association with poor prognosis in ERα-positive breast cancer.

Aim:Tamoxifen (TAM) resistance remains a clinical issue in breast cancer. The authors previously reported that 15-hydroxyprostaglandin dehydrogenase (HPGD) was significantly downregulated in tamoxifen-resistant (TAMr) breast cancer cell lines. Here, the authors investigated the relationship between HPGD expression, TAM resistance and prediction of outcome in breast cancer.Methods:HPGD overexpression and silencing studies were performed in isogenic TAMr and parental human breast cancer cell lines to establish the impact of HPGD expression on TAM resistance. HPGD expression and clinical outcome relationships were explored using immunohistochemistry and in silico analysis.Results:Restoration of HPGD expression and activity sensitised TAMr MCF-7 cells to TAM and 17β-oestradiol, whilst HPGD silencing in parental MCF-7 cells reduced TAM sensitivity. TAMr cells released more prostaglandin E2 (PGE2) than controls, which was reduced in TAMr cells stably transfected with HPGD. Exogenous PGE2 signalled through the EP4 receptor to reduce breast cancer cell sensitivity to TAM. Decreased HPGD expression was associated with decreased overall survival in ERα-positive breast cancer patients.Conclusions:HPGD downregulation in breast cancer is associated with reduced response to TAM therapy via PGE2-EP4 signalling and decreases patient survival. The data offer a potential target to develop combination therapies that may overcome acquired tamoxifen resistance.

The Role of Long Noncoding RNAs in Antiestrogen Resistance in Breast Cancer: An Overview and Update.

As a standard treatment, endocrine therapy has dramatically enhanced the prognosis of patients with estrogen receptor (ER)-positive breast cancer, which accounts for nearly 70% of all breast cancers. Antiestrogen drugs such as tamoxifen and aromatase inhibitors are the standard treatment options for ERα-positive breast cancer. However, acquired antiestrogen resistance is still the leading cause of disease recurrence and progression. Evidence has shown that long noncoding RNAs (lncRNAs) play an essential role in the development of antiestrogen resistance in ER-positive breast cancer and can serve as biomarkers or potential therapeutic targets. This review highlights the role of lncRNAs in the development of antiestrogen resistance in breast cancer.

Novel nano-pomegranates based on astragalus polysaccharides for targeting ERα-positive breast cancer and multidrug resistance

Chemotherapy is an important method for treating breast cancer. However, multidrug resistance is one of the major challenges in breast cancer chemotherapy. There is an urgent need to develop novel, effective antitumor strategies that will perfect existing therapeutic regimens. In this study, the double-targeted nanocarrier, Quercetin-3’3-dithiodipropionic acid-Astragalus polysaccharides-Folic acid (QDAF), was successfully synthesized and self-assembled into a neoteric nano-targeted delivery strategy, named nano-pomegranates, and which were utilized to effectively inhibit multidrug resistance in estrogen receptor α (ERα)-positive breast tumor. The outstanding abilities of nano-pomegranates to release the drug in a reducing environment was determined by in vitro release assay. The cellular studies in MCF-7 cells were examined that nano-pomegranates have remarkable efficiencies of enhancing cellular uptake, inhibition and necrosis and apoptosis. In vivo antitumor experiments showed that nano-pomegranates have better anti-tumor effects and lower systemic toxicity than free Cur. In conclusion, nano-pomegranates have great potential in anti-breast cancer treatment.

PAK4 phosphorylating RUNX1 promotes ERα-positive breast cancer-induced osteolytic bone destruction.

The biological function of nuclear PAK4 in ERα-positive breast cancer osteolytic bone destruction remains unclear. Here, we find that the nuclear PAK4 promotes osteoclastogenesis and tumor-induced osteolysis via phosphorylating RUNX1. We show that nuclear PAK4 interacts with and phosphorylates RUNX1 at Thr-207, which induces its localization from the nucleus to the cytoplasm and influences direct interaction with SIN3A/HDAC1 and PRMT1. Furthermore, we reveal that RUNX1 phosphorylation by PAK4 at Thr-207 promotes osteolytic bone destruction via targeting downstream genes related to osteoclast differentiation and maturation. Importantly, we verify changes in RUNX1 subcellular localization when nuclear PAK4 is positive in breast cancer bone metastasis tissues. Functionally, we demonstrate that RUNX1 phosphorylation promotes osteolytic bone maturation and ERα-positive breast cancer-induced osteolytic bone damage in the mouse model of orthotopic breast cancer bone metastasis. Our results suggest PAK4 can be a therapeutic target for ERα-positive breast cancer osteolytic bone destruction.

Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α

Proteolysis-targeting chimera (PROTAC) could selectively degrade target protein and may become a promising strategy for treating estrogen receptor α (ERα) positive breast cancers. Here, we designed penetrated peptide-based PROTACs by constructing an N-terminal lactam cyclic to improve proteolytic stability and cell penetration. We used a lactam cyclic peptide as ERα binding ligand, 6-aminocaproic acid as a linker, and a hydroxylated pentapeptide structure for recruiting E3 ligase to obtain heterobifunctional compounds. The resulting optimized compound I-6 selectively recruited ERα to the E3 ligase complex for promoting the degradation of ERα. Compound I-6 possessed strong effect on MCF-7 cell toxicity (IC50 ∼9.7 μM) and significantly enhanced activities in inducing ERα degradation. Meanwhile, I-6 performed much stronger potency in inhibition of tumors growth than tamoxifen. This work is a successful template to construct PROTACs based on cell-permeable peptides, which could extend the chemical space of PROTACs.

Distinct Roles for BET Family Members in Estrogen Receptor α Enhancer Function and Gene Regulation in Breast Cancer Cells.

The bromodomain family member proteins (BRD; BET proteins) are key coregulators for estrogen receptor alpha (ERα)-mediated transcriptional enhancers. The use of BRD-selective inhibitors has gained much attention as a potential treatment for various solid tumors, including ER-positive breast cancers. However, the roles of individual BET family members have largely remained unexplored. Here, we describe the role of BRDs in estrogen (E2)-dependent gene expression in ERα-positive breast cancer cells. We observed that chemical inhibition of BET family proteins with JQ1 impairs E2-regulated gene expression and growth in breast cancer cells. In addition, RNAi-mediated depletion of each BET family member (BRDs 2, 3, and 4) revealed partially redundant roles at ERα enhancers and for target gene transcription. Furthermore, we found a unique role of BRD3 as a molecular sensor of total BET family protein levels and activity through compensatory control of its own protein levels. Finally, we observed that BRD3 is recruited to a subset of ERα-binding sites (ERBS) that are enriched for active enhancer features, located in clusters of ERBSs likely functioning as "super enhancers," and associated with highly E2-responsive genes. Collectively, our results illustrate a critical and specific role for BET family members in ERα-dependent gene transcription. IMPLICATIONS: BRD3 is recruited to and controls the activity of a subset ERα transcriptional enhancers, providing a therapeutic opportunity to target BRD3 with BET inhibitors in ERα-positive breast cancers.