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Background: Breast cancer is the most prevalent cancer in women globally. Over-activated estrogen receptor (ER) α signaling is considered the main factor in luminal breast cancers, which can be effectively managed with selective estrogen receptor modulators (SERMs) like tamoxifen. However, approximately 30-40% of ER + breast cancer cases are recurrent after tamoxifen therapy. This implies that the treatment of breast cancer is still hindered by resistance to tamoxifen. Recent studies have suggested that post-translational modifications of ERα play a significant role in endocrine resistance. The stability of both ERα protein and its transcriptome is regulated by a balance between E3 ubiquitin ligases and deubiquitinases. According to the current knowledge, approximately 100 deubiquitinases are encoded in the human genome, but it remains unclear which deubiquitinases play a critical role in estrogen signaling and endocrine resistance. Thus, decoding the key deubiquitinases that significantly impact estrogen signaling, including the control of ERα expression and stability, is critical for the improvement of breast cancer therapeutics., Methods: We used several ER positive breast cancer cell lines, DUB siRNA library screening, xenograft models, endocrine-resistant (ERα-Y537S) model and performed immunoblotting, real time PCR, RNA sequencing, immunofluorescence, and luciferase activity assay to investigate the function of USP36 in breast cancer progression and tamoxifen resistance., Results: In this study, we identify Ubiquitin-specific peptidase 36 (USP36) as a key deubiquitinase involved in ERα signaling and the advancement of breast cancer by deubiquitinases siRNA library screening. In vitro and in vivo studies showed that USP36, but not its catalytically inactive mutant (C131A), could promote breast cancer progression through ERα signaling. Conversely, silencing USP36 inhibited tumorigenesis. In models resistant to endocrine therapy, silencing USP36 destabilized the resistant form of ERα (Y537S) and restored sensitivity to tamoxifen. Molecular studies indicated that USP36 inhibited K48-linked polyubiquitination of ERα and enhanced the ERα transcriptome. It is interesting to note that our results suggest USP36 as a novel biomarker for treatment of breast cancer., Conclusion: Our study revealed the possibility that inhibiting USP36 combined with tamoxifen could provide a potential therapy for breast cancer., (© 2024. The Author(s).)

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients., Competing Interests: Declaration of Competing Interest All authors declare no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Estrogen receptor alpha (ERα) is expressed in approximately 70% of breast cancer cases and determines the sensitivity and effectiveness of endocrine therapy. 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase3 (PFKFB3) is a glycolytic enzyme that is highly expressed in a great many human tumors, and recent studies have shown that it plays a significant role in improving drug sensitivity. However, the role of PFKFB3 in regulating ERα expression and the underlying mechanism remains unclear. Here, we find by using immunohistochemistry (IHC) that PFKFB3 is elevated in ER-positive breast cancer and high expression of PFKFB3 resulted in a worse prognosis. In vitro and in vivo experiments verify that PFKFB3 promotes ER-positive breast cancer cell proliferation. The overexpression of PFKFB3 promotes the estrogen-independent ER-positive breast cancer growth. In an estrogen-free condition, RNA-sequencing data from MCF7 cells treated with siPFKFB3 showed enrichment of the estrogen signaling pathway, and a luciferase assay demonstrated that knockdown of PFKFB3 inhibited the ERα transcriptional activity. Mechanistically, down-regulation of PFKFB3 promotes STUB1 binding to ERα, which accelerates ERα degradation by K48-based ubiquitin linkage. Finally, growth of ER-positive breast cancer cells in vivo was more potently inhibited by fulvestrant combined with the PFKFB3 inhibitor PFK158 than for each drug alone. In conclusion, these data suggest that PFKFB3 is identified as an adverse prognosis factor for ER-positive breast cancer and plays a previously unrecognized role in the regulation of ERα stability and activity. Our results further explores an effective approach to improve fulvestrant sensitivity through the early combination with a PFKFB3 inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER + ) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER + BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER + BC progression., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Breast cancer (BC) is a leading cause of global cancer-related mortality in women, necessitating accurate tumor classification for timely intervention. Molecular and histological factors, including PAM50 classification, estrogen receptor α (ERα), breast cancer type 1 susceptibility protein (BRCA1), progesterone receptor (PR), and HER2 expression, contribute to intricate BC subtyping. In this work, through a combination of bioinformatic and wet lab screenings, followed by classical signal transduction and cell proliferation methods, and employing multiple BC cell lines, we identified enhanced sensitivity of ERα-positive BC cell lines to ALK and MELK inhibitors, inducing ERα degradation and diminishing proliferation in specific BC subtypes. MELK inhibition attenuated ERα transcriptional activity, impeding E2-induced gene expression, and hampering proliferation in MCF-7 cells. Synergies between MELK inhibition with 4OH-tamoxifen (Tam) and ALK inhibition with HER2 inhibitors revealed potential therapeutic avenues for ERα-positive/PR-positive/HER2-negative and ERα-positive/PR-negative/HER2-positive tumors, respectively. Our findings propose MELK as a promising target for ERα-positive/PR-positive/HER2-negative BC and highlight ALK as a potential focus for ERα-positive/PR-negative/HER2-positive BC. The synergistic anti-proliferative effects of MELK with Tam and ALK with HER2 inhibitors underscore kinase inhibitors' potential for selective treatment in diverse BC subtypes, paving the way for personalized and effective therapeutic strategies in BC management., (© 2024. The Author(s).)

Background: The current study recognizes the significance of estrogen receptor alpha (ERα) as a member of the nuclear receptor protein family, which holds a central role in the pathophysiology of breast cancer. ERα serves as a valuable prognostic marker, with its established relevance in predicting disease outcomes and treatment responses., Methods: In this study, computational methods are utilized to search for suitable drug-like compounds that demonstrate analogous ligand binding kinetics to ERα., Results: Docking-based simulation screened out the top 5 compounds - ZINC13377936, NCI35753, ZINC35465238, ZINC14726791, and NCI663569 against the targeted protein. Further, their dynamics studies reveal that the compounds ZINC13377936 and NCI35753 exhibit the highest binding stability and affinity., Conclusion: Anticipating the competitive inhibition of ERα protein expression in breast cancer, we envision that both ZINC13377936 and NCI35753 compounds hold substantial promise as potential therapeutic agents. These candidates warrant thorough consideration for rigorous In vitro and In vivo evaluations within the context of clinical trials. The findings from this current investigation carry significant implications for the advancement of future diagnostic and therapeutic approaches for breast cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

HAX1 is a multifunctional protein involved in the antagonism of apoptosis in cellular response to oxidative stress. In the present study we identified HAX1 as a novel binding partner for Che-1/AATF, a pro-survival factor which plays a crucial role in fundamental processes, including response to multiple stresses and apoptosis. HAX1 and Che-1 proteins show extensive colocalization in mitochondria and we demonstrated that their association is strengthened after oxidative stress stimuli. Interestingly, in MCF-7 cells, resembling luminal estrogen receptor (ER) positive breast cancer, we found that Che-1 depletion correlates with decreased HAX1 mRNA and protein levels, and this event is not significantly affected by oxidative stress induction. Furthermore, we observed an enhancement of the previously reported interaction between HAX1 and estrogen receptor alpha (ERα) upon H 2 O 2 treatment. These results indicate the two anti-apoptotic proteins HAX1 and Che-1 as coordinated players in cellular response to oxidative stress with a potential role in estrogen sensitive breast cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Estrogens have been implicated in the pathogenesis of various cancers, with increasing concern regarding the overall rising incidence of disease and exposure to environmental estrogens. Estrogens, both endogenous and environmental, manifest their actions through intracellular and plasma membrane receptors, named ERα, ERβ, and GPER. Collectively, they act to promote a broad transcriptional response that is mediated through multiple regulatory enhancers, including estrogen response elements (EREs), serum response elements (SREs), and cyclic AMP response elements (CREs). Yet, the design and rational assignment of antiestrogen therapy for breast cancer has strictly relied upon an endogenous estrogen-ER binary rubric that does not account for environmental estrogens or GPER. New endocrine therapies have focused on the development of drugs that degrade ER via ER complex destabilization or direct enzymatic ubiquitination. However, these new approaches do not broadly treat all cancer-involved receptors, including GPER. The latter is concerning since GPER is directly associated with tumor size, distant metastases, cancer stem cell activity, and endocrine resistance, indicating the importance of targeting this receptor to achieve a more complete therapeutic response. This review focuses on the critical importance and value of GPER-targeted therapeutics as part of a more holistic approach to the treatment of estrogen-driven malignancies.

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., Competing Interests: Declaration of competing interest On behave of all authors, I, Zhaoyu Li, corresponding author, to declare that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Estrogen receptor (ER) is a transcription factor that affects the expression of some genes involved in the progression and development of breast cancer (BC). Hesperetin (Hst) is a flavonoid that inhibits the proliferation of BC cells. In this study, we investigated the effect of Hst on the cell viability of MCF-7 cells and the gene expression of the ERα, ERβ, IL-6, Ps2, and Cyclin D1., Methods: In this study, cell viability was determined by MTT assay. The cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, 200, and 400 µM) for 24 h, and IC50 was calculated. Real-time PCR was used to assess the expression of ERα, ERβ, pS2, Cyclin D1, and IL-6 mRNA. MCF-7 cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, and 200 µM) for 24 h. Real-time PCR was carried out using a Step One Real-Time PCR System (ABI, USA) and Amplicon SYBR Green reagents., Results: The MTT assay revealed increased cytotoxicity with higher concentrations of Hst, and the IC 50 was calculated at 200 µM. Real-time PCR analysis following treatment with Hst showed a significant increase in ERα gene expression at 25 µM of Hst and a decrease in expression at 50, 100, and 200 µM of Hst (p < 0.0001). ERβ gene expression significantly decreased across all concentrations of Hst (p < 0.0001), while IL-6 gene expression decreased significantly in all concentrations (p < 0.0001). pS2 gene expression increased significantly with all concentrations of Hst (p < 0.0001), while Cyclin D1 gene expression did not significantly decrease upon Hst exposure (p > 0.05)., Conclusions: The results of our study demonstrate that Hst has the ability to induce cell death in MCF-7 cells. Furthermore, it was observed that Hst reduces the expression of the ER gene and enhances its activity, which can affect the downstream pathways of the ER., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance. NEW & NOTEWORTHY The development of resistance to hormone therapy caused by both fulvestrant and tamoxifen promotes autophagy with concomitant apoptosis evasion, rendering cells capable of surviving and growing. The fact that resistance also triggers ERBB family signaling pathways, which are poorly inhibited by tyrosine kinase inhibitors might attribute to cells' aggressiveness. It is obvious that the development of endocrine therapy resistance involves a complex interplay between deregulated ERBB signaling and autophagy that may be considered in clinical practice.

Perfluoroalkyl substances (PFASs) are a classic environmental endocrine disruptor with carcinogenic risk. Epidemiological studies have shown that PFASs contamination is associated with breast cancer development, but the mechanism remains largely unknown. This study first obtained complex biological information about PFASs-induced breast cancer through the comparative toxicogenomics database (CTD). The Protein-Protein Interaction (PPI) network, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis were utilized to investigate molecular pathways. The ESR1 and GPER expression levels at different pathological stages and the prognosis of Breast Cancer patients were confirmed using the Cancer Genome Atlas (TCGA) database. Furthermore, we verified this by cellular experiments and the results showed breast cancer cell migration and invasion were promoted by PFOA. Two estrogen receptors (ER), ERα and G protein-coupled estrogen receptor (GPER), mediated the promoting effects of PFOA by activating MAPK/Erk and PI3K/Akt signaling pathways. These pathways were regulated by ERα and GPER in MCF-7 cells or independently by GPER in MDA-MB-231 cells. Overall, our study provides a better overview of the mechanisms associated with PFASs-induced breast cancer development and progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate CITED1 as a potential biomarker of anti-endocrine response and breast cancer recurrence, given its previously determined role in mediating estrogen-dependant transcription. The study is a continuation of earlier work establishing the role of CITED1 in mammary gland development., Results: CITED1 mRNA is associated with estrogen-receptor positivity and selectively expressed in the GOBO dataset of cell lines and tumours representing the luminal-molecular subtype. In patients treated with tamoxifen, higher CITED1 correlated with better outcome, suggesting a role in anti-estrogen response. The effect was particularly evident in the subset of estrogen-receptor positive, lymph-node negative (ER+/LN-) patients although noticeable divergence of the groups was apparent only after five years. Tissue microarray (TMA) analysis further validated the association of CITED1 protein, by immunohistochemistry, with favourable outcome in ER+, tamoxifen-treated patients. Although we also found a favourable response to anti-endocrine treatment in a larger TCGA dataset, the tamoxifen-specific effect was not replicated. Finally, MCF7 cells overexpressing CITED1 showed selective amplification of AREG but not TGFα suggesting that maintenance of specific ERα-CITED1 mediated transcription is important for the long-term response to anti-endocrine therapy. These findings together confirm the proposed mechanism of action of CITED1 and support its potential use as a prognostic biomarker., (© 2023. The Author(s).)

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., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Malignant breast cancer (BC) remains incurable mainly due to the cancer cell metastasis, which is mostly related to the status of Estrogen receptor alpha (ERα). However, our understanding of the mechanisms through which ERα regulates cancer cell metastasis remains limited. Here we identified a miR-29a-PTEN-AKT axis as a downstream signaling pathway of ERα governing breast cancer progression and metastasis. Two estrogen response element (ERE) half sites were identified in the promoter and enhancer regions of miR-29a, which mediated transcriptional regulation of miR-29a by ERα. Low level of miR-29a showed association with reduced metastasis and better survival in ERα+ luminal subtype of BC. In contrast, high level of miR-29a was detected in ERα- triple negative breast cancer (TNBC) in association with distant metastasis and poor survival. miR-29a overexpression in BC tumors increased the number of circulating tumor cells and promoted lung metastasis in mice. Targeted knockdown of miR-29a in TNBC cells in vitro or administration of a nanotechnology-based anti-miR-29a delivery in TNBC tumor-bearing mice in vivo suppressed cellular invasion, EMT and lung metastasis. PTEN was identified as a direct target of miR-29a, inducing EMT and metastasis via AKT signaling. A small molecular inhibitor of AKT attenuated miR-29a-induced EMT. These findings demonstrate a novel mechanism responsible for ERα-regulated breast cancer metastasis, and reveal the combination of ERα status and miR-29a levels as a new risk indicator in BC., (© 2023. The Author(s).)

The molecular mechanisms regulating oestrogen homeostasis have been primarily studied in the mammary gland, which is the focus of this review. In the non-pregnant adult, the mammary gland undergoes repeated cycles of proliferation and apoptosis in response to the fluctuating levels of oestrogen that occur during the reproductive stage. Oestrogen actions are mediated through the steroid hormone receptors, oestrogen receptor α and β and through a G-protein coupled receptor. In the mammary gland, ERα is of particular importance and thus will be highlighted. Mechanisms regulating oestrogen-induced responses through ERα are necessary to maintain homeostasis given that the signalling pathways that are activated in response to ERα-mediated transcription can also induce transformation. ERK1/2 and its downstream effector, p90 ribosomal S6 kinase (RSK), control homeostasis in the mammary gland by limiting oestrogen-mediated ERα responsiveness. ERK1/2 drives degradation coupled ERα-mediated transcription, whereas RSK2 acts as a negative regulator of ERK1/2 activity to limit oestrogen responsiveness. Moreover, RSK2 acts as a positive regulator of translation. Thus, RSK2 provides both positive and negative signals to maintain oestrogen responsiveness. In addition to transmitting signals through tyrosine kinase receptors, ERK1/2-RSK engages with hedgehog signalling to maintain oestrogen levels and with the HIPPO pathway to regulate ERα-mediated transcription. Additionally, ERK1/2-RSK controls the progenitor populations within the mammary gland to maintain the ERα-positive population. RSK2 is involved in increased breast cancer risk in individuals taking oral contraceptives and in parity-induced protection against breast cancer. RSK2 and ERα may also co-operate in diseases in tissues outside of the mammary gland., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Breast cancer (BC), a well-known estrogen-dependent cancer, is the most common cancer among women and the leading cause of cancer deaths. One of the most important therapeutic approaches for BC is endocrine therapy targeting estrogen receptor alpha (ERα) and thus blocking the estrogen receptor signaling pathway. Drugs, such as tamoxifen or fulvestrant, are developed based on this theory and have benefited numerous patients with BC for many years. However, many patients with advanced BC, such as tamoxifen-resistant BC, cannot benefit from these developed drugs anymore. Therefore, new drugs targeting ERα are urgently needed by patients with BC. Recently, elacestrant, a novel selective estrogen receptor degrader (SERD), was approved by the United States Food and Drug Administration (FDA), highlighting the importance of ERα degradation in endocrine therapy. Proteolysis targeting chimera (PROTAC) has been considered a powerful technique for targeting protein degradation (TPD). In this regard, we developed and studied a novel ERα degrader, which is a PROTAC-like SERD named 17e. We found that compound 17e can inhibit the growth of BC both in vitro and in vivo and induce the cell cycle arrest of BC. Importantly, 17e displayed no apparent toxicity toward healthy kidney and liver cells. Moreover, we observed that the presence of 17e led to a dramatic increase in the autophagy-lysosome pathway in an ERα-independent manner. Finally, we revealed that a decrease in MYC, a frequent deregulation oncogene in human cancers, was mediated by both ERα degradation and autophagy activation in the presence of 17e. Collectively, we discovered that compound 17e induced ERα degradation and exerts significant anti-cancer effects on BC mainly through promoting the autophagy-lysosome pathway and decreasing MYC level., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: I would like to declare that the work described was original that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Estrogen receptor α (ERα), encoded by the ESR1 gene, is a key prognostic and predictive biomarker firmly established in routine diagnostics and as a therapeutic target of breast cancer, and it has a central function in breast cancer biology. Genetic variants at 6q25.1, containing the ESR1 gene, were found to be associated with breast cancer susceptibility. The rs2046210 and rs9383590 single nucleotide variants (SNVs) are located in the same putative enhancer region upstream of ESR1 and were separately identified as candidate causal variants responsible for these associations. Here, both SNVs were genotyped in a hospital-based case-control study of 409 female breast cancer patients and 422 female controls of a Central European (Austrian) study population. We analyzed the association of both SNVs with the risk, age at onset, clinically and molecularly relevant characteristics and prognosis of breast cancer. We also assessed the concordances between both SNVs and the associations of each SNV conditional on the other SNV. The minor alleles of both SNVs were found to be non-significantly associated with an increased breast cancer risk. Significant associations were found in specific subpopulations, particularly in patients with an age younger than 55 years. The minor homozygotes of rs2046210 and the minor homozygotes plus heterozygotes of rs9383590 exhibited a several-years-younger age at onset than the common homozygotes, which was more pronounced in ER-positive and luminal patients. Importantly, the observed associations of each SNV were not consistently nullified upon correction for the other SNV nor upon analyses in common homozygotes for the other SNV. We conclude that both SNVs remain independent candidate causal variants.

Estrogen receptor α (ERα) is the most common clinical marker used for breast cancer prognosis and the classification of breast cancer subtypes. Clinically, patients with estrogen receptor-positive breast cancer can receive endocrine therapy. However, resistance to endocrine therapy has become an urgent clinical problem. A large number of previous studies have proven that posttranslational modification of the estrogen receptor is significantly related to endocrine therapy resistance. RNF2 is a member of the RING finger protein family that functions as an E3 ubiquitin ligase. Several studies have clarified that RNF2 is a critical regulator of ERα transcriptional regulation. In our current study, we identified RNF2 as an important posttranslational modification regulator of the estrogen receptor. RNF2 depletion inhibited breast cancer cell progression and ERα signaling activity. TCGA data analysis indicated that RNF2 was elevated in breast malignancies, while RNF2 depletion could drastically inhibit estrogen response gene expression on a whole-genome scale. TCGA data analysis revealed that RNF2 was positively correlated with ERα target gene expression. Further mechanistic studies showed that RNF2 was mainly localized in the nucleus and associated with ERα. The association increased ERα stability by inhibiting ERα K48-linked polyubiquitination. In conclusion, our study implicates nongenomic regulation by RNF2 on ERα protein stability and suggests that targeting RNF2 could be a promising strategy for breast cancer treatments., (© 2022. The Author(s).)

Estrogen receptor-α (ERα) promotes breast cancer, and ER-positive cancer accounts for ~ 80% of breast cancers. This subtype responds positively to hormone/endocrine therapies involving either inhibition of estrogen synthesis or blockade of estrogen action. Carbidopa, a drug used to potentiate the therapeutic efficacy of L-DOPA in Parkinson's disease, is an agonist for aryl hydrocarbon receptor (AhR). Pharmacotherapy in Parkinson's disease decreases the risk for cancers, including breast cancer. The effects of carbidopa on ER-positive breast cancer were evaluated in cell culture and in mouse xenografts. The assays included cell proliferation, apoptosis, cell migration/invasion, subcellular localization of AhR, proteasomal degradation, and tumor growth in xenografts. Carbidopa decreased proliferation and migration of ER-positive human breast cancer cells in vitro with no significant effect on ER-negative breast cancer cells. Treatment of ER-positive cells with carbidopa promoted nuclear localization of AhR and expression of AhR target genes; it also decreased cellular levels of ERα via proteasomal degradation in an AhR-dependent manner. In vivo, carbidopa suppressed the growth of ER-positive breast cancer cells in mouse xenografts; this was associated with increased apoptosis and decreased cell proliferation. Carbidopa has therapeutic potential for ER-positive breast cancer either as a single agent or in combination with other standard chemotherapies., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element ( ERE ) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs -associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.

The ubiquitination process, which involves that binding of an ubiquitin protein to certain substrates, regulates several human biological processes and human cancers. Several studies report that the abnormal expression of quite a few E3 ubiquitin ligases could play critical role in carcinogenic process and cancer progression. In our current study, we identify UHRF1 (Ubiquitin Like with PHD And Ring Finger Domain 1) is an important regulator for breast cancer growth. UHRF1 depletion significantly decreases breast cancer growth in vitro and in vivo. Clinical data analysis reveals that UHRF1 is dramatically elevated in breast cancer, compared to normal breast tissue. UHRF1 correlates with poor survival in luminal type of breast cancer patients, but not in ER-negative groups. The molecular biological studies show that UHRF1 localizes in the nuclear and interact with ERα via its SRA domain, which subsequently inhibits K48-linked ubiquitination of ERα and enhances ERα stability. Our study provides a novel function of UHRF1 in regulation estrogen signaling in breast cancer and a promising target for breast cancer therapeutics., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Estrogen signaling plays important roles in diverse physiological and pathophysiological processes. However, the relationship between estrogen signaling and epigenetic regulation is not fully understood. Here, we explored the effect of estrogen signaling on the expression of Ten-Eleven Translocation (TET) family genes and DNA hydroxylmethylation in estrogen receptor alpha positive (ERα+) breast cancer cells. By analyzing the RNA-seq data, we identified TET2 as an estradiol (E2)-responsive gene in ERα+ MCF7 cells. RT-qPCR and Western blot analyses confirmed that both the mRNA and protein levels of TET2 gene were upregulated in MCF7 cells by E2 treatment. ChIP-seq and qPCR analyses showed that the enrichment of ERα and H3K27ac on the upstream regulatory regions of TET2 gene was increased in MCF7 cells upon E2 treatment. Moreover, E2 treatment also led to a significant increase in the global 5-hydroxymethylcytosine (5hmC) level, while knockout of TET2 abolished such E2-induced 5hmC increase. Conversely, treatment with ICI 182780, a potent and selective estrogen receptor degrader (SERD), inhibited TET2 gene expression and down-regulated the 5hmC level in MCF7 cells. Taken together, our study identified an ERα/TET2/5hmC epigenetic pathway, which may participate in the estrogen-associated physiological and pathophysiological processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Menin, encoded by the MEN1 gene, was recently reported to be involved in breast cancers, though the underlying mechanisms remain elusive. In the current study, we sought to further determine its role in mammary cells., Methods: Menin expression in mammary lesions from mammary-specific Men1 mutant mice was detected using immunofluorescence staining. RT-qPCR and western blot were performed to determine the role of menin in ERα expression in human breast cancer cell lines. ChIP-qPCR and reporter gene assays were carried out to dissect the action of menin on the proximal ESR1 promoter. Menin expression in female patients with breast cancer was analyzed and its correlation with breast cancer subtypes was investigated., Results: Immunofluorescence staining revealed that early mammary neoplasia in Men1 mutant mice displayed weak ERα expression. Furthermore, MEN1 silencing led to both reduced ESR1 mRNA and ERα protein expression in MCF7 and T47D cells. To further dissect the regulation of ESR1 transcription by menin, we examined whether and in which way menin could regulate the proximal ESR1 promoter, which has not been fully explored. Using ChIP analysis and reporter gene assays covering - 2500 bp to + 2000 bp of the TSS position, we showed that the activity of the proximal ESR1 promoter was markedly reduced upon menin downregulation independently of H3K4me3 status. Importantly, by analyzing the expression of menin in 354 human breast cancers, we found that a lower expression was associated with ER-negative breast cancer (P = 0.041). Moreover, among the 294 ER-positive breast cancer samples, reduced menin expression was not only associated with larger tumors (P = 0.01) and higher SBR grades (P = 0.005) but also with the luminal B-like breast cancer subtype (P = 0.006). Consistent with our clinical data, we demonstrated that GATA3 and FOXA1, co-factors in ESR1 regulation, interact physically with menin in MCF7 cells, and MEN1 knockdown led to altered protein expression of GATA3, the latter being a known marker of the luminal A subtype, in MCF7 cells., Conclusion: Taken together, our data provide clues to the important role of menin in ERα regulation and the formation of breast cancer subtypes., (© 2021. The Author(s).)

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response., Competing Interests: Declaration of interests D.R. is a shareholder of eFFECTOR Therapeutics, Inc. and is a member of its scientific advisory board., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported., Methods and Results: The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expression compared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17β-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα., Conclusion: Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The development of new biomarkers with diagnostic, prognostic and therapeutic prominence will greatly enhance the management of breast cancer (BC). Several reports suggest the involvement of the histone acetyltransferases CREB-binding protein (CBP) and general control non-depressible 5 (GCN5) in tumor formation; however, their clinical significance in BC remains poorly understood. This study aims to investigate the value of CBP and GCN5 as markers and/or targets for BC prognosis and therapy. Expression of CBP, GCN5, estrogen receptor α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in BC was analyzed in cell lines by western blot and in patients' tissues by immunohistochemistry. The gene amplification data were also analyzed for CBP and GCN5 using the publicly available data from BC patients., Results: Elevated expression of CBP and GCN5 was detected in BC tissues from patients and cell lines more than normal ones. In particular, CBP was more expressed in luminal A and B subtypes. Using chemical and biological inhibitors for CBP, ERα and HER2 showed a strong association between CBP and the expression of ERα and HER2. Moreover, analysis of the CREBBP (for CBP) and KAT2A (for GCN5) genes in a larger number of patients in publicly available databases showed amplification of both genes in BC patients. Amplification of CREBBP gene was observed in luminal A, luminal B and triple-negative but not in HER2 overexpressing subtypes. Furthermore, patients with high CREBBP or KAT2A gene expression had better 5-year disease-free survival than the low gene expression group (p = 0.0018 and p < 0.00001, respectively)., Conclusions: We conclude that the persistent amplification and overexpression of CBP in ERα- and PR-positive BC highlights the significance of CBP as a new diagnostic marker and therapeutic target in hormone-positive BC.

Background: Gli is an oncogenic transcription factor family thought to be involved in breast cancer (BrCa) cell growth. Gli activity is regulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling. In prostate cancer cells, however, Gli activation is mediated by an interaction of active androgen receptor proteins with Gli3 that stabilizes Gli3 in its un-proteolyzed form. Here we show that the estrogen receptor (ER), ERα, also binds Gli3 and activates Gli in BrCa cells. Moreover, we show that ER + BrCa cells are dependent on Gli3 for cancer cell growth., Methods: Transfection with Gli-luciferase reporter was used to report Gli activity in 293FT or BrCa cells (MCF7, T47D, MDA-MB-453) with or without steroid ligands. Co-immunoprecipitation and proximity ligation were used to show association of Gli3 with ERα. Gli3 stability was determined by western blots of BrCa cell extracts. ERα knockdown or destabilization (by fulvestrant) was used to assess how loss of ERα affects estradiol-induced Gli reporter activity, formation of intranuclear ERα-Gli3 complexes and Gli3 stability. Expression of Gli1 and/or other endogenous Gli-target genes in BrCa cells were measured by qPCR in the presence or absence of estradiol. Gli3 knockdown was assessed for effects on BrCa cell growth using the Cyquant assay., Results: ERα co-transfection increased Gli reporter activity in 293FT cells that was further increased by estradiol. Gli3 co-precipitated in ERα immunoprecipitates. Acute (2 h) estradiol increased Gli reporter activity and the formation of intranuclear ERα-Gli3 complexes in ER + BrCa cells but more chronic estradiol (48 h) reduced ERα-Gli complexes commensurate with reduced ERα levels. Gli3 stability and endogenous activity was only increased by more chronic estradiol treatment. Fulvestrant or ERα knockdown suppressed E2-induction of Gli activity, intranuclear ERα-Gli3 complexes and stabilization of Gli3. Gli3 knockdown significantly reduced the growth of BrCa cells., Conclusions: ERα interacts with Gli3 in BrCa cells and estradiol treatment leads to Gli3 stabilization and increased expression of Gli-target genes. Furthermore, we found tthat Gli3 is necessary for BrCa cell growth. These results support the idea that the ERα-Gli interaction and Gli3 may be novel targets for effective control of BrCa growth., (Copyright © 2020. Published by Elsevier B.V.)

CDATA[The inherited mutations and underexpression of BRCA1 in sporadic breast cancers resulting in the loss or functional inactivation of BRCA1 may contribute to a high risk of breast cancer. Recent researchers have identified small molecules (BRCA1 mimetics) that fit into a BRCA1 binding pocket within Estrogen Receptor alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity, and overcome antiestrogen resistance. Studies indicate that most of the BRCA1 breast cancer cases are associated with p53 mutations. It indicates that there is a potential connection between BRCA1 and p53. Most p53 mutations are missense point mutations that occur in the DNA-binding domain. Structural studies have demonstrated that mutant p53 core domain misfolding, especially p53-R175H, is reversible. Mutant p53 reactivation with a new class of zinc metallochaperones (ZMC) restores WT p53 structure and functions by restoring Zn 2+ to Zn 2+ deficient mutant p53. Considering the role of WT BRCA1 and reactivation of p53 in tumor cells, our hypothesis is to target both tumor suppressor proteins by a novel biomolecule (ZMC). Since both proteins are present in the same cell and are functionally inactive, this state may be a novel efficacious therapeutic regime for breast cancer therapy. In addition, we propose to use Albumin Nanovector (ANV) formulation for target drug release., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Breast cancer is the most commonly diagnosed malignancy in female worldwide, over 70% of which are estrogen receptor α (ERα) positive. ERα has a crucial role in the initiation and progression of breast cancer and is an indicator of endocrine therapy, while endocrine resistance is an urgent problem in ER-positive breast cancer patients. In the present study, we identify a novel E3 ubiquitin ligase TRIM11 function to facilitate ERα signaling. TRIM11 is overexpressed in human breast cancer, and associates with poor prognosis. The protein level of TRIM11 is highly correlated with ERα. RNA-seq results suggest that ERα signaling may be an underlying target of TRIM11. Depletion of TRIM11 in breast cancer cells significantly decreases cell proliferation and migration. And the suppression effects can be reversed by overexpressing ERα. In addition, ERα protein level, ERα target genes expression and estrogen response element activity are also dramatically decreased by TRIM11 depletion. Further mechanistic analysis indicates that the RING domain of TRIM11 interacted with the N terminal of ERα in the cytoplasm and promotes its mono-ubiquitination, thus enhances ERα protein stability. Our study describes TRIM11 as a modulating factor of ERα and increases ERα stability via mono-ubiquitination. TRIM11 could be a promising therapeutic target for breast cancer treatment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer is a disease where cells in the tissue of the breast, grow and divide without normal control. Breast cancer is second major cause for death in world wide. Importance of natural product increase due to adverse effect of existing synthetic drugs. Asparagus racemosus comprises phytoestrogens which can be used for the treatment of breast cancer. In the current study, In vitro antiproliferative activity of the extracts of A. racemosus is performed in T47D cancer cell lines. Outcomes of the result indicated that aqueous methanol and methanol extract showed excellent antiproliferative activity as compared to bazedoxifene (standard), ethyl acetate and petroleum ether extract . In silico study of reported phytochemical constituents of A. racemosus was performed for understand the molecular mechanism and prospect pharmacophore development. Furthermore, compound 26 (rutin) which has been earlier reported and isolated from alcoholic extract exhibited the remarkable binding profile with estrogen receptor α. For that reason, our study proposed that A. racemosus could be used as a new source for the treatment of breast cancer.

Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3-ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3-ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3-ERα complex and substitution of the occupancy on the promoter by the p53-p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3-ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

The incidence and mortality of breast cancer (BCa) are the highest among female cancers. There are approximate 70% BCa that are classified as estrogen receptor alpha (ERα) positive. Therefore, targeting ERα is the most significantly therapeutic schedule. However, patients with breast cancer develop resistance to ERα or estrogen (E2) antagonists such as fulvestrant and tamoxifen. In the present study, we found that L-Tetrahydropalmatine (L-THP) significantly suppressed cell proliferation in ERα + BCa cells via inducing cell cycle arrest rather than apoptosis. Additionally, L-THP enhanced the sensitivity of ERα + BCa cells to tamoxifen and fulvestrant. Mechanically, the application of L-THP promotes ERα degradation through accumulating ubiquitin chains on ERα. Overexpressing ERα abrogates L-THP induced-antiproliferation in ERα + BCa cells. Collectively, our work indicates that L-THP may represent a potentially novel therapeutic medicine for ERα + breast cancer patient., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Chromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Frequent long-range chromatin interactions between transcription factors (TFs) and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Although studies have proposed models to explain the initiation of chromothripsis, few discussed how TFs influence this process for tumor progression., Methods: This study focused on genomic alterations in amplification associated regions within chromosome 17. Inter-/intra-chromosomal rearrangements were analyzed using whole genome sequencing data of breast tumors in the Cancer Genome Atlas (TCGA) cohort. Common ERα binding sites were defined based on MCF-7, T47D, and MDA-MB-134 breast cancer cell lines using univariate K-means clustering methods. Nanopore sequencing technology was applied to validate frequent rearrangements detected between ATC loci on 17q23 and an ERα hub on 20q13. The efficacy of pharmacological inhibition of a potentially druggable target gene on 17q23 was evaluated using breast cancer cell lines and patient-derived circulating breast tumor cells., Results: There are five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter-/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the locations of the rearrangements were often mapped within or close to dense ERα binding sites localized on these five 17q regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ERα hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells., Conclusion: This study demonstrates a new approach for identifying tumorigenic drivers from genomic regions highly susceptible to ERα-related chromothripsis. We found a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts.

Background: Some stemness-associated transcription factors consistently play essential roles in the maintenance of pluripotency or induce the differentiation of cancer stem cells (CSCs). However, the regulatory mechanism of CSC stemness mediated by transcription factors has not been extensively explored. Here, we show that two transcription factors (YB-1 and ERα), which are simultaneously highly expressed in estrogen receptor (ER)-positive CSCs, interact with each other to regulate the stemness and differentiation of ER-positive CSCs. Methods: The expression of YB-1 was examined in ER-positive CSCs and patient specimens. Western blot, real-time PCR, cell viability analysis, tumorsphere formation assay and subcutaneous tumorigenesis assays were used to study the stemness functions of YB-1 and ERα in CSCs. The relationship between YB-1 and ERα in cells was studied by promoter activity analysis, the electrophoretic mobility shift assay (EMSA) and the Co-IP assay. The mechanisms and functional significance of YB-1 in the sensitivity of CSCs to tamoxifen were further investigated with both in vitro and in vivo models. Results: YB-1 was aberrantly upregulated in the cancerous tissue of ER-positive breast cancer patients and in CSCs. Knockdown of YB-1 in ER-positive CSCs significantly inhibited cell stemness and induced differentiation, and the expression of YB-1 could be regulated by estrogen signaling and ERα in ER-positive breast CSCs. The Co-IP results showed that YB-1 interacted directly with ERα specifically in ER-positive non-CSCs and that YB-1 induced ERα degradation by ubiquitination via direct interaction in differentiated cells. Cell differentiation induced by FBS could inhibit YB-1 phosphorylation and promote YB-1 protein transfer from the nucleus to the cytoplasm. Moreover, cell differentiation induced by targeting inhibited the expression of YB-1 in ER-positive CSCs, which increased the sensitivity of cells to tamoxifen in vitro and in vivo. Conclusion: The ERα/YB-1 axis has an important role in the regulation of ER-positive breast cancer stemness. The dephosphorylation of YB-1 and the interaction between YB-1 and ERα may be the switch that initiates the differentiation of ER-positive CSCs. Targeting YB-1 to sensitize ER-positive CSCs to antiestrogen therapy might represent a new therapeutic strategy that warrants further exploration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: Although tamoxifen remains the frontline treatment for ERα-positive breast cancers, resistance to this drug limits its clinical efficacy. Most tamoxifen-resistant patients retain ERα expression which may support growth and progression of breast cancers. Therefore, we investigated epigenetic regulation of ERα that may provide a rationale for targeting ERα in these patients., Methods: Expression levels of the mixed-lineage leukemia (MLL) family of proteins in tamoxifen-resistant breast cancer cells and publicly available breast cancer patient data sets were analyzed. Histone methylation levels in ERα promoter regions were assessed using chromatin immunoprecipitation. Expression levels of ERα and its target gene were analyzed using western blotting and real-time qPCR. Cell-cycle was analyzed by flow cytometry., Results: The expression of MLL3 and SET-domain-containing 1A (SET1A) were increased in tamoxifen-resistant breast cancers. An MLL3 chromatin immunoprecipitation-sequencing data analysis and chromatin immunoprecipitation experiments for MLL3 and SET1A suggested that these proteins bound to enhancer or intron regions of the ESR1 gene and regulated histone H3K4 methylation status. Depletion of MLL3 or SET1A downregulated the expression level of ERα and inhibited the growth of tamoxifen-resistant breast cancer cells. Additional treatment with fulvestrant resulted in a synergistic reduction of ERα levels and the growth of the cells., Conclusions: The enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported the ERα-dependent growth of these cells by increasing ERα expression. Our results suggest that targeting these histone methyltransferases might provide an attractive strategy to overcome endocrine resistance.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Pregnancy-associated breast cancer (PABC) is usually diagnosed at an advanced stage in comparison to non-pregnant women. The placenta secretes hormones and cytokines, which affect breast cancer progression. Previously, we demonstrated that human placental secretome facilitates the survival and migration of ERα+ breast cancer cells (BCCL), but pregnant women have a relatively high frequency of ERα-negative tumors. In the current study, we analyzed the effect of placental secretome on ERα-negative BCCL., Methods: BCCL [MCF-7(estrogen/progesterone receptor positive (ERα+/PR+), ERα reduced MCF-7 (siRNA, MCF-7 ERα-), HS-578 and BT-549 cells (both ER-/PR-)] were exposed to supernatants (collected from first trimester human placental explants and from control BCCL) or to E2 + P4 (estrogen + progesterone) in placental supernatant concentrations and then tested for cell proliferation (number, cell cycle, PCNA), cell-death, cell migration, STAT3 pathway activation and functionality., Results: Silencing ERα in the MCF-7 cells negated the placental supernatant and E2 + P4 enhancement of cell migration (> 130%, p < 0.05), number (> 120%) and survival (~ 130%). However, it had no such effect on MCF-7-ER- migration, which was still elevated in the presence of placental secretome. ER-/PR- BCCL were unaffected by the hormones, but placental secretome significantly elevated their migration (115%), number (140-170%), STAT3 phosphorylation (~ 180%) and BT-549 STAT3 level. These effects were negated by the STAT3 inhibitor., Conclusions: Placental supernatant facilitates BCCL malignant characteristics by activating ERα in estrogen responsive cells and STAT3 in ERα- BCCL. This indicates a possible mechanism that may underlie PABC's advanced state and suggests STAT3 pathway as a therapeutic target for PABC.

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an in vivo proximity-dependent labeling (BioID) technique, we identified YAP1 and TEAD4 protein as co-regulators of ERα on enhancers. The binding of YAP1/TEAD4 to ERα-bound enhancers is augmented upon E 2 stimulation and is required for the induction of E 2 /ERα target genes and E 2 -induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. Our data reveal a non-canonical function of YAP1 and TEAD4 as ERα cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERα + breast cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically. Methods : Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information (RNA-seq, ATAC-seq, Gene microarray, 450k methylation chip, GRO-seq), forming a multi-dimension network to illuminate ERα associated transcription. Results : We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers. Conclusions : The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors (NR2F2, FOXA1 and GATA3).The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

MicroRNA-26a-5p (miR-26a-5p) has been reported to be involved in the tumorigenesis of several tumors, but its function in breast cancer is still unknown. In this study, miR-26a-5p was found significantly downregulated in both of the breast cancer tissues and cell lines, and low expression of miR-26a-5p predicted a poor prognosis for breast cancer patients. Overexpression of miR-26a-5p could significantly inhibit breast cancer cell growth. Further studies revealed that overexpression of miR-26a-5p downregulated the protein levels of Cyclin D1, CDK4, and CDK6, but upregulated the expression levels of p21, p27, and p53. In mechanism, miR-26a-5p targeted the 3'UTR of ring finger protein 6 (RNF6) mRNA and inhibited RNF6 expression in breast cancer cells. Moreover, overexpression of miR-26a-5p inhibited RNF6/ERα/Bcl-xL axis in breast cancer cells. In contrast, inhibiting miR-26a-5p upregulated RNF6/ERα/Bcl-xL axis. Further studies indicated that miR-26a-5p mediated RNF6/ERα/Bcl-xL axis through regulating the stability of ERα protein. Collectively, downregulation of miR-26a-5p plays essential roles in breast cancer by mediating RNF6/ERα/Bcl-xL axis, which might provide important implications for the therapeutics of breast cancer., (© 2019 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Objective: POU5F1 (OCT4) is implicated in cancer stem cell self-renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear., Materials and Methods: RT-PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer., Results: OCT4 was down-regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA-MB-231 cell proliferation and inhibited the proliferation of MCF-7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5-aza-dC and zebularine) suppressed OCT4-induced MDA-MB-231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF-7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4-induced proliferation in MCF-7 cells via the activation of ERK signalling pathway., Conclusions: OCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis., (© 2019 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α-positive (ERα + ) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2's cis -regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Purpose: Low expression of long intergenic non-coding RNA LINC00472 in breast cancer is associated with aggressive tumors and unfavorable disease outcomes in multiple clinical datasets, but the reasons for these associations were unknown., Methods: To study the mechanisms underlying the lncRNA's connection to breast cancer, we investigated the molecular targets and regulation of LINC00472 in breast cancer cells, and analyzed relevant molecular features in relation to patient survival. Gene expression profiles of breast cancer cells overexpressing LINC00472 were analyzed for its regulatory pathways and downstream targets. Effects of LINC00472 overexpression on cell behaviors were evaluated in vitro and in vivo. Meta-analysis was performed using online datasets and our own study., Results: Analysis of LINC00472 transcriptome revealed ERα upregulation of LINC00472 expression, and an ERα-binding site in the LINC00472 promoter was identified. Evaluation of LINC00472 overexpression also indicated a possible link between LINC00472 and NF-κB. Cell experiments confirmed that LINC00472 suppressed the phosphorylation of p65 and IκBα through binding to IKKβ, inhibiting its phosphorylation. High LINC00472 expression inhibited tumor growth both in vitro and in vivo and suppressed aggressive tumor cell behaviors in vitro. Suppressing LINC00472 expression in ER-positive tumor cells increased cell aggressive behaviors. Tamoxifen treatment of ER-positive cells inhibited ERα and LINC00472 expression and increased p65 and IκBα phosphorylation. Meta-analysis showed that LINC00472 expression were higher in ER-positive than ER-negative tumors and that high expression was associated with better disease outcomes in ER-positive patients., Conclusions: The study demonstrates that ERα upregulates LINC00472 which suppresses the phosphorylation of NF-κB, and suggests that endocrine treatment may lower LINC00472 and increase NF-κB activities, leading to tumor progression and disease recurrence.

Background: Breast cancer is the most common cancer among women worldwide, and approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. ERα has been identified to promote the growth of primary breast cancer, however, it can also antagonize signaling pathways that lead to epithelial-mesenchymal transition (EMT), including transforming growth factor-β (TGF-β) signaling. miRNA alteration or dysfunction is involved in cancer development and progression. Although miR-1271 has identified as a tumor suppressor in various cancers, the role of miR-1271 in breast cancer is still limited., Methods: The effect of miR-1271 on breast cancer progression was investigated both in vitro and in vivo. The EMT-related protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Chromatin immunoprecipitation and dual-luciferase reporter assays were used to validate the regulation of ERα-miR-1271-SNAI2 feedback loop., Results: miR-1271 suppresses breast cancer progression and EMT phenotype both in vitro and in vivo by targeting SNAI2. Estrogen reverses TGF-β-induced EMT in a miR-1271 dependent manner. Furthermore, ERα transactivates the miR-1271 expression and is also transcriptionally repressed by SNAI2., Conclusions: Our data uncover the ERα-miR-1271-SNAI2 feedback loop and provide a mechanism to explain the TGF-β network in breast cancer progression.

Approximately two thirds of all breast cancer cases are estrogen receptor (ER)-positive. The treatment of this breast cancer subtype with endocrine therapies is effective in the adjuvant and recurrent settings. However, their effectiveness is compromised by the emergence of intrinsic or acquired resistance. Thus, identification of new molecular targets can significantly contribute to the development of novel therapeutic strategies. In recent years, many studies have implicated aberrant levels of translation initiation factors in cancer etiology and provided evidence that identifies these factors as promising therapeutic targets. Accordingly, we observed reduced levels of the eIF3 subunit eIF3f in ER-positive breast cancer cells compared with ER-negative cells, and determined that low eIF3f levels are required for proper proliferation and survival of ER-positive MCF7 cells. The expression of eIF3f is tightly controlled by ERα at the transcriptional (genomic pathway) and translational (nongenomic pathway) level. Specifically, estrogen-bound ERα represses transcription of the EIF3F gene, while promoting eIF3f mRNA translation. To regulate translation, estrogen activates the mTORC1 pathway, which enhances the binding of eIF3 to the eIF4F complex and, consequently, the assembly of the 48S preinitiation complexes and protein synthesis. We observed preferential translation of mRNAs with highly structured 5'-UTRs that usually encode factors involved in cell proliferation and survival ( e.g. cyclin D1 and survivin). Our results underscore the importance of estrogen-ERα-mediated control of eIF3f expression for the proliferation and survival of ER-positive breast cancer cells. These findings may provide rationale for the development of new therapies to treat ER-positive breast cancer., (© 2019 Cuesta et al.)

Heading Aims: This topic aims to clarify whether miR-22 directly targets and downregulates the expression of ERα and NK1R-Tr to inhibit the malignant behaviors of breast cancer cells., Materials and Methods: RT-PCR and Western Blotting were used to detect the expression profile of miR-22, NK1R-Tr and ERα. Luciferase reporter assay and CHIP experiment were conducted to investigate the regulation network between miR-22, NK1R-Tr and ERα. MCF-7-ERαI and MDA-MB-231-ERα cell lines were constructed to study the biological behaviors. The SP-NK1R-ERK1/2 signaling pathway was analyzed using Western Blotting. The subcutaneous and metastases tumor models were employed to study the effects of miR-22 on cell proliferation and metastasis of breast cancer cells in vivo., Key Findings: MiR-22 expression level was significantly lower in breast cancerous tissues and cell lines than the adjacent normality, while that of NK1R-Tr increased. The ERα could positively regulate NK1R-Tr expression at DNA level. The descent degree of NK1R-Tr in MCF-7-ERαI cells was far less than that in wild MCF-7 cells, while the findings in MDA-MB-231-ERα cells was more apparent than wild MDA-MB-231 cells. The malignant phenotype was decreased in miR-22 overexpressing cells compared with the wild type. The peak of ERK1/2 phosphorylation was delayed and weakened in miR-22 overexpressing MCF-7 cells, which was agreed with the findings using NK1R-Tr antagonist. The size and number of metastatic tumors declined compared to the controls., Significance: MiR-22 downregulated the expression of NK1R-Tr and ERα to delay and weaken phosphorylation of ERK1/2 to inhibit proliferation and metastasis of breast cancer cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Up to 80% of endometrial and breast cancers express oestrogen receptor alpha (ERα). Unlike breast cancer, anti-oestrogen therapy has had limited success in endometrial cancer, raising the possibility that oestrogen has different effects in both cancers. We investigated the role of oestrogen in endometrial and breast cancers using data from The Cancer Genome Atlas (TCGA) in conjunction with cell line studies. Using phosphorylation of ERα (ERα-pSer118) as a marker of transcriptional activation of ERα in TCGA datasets, we found that genes associated with ERα-pSer118 were predominantly unique between tumour types and have distinct regulators. We present data on the alternative and novel roles played by SMAD3, CREB-pSer133 and particularly XBP1 in oestrogen signalling in endometrial and breast cancer.

Selective phosphatidylinositol 3 kinase (PI3K) inhibitors are being actively tested in clinical trials for ERα-positive (ER+) breast cancer due to the presence of activating PIK3CA mutations. However, recent studies have revealed that increased ERα transcriptional activity limits the efficacy of PI3K inhibitor monotherapy for ER + breast cancers. Herein, we report the identification of BTF3 as an oncogenic transcription factor that regulates ERα expression in luminal breast cancers. Our TCGA analysis reveals high expression levels of BTF3 in luminal/ER + breast cancer and cell line models harboring ERα overexpression. Concordantly, BTF3 expression is highly and strongly associated with ESR1 expression in multiple breast cancer cohorts. We further show that BTF3 promotes the proliferation, survival and migration of ER + breast cancer cells by modulating ESR1 expression and ERα-dependent transcription. Moreover, BTF3 knockdown sensitizes ER + breast cancer cells to the PI3Kα inhibitor BYL-719 in both in vitro and in vivo models. Together, our findings highlight a novel role of BTF3 in modulation of ERα-dependent transcriptional activity and its potential as a predictive marker for the response to PI3K-targeted therapy in ER + breast cancer., (Copyright © 2018 The Second Hospital of Dalian Medical University, Dalian Medical University. Published by Elsevier B.V. All rights reserved.)