Estrogen Receptor Signaling Pathways Research Articles

Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression

BackgroundBreast cancer is the most common malignancy in women, and is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers express normal levels of REST (repressor element 1 silencing transcription factor) mRNA but lack functional REST protein. Loss of REST function is seen in ~ 20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer.MethodsTCGA data was analyzed for the expression of REST target genes in breast cancer patient samples. We then utilized gene knockdown in MCF-7 cells in the presence or absence of steroid hormones estrogen and/ progesterone followed by RNA sequencing, as well as chromatin immunoprecipitation and PCR in an attempt to understand the tumor suppressor role of REST in breast cancer.ResultsWe show that REST directly regulates CEMIP (cell migration-inducing and hyaluronan-binding protein, KIAA1199) and MMP24 (matrix metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cells leads to significant upregulation of CEMIP and MMP24. In addition, we found REST binds to RE-1 sites (repressor element-1) within the genes and influences their transcription. Furthermore, we found that the estrogen receptor (ESR1) signaling pathway is activated in the absence of REST, regardless of hormone treatment.ConclusionsWe demonstrate a critical role for the loss of REST in aggressive breast cancer pathogenesis and provide evidence for REST as an important diagnostic marker for personalized treatment plans.

Abstract P5-04-05: Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression

Abstract Breast cancer is the most common malignancy in women and is the second leading cause of cancer-related deaths. Breast cancer is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers expresses normal levels of REST (Repressor Element 1 Silencing Transcription factor) mRNA but lacks functional REST protein. Loss of REST function is seen in ~20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. TCGA data was analyzed for the expression of REST and its target genes in breast cancer patient samples. MCF7, an estrogen and progesterone receptor positive breast cancer cell line, and MDA-MB-231, a triple-negative breast cancer cell line were used for in vitro studies to understand the function of REST. To evaluate the role of REST in the regulation of hormone receptor pathways, RNA sequencing was carried out on total RNA from MCF7 cells after siRNA knockdown of REST followed by treatment of hormones estrogen and progesterone. Chromatin Immunoprecipitation and PCR were performed on REST knockdown cells to evaluate gene regulation by REST and understand its tumor suppressor role in breast cancer. We show that REST directly regulates CEMIP (Cell Migration-Inducing and Hyaluronan-Binding Protein, KIAA1199) and MMP24 (Matrix Metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cell lines leads to significant upregulation of CEMIP and MMP24. In addition, we found that REST binds to RE-1 sites (Repressor Element-1) within the genes and influences their transcriptional regulation. Aberrant expression of CEMIP and MMP24 results in CD44 dependent cell signaling that promotes cell proliferation and survival. We also found that the estrogen receptor signaling pathway is activated in the absence of REST regardless of hormone treatment. Our studies show that REST plays a significant role in altering the estrogen signaling pathway in hormone-sensitive breast cancers. REST transcriptionally regulates genes known to affect cell invasion and metastasis. Importantly, we demonstrate that loss of REST promotes aggressive breast cancer and see REST as an attractive therapeutic target. Citation Format: Aditya M. Vargheese, Ashley S. Cloud, Sumedha Gunewardena, Raeann M. Shimak, Sornakala Ganeshkumar, Easwari Kumaraswamy, Roy A. Jensen, Vargheese M. Chennathukuzhi. Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-04-05.

Abstract TF1-1: Leveraging our most contemporary understanding of estrogen signaling in breast cancer to inform the development of next generation endocrine therapies

Abstract Endocrine therapies designed to block estrogen-dependent activation of the estrogen receptor (ER) signaling pathway have, and will continue to be, the cornerstone of therapeutic approaches to treat luminal breast cancer. These agents work by either inhibiting the synthesis of gonadal/extragonadal estrogens or by directly blocking the activity of ER and have had a very significant impact on mortality and morbidity in patients with breast cancer, when used in the preventative, adjuvant, or treatment settings. However, as with most targeted therapies, de novo and acquired resistance remain impediments to durable clinical responses. Whereas the mechanisms underlying resistance to endocrine therapies are complex and multifactorial, most can be attributed to changes in the biochemical properties of the receptor or to adaptive changes in cancer cells that alter the way ER-ligand complexes are recognized. Mutations in ER that permit it to activate transcription in a ligand-independent manner and/or which enable pathological neomorphic activities have received considerable attention of late. These mutations, which occur infrequently in primary tumors are present in ~30-40% of metastatic lesions. Whereas it remains to be determined if these mutations are causally responsible for endocrine therapy failure their presence is at least a surrogate for the acute ER dependence of a tumor. Notable also are emerging data suggesting that ~1-2% of lesions from patients with advanced disease harbor gene arrangements that result in the formation of pathological ESR1-fusions. There is abundant evidence from both preclinical and clinical studies that resistance to endocrine therapies may result from changes in the absolute expression or activity of receptor-associated coregulators (coactivators or corepressors) and/or to dysregulated activity of signaling pathways that converge on ER-coregulator complexes. These activities negate the inhibitory effects of aromatase inhibitors and competitive antagonists like tamoxifen. A key feature of the primary resistance mechanisms that have been described to date is the continued dependence of tumors on ER signaling. This observation, together with the positive clinical activity of first generation Selective Estrogen Receptor Downregulators (SERDs), has encouraged the development of the next generation of endocrine therapies. Currently, there are 13 SERDs in clinical development for breast cancer, several of which have already shown efficacy in early clinical studies. It is likely that several of these SERDs, which accomplish ER downregulation/degradation by different mechanisms, will be approved for clinical use and that it will be issues associated with tolerability, effects on bone and cognitive function, and the ability to manage off-target activities which will differentiate these drugs. In addition to these drugs which target ER directly there is considerable enthusiasm for emerging new classes of drugs that directly target ER-coregulators or exploit collateral vulnerabilities that emerge in cancer cells as they become therapy resistant. Most endocrine therapies have been developed with the goal of inhibiting the cancer cell intrinsic actions of ER. However, we believe that it is important to consider that drugs of this class also inhibit cancer cell extrinsic actions of ER (a) in stromal cells within tumors and (b) at sites distal to the tumor (i.e. bone), that may impact tumor biology. Understanding these complexities. will inform the best use of current medicines and will be instructive as to the development of the next generation of endocrine therapies. Citation Format: DP McDonnell. Leveraging our most contemporary understanding of estrogen signaling in breast cancer to inform the development of next generation endocrine therapies. [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr TF1-1.

Abstract OT2-11-02: Ameera-1 Arm 4: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), with everolimus in postmenopausal women with ER+/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer

Abstract Background Amcenestrant (SAR439859) is an optimized oral SERD with potent dual activity that antagonizes and degrades the ER, resulting in inhibition of the ER signaling pathway. Amcenestrant, as monotherapy or in combination with palbociclib, has shown antitumor activity and a favorable safety profile in postmenopausal women with heavily pretreated ER+/HER2- advanced breast cancer. Published data support the addition of targeted therapy to endocrine therapy for patients with ER+/HER2- advanced breast cancer. The objective of Arm 4 of the AMEERA-1 study is to evaluate safety and antitumor activity of amcenestrant in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus for patients with ER+/HER2- advanced breast cancer. Methods AMEERA-1 (NCT03284957) is an open-label, non-comparative, dose escalation and dose expansion Phase 1/2 study of amcenestrant as monotherapy, then in combination with other anti-cancer targeted therapies. Arm 4 investigates dose escalation (Part H) and dose expansion (Part I), of amcenestrant in combination with everolimus. Postmenopausal women with ER+/HER2- advanced breast cancer, ECOG performance status 0-1, and ≥ 6 months prior endocrine therapy are eligible. In Arm 4 (Parts H and I), ≤ 1 prior line of a single endocrine therapy for advanced disease is allowed. Patients must have progressed on a non-steroidal aromatase inhibitor plus cyclin-dependent kinase 4/6 inhibitor as first-line therapy for advanced disease. Prior treatment with fulvestrant or any other SERD is not allowed. Part H allows ≤ 1 prior chemotherapy for advanced disease; no prior chemotherapy for advanced disease is allowed in Part I. Exclusion criteria in Arm 4 include prior drugs targeting the phosphoinositide 3-kinase axis; history of or concurrent pneumonitis; history of severe cutaneous reactions; type 1 diabetes; uncontrolled type 2 diabetes; uncontrolled hypercholesterolemia, hypertriglyceridemia, and hyperglycemia; any uncontrolled infection; uncontrolled stomatitis, angioedema due to angiotensin-converting enzyme inhibitors, and impaired wounds. Part H evaluates the selected amcenestrant dose for combination therapy plus everolimus 10 mg once daily (QD) (the approved standard dose) or everolimus 5 mg QD, taken in 28-day cycles. Additional amcenestrant doses may be explored based on safety and pharmacokinetics (PK). The objective of Part H is to determine the recommended dose (RD) of everolimus in combination with the selected amcenestrant dose for combination therapy, based on preliminary safety, PK, and antitumor activity data. The primary endpoint in Part H is the incidence of treatment-related dose-limiting toxicities (DLTs) at Cycle 1. Approximately up to 12 DLT-evaluable patients will be needed to establish the RD of everolimus in combination with amcenestrant in Part H. In Part I, approximately 12 patients will be treated at the RD of everolimus for combination therapy with amcenestrant, the primary endpoint being safety and tolerability. Secondary endpoints include PK and antitumor activity. Funding: Sanofi. Citation Format: Mario Campone, Patrick Neven, Katarina Petrakova, Sofia Braga, Marina Celanovic, Patrick Cohen, Alice Gosselin, Sylvaine Cartot-Cotton, Vasiliki Pelekanou, Valentina Boni. Ameera-1 Arm 4: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), with everolimus in postmenopausal women with ER+/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-11-02.

Abstract OT2-11-04: Ameera-1 Arm 5: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), with abemaciclib in postmenopausal women with ER+/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer

Abstract Background Endocrine therapy in combination with a targeted cyclin-dependent kinase (CDK) 4/6 inhibitor is the clinical standard for treatment of ER+/HER2- advanced breast cancer. Amcenestrant (SAR439859) is an optimized oral SERD with potent dual activity that antagonizes and degrades the ER, resulting in inhibition of the ER signaling pathway. In previous arms of the AMEERA-1 study, amcenestrant, as monotherapy or in combination with the CDK4/6 inhibitor palbociclib, demonstrated antitumor activity and a favorable safety profile in postmenopausal women with heavily pretreated ER+/HER2- advanced breast cancer. The objective of Arm 5 of the AMEERA-1 study is to evaluate safety and antitumor activity of amcenestrant in combination with the CDK4/6 inhibitor abemaciclib for patients with ER+/HER2- advanced breast cancer. Methods AMEERA-1 (NCT03284957) is an open-label, non-comparative, dose escalation and dose expansion Phase 1/2 study of amcenestrant as monotherapy, then in combination with other anti-cancer targeted therapies. Arm 5 investigates dose escalation (Part J) and dose expansion (Part K), of amcenestrant in combination with abemaciclib. Postmenopausal women with ER+/HER2- advanced breast cancer, ECOG performance status 0-1, and ≥ 6 months prior endocrine therapy are eligible. In Arm 5 (Parts J and K), ≤ 1 prior line of a single endocrine therapy for advanced disease is allowed. Prior treatment with fulvestrant or any other SERD is not allowed; in addition, prior therapy with CDK4/6 inhibitors for advanced disease is not allowed. Part J allows ≤ 1 prior chemotherapy for advanced disease, while prior chemotherapy for advanced disease is not allowed in Part K. Additional exclusion criteria in Arm 5 are prior drugs targeting the phosphoinositide 3-kinase axis; history of or concurrent pneumonitis; and history of or concurrent venous thromboembolism (i.e., deep vein thrombosis, pulmonary embolism, or cerebral venous sinus thrombosis). Part J evaluates the selected amcenestrant dose for combination therapy plus abemaciclib 150 mg twice daily (BID) (the approved standard dose) or abemaciclib 100 mg BID, taken in 28-day cycles. Additional dose levels of amcenestrant may be explored based on safety and pharmacokinetics (PK). The objective of Part J is to determine the recommended dose (RD) of abemaciclib in combination with the selected amcenestrant dose for combination therapy, based on preliminary safety, PK, and antitumor activity data. The primary endpoint in Part J is the incidence of treatment-related dose-limiting toxicities (DLTs) at Cycle 1. Approximately up to 12 DLT-evaluable patients will be needed to establish the RD of abemaciclib in combination with amcenestrant in Part J. In Part K, approximately 20 patients will be treated at the RD of abemaciclib for combination therapy with amcenestrant, the primary endpoint being safety and tolerability. Secondary endpoints include PK and antitumor activity. Funding: Sanofi. Citation Format: Mario Campone, Sarat Chandarlapaty, Aditya Bardia, Patrick Neven, Katarina Petrakova, Peter Kabos, Valentina Boni, Sofia Braga, Marina Celanovic, Patrick Cohen, Alice Gosselin, Sylvaine Cartot-Cotton, Vasiliki Pelekanou, Hannah Linden. Ameera-1 Arm 5: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), with abemaciclib in postmenopausal women with ER+/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-11-04.

Chlorobisphenol A activated kisspeptin/GPR54-GnRH neuroendocrine signals through ERα and GPER pathway in neuronal GT1-7 cells

Chlorobisphenol A (ClxBPA) is a kind of novel estrogenic compounds. The present study aims to investigate the effects of three ClxBPA compounds on the kisspeptin/G protein-coupled receptor 54 (GPR54, also named KissR1)-gonadotropin-releasing hormone (GnRH) (KGG) system in neuronal GT1–7 cells with mechanistic insights by estrogen receptor signaling pathways. The study demonstrated that low-concentration ClxBPA induced the cell proliferation, promoted GnRH secretion, upregulated the expression of KGG neuroendocrine signal-related proteins (KissR1, GnRH1 and kisspeptin) and genes including Kiss1, GnRH1, KissR1, luteinizing hormone receptor (Lhr) and follicle-stimulating hormone receptor (Fshr) in GT1–7 cells. Additionally, ClxBPA activated nuclear estrogen receptor alpha (ERα) and member estrogen receptor G protein-coupled estrogen receptor (GPER)-regulated phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinase (Erk1/2) signaling pathways. Pretreatment of GT1–7 cells with GPER inhibitor G15 and ERα inhibitor ICI reduced the expression of KissR1, GnRH1 and kisspeptin proteins, attenuated mRNA levels of Kiss1, GnRH1, KissR1, Fshr and Lhr genes, and decreased ClxBPA-induced GT1–7 cell proliferation. The results suggested that ClxBPA activated the KGG neuroendocrine signals and induced the proliferation of GT1–7 cells via ERα and GPER signaling pathways. This study provides a new perspective to explore the neuroendocrine toxicity mechanism of ClxBPA. CapsuleClxBPA activated KGG neuroendocrine signaling pathway via ERα and GPER and induced the proliferation of GT1–7 cells.

Maternal Perinatal Exposure to Dibutyl Phthalate Promotes Ovarian Dysfunction in Adult Female Offspring via Downregulation of TGF-β2 and TGF-β3

Maternal exposure to dibutyl phthalate (DBP) may result in ovarian dysfunction in female offspring. However, the underlying mechanisms remain elusive. Pregnant Sprague-Dawley rats were intraperitoneally injected with different doses of DBP, estradiol, and corn oil from gestational day 7 until the end of lactation. The reproductive characteristics, mRNA, and protein expression of ovaries for the adult female offspring were compared. KGN cells were cultured in vitro with DBP, estrogen receptor antagonist, or ALK-5 inhibitor. Genes, proteins, estradiol, and progesterone expressed by KGN, cell proliferation, and apoptosis were measured respectively. Maternal perinatal exposure to DBP induced prolonged estrous period, increased secondary follicles, significant decreased mRNA, and protein levels of TGF-β2, TGF-β3, and TGF-βRII in ovaries of the adult female offspring, but none difference for serum levels of sex hormones, ovarian TGF-β1, and estrogen receptor. The mRNA levels of LHR, FSHR, and CYP19a in ovaries were also decreased. DBP might decrease the mRNA of TGF-β2, TGF-β3, and TGF-βR II of KGN. DBP can inhibit the mRNA of CYP19 at 24h, which might be blocked by the estrogen receptor antagonist, whose effects were attenuated at 48h. DBP combined with FSH might time-dependently regulate the gene expression of TGF-βR II, inhibitory at 24h, but stimulative at 48h, which could be blocked by the ALK5 inhibitor. However, the protein expressed by KGN was not influenced by DBP. DBP stimulated the proliferation of KGN at 24h, which could be blocked by estrogen receptor antagonist, but attenuated at 48h. The progesterone in culture medium secreted by KGN was decreased by DBP at 24h. Maternal perinatal exposure to DBP induced decreased gene expression of TGF-β signaling and functional proteins in ovaries of the adult female offspring. Molecular cross-talk between estrogen receptor and TGF-β signaling pathway may play role in the mechanism of granulosa dysfunction induced by DBP.

The ER\u03b1/KDM6B regulatory axis modulates osteogenic differentiation in human mesenchymal stem cells

Osteoporosis is a highly prevalent public health burden associated with an increased risk of bone fracture, particularly in aging women. Estrogen, an important medicinal component for the preventative and therapeutic treatment of postmenopausal osteoporosis, induces osteogenesis by activating the estrogen receptor signaling pathway and upregulating the expression of osteogenic genes, such as bone morphogenetic proteins (BMPs). The epigenetic regulation of estrogen-mediated osteogenesis, however, is still unclear. In this report, we found that estrogen significantly induced the expression of lysine-specific demethylase 6B (KDM6B) and that KDM6B depletion by shRNAs led to a significant reduction in the osteogenic potential of DMSCs. Mechanistically, upon estrogen stimulation, estrogen receptor-α (ERα) was recruited to the KDM6B promoter, directly enhancing KDM6B expression. Subsequently, KDM6B was recruited to the BMP2 and HOXC6 promoters, resulting in the removal of H3K27me3 marks and activating the transcription of BMP2 and HOXC6, the master genes of osteogenic differentiation. Furthermore, we found that estrogen enhanced DMSC osteogenesis during calvarial bone regeneration and that estrogen’s pro-osteogenic effect was dependent on KDM6B in vivo. Taken together, our results demonstrate the vital role of the ERα/KDM6B regulatory axis in the epigenetic regulation of the estrogen-dependent osteogenic response.

Regulation of Bcl-2 Family Proteins in Estrogen Receptor-Positive Breast Cancer and Their Implications in Endocrine Therapy.

Simple SummaryBreast cancer is the leading cause of cancer-related deaths in the female population, with estrogen receptor (ER)-positive breast cancer accounting for two-third of incidents. Endocrine therapy has proven beneficial in treating ER-positive breast cancer. However, resistance acquired toward therapy remains a drawback in treatment. The activation of signaling pathways regulated by estrogen receptors has been linked with the evasion of cell death and drug resistance. An important role in regulating signals determining cell survival or death is assigned to the Bcl-2 family of proteins. Since the upregulation of anti-survival Bcl-2 proteins has been associated with decreased endocrine therapy efficacy and resistance, this review focuses on the molecular regulation of this group of proteins in ER-positive breast cancer and their implications in endocrine therapy treatment. Furthermore, advancements in the development of agents targeting the Bcl-2 family proteins have been overviewed, and their application in ER-positive breast cancer is presented.Estrogen receptor (ER)-positive breast cancer accounts for around two-thirds of breast cancer occurrences, with endocrine therapy serving as first-line therapy in most cases. Targeting estrogen signaling pathways, which play a central role in regulating ER+ breast cell proliferation and survival, has proven to improve patient outcomes. However, despite the undeniable advantages of endocrine therapy, a subset of breast cancer patients develop acquired or intrinsic resistance to ER-targeting agents, limiting their efficacy. The activation of downstream ER signaling pathways upregulates pro-survival mechanisms that have been shown to influence the response of cells to endocrine therapy. The Bcl-2 family proteins play a central role in cell death regulation and have been shown to contribute to endocrine therapy resistance, supporting the survival of breast cancer cells and enhancing cell death evasion. Due to the overexpression of anti-apoptotic Bcl-2 proteins in ER-positive breast cancer, the role of these proteins as potential targets in hormone-responsive breast cancer is growing in interest. In particular, recent advances in the development of BH3 mimetics have enabled their evaluation in preclinical studies with ER+ breast cancer models, and BH3 mimetics have entered early ER+ breast cancer clinical trials. This review summarizes the molecular mechanisms underlying the regulation of Bcl-2 family proteins in ER+ breast cancer. Furthermore, an overview of recent advances in research regarding the efficacy of BH3 mimetics in ER+ breast cancer has been provided.

Ginsenoside Re, Produces Multi-targeted Potent Antidiabetic Effects Via Estrogen Receptor Signaling Pathway in Rat, an Alternative Multi-Targeted Therapeutic for Type 2 Diabetes

Aims: To identify more effective ginsenoside for type 2 diabetes (T2D) and clarify whether the ginsenoside characterizing estrogenic multi-targeted antidiabetic effects.
 Study Design: Identifying more effective ginsenoside through preclinical evaluation of antidiabetic effects of representative ginsenosides with T2D rat model, and further test pharmacological mechanism underlying the potent antidiabetic effects of the ginsenoside in the same model.
 Place and Duration of Study: Key laboratory for Pharmacy, Inner Mongolia Medical University, March 2018 to November 2020.
 Methodology: Used a total of 240 female adult rats. Rat model of T2D induced by high-fat diet fed and streptozotocin. Five tapes of representative ginsenosides (Rb1, Rd, Rg3, Re, Rg1) administrated at low (20 mg/kg daily) and high (40 mg/ kg daily) doses to T2D rats with orally for 4 weeks. Detect testing indexes with biochemical, histological, Quantitative Real-Time PCR, and western blots analysis.
 Results: Ginsenoside Re (Re), very significantly lowered blood glucose (P<0.01), lipids (P<0.001), free fatty acid (P<0.001), and glucagon (P<0.01) levels, markedly improved impaired insulin sensitivity (P<0.01), ameliorated oxidative stress (P<0.01) and inflammation (P<0.01) in T2D rats, exhibited potent antidiabetic effects. Moreover, Re, phosphorylate serine/threonine kinase (Akt) (P<0.01) and endothelial nitric oxide synthase (eNOS) (P<0.01), up regulates B-cell lymphoma-2 (P<0.01) and insulin gene expression (P<0.01), down regulates glucagon gene expression(P<0.01), reverse impaired glucagon-like peptide 1 (P<0.01); exhibits multi-targeted effects; these effects of Re were inhibited by estrogen receptor (ER) inhibitor (ICI-182,780) (P<0.01). Functionally, the antidiabetic effects of Re were sequentially inhibited by inhibitor of ER, Akt, and eNOS, respectively (P<0.01).
 Conclusion: These findings, revealed a novel pharmacological property of Re that characterized in multi-targeted potent antidiabetic effects mediated by ER/Akt/eNOS/NO signaling pathway, provide the first evidence for the potential use of Re, as a multi-targeted therapeutic for T2D, particularly, a novel candidate for replacement of estrogen therapy and NO therapy in diabetes.

TP53 Pathway Function, Estrogen Receptor Status, and Breast Cancer Risk Factors in the Carolina Breast Cancer Study.

TP53 and estrogen receptor (ER) both play essential roles in breast cancer development and progression, with recent research revealing cross-talk between TP53 and ER signaling pathways. Although many studies have demonstrated heterogeneity of risk factor associations across ER subtypes, associations by TP53 status have been inconsistent. This case-case analysis included incident breast cancer cases (47% Black) from the Carolina Breast Cancer Study (1993-2013). Formalin-fixed paraffin-embedded tumor samples were classified for TP53 functional status (mutant-like/wild-type-like) using a validated RNA signature. For IHC-based TP53 status, mutant-like was classified as at least 10% positivity. We used two-stage polytomous logistic regression to evaluate risk factor heterogeneity due to RNA-based TP53 and/or ER, adjusting for each other and for PR, HER2, and grade. We then compared this with the results when using IHC-based TP53 classification. The RNA-based classifier identified 55% of tumors as TP53 wild-type-like and 45% as mutant-like. Several hormone-related factors (oral contraceptive use, menopausal status, age at menopause, and pre- and postmenopausal body mass index) were associated with TP53 mutant-like status, whereas reproductive factors (age at first birth and parity) and smoking were associated with ER status. Multiparity was associated with both TP53 and ER. When classifying TP53 status using IHC methods, no associations were observed with TP53. Associations observed with RNA-based TP53 remained after accounting for basal-like subtype. This case-case study found breast cancer risk factors associated with RNA-based TP53 and ER. RNA-based TP53 and ER represent an emerging etiologic schema of interest in breast cancer prevention research.

Exploration the potential mechanism of the SIRT1 and its target gene FOXO1/PPARGC1A in uteropelvic junction obstruction.

BackgroundUteropelvic junction obstruction (UPJO) is a common surgical condition, which refers to the blockage of urine flowing through kidney into proximal upper ureter. However, the underlying mechanism of UPJO is poorly understood, especially the regulated and targeted genes of sirtuin 1 in UPJO.MethodsWe sequenced three renal tissues on the obstructed side of independent children with <20% differential renal function (DRF) and three samples with >40% DRF. Gene expression values were obtained and compared for differentially expressed genes (DEGs). Protein-protein interaction (PPI) analysis was conducted to identify the overlapping proteins of DEGs and Sirtuin 1 (SIRT1). The co-expression genes of overlapped genes were computed using Pearson correlation coefficient. The potential role of SIRT1 gene in UPJO was explored by resequencing 3 microarray data from RNA interference (RNAi) SIRT1 lines of renal tubular epithelial (NRK52E) cells in rat and three control datasets were sequenced again. The DEGs were obtained as parallel. GO/KEGG enrichment analysis and co-expression network were conducted to explore the underlying mechanism, particularly shared pathways or function in GO/KEGG enrichment analysis results.ResultsA total of 427 up-regulated genes and 1,099 down-regulated genes were identified among 3 mRNA-seq of renal tissue on the obstructed side of the independent children with <20% DRF and 3 samples with >40% DRF. According to prediction using the Search Tool for Retrieval of Interacting Genes/Proteins, 2 PPIs, FOXO1 and PPARGC1A, were identified among 2,524 DEGs, predicted as targets of SIRT1. Gene set enrichment analysis (GSEA) of their co-expression genes showed they may co-participate in biological activities including fatty acid degradation, regulation of signal transduction by p53 mediator. Moreover, GSEA results of DEGs was confirmed through RNAi SIRT1 lines of rat renal tubular epithelial (NRK52E) cells.ConclusionsUPJO may cause abnormal phenotypic changes of renal tubular epithelial cells through SIRT1/FOXO1 mediated protein transport, establishment of protein localization, and intracellular transport. In addition, UPJO is involved in regulation of signal transduction, regulation of intracellular estrogen receptor signaling pathways, and nucleoprotein localization through SIRT1/PPARGC1A-mediated p53 mediators, causing abnormal phenotypic changes in renal tubular epithelial cells.

A Short S-Equol Exposure Has a Long-Term Inhibitory Effect on Adipogenesis in Mouse 3T3-L1 Cells

In the search for new drugs against obesity, the chronic disease that threatens human health worldwide, several works have focused on the study of estrogen homologs because of the role of estrogen receptors (ERs) in adipocyte growth. The isoflavone equol, an ERβ agonist, has shown beneficial metabolic effects in in vivo and in vitro assays; however, additional studies are required to better characterize its potential for body weight control. Here, we showed that the treatment of 3T3-L1 cells with 10 μM of S-equol for the first three days of the adipocyte differentiation protocol was able to prevent cells becoming semi-rounded and having a lipid droplet formation until the seventh day of culture; moreover, lipid accumulation was reduced by about 50%. Congruently, S-equol induced a reduction in mRNA expression of the adipogenic markers C/EBPα and PPARγ, and adipokines secretion, mainly Adiponectin, Leptin, Resistin, and MCP-1, while the release of PAI-1 was augmented. Moreover, it also reduced the expression of ERα and attenuated the subexpression of ERβ associated with adipogenesis. Altogether, our data suggested that S-equol binding to ERβ affects the transcriptional program that regulates adipogenesis and alters adipocyte functions. Future efforts will focus on studying the impact of S-equol on ER signaling pathways.

Synaptotagmin 13 Is Highly Expressed in Estrogen Receptor-Positive Breast Cancer.

Background: Accumulating evidence indicates tumor-promoting roles of synaptotagmin 13 (SYT13) in several cancers; however, no studies have investigated its expression in breast cancer (BC). This study aimed to clarify the significance of SYT13 in BC. Methods: SYT13 mRNA expression levels were evaluated in BC cell lines. Polymerase chain reaction (PCR) array analysis was conducted to determine the correlation between expression levels of SYT13 and other tumor-associated genes. Then, the association of SYT13 expression levels in the clinical BC specimens with patients’ clinicopathological factors was evaluated. These findings were subsequently validated using The Cancer Genome Atlas (TCGA) database. Results: Among 13 BC cell lines, estrogen receptor (ER)-positive cells showed higher SYT13 mRNA levels than ER-negative cells. PCR array analysis revealed positive correlations between SYT13 and several oncogenes predominantly expressed in ER-positive BC, such as estrogen receptor 1, AKT serine/threonine kinase 1, and cyclin-dependent kinases 4. In 165 patients, ER-positive specimens exhibited higher SYT13 mRNA expression levels than ER-negative specimens. The TCGA database analysis confirmed that patients with ER-positive BC expressed higher SYT13 levels than ER-negative patients. Conclusion: This study suggests that SYT13 is highly expressed in ER-positive BC cells and clinical specimens, and there is a positive association of SYT13 with the ER signaling pathways.

Estrogen-regulated AGR3 activates the estrogen receptor signaling pathway to promote tamoxifen resistance in breast cancer.

Anterior gradient 3 (AGR3) is associated with breast cancer progression, but its relationship with estrogen and tamoxifen resistance in breast cancer is still unclear. This study was designed to investigate the correlation of ARG3 and estrogen as well as the roles of ARG3 in tamoxifen resistance in breast cancer. Online database including GEPIA, UALCAN, and TCGA and rVista predictive tool were applied to analyze the expression patterns of AGR3 and its relationship with estrogen receptor 1. AGR3 knockdown and overexpression cell models were constructed. Luciferase reporter assay and ChIP were performed to investigate intermolecular interactions. Western blotting and qPCR were applied to assess targets at mRNA and protein levels, respectively. Cell counting and MTT assay were applied to determine the cell proliferation. An elevation of AGR3 was observed in patients with breast cancer, especially in the patients with estrogen receptor (ER)-positive breast cancer. The TCGA dataset and in vitro data supported that AGR3 was positively correlated to ER. Further results demonstrated that ER protein bound to AGR3 promoter sites. AGR3 expression exhibited a positive correlation to cell viability. Besides, AGR3 promoted tamoxifen resistance in breast cancer. AGR3 is associated with estrogen and promotes tamoxifen resistance in breast cancer.

Hesperidin and Chlorogenic Acid Synergistically Inhibit the Growth of Breast Cancer Cells via Estrogen Receptor/Mitochondrial Pathway.

Breast cancer is the most common cancer in women worldwide. Hesperidin (Hes) and chlorogenic acid (CA) are traditional medicinal molecules that abundantly exist in natural plants or foods. These compounds have been shown to prevent and suppress various cancers and therefore can be utilized as adjunctive therapies to aid cancer treatment. Here, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays show a greater synergistic inhibitory effect on the growth of breast cancer cells, MCF-7, but not normal breast cells, MCF-10A, than hesperidin or chlorogenic acid alone. We present the possible molecular signaling pathways in MCF-7 cells with or without herbal molecule treatments via proteomic approaches. The data were further analyzed by Ingenuity Pathway Analysis (IPA) and confirmed by quantifying mRNA associated with the estrogen-receptor signaling pathway and mitochondrial functions. We demonstrated that the expression of CYC1, TFAM, ATP5PB, mtATP6, mtDNA, and NRF-1 were decreased upon 12 h treatment, and subsequent ATP production was also significantly decreased at 24 h. These results identified a synergistic effect induced by combinational treatment with hesperidin and chlorogenic acid, which can regulate mitochondria and ATP production through the estrogen receptor pathway in MCF-7 cells. However, none of the treatments induced the generation of reactive oxygen species (ROS), suggesting that ROS likely plays no role in the observed pharmacological activities. Overall, our study sheds light on the adequacy of hesperidin and chlorogenic acid to serve as an adjunctive therapy when co-administrated with chemotherapy drugs in breast cancer patients.

Amelioration of oestradiol valerate-induced endometrial hyperplasia in female rats by methanol fraction ofMangifera indicaLinn. through modulation of oestrogen receptor signalling pathway

Objectives:Mangifera indicais a medicinal plant that is folklorically used in the treatment of certain disorders connected with women reproductive organs, especially, uterine fibroids. This study investigated the effect of methanol fraction ofM. indica(MFMI) extract on oestradiol valerate (OV)-induced endometrial hyperplasia (EH).Materials and Methods:The animals were randomly divided into four groups of seven rats each. These include a control group, an MFMI-alone group, a model (OV-alone) group and MFMI treatment (OV+MFMI) group. The EH was induced by intraperitoneal injection of OV. The levels of oestrogen (E2), progesterone (PG) and total cholesterol (TC) were determined using ELISA technique. The uterine histological and immunohistochemical assessments of oestrogen receptor, β-catenin and Ki-67 were carried out. Fibroblast cell count/μm2using histomorphometry as well as gas chromatography–mass spectrometry (GCMS) analysis of MFMI was carried out.Results:Severe EH was induced on oestradiol valerate administration. The MFMI was able to improve the pathological features of the animal model. Furthermore, the levels of oestrogen, PG and TC were reduced by MFMI. The immune reactive expression of oestrogen receptor alpha, β-catenin and Ki-67 was downregulated by MFMI coadministration. The histomorphometric analysis of the fibroblast cell count/μm2showed increased cell count density in the OV-treated group which was significantly ameliorated by MFMI coadministration. The GC– MS analysis revealed the presence of some pharmacologically relevant phytochemicals.Conclusion:This study suggests that MFMI contains phytochemicals that can ameliorate OV-induced EH in female Wistar and the possible mechanism of action involves modulation of oestrogen signalling pathway.

Perfluorooctane sulfonate interferes with non-genomic estrogen receptor signaling pathway, inhibits ERK1/2 activation and induces apoptosis in mouse spermatocyte-derived cells

Perfluorooctane sulfonate (PFOS) is a widespread persistent organic pollutant. Both epidemiological survey and our previous in vivo study have revealed the associations between PFOS exposure and spermatogenesis disorder, while the underlying mechanisms are far from clear. In the present study, GC-2 cells, a mouse spermatocyte-derived cell line, was used to investigate the toxic effects of PFOS and its hypothetical mechanism of action. GC-2 cells were treated with PFOS (0, 50, 100 and 150 μM) for 24 h or 48 h. Results demonstrated that PFOS dose-dependently inhibited cell viability, induced G0/G1 cell cycle arrest and triggered apoptosis, which might be partly explained by the decrease in cyclin D1, PCNA and Bcl-2 protein expression; increase in Bax protein expression; and activation of caspase-9, -3. In addition, PFOS did not directly transactivate or repress estrogen receptors (ERs) in gene reporter assays, whereas the protein levels of both ERα and ERβ were significantly altered and the downstream ERK1/2 phosphorylation was inhibited by PFOS. Furthermore, pretreatment with specific ERα agonist PPT (1 μM) significantly attenuated the above PFOS-induced effects while specific ERβ agonist DPN (1 μM) accelerated them. These results suggest that PFOS may induce growth inhibition and apoptosis via non-genomic estrogen receptor/ERK1/2 signaling pathway in GC-2 cells, which provides a novel insight regarding the potential role of ERs in mediating PFOS-triggered spermatocyte toxicity.

Predictive and Prognostic Biomarker Identification in a Large Cohort of Androgen Receptor-Positive Salivary Duct Carcinoma Patients Scheduled for Combined Androgen Blockade.

Simple SummaryAndrogen receptor signaling seems pivotal in aggressive salivary duct carcinoma. However, androgen deprivation therapy (ADT) frequently fails, emphasizing the need for biomarkers to predict treatment response. Here, the activities of several possible tumor-driving pathways were quantified and related to clinical outcome in a large cohort of SDC patients. Our results indicated that AR pathway activity and SRD5A1 expression levels can be used to predict response to ADT, which could prevent overtreatment in clinical practice.Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

Abstract 876: TP53 pathway function, estrogen receptor status, and breast cancer risk factors in the Carolina Breast Cancer Study

Abstract Purpose: TP53 and estrogen receptor (ER) both play essential roles in breast cancer development and progression, with recent research revealing crosstalk between TP53 and ER signaling pathways. While many studies have demonstrated heterogeneity of risk factor associations across ER subtypes, TP53 status has been inconsistently linked to breast cancer risk factors. This may be due to few studies evaluating TP53 effects in the context of ER. Additionally, studies have generally classified TP53 status using immunohistochemistry (IHC) staining or DNA sequencing, which are prone to misclassification of TP53 pathway function. RNA-based methods of measuring TP53 pathway activity may reduce misclassification and clarify etiologic associations. Methods: This case-only analysis included 4,466 incident breast cancer cases from the Carolina Breast Cancer Study (1993-2013). Using RNA expression previously quantified using NanoString assays on FFPE tumor samples, tumors were classified for TP53 functional status (mutant-like or wildtype-like) using a validated 52-gene RNA signature. We used a two-stage polytomous logistic regression model to evaluate sources of risk factor heterogeneity due to RNA-based TP53 or ER, adjusting for each other and for PR, HER2, and grade, while accounting for missing tumor marker data. Risk factor heterogeneity was also evaluated for IHC-based TP53 and ER. For each risk factor, joint effects of TP53 and ER were evaluated by allowing for an interaction between the two markers. Results: When classifying TP53 status using the RNA signature, the effects of several hormone-related factors (oral contraceptive use, menopausal status, age at menopause, and pre- and post-menopausal BMI) were heterogeneous across TP53 subtypes, while heterogeneity of reproductive factors (age at first birth and parity) and smoking status was observed across ER subtypes. The effect of number of births was heterogenous across both TP53 and ER-based subtype definitions. Additionally, we observed an interaction between RNA-based TP53 and ER status with family history of breast cancer in a first-degree relative (p=0.05). When classifying TP53 status using IHC, the TP53 effects were not recapitulated but there was minimal change in the ER effects. We observed an interaction between IHC-based TP53 and ER status with age at menarche (p=0.02), smoking status (p=0.05), and alcohol use (p=0.06). Conclusions: This study demonstrates that TP53 and ER are valuable for defining etiologic subtypes of breast cancer, although IHC measures may not have as much value as RNA. Analyses of the joint effects of TP53 and ER with breast cancer risk factors revealed intriguing findings that could lead to new hypotheses. However, larger studies incorporating multiple correlated tumor characteristics will be required to confirm our findings to conclusively define etiologically relevant subtypes of breast cancer. Citation Format: Amber N. Hurson, Mustapha Abubakar, Kathleen Conway-Dorsey, Katherine Hoadley, Michael I. Love, Andrew F. Olshan, Montserrat Garcia-Closas, Melissa A. Troester. TP53 pathway function, estrogen receptor status, and breast cancer risk factors in the Carolina Breast Cancer Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 876.