Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

LncRNA-SERB promotes vasculogenic mimicry (VM) formation and tumor metastasis in renal cell carcinoma.

A growing body of evidence shows that vasculogenic mimicry (VM) is closely related to the invasion and metastasis of many tumor cells. Although the estrogen receptor (ER) can promote initiation and progression of renal cell carcinoma (RCC), how the downstream biomolecules are involved, and the detailed mechanisms of how ER expression is elevated in RCC remain to be further elucidated. Here, we discovered that long noncoding RNA (LncRNA)-SERB is highly expressed in tumor cells of RCC patients. We used multiple RCC cells and an in vivo mouse model for our study, and results indicated that LncRNA-SERB could boost RCC VM formation and cell invasion in vitro and in vivo. Although a previous report showed that ERß can affect the VM formation in RCC, it is unclear which factor could upregulate ERß. This is the first study to show LncRNA-SERB can be the upstream regulator of ERß to control RCC progression. Mechanistically, LncRNA-SERB may increase ERß via binding to the promoter area, and ERß functions through transcriptional regulation of zinc finger E-box binding homeobox 1 (ZEB1) to regulate VM formation. These results suggest that LncRNA-SERB promotes RCC cell VM formation and invasion by upregulating the ERß/ZEB1 axis and that therapeutic targeting of this newly identified pathway may better inhibit RCC progression.

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair.

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERß subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERß attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERß promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and ß-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERß were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERß subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

NLRC5 exerts anti-endometriosis effects through inhibiting ERß-mediated inflammatory response.

BACKGROUND: Endometriosis is well known as a chronic inflammatory disease. The development of endometriosis is heavily influenced by the estrogen receptor ß (ERß), while NOD-like receptors (NLRs) family CARD domain-containing 5 (NLRC5) exhibits anti-inflammatory properties during endometriosis. However, whether NLRC5-mediated anti-inflammation is involved in the ERß-mediated endometriosis is still uncertain. This study aimed to assess that relation. METHODS: Nine cases of eutopic endometrial tissue and ten cases of ectopic endometrial tissue were collected from patients with endometriosis, and endometrial samples from ten healthy fertile women were analyzed, and the expression levels of ERß were quantified using immunohistochemistry (IHC). Subsequently, we constructed mouse model of endometriosis by intraperitoneal injection. We detected the expression of ERß, NLRC5, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-10 and measured the volume of ectopic lesions in mice with endometriosis. In vitro, human endometrial stromal cells (hESCs) were transfected respectively with ERß-overexpressing and NLRC5-overexpressing plasmids. We then assessed the expression of ERß and NLRC5 using quantitative real-time PCR (qRT-PCR) and western blot analysis. Furthermore, we measured the concentrations of TNF-α, IL-6, and IL-10 in the cell culture supernatant through enzyme-linked immunosorbent assay (ELISA). Additionally, we evaluated the migration and invasion ability of hESCs using transwell and wound healing assays. RESULTS: Inhibition of NLRC5 expression promotes the development of ectopic lesions in mice with endometriosis, upregulates the expression of pro-inflammatory factors TNF-α and IL-6, and downregulates the expression of anti-inflammatory factor IL-10. The high expression of NLRC5 in endometriosis depended on the ERß overexpression. And ERß promoted the migration of hESCs partially depend on inflammatory microenvironment. Lastly, NLRC5 overexpression inhibited ERß-mediated development and inflammatory response of endometriosis. CONCLUSIONS: Our results suggest that the innate immune molecule NLRC5-mediated anti-inflammation participates in ERß-mediated endometriosis development, and partly clarifies the pathological mechanism of endometriosis, expanding our knowledge of the specific molecules related to the inflammatory response involved in endometriosis and potentially providing a new therapeutic target for endometriosis.

Inhibition of neuroinflammation and neuronal damage by the selective non-steroidal ERß agonist AC-186.

BACKGROUND: AC-186 (4-[4-4-Difluoro-1-(2-fluorophenyl) cyclohexyl] phenol) is a neuroprotective non-steroidal selective oestrogen receptor modulator. This study investigated whether inhibition of neuroinflammation contributed to neuroprotective activity of this compound. METHODS: BV-2 microglia were treated with AC-186 (0.65-5 µM) prior to stimulation with LPS (100 ng/mL). Levels of pro-inflammatory mediators and proteins were then evaluated. RESULTS: Treatment of LPS-activated BV-2 microglia with AC-186 resulted in significant (p < 0.05) reduction in TNFα, IL-6, NO, PGE2, iNOS and COX-2. Further investigations showed that AC-186 decreased LPS-induced elevated levels of phospho-p65, phospho-IκBα and acetyl-p65 proteins, while blocking DNA binding and luciferase activity of NF-κB. AC-186 induced significant (p < 0.05) increase in protein expression of ERß, while enhancing ERE luciferase activity in BV-2 cells. Effects of the compound on oestrogen signalling in the microglia was confirmed in knockdown experiments which revealed a loss of anti-inflammatory activity following transfection with ERß siRNA. In vitro neuroprotective activity of AC-186 was demonstrated by inhibition of activated microglia-mediated damage to HT-22 neurons. CONCLUSIONS: This study established that AC-186 produces NF-κB-mediated anti-inflammatory activity, which is proposed as a contributory mechanism involved in its neuroprotective actions. It is suggested that the anti-inflammatory activity of this compound is linked to its agonist effect on ERß.

Estrogen receptor beta (ERß) in esophageal cancer - a systematic review and meta-analysis.

BACKGROUND: Esophageal cancer is the eighth most common cause of cancer-related deaths worldwide. There are two main histological subtypes of esophageal cancer: adenocarcinoma and squamous cell carcinoma. Among the factors associated with the development of esophageal cancer, estrogen receptor beta (ERß) has been found to have a clinical significance. AIM: To investigate the relationship between ERß expression and esophageal cancer. METHODS: English Medical literature searches were conducted for ERß expression in patients with esophageal cancer versus healthy controls. Searches were performed up to August 31, 2023, using MEDLINE, PubMed, Embase and Google Scholar. Meta-analysis was performed by using Comprehensive meta-analysis software (Version 4, Biostat Inc., Englewood, NJ, USA). Pooled odds ratios (ORs) and 95% confidence intervals (95%CIs) were calculated. Heterogeneity was evaluated using Cochrane Q-test, and it was considered present if the Q-test P value was less than 0.10. I2 statistic was used to measure the proportion of inconsistency in individual studies, with I2 > 50% representing heterogeneity. We also calculated a potential publication bias. RESULTS: Ten studies representing 11 substudies were selected according to the inclusion criteria. The odds ratio of ERß expression in fixed effect analysis was 0.448, 95% CI: 0.237 to 0.846, 55.2% lower in esophageal cancer than in normal mucosa. Heterogeneity and inconsistency were low, and no publication bias was demonstrated. CONCLUSION: This meta-analysis showed that ERß expression is lower in esophageal cancer biopsy specimens than in healthy controls, this finding may have a significant effect on survival and can lead to new therapeutic avenues.

FoxA2 represses ERß-mediated pyroptosis in endometriosis by transcriptionally inhibiting IGF2BP1.

BACKGROUND: Endometriosis is a severe disease which is associated with excessive activation of pyroptosis. Our present research aimed to investigate the function of Forkhead Box A2 (FoxA2) in regulating pyroptosis in endometriosis. METHODS: IL-1ß and IL-18 concentrations were assessed using ELISA. Cell pyroptosis was analyzed using flow cytometry. TUNEL staining was performed to determine human endometrial stromal cells (HESC) death. Moreover, ERß mRNA stability was assessed using RNA degradation assay. Finally, the binding relationships between FoxA2, IGF2BP1 and ERß were verified by dual-luciferase reporter system, ChIP, RIP and RNA pull-down assays. RESULTS: Our results revealed that IGF2BP1 and ERß were significantly upregulated in ectopic endometrium (EC) tissues of endometriosis patients compared to that in eutopic endometrium (EU) tissues as well as IL-18 and IL-1ß levels. Loss-of-function experiments subsequently demonstrated that either IGF2BP1 knockdown or ERß knockdown could repress HESC pyroptosis. In addition, IGF2BP1 upregulation promoted the pyroptosis in endometriosis by binding to ERß and promoting ERß mRNA stability. Our further research displayed that FoxA2 upregulation suppressed HESC pyroptosis by interacting with IGF2BP1 promoter. CONCLUSION: Our research proved that FoxA2 upregulation downregulated ERß by transcriptionally inhibiting IGF2BP1, thereby repressing pyroptosis in endometriosis.

Cisplatin-activated ERß/DCAF8 positive feedback loop induces chemoresistance in non-small cell lung cancer via PTEN/Akt axis.

High levels of the estrogen receptor ß (ERß) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERß remains elusive. In this study, we demonstrated that targeting ERß could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERß, which facilitates its homodimerization and nuclear translocation. ERß activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERß, leading to ERß accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERß are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERß-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.

The last stage of development: The restructuring and plasticity of the cortex during adolescence especially at puberty.

There is considerable evidence of reorganization in the prefrontal cortex during adolescence in humans, as well as in rodents, where the cellular basis can be explored. Studies from my laboratory in the rat medial prefrontal cortex are reviewed here. In general, growth predominates before puberty. Pruning mainly occurs at puberty and after with decreases in the number of synapses, dendrites, and neurons. Perineuronal nets, extracellular structures that control plasticity, are pruned peripubertally only in female rats, which may further open the adolescent prefrontal cortex to environmental influences. This is supported by our recent evidence that exposure to mild stress early, but not late, in adolescence decreases prepulse inhibition. Additionally, exposure to methamphetamine in females early in adolescence increases the number of a major class of inhibitory interneurons, parvalbumin neurons, while the opposite occurs late in adolescence. In females, even estrogen receptor beta mRNA decreases at puberty in the prefrontal cortex. Interestingly, rats of both sexes perform better after puberty on a test of cognitive flexibility in the water maze. Thus, evidence is accruing that adolescence is not a single entity but rather an ongoing set of processes, and environmental effects will differ depending on timing and sex.

ERß activation improves nonylphenol-induced depression and neurotransmitter secretion disruption via the TPH2/5-HT pathway.

The aim of this study is to investigate the role of estrogen receptor ß (ERß) in nonylphenol (NP) - induced depression - like behavior in rats and its impact on the regulation of the TPH2/5-HT pathway. In the in vitro experiment, rat basophilic leukaemia cells (RBL-2H3) cells were divided into the four groups: blank group, NP group (20 µM), ERß agonist group (0.01 µM), and NP＋ERß agonist group (20 µM＋0.01 µM). For the in vivo experiment, 72 adult male Sprague-Dawley rats were randomly divided into following six groups: the Control, NP (40 mg/kg) group, ERß agonist (2 mg/kg, Diarylpropionitrile (DPN)) group, ERß inhibitor (0.1 mg/kg, 4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl) phenol (PHTPP)) group, NP+ERß agonist (40 mg/kg NP ＋ 2 mg/kg DPN) group, and NP+ERß inhibitor (40 mg/kg NP + 0.1 mg/kg PHTPP) group, with 12 rats in each group. Each rat in drug group were given NP by gavage and/or received a single intraperitoneal injection of DPN 2 mg/kg or PHTPP 0.1 mg/kg. Both in vivo and in vitro, NP group showed a decrease in the expression levels of ERß, tryptophan hydroxylase (TPH1), and tryptophan hydroxylase-2 (TPH2) genes and proteins, and reduced levels of DA, NE, and 5-hydroxytryptophan (5-HT) neurotransmitters. RBL-2H3 cells showed signs of cell shrinkage, with rounded cells, increased suspension and more loosely arranged cells. The effectiveness of the ERß agonist stimulation exhibited an increase exceeding 60% in RBL-2H3 cells. The application of ERß agonist resulted in an alleviation the aforementioned alterations. ERß agonist activated the TPH2/5-HT signaling pathways. Compared to the control group, the NP content in the brain tissue of the NP group was significantly increased. The latency to eat for the rats was longer and the amount of food consumed was lower, and the rats had prolonged immobility time in the behavioral experiment of rats. The expression levels of ERß, TPH1, TPH2, 5-HT and 5-HITT proteins were decreased in the NP group, suggesting NP-induced depression-like behaviours as well as disturbances in the secretion of serum hormones and monoamine neurotransmitters. In the NP group, the midline raphe nucleus showed an elongated nucleus with a dark purplish-blue colour, nuclear atrophy, displacement and pale cytoplasm. ERß might ameliorate NP-induced depression-like behaviors, and secretion disorders of serum hormones and monoamine neurotransmitters via activating TPH2/5-HT signaling pathways.

Mode of action exploration of reproductive toxicity induced by bisphenol S using human normal ovarian epithelial cells through ERß-MAPK signaling pathway.

BACKGROUND: In the plastics production sector, bisphenol S (BPS) has gained popularity as a replacement for bisphenol A (BPA). However, the mode of action (MOA) of female reproductive toxicity caused by BPS remains unclear and the safety of BPS is controversial. METHODS: Human normal ovarian epithelial cell line, IOSE80, were exposed to BPS at human-relevant levels for short-term exposure at 24 h or 48 h, or for long-term exposure at 28 days, either alone or together with five signaling pathway inhibitors: ICI 18,2780 (estrogen receptor [ER] antagonist), G15 (GPR30 specific inhibitor), U0126 (extracellular regulated protein kinase [ERK] 1/2 inhibitor), SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) or SB203580 (p38 mitogen­activated protein kinase [p38MAPK] inhibitor). MOA through ERß-MAPK signaling pathway interruption was explored, and potential thresholds were estimated by the benchmark dose method. RESULTS: For short-term exposure, BPS exposure at human-relevant levels elevated the ESR2 and MAPK8 mRNA levels, along with the percentage of the G0/G1 phase. For long-term exposure, BPS raised the MAPK1 and EGFR mRNA levels, the ERß, p-ERK, and p-JNK protein levels, and the percentage of the G0/G1 phase, which was partly suppressed by U0126. The benchmark dose lower confidence limit (BMDL) of the percentage of the S phase after 24 h exposure was the lowest among all the BMDLs of a good fit, with BMDL5 of 9.55 µM. CONCLUSIONS: The MOA of female reproductive toxicity caused by BPS at human-relevant levels might involve: molecular initiating event (MIE)-BPS binding to ERß receptor, key event (KE)1-the interrupted expression of GnRH, KE2-the activation of JNK (for short-term exposure) and ERK pathway (for long-term exposure), KE3-cell cycle arrest (the increased percentage of the G0/G1 phase), and KE4-interruption of cell proliferation (only for short-term exposure). The BMDL of the percentage of the S phase after 24 h exposure was the lowest among all the BMDLs of a good fit, with BMDL5 of 9.55 µM.

Effects of matcha tea extract on cell viability and estrogen receptor-ß expression on MCF-7 breast cancer cells.

PURPOSE: In the following work, we investigated the effect of matcha green tea extract (MTE) on MCF-7 breast cancer cell viability and estrogen receptor-beta expression (ERß). METHODS: MCF-7 cells were stimulated with MTE at concentrations of 5 and 10 µg/ml. Cell viability was assessed using a water-soluble tetrazolium assay (WST-1 assay) after an incubation time of 72 h. ERß was quantified at gene level by real-time polymerase chain reaction (PCR). A western blot (WB) was carried out for the qualitative assessment of the expression behavior of on a protein level. RESULTS: The WST-1 test showed a significant inhibition of viability in MFC-7 cells after 72 h at 10 µg/ml. The WB demonstrated a significant quantitative decrease of ERß at protein level with MTE concentrations of 10 µg/ml. In contrast, the PCR did not result in significant downregulation of ERß. CONCLUSION: MTE decreases the cell viability of MCF-7 cells and furthermore leads to a decrease of ERß at protein level.

Ovarian ERß cistrome and transcriptome reveal chromatin interaction with LRH-1.

BACKGROUND: Estrogen receptor beta (ERß, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized. RESULTS: We here performed ERß ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERß knockout ovaries. By integrating the ERß cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERß loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERß and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERß and LRH-1 co-binding at the ERß-repressed gene Greb1 but not at the ERß-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERß and LRH-1 can inhibit their respective transcriptional activity at classical response elements. CONCLUSIONS: By characterizing the genome-wide endogenous ERß chromatin binding, gene regulations, and extensive crosstalk between ERß and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

N-Myc and STAT Interactor is an Endometriosis Suppressor.

In patients with endometriosis, refluxed endometrial fragments evade host immunosurveillance, developing into endometriotic lesions. However, the mechanisms underlying this evasion have not been fully elucidated. N-Myc and STAT Interactor (NMI) have been identified as key players in host immunosurveillance, including interferon (IFN)-induced cell death signaling pathways. NMI levels are markedly reduced in the stromal cells of human endometriotic lesions due to modulation by the Estrogen Receptor beta/Histone Deacetylase 8 axis. Knocking down NMI in immortalized human endometrial stromal cells (IHESCs) led to elevated RNA levels of genes involved in cell-to-cell adhesion and extracellular matrix signaling following IFNA treatment. Furthermore, NMI knockdown inhibited IFN-regulated canonical signaling pathways, such as apoptosis mediated by Interferon Stimulated Gene Factor 3 and necroptosis upon IFNA treatment. In contrast, NMI knockdown with IFNA treatment activated non-canonical IFN-regulated signaling pathways that promote proliferation, including ß-Catenin and AKT signaling. Moreover, NMI knockdown in IHESCs stimulated ectopic lesions' growth in mouse endometriosis models. Therefore, NMI is a novel endometriosis suppressor, enhancing apoptosis and inhibiting proliferation and cell adhesion of endometrial cells upon IFN exposure.

Bone-Differentiation-Associated Circ-Spen Regulates Death of Mouse Bone Marrow Mesenchymal Stem Cells by Inhibiting Apoptosis and Promoting Autophagy.

The role of estrogen receptor ß (ERß) in bone health is closely associated with its function in vivo, and ERß-/- mice have been widely utilized to explore the related influences. In this study, ERß-/- female mice were established to investigate the differential expression of circular RNAs (circRNAs) by RNA-Sequencing (RNA-Seq). Among these circRNAs, mmu_circ_0011379 (named Circ-Spen) exhibited high expression in ERß-/- female mice. However, the precise mechanism by which Circ-Spen regulates bone health remained unclear. This study identified Circ-Spen as a positive regulator of mouse bone marrow mesenchymal stem cell (mBMSC) viability. The expression of Circ-Spen was markedly increased in ERß-/- mice femurs tested by RT-qPCR. Moreover, Circ-Spen exhibited an enhanced expression during the bone formation process of mBMSCs. Qualitative experiments also demonstrated that Circ-Spen possessed a circular structure and was localized within the nucleus of mBMSCs. Functionally, it inhibited apoptosis via caspase-3, BCL-2, and BAX, while also promoting autophagy through BECN1 and P62 in mBMSCs tested by MTT assays, transmission electron microscopy (TEM), and Western blotting. These findings reveal the potential of targeting Circ-Spen as a promising therapeutic strategy for rejuvenating senescent mBMSCs and enhancing the efficiency of mBMSC transplantation, which lays the foundation for advancements in the field of bone therapy.

Therapeutic targeting of angiopoietins in tumor angiogenesis and cancer development.

The formation and progression of tumors in humans are linked to the abnormal development of new blood vessels known as neo-angiogenesis. Angiogenesis is a broad word that encompasses endothelial cell migration, proliferation, tube formation, and intussusception, as well as peri-EC recruitment and extracellular matrix formation. Tumor angiogenesis is regulated by angiogenic factors, out of which some of the most potent angiogenic factors such as vascular endothelial growth factor and Angiopoietins (ANGs) in the body are produced by macrophages and other immune cells within the tumor microenvironment. ANGs have a distinct function in tumor angiogenesis and behavior. ANG1, ANG 2, ANG 3, and ANG 4 are the family members of ANG out of which ANG2 has been extensively investigated owing to its unique role in modifying angiogenesis and its tight association with tumor progression, growth, and invasion/metastasis, which makes it an excellent candidate for therapeutic intervention in human malignancies. ANG modulators have demonstrated encouraging outcomes in the treatment of tumor development, either alone or in conjunction with VEGF inhibitors. Future development of more ANG modulators targeting other ANGs is needed. The implication of ANG1, ANG3, and ANG4 as probable therapeutic targets for anti-angiogenesis treatment in tumor development should be also evaluated. The article has described the role of ANG in tumor angiogenesis as well as tumor growth and the treatment strategies modulating ANGs in tumor angiogenesis as demonstrated in clinical studies. The pharmacological modulation of ANGs and ANG-regulated pathways that are responsible for tumor angiogenesis and cancer development should be evaluated for the development of future molecular therapies.

The effect of hesperetin on estrogen receptor gene expression and its relationship with the downstream pathways of estrogen receptor alpha.

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, ERß, 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, ERß, 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 IC50 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). ERß 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.

ERß1 Sensitizes and ERß2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERß1 and ERß2 (isoforms of ERß), the second ER isotype. At present, the impact of ERß isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERß1 or ERß2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERß1 and MCF7-ERß2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERß1 cells and cancer-promoting effects in MCF7-ERß2 cells. Our data are favorable to ERß1 being a marker of responsiveness and ERß2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

The Co-Expression of Estrogen Receptors ERα, ERß, and GPER in Endometrial Cancer.

Estrogens have important roles in endometrial cancer (EC) and exert biological effects through the classical estrogen receptors (ERs) ERα and ERß, and the G-protein-coupled ER, GPER. So far, the co-expression of these three types of ERs has not been studied in EC. We investigated ERα, ERß, GPER mRNA and protein levels, and their intracellular protein distributions in EC tissue and in adjacent control endometrial tissue. Compared to control endometrial tissue, immunoreactivity for ERα in EC tissue was weaker for nuclei with minor, but unchanged, cytoplasmic staining; mRNA and protein levels showed decreased patterns for ERα in EC tissue. For ERß, across both tissue types, the immunoreactivity was unchanged for nuclei and cytoplasm, although EC tissues again showed lower mRNA and protein levels compared to adjacent control endometrial tissue. The immunoreactivity of GPER as well as mRNA levels of GPER were unchanged across cancer and control endometrial tissues, while protein levels were lower in EC tissue. Statistically significant correlations of estrogen receptor α (ESR1) versus estrogen receptor ß (ESR2) and GPER variant 3,4 versus ESR1 and ESR2 was seen at the mRNA level. At the protein level studied with Western blotting, there was significant correlation of ERα versus GPER, and ERß versus GPER. While in clinical practice the expression of ERα is routinely tested in EC tissue, ERß and GPER need to be further studied to examine their potential as prognostic markers, provided that specific and validated antibodies are available.

Methylation of ERß 5'-untranslated region attenuates its inhibitory effect on ERα gene transcription and promotes the initiation and progression of papillary thyroid cancer.

Normal thyroid tissue displays a prevalent expression of ERß than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERß were coexpressed in human thyroid tissues and cells. ERα mRNA (A-1) and ERß mRNA (0N-1), transcribed from Promoter A of ERα gene and Promoter 0N of ERß gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERß mRNA in human thyroid-derived cell lines and tissues. The expression levels of ERα mRNA (A-1) and total ERα mRNA were gradually increased, and those of ERß mRNA (0N-1) and total ERß mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5'-untranslated region was involved in its up-regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERß gene was found to be involved in its down-regulation in the initiation and progression of PTC. ERß can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A-specific region in thyroid-derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERß gene leads to a decreased ERß gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.