ERα Expression Research Articles

PFKFB3 regulates breast cancer tumorigenesis and Fulvestrant sensitivity by affecting ERα stability

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.

The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

Just like the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using 2 human PCa tissue microarray cohorts, we first demonstrate that nuclear ERα expression was heterogeneous among patients, being detected in only half of the tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimicked the androgen transcriptional response and activated specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprogrammed PCa metabolism, was associated with disease progression, and could be targeted for therapeutic purposes.

Estrogen receptors alpha and beta expression in different canine cancer types with an emphasis on hematopoietic malignancies.

Estrogen receptors (ERs) are located in both healthy and neoplastic tissues. The type of estrogen receptor expressed varies depending on its location, tumor type, and species. Estrogen action is mediated by binding to ER and activating the transcriptional and signaling processes that result in the control of gene expression. There are two main types of estrogen receptors: ER alpha (ERα) and ER beta (ERβ). Both receptors are functionally different, they may act antagonistically and are distributed in different tissues but their structure is similar - as they are composed of 5 different domains: A/B, C, D, E, and F. The signaling pathway and hence regulation of the gene expression by ERs is a complex and multifactorial process that involves both genomic and nongenomic actions. In the human reproductive tract, both ERα and β are present, with predominant expression of ERβ, while there are no satisfactory data distinguishing the type of ERs expressed in the canine reproductive tract. In mammary gland neoplasia, a decreased or lacking ERα expression in humans is associated with a poorer prognosis. This is similar to dogs, where higher ERα expression intensity was noted in benign tumors than in carcinomas. In human hematopoietic malignancies, ERβ is a predominant receptor. Selective and non-selective ERβ agonists have an antiproliferative and pro-apoptotic effect on human lymphoma cell lines and may be effective in the therapy of ERβ positive lymphomas and leukemias. In canine lymphoma tissues, none or only marginal expression of ERs was detected over the decades. Considering available data, we conducted preliminary studies proving that, in contrast to humans, the dominant ER expressed in canine hematopoietic tumors is ERα.

Ge-gen decoction alleviates primary dysmenorrhoea symptoms in a rat model

Background Existing treatments for primary dysmenorrhoea (PD), such as NSAIDs, impart side effects. Ge-Gen decoction (GGD), a traditional Chinese medicine, has shown promise in treating PD, but its exact mechanisms remain unclear. Here, we aimed to investigate the efficiency of GGD in alleviating PD using a rat model to understand its precise mechanism of action. Methods We established a rat model of dysmenorrhoea induced by oestradiol and oxytocin. The PD rats were administered GGD or Ibuprofen (positive control) intragastrically once daily for seven consecutive days. Serum levels of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2α), β-endorphin (β-EP), thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-keto-PGF1α) were determined using an enzyme-linked immunosorbent assay (ELISA). The expression levels of oestrogen receptor alpha (ERα) and cyclooxygenase-2 (COX-2) in uterine tissue were measured using immunohistochemical assays, and those of phosphorylated and total extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were assessed using western blot analysis. Results Treatment with GGD significantly reduced writhing behaviour, histopathological scores, and levels of COX-2, PGE2, and PGF2α in the serum of PD rats. Additionally, GGD increased β-EP content and inhibited ERK1/2 activation and ERα expression in uterine tissues. Conclusions The results of this study suggest that GGD alleviates PD in rats by suppressing the COX-2-mediated release of PGE2 and PGF2α, modulating the ERα/ERK1/2/COX-2 pathway, and increasing β-EP content. These results provide insights into the potential mechanisms of GGD in treating PD and support its further investigation as an alternative therapy for this condition.

Risk factors for head and neck squamous cell carcinoma in disease pathogenesis and prognosis: the importance of smoking cessation and the concept of harm reduction

Introduction. Cigarette smoking along with alcohol consumption, infection with human papilloma (HPV) and the Epstein–Barr viruses, is one of the main risk factors for head and neck squamous cell carcinoma. It can also contribute to virus-associated carcinogenesis and affect the clinical and prognostic features of the tumor. Continued smoking at diagnosis of head and neck squamous cell carcinoma is a negative prognostic factor and contributes to reduced patient survival rates. Aim. To analyze the smoking status and the effect of etiopathogenesis on molecular and clinical features in patients with oropharyngeal squamous cell carcinoma (OpSCC). Materials and methods. In patients with OPSCC, the smoking status and the level of motivation to its cessation were clarified. Immunohistochemical analysis of tumor sections from paraffin blocks from the ThermoScentific immunohis-tosteiner was performed using monoclonal antibodies to estrogen receptors α (ERα), programmed death-ligand 1 (pD-L1), Bcl-2, p53 and p16. The percentage of tumor-infiltrating lymphocytes (TILs) in the stromal component of the tumor (hematoxylin and eosin staining) was also analyzed. Results. Of 113 patients with OpSCC, 80 % of patients were current smokers, including 75 % of patients with Hpv-positive tumor status. men with OpSCC were statistically significantly more likely to smoke than women (p = 0.001), as were men with Hpv-positive tumor (p = 0.015). Smoking patients were characterized by a larger size of the primary tumor (T3–4), including those with Hpv (p = 0.015). positive Hpv status of the tumor is associated with autoimmune diseases (autoimmune thyroiditis, psoriasis) (p ˂ 0.05). A high level of TILs is characteristic of smokers, including those with p16+-status (p ˂0.05), but not for all patients with this status. The positive expression of ERα is associated with p16+-status, and the latter is associated with the absence of p53 expression (p ˂0.05). During conservative therapy, tumor regression is associated with the primary tumor size T1–2, p16 + status, female sex, age less than 55 years, TILs level ≥ 57.5 %, pD-L1 expression levels on tumor cells ≥2.2 %, Bcl-2 ≥5 % and ERα ≥65 % (p ˂ 0.05). At the same time, quality of good predictive model for tumor regression showed a TILs level of ≥57.5 % (area under curve, AuC) 0.796; sensitivity 78.6 %; specificity 75 %) and ERα (AuC 0.725; sensitivity 65 %; specificity 83 %). The level of motivation to quit smoking ranged from 1 to 7 points according the 10-point motivation scale in 89 % of respondents. Only 10 patients were ready to quit smoking immediately (10 points on the motivation scale). Conclusion. Molecular heterogeneity of OPSCC was revealed, including in the group of p16+-tumors. Correlation of clinical and molecular parameters with response to conservative therapy can be used in planning of primary therapy and earlier use of surgical treatment. The level of motivation to quit smoking in patients with squamous cell carcinoma of the head and neck may be increased by drawing attention to the problem at each doctor’s appointment as well as by informing about how to quit smoking and therapeutic approaches. In unmotivated patients, the concept of reducing the harm of smoking can be used involving the replacement of cigarettes with electronic tobacco heating systems, eliminating the carcinogenic effect of its combustion products.

Exploration and biological evaluation of 20-vinyl pregnenes: A step forward toward selective modulators of the estrogen receptor α signaling for breast cancer treatment.

A series of D-ring modified steroids bearing a vinyl ketone pendant were synthesized and evaluated for antiproliferative activity against breast cancer cell line and cytochromes P450. The lead compound, 21-vinyl 20-keto-pregnene (2f) (IC50 = 2.4 µM), was shown to be a promising candidate for future anticancer drug design, particularly against estrogen receptor α (ERα)-positive breast cancer. The lead compound was found to have a significant effect on the signaling pathways in parental and 4-hydroxytamoxifen-resistant cells. Compound 2f modulated the ERK, cyclin D1, and CDK4 pathways and blocked the expression of ERα, the main driver of breast cancer growth. Compound 2f significantly reduced 17β-estradiol-induced progesterone receptor expression. Accumulation of cleaved poly(ADP-ribose) polymerase in cells treated with compound 2f indicated induction of apoptosis. The selectivity analysis showed that lead compound 2f produces no significant effects on cytochromes P450, CYP19A1, CYP21A2, and CYP7B1.

The Estrogenic Effect of Lysiphyllum strychnifolium (Craib) A. Schmitz Leaf Water Extract in MCF-7 Cells

Lysiphyllum strychnifolium (Craib) A. Schmitz (LS) has been widely used as traditional medicine in the northeast region of Thailand to stimulate the production of breast milk. There are no known studies on the estrogenic action of LS on the stimulation of breast milk production. Hence, the present study aimed to investigate the estrogenic effect of LS leaf water extract compared with quercetin, one of the compounds in LS, in human breast cancer cells (MCF-7). The effect of LS leaf water extract and quercetin on cell viability of MCF-7 cells was studied by MTT assay at a concentration range of 0 to 500 µg/mL. The expression of estrogen-dependent genes, the pS2, ERα, and ERβ was also examined by real-time RT-PCR, and the expression of ERα protein was detected by Western blotting. The quercetin content in the LS water extract was 285.67 ± 0.11 µg/g of dry extract. MCF-7 cells treated with LS leaf water extract (20 µg/mL) showed an upregulation of pS2 gene expression similar to that of the treatment with E2 (10−9 M) compared with that of the untreated control. The ERα gene expression was found to be upregulated by quercetin (0.16 µg/mL) and E2 (10−5 M) compared with the untreated control. In addition, quercetin (0.16 µg/mL) and LS extract (0.8, 4, and 20 µg/mL) decreased the phosphorylation of ERα at Ser167. LS extract (20 µg/mL) decreased ERα, but there was no significant effect on the ERα at Ser118 protein expression. This study provided scientific evidence for the potential estrogenic activities of LS leaf water extract. This indicates that ER-dependent pathways in MCF-7 cells served as mediators, facilitating the estrogenic properties of LS and its compounds to function effectively. Since LS induced pS2 gene transcription, it was confirmed that this could affect the transcription of estrogen-responsive genes, causing estrogenic effects. HIGHLIGHTSThis is the first study to demonstrate the estrogenic effect of Lysiphyllum strychnifolium leaf water extract. L. strychnifolium leaf water extract induced pS2 gene transcription, and it was confirmed that this could affect the transcription of estrogen-responsive genes which cause estrogenic effects. The results of this study support the mechanism and scientific evaluation on the use of L. strychnifolium for stimulation of breast milk in folk medicine. GRAPHICAL ABSTRACT

Abstract 2994: MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling

Abstract Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported. Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients. Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation. Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer. Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2994.

Abstract 2084: Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3

Abstract Vitamin D3 supplementation is becoming widely accepted as a therapeutic in many common estrogen responsive cancers. Previous studies in breast cancer showed that 24R,25-dihydroxyvitamin D3 (24,25) regulates tumorigenesis via a caveolae membrane-associated signaling pathway involving phospholipase D [PLD] and palmitoylation of an unknown receptor. The goal of this study was to examine the effects of 24,25 in estrogen responsive laryngeal cancer tumorigenesis, investigate the mechanisms involved, and determine the ability of the tumor cells to produce 24,25 locally. Expression of CYP24A1 and CYP27B1 was measured in ERα66+ UM-SCC-12 (UM12) and ERα66- UM-SCC-11A (UM11A) laryngeal cancer cells. Cells were treated with 25(OH)D3, and production of the vitamin D3 metabolites 1,25(OH)2D3, 24,25(OH)2D3, and 1,24,25(OH)3D3 was measured using HPLC. The therapeutic potential of 24,25 was then assessed by treating the cells with 24,25 and measuring proliferation and apoptosis. In vivo, UM12 and UM11A cells were implanted into the cheek of NSG mice; the mice were treated with 24,25, and evaluated for survival and tumor volume. The signaling pathways involving PLD, palmitoylation, and caveolin-1 were investigated as driving mechanisms. CYP24A1 and CYP27B1 were present in ERα66+ UM12 and ERα66- UM11A cells, both of which are responsive to estrogen. When treated with 25(OH)D3, UM11A cells produced more 24,25 than UM12s, comparable to the physiologically relevant levels in blood. Both cells produced similar levels of 1,25. 24,25 increased proliferation and inhibited apoptosis in ERα66+ UM12 cells in vitro, but had the opposite effect in ERα66- UM11A cells. 24,25 increased PLD activity in UM12 cells but decreased PLD activity in UM11A cells. Inhibition of PLD activity or palmitoylation, as well as silencing expression of caveolin-1, reduced the effects of 24,25 on P53 in both cell types, indicating they played a role. Mice with UM11A tumors given 24,25 exhibited reduced tumor burden and increased survival compared to vehicle-treated controls. In contrast, mice with UM12 tumors given 24,25 had an increased tumor burden and reduced survival compared to controls. The presence of CYP24A1 and CYP27B1, local production of 24,25, and demonstration that laryngeal cancer cells respond to 24,25, indicate that 24,25 acts in an autocrine/paracrine manner. 24,25 was pro-tumorigenic in ERα66+ laryngeal cancer cells while having the opposite effect in ERα66- cells, both in vitro and in vivo, demonstrating a dependence on ERα66 expression. 24,25 acts via a mechanism involving PLD, caveolae, and palmitoylation. These data show that 24,25 regulates laryngeal cancer tumorigenesis, which is mediated by membrane associated signaling pathways that may depend on ERα66 level, and suggest a major role of 24R,25-dihydroxyvitamin D3 in laryngeal cancer. Citation Format: Cydney D. Dennis, D. Joshua Cohen, Jonathan T. Dillon, Barbara D. Boyan, Zvi Schwartz. Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2084.

Abstract 5873: ERα coordinates with squalene synthase to confer statin resistance in lung cancer

Abstract Statins are widely used to treat hypercholesterolemia by inhibiting hydroxymethylglutaryl coenzyme A reductase (HMGCR). Accumulating evidence suggests that statins also have protective effects against cancer recurrence. However, the role of statins in preventing lung cancer and biomarkers for predicting statin treatment response remains lacking. This study analyzes molecular features that dictate statin sensitivity in lung cancer by correlating large-scale statin sensitivity data and basal gene expression in a collection of 125 lung cancer patients. We identified a gene signature associated with lovastatin sensitivity and showed that gene expression in mesenchymal molecular features, the cholesterol biosynthesis pathway, and estrogen receptor alpha (ERα) signaling were key determinants of lovastatin sensitivity in lung cancer cells. To this end, we further discovered that overexpression of a committed step enzyme in cholesterol biosynthesis farnesyl-diphosphate farnesyltransferase 1 (FDFT1), also known as squalene synthase (SQS), is capable of activating ERα and is associated with increased statin tolerance. In clinical specimens, overexpression of SQS positively correlated with nuclear expression of ERα in lung cancer patients, and the combination of SQS and ERα is a powerful prognostic predictor for lung cancer. Activation of the SQS-ERα signaling axis and epithelial-to-mesenchymal transition (EMT) status represents two important features in determining statin sensitivity in lung cancer. Keywords: Statin, cholesterol biosynthesis, squalene synthase, estrogen receptor α, Epithelial-mesenchymal transition Citation Format: Yi-Fang Yang. ERα coordinates with squalene synthase to confer statin resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5873.

Abstract 6279: Estrogen signaling in low-grade serous ovarian carcinoma

Abstract High level of estrogen in the blood of women has been linked to the progression and possibly development of ovarian cancer. Almost all low-grade serous ovarian cancer (LGSOC) expresses estrogen receptor alpha (ERα protein encoded by the gene ESR1) and develops during reproductive age of women. However, recent studies suggested that a higher expression of ERα correlates with better survival. In a phase II clinical trial, hormonal therapies have been shown to be associated with a clinical benefit rate of 61% for recurrent ER-positive LGSOC patients. The study also indicated that women with recurrent LGSOC who received hormonal therapies had a significantly longer progression-free survival but not overall survival. Endocrine therapies have proven to be effective in treating breast cancer. Unfortunately, in ovarian cancer clinical trials (GOG0281 and PARAGON), these treatments have not achieved comparable results, with an objective response rate of only approximately 14%. To understand the ESR1 regulation in LGSOC, we have performed RNAseq and ChIPseq analyses of LGSOC tumor tissues. We also forced over-expression of ESR1 in LGSOC cell lines. Using RNAseq data, we aligned the sequencing reads to all the known ESR1 transcript variants. We found that ESR1 transcript variant 1, 2, 3 and X11 were the major transcripts expressed. Variants 1, 2 and 3 encode the regular 66 kDa ESR1 protein, while the X11 variant encodes a 52 kDa ESR1 protein. Both 66 kDa and 52 kDa ESR1 proteins were also detected in LGSOC tumor tissues by western blots. To further explore the function of ESR1 66kDa in LGSOC, we over-expressed ESR1 variant 1 in two LGSOC cell lines (HOC-7 and HCC5075) by transfecting with an expression plasmid. Total proteins were extracted from the mock control and transfected cells for functional proteomic analysis by reverse phase protein arrays (RPPA). From the RPPA data, overexpressing ESR1 in LGSOC cell lines suppressed DNA repair pathways but activated cell cycle and mitosis pathways. To further delineate the ESR1 DNA binding sites in LGSOC, we performed ChIPseq analysis of these tumor samples as well as two ovarian cancer cell lines that express ESR1 protein. Our results demonstrated that ESR1 regulated “cistromes” in ER positive ovarian carcinoma is very different from that in ER positive breast cancer as much less ESR1 binding sites were found in LGSOC. There are ESR1 binding sites common to both types of cancers, but we found quite a few unique ESR1 binding sites. We are trying to integrate the ChIPseq and RNAseq data and hopefully identify, for the first time, an ovarian cancer specific ERα cistrome. ER expression may not always correlate with activation of ESR1 signaling pathways, which may be one of the reasons underlying endocrine therapy resistance in ovarian cancer. The results of an integrated analyses will also give us insight on the management of ER positive LGSOC by identifying new genetic targets that may be used in combination with endocrine therapy. Citation Format: Yvonne TM Tsang, Chun Wai Oscar Ng, David Gershenson, K-Kwok wong. Estrogen signaling in low-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6279.

Abstract 3046: The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression

Abstract Just as the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet, the incomplete understanding of ERα cellular functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for treatment of this disease. Using two human PCa tissue microarray cohorts, we first demonstrated that nuclear ERα expression was heterogeneous between patients, being only detected in half of tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single cell RNA-seq analyses revealed that estrogens partially mimic the androgen transcriptional response and induce specific biological pathways linked to cellular proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprograms PCa metabolism, is associated with disease progression, and could be targeted for therapeutic purposes. Citation Format: Camille Lafront, Lucas Germain, Gabriel H. Campolina-Silva, Cindy Weidmann, Line Berthiaume, Hélène Hovington, Hervé Brisson, Cynthia Jobin, Lilianne Frégeau-Proulx, Raul Cotau, Kevin Gonthier, Aurélie Lacouture, Patrick Caron, Claire Ménard, Chantal Atallah, Julie Riopel, Éva Latulippe, Alain Bergeron, Paul Toren, Chantal Guillemette, Martin Pelletier, Yves Fradet, Clémence Belleannée, Frédéric Pouliot, Louis Lacombe, Éric Lévesque, Étienne Audet-Walsh. The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3046.

Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.

Estrogen and Progesterone Receptors Are Dysregulated at the BPH/5 Mouse Preeclamptic-Like Maternal-Fetal Interface.

The etiopathogenesis of preeclampsia, a leading hypertensive disorder of pregnancy, has been proposed to involve an abnormal circulating sex hormone profile and misexpression of placental estrogen and progesterone receptors (ER and PR, respectively). However, existing research is vastly confined to third trimester preeclamptic placentas. Consequently, the placental-uterine molecular crosstalk and the dynamic ER and PR expression pattern in the peri-conception period remain overlooked. Herein, our goal was to use the BPH/5 mouse to elucidate pre-pregnancy and early gestation Er and Pr dynamics in a preeclamptic-like uterus. BPH/5 females display low circulating estrogen concentration during proestrus, followed by early gestation hypoestrogenemia, hyperprogesteronemia, and a spontaneous preeclamptic-like phenotype. Preceding pregnancy, the gene encoding Er alpha (Erα, Esr1) is upregulated in the diestrual BPH/5 uterus. At the peak of decidualization, Esr1, Er beta (Erβ, Esr2), and Pr isoform B (Pr-B) were upregulated in the BPH/5 maternal-fetal interface. At the protein level, BPH/5 females display higher percentage of decidual cells with nuclear Erα expression, as well as Pr downregulation in the decidua, luminal and glandular epithelium. In conclusion, we provide evidence of disrupted sex hormone signaling in the peri-conception period of preeclamptic-like pregnancies, potentially shedding some light onto the intricate role of sex hormone signaling at unexplored timepoints of human preeclampsia.

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

BackgroundUbiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC.MethodsBioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR.ResultsUCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner.ConclusionUCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC.

Investigating the effect of hesperetin on estrogen receptor alpha (ERα) expression, phosphorylation and activity in MCF-7 cells

PurposeThe most common malignancy among women worldwide is breast cancer. The estrogen receptor plays a vital role in this cancer. One of the most well-known mechanisms that affects the activity of this receptor is its phosphorylation by protein kinase pathways. Hesperetin, a flavonoid abundant in citrus species such as lemons, grapefruits, and oranges, is the aglycone form of hesperidin. It has undergone thorough evaluation for its potential anti-cancer properties, particularly in the context of breast cancer. Studies have shown that hesperetin has an effect on intracellular kinase pathways. The aim of this study was to investigate the effect of hesperetin on the expression, phosphorylation and activity of estrogen receptor alpha (ERα) in MCF-7 breast cancer cell line. Study design and methodsMCF-7 cells were cultured in RPMI-1640 phenol red-free medium supplemented with charcoal-stripped FBS and treated with hesperetin. The MTT method was used to evaluate cell survival. The levels of the ERα protein and its phosphorylated form (Ser118) were determined via western blotting. A luciferase reporter vector was used to evaluate ERE activity. ResultsThe results of this study indicated that hesperetin reduced the survival of MCF-7 cells in a dose-dependent manner. The expression and phosphorylation (at Ser118) of the ERα significantly increased and decreased, respectively, in the groups treated with hesperetin. Hesperetin increased the activity of the ERα in the absence of E2, although these differences were not statistically significant. Conversely, in the presence of E2, hesperetin caused a significant decrease in receptor activity. ConclusionBased on the results of this study, it can be concluded that hesperetin has a significant effect on ERα expression, phosphorylation and activity.

EB1089 Increases the Antiproliferative Response of Lapatinib in Combination with Antiestrogens in HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is associated with aggressive behavior and reduced survival rates. Calcitriol restores the antiproliferative activity of antiestrogens in estrogen receptor (ER)-negative breast cancer cells by re-expressing ERα. Furthermore, calcitriol and its analog, EB1089, enhance responses to standard anti-cancer drugs. Therefore, we aimed to investigate EB1089 effects when added to the combined treatment of lapatinib and antiestrogens on the proliferation of HER2-positive breast cancer cells. BT-474 (ER-positive/HER2-positive) and SK-BR-3 (ER-negative/HER2-positive) cells were pre-treated with EB1089 to modulate ER expression. Then, cells were treated with EB1089 in the presence of lapatinib with or without the antiestrogens, and proliferation, phosphorylation array assays, and Western blot analysis were performed. The results showed that EB1089 restored the antiproliferative response to antiestrogens in SK-BR-3 cells and improved the inhibitory effects of the combination of lapatinib with antiestrogens in the two cell lines. Moreover, EB1089, alone or combined, modulated ERα protein expression and reduced Akt phosphorylation in HER2-positive cells. EB1089 significantly enhanced the cell growth inhibitory effect of lapatinib combined with antiestrogens in HER2-positive breast cancer cells by modulating ERα expression and Akt phosphorylation suppression. These results highlight the potential of this therapeutic approach as a promising strategy for managing HER2-positive breast cancer.

Neuropeptide W protects against ovarian oxidative injury and reinforces ovarian steroidogenic activity via the upregulation of ERα expression.

The aim of this study was to investigate the protective effect of neuropeptide W (NPW) on ovarian ischemia-reperfusion-induced oxidative injury and ovarian steroid metabolism. Rats were randomly divided into control and ischemia groups that received either saline or NPW (0.1 or 5 μg/kg/day). Bilateral ovarian ischemia was performed for 3 h followed by a 72-h reperfusion. Blood, ovary, and uterus samples were collected for biochemical and histological assessments. Treatment with either dose of NPW alleviated oxidative injury of the ovaries with a significant suppression in free radical formation and decreased histopathological injury in both the ovarian and uterine tissues, along with reduced lipid peroxidation and neutrophil accumulation in the uterus. Moreover, NPW treatment reversed the decrease in aromatase expression with a concomitant reduction in the expression of the inactivity enzyme estrogen sulfotransferase. Also, downregulation of estrogen receptor-α (ERα) expression in the injured ovarian tissue was abolished by NPW treatment, which implicates that the protective effect of NPW on the female reproductive system may involve the upregulation of the ERα-mediated signaling pathway. Our study demonstrated for the first time that NPW protects against ovarian oxidative injury and reinforces ovarian steroidogenic activity, which is accompanied by the upregulation of ERα expression in the ovaries.

Anticancer activity of Hedyotis corymbosa nanoliposomes targeting estrogen receptor-alpha in breast cancer cells: In silico and in vitro studies

Context: Breast cancer expresses high levels of the estrogen receptor alpha (ER-α). Aims: To investigate the potential anticancer activity of nanoliposome Hedyotis corymbosa in breast cancer cells by specifically targeting the ER-α through a combination of in silico and in vitro studies. Methods: This study used molecular docking to analyze the interaction between ER-α and active compounds from H. corymbosa. In vitro studies, the H. corymbosa extract was converted into nanoliposomes. The nanoliposomes' characteristics were examined, combined with tamoxifen, and applied to breast cancer cell cultures. Viability, proliferation, and ER-α expression were assessed, and statistical analysis was performed (p<0.05). Results: The findings show that H. corymbosa compounds, especially rutin, inhibit ER-α binding and act as a substrate for P-glycoprotein. Extract H. corymbosa selectively decreases viability in breast cancer cells (T47D) compared to non-cancerous cells (NIH3T3). Combining extracts with tamoxifen at 10 mg/mL enhances treatment efficacy. The combined treatment is effective for up to 72 h, reducing T47D breast cancer cell proliferation. Nanoliposomes derived from extract exhibit favorable characteristics, including a spherical shape, uniform size distribution, and stability. Using these nanoliposomes at 100 μg/mL with tamoxifen significantly reduces ER-α expression (p<0.05) without affecting cell viability. Conclusions: Compounds from H. corymbosa, especially rutin, inhibit ER-α binding. H. corymbosa nanoliposomes are stable for drug delivery. Combining tamoxifen with nanoliposomes at 100 μg/mL reduces ER-alpha expression. This shows potential for breast cancer treatment, but validation through further research and clinical trials is necessary.

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

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