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OZADJE Hormonsko odvisne oblike rakov, med katere spadajo tudi rak dojke, rak endometrija in rak jajčnika, predstavljajo 25 % vseh rakov pri ženskah. Rak endometrija je peti najpogostejši rak. Rak jajčnika je sicer manj pogost, ima pa slabšo prognozo in večjo smrtnost. Estrogeni so steroidni hormoni, ki uravnavajo celično rast, proliferacijo in fiziologijo reproduktivnih procesov ter so med drugim vpleteni tudi v patofiziologijo rakavih obolenj. Učinki estrogenov so posledica aktivacije ER? in ERß, ki sprožita prepis tarčnih genov, medtem ko so negenomski učinki posledica interakcije z membransko vezanim ER in z vezavo na receptor GPER. Encimi iz proteinske naddružine aldo-keto reduktaz (AKR) so vpleteni v številne pomembne biokemijske procese, med drugim v biosintezo, metabolizem in detoksifikacijo endogenih in eksogenih spojin. Pri hormonsko odvisnih rakih imajo pomembno vlogo encimi AKR1C3, AKR1B1 in AKR1B10. Ti trije encimi uravnavajo proliferacijsko aktivnost celic in sposobnost diferenciacije ter so vpleteni v presnovo in rezistenco na kemoterapevtike ter tudi v metabolizem steroidnih hormonov. V okviru doktorske naloge smo preverili ali so encimi AKR1C3, AKR1B1 in AKR1B10 ter receptorji ER?, ERß in GPER biokemijski označevalci za napoved poteka bolezni in napoved verjetnosti razvoja rezistence na posamezne kemoterapevtike. METODE V preiskovano skupino smo vključili 233 bolnic, ki so se zdravile zaradi raka jajčnika ali raka endometrija. Na vzorcih v parafin vklopljenega rakastega tkiva smo izvedli imunohistokemijska barvanja z validiranimi protitelesi in po optimiziranih protokolih in tako preverili prisotnost izbranih proteinov AKR1C3, AKR1B1, AKR1B10, ER?, ERß in GPER. Ocenili smo delež, vzorec in intenziteto barvanja. S statističnimi metodami smo preverili možne povezave med izražanjem proučevanih proteinov s preživetjem in z drugimi klinično-patološkimi podatki. Dodatno smo analizirali podatke o izražanju ER?, ERß in GPER na ravni RNA (RNA-Seq normalizirano v seriji) in podatke ekspresije na proteinski ravni (proteinske mikromreže z reverzno fazo) iz prosto dostopne podatkovne baze Portal cBioPortal (https://www.cbioportal.org/). REZULTATI Rak endometrija Pri bolnicah z rakom endometrija smo zaznali značilno višje izražanje AKR1C3, AKR1B1 in AKR1B10 v okolnem ne-neoplastičnem endometriju v primerjavi z endometrioidnim rakom endometrija. Analiza preživetja je pri bolnicah z endometrioidnim rakom endometrija pokazala, da visoko izražanje AKR1C3 ter izražanje AKR1B1 in AKR1B10 nad mediano vrednostjo korelira z boljšim celokupnim preživetjem in s preživetjem brez ponovitve bolezni. Pri analizi receptorjev za estrogene smo ugotovili, da je v primerjavi s kontrolnim tkivom endometrija imunoreaktivnost za ER? v jedrih šibkejša pri raku endometrija v citoplazmi ni bilo razlik. Za ERß je bila jedrna in citoplazemska imunoreaktivnost nespremenjena v rakavem tkivu v primerjavi s kontrolnim tkivom. Imunoreaktivnost GPER je bila nespremenjena pri raku in kontrolnem tkivu endometrija. Analiza preživetja je pri bolnicah z endometrioidnim rakom endometrija z višjo ravnjo ER? pokazala trend daljšega preživetja. Pri bolnicah z rakom endometrija (tipa 1 in tipa 2) je značilno daljše preživetje pri raku z višjo ravnjo ER? in višjim izražanjem genov ESR1 in GPER. Izražanje gena ESR2 na preživetje nima vpliva. Izražanje genov ESR1, ESR2 in GPER ter proteina ER? ni imelo statistično značilnega vpliva na preživetje brez ponovitve bolezni. Rak jajčnika Pri bolnicah z rakom jajčnika ni bilo korelacije med izražanjem AKR1C3 in celokupnim preživetjem ali preživetjem brez ponovitve bolezni ali odzivom na kemoterapijo. Višja imunohistokemijska raven AKR1B1 je bila povezana z boljšim celotnim preživetjem in preživetjem brez ponovitve bolezni pri bolnicah s seroznim rakom jajčnika visokega gradusa, medtem ko raven AKR1B10 ni pokazala pomembnih razlik. Povezave med AKR1B1 in AKR1B10 z različnimi odzivi na kemoterapijo nismo zaznali. Analiza preživetja je pokazala, da pri bolnicah s seroznim rakom jajčnika visokega gradusa ni statistično značilne povezave med celokupnim preživetjem in med ravnjo izražanja genov ESR1, ESR2 in GPER ali proteina ER?. Izražanje gena ESR2 nad mediano pri seroznem raku jajčnika visokega gradusa je bilo povezano z daljšim preživetjem brez ponovitve bolezni, medtem ko izražanje genov ESR1 in GPER ni vplivalo na preživetje brez ponovitve bolezni. ZAKLJUČEK Z rezultati doktorske disertacije smo prispevali k boljšemu poznavanju patofiziologije raka endometrija in raka jajčnika. Določili smo izražanje receptorjev za estrogene in prvi okarakterizirali aldo-keto reduktaze AKR1C3, AKR1B1 in AKR1B10 v večjem številu vzorcev raka endometrija in raka jajčnika. Pri obeh malignih obolenjih smo pokazali povezavo aldo-keto reduktaz s patogenezo in opisali povezavo različnih ravni proučevanih proteinov s preživetjem. Z našimi rezultati smo opredelili biokemijske označevalce s prognostičnim potencialom, kar odpira možnosti za prihodnje študije in morebiten prenos v klinično prakso. BACKGROUND Hormone-dependent cancers, including breast, endometrial and ovarian cancers, account for 25% of all cancers in women. Endometrial cancer is the fifth most common cancer. Ovarian cancer is less common, but has a worse prognosis and higher mortality. Estrogens are steroid hormones that regulate cell growth, proliferation and the physiology of reproductive processes and are also involved in the pathophysiology of cancer. Estrogens effects are caused by the activation of ERα and ERβ, which trigger the transcription of target genes, while the non-genomic effects are caused by interaction with the membrane-bound ER and by binding to the GPER receptor. Enzymes from the aldo-keto reductase (AKR) protein superfamily are involved in many important biochemical processes, including the biosynthesis, metabolism and detoxification of endogenous and exogenous compounds. AKR1C3, AKR1B1 and AKR1B10 enzymes play an important role in hormone-dependent cancers. Among other, these three enzymes regulate cell proliferation, differentiation, and are involved in metabolism and resistance to chemotherapeutic agents, as well as in the metabolism of steroid hormones. We investigated whether the enzymes AKR1C3, AKR1B1 and AKR1B10 and receptors ERα, ERβ and GPER are prognostic biochemical markers for estimating the course of the disease and the probability of developing resistance to individual chemotherapeutic agents. METHODS In study we included 233 patients who were treated for ovarian cancer or endometrial cancer. We performed immunohistochemical staining on paraffin-embedded cancerous tissue samples using validated antibodies according to optimized protocols and analyzed the presence of selected proteins AKR1C3, AKR1B1, AKR1B10, ERα, ERβ and GPER. We evaluated the proportion, pattern and intensity of staining. Statistical methods were used to check possible correlations between the expression of the studied proteins with survival and with other clinical and pathological data. We additionally analyzed the expression data for ERα, ERβ and GPER at the RNA level (RNA-Seq normalized in series) and protein-level expression data (reverse-phase protein microarrays) from the open access database Portal cBioPortal (https://www.cbioportal.org/). RESULTS Endometrial cancer In patients with endometrial cancer, we detected a significantly higher expression of AKR1C3, AKR1B1 and AKR1B10 in the adjacent non-neoplastic endometrium compared to endometrioid endometrial cancer. Survival analysis in patients with endometrioid endometrial cancer showed that high expression of AKR1C3 and expression of AKR1B1 and AKR1B10 above the median value correlates with better overall survival and disease-free survival. When analyzing estrogen receptors, we found that nuclear immunoreactivity for ERα in is weaker in endometrial cancer compared to control endometrial tissue there were no differences in the cytoplasm. For ERβ, nuclear and cytoplasmic immunoreactivity was unchanged in cancer tissue compared to control tissue. GPER immunoreactivity was unchanged in cancer and control endometrial tissue. Survival analysis showed a trend for longer survival in patients with endometrioid endometrial cancer with higher ERα levels. In patients with endometrial cancer (type 1 and type 2), statistically significant longer survival was found in patients with a higher level of ERα and higher expression of the ESR1 and GPER genes, while the expression of the ESR2 gene had no effect on survival. Expression of ESR1, ESR2 and GPER genes and ERα protein had no statistically significant effect on disease free survival. Ovarian cancer In patients with ovarian cancer, there was no correlation between AKR1C3 expression and overall or disease-free survival or response to chemotherapy. A higher immunohistochemical level of AKR1B1 was associated with better overall survival and disease-free survival in patients with high-grade serous ovarian cancer, whereas AKR1B10 level showed no significant differences. We did not detect an association between AKR1B1 and AKR1B10 with different responses to chemotherapy. Survival analysis showed that in patients with high-grade serous ovarian cancer, there was no statistically significant association between overall survival and high or low expression levels of ESR1, ESR2 and GPER genes or ERα protein. ESR2 gene expression above the median in high-grade serous ovarian cancer was associated with longer disease-free survival, whereas ESR1 and GPER gene expression did not affect disease free survival. CONCLUSION With the results of the doctoral dissertation, we contributed to better understanding of the pathophysiology of endometrial and ovarian cancer. We determined the expression of estrogen receptors and were the first to characterize the aldo-keto-reductases AKR1C3, AKR1B1 and AKR1B10 in a large number of endometrial and ovarian cancer samples. In both malignant diseases, we showed the connection of aldo-keto-reductases with pathogenesis and described the association of different levels of the studied proteins with survival. With our results, we identified biochemical markers with prognostic potential, which opens up possibilities for future studies and possible translation into clinical practice.

Estrogens have important roles in endometrial cancer (EC) and exert biological effects through the classical estrogen receptors (ERs) ERα and ERβ, 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, ERβ, 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 ERβ, 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 β (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 ERβ versus GPER. While in clinical practice the expression of ERα is routinely tested in EC tissue, ERβ and GPER need to be further studied to examine their potential as prognostic markers, provided that specific and validated antibodies are available.

The expression of miRNAs is one of the main epigenetic mechanisms responsible for the regulation of gene expression in mammals, and in cancer, miRNAs participate by regulating the expression of protein-coding cancer-associated genes. In canine mammary tumors (CMTs), the ESR1 gene encodes for ERα, and represents a major target gene for miR-18a and miR-18b, previously found to be overexpressed in mammary carcinomas. A loss in ERα expression in CMTs is commonly associated with poor prognosis, and it is noteworthy that the downregulation of the ESR1 would appear to be more epigenetic than genetic in nature. In this study, the expression of ESR1 mRNA in formalin-fixed, paraffin-embedded (FFPE) canine mammary tumors (CMTs) was evaluated and compared with the expression levels of miR18a and miR18b, both assessed via RT-qPCR. Furthermore, the possible correlation between the miRNA expression data and the immunohistochemical prognostic factors (ERα immunoexpression; Ki67 proliferative index) was explored. A total of twenty-six FFPE mammary samples were used, including 22 CMTs (7 benign; 15 malignant) and four control samples (three normal mammary glands and one case of lobular hyperplasia). The obtained results demonstrate that miR-18a and miR-18b are upregulated in malignant CMTs, negatively correlating with the expression of target ESR1 mRNA. Of note, the upregulation of miRNAs strictly reflects the progressive loss of ERα immunoexpression and increased tumor cell proliferation as measured using the Ki67 index. The results suggest a central role of miR-18a and miR-18b in the pathophysiology of canine mammary tumors as potential epigenetic mechanisms involved in ERα downregulation. Moreover, as miRNA expression reflects ERα protein status and a high proliferative index, miR-18a and miR-18b may represent promising biomarkers with prognostic value. More detailed investigations on a larger number of cases are needed to better understand the influence of these miRNAs in canine mammary tumors.

Isocitrate dehydrogenase (IDH) mutations increase (R)-2-hydroxyglutarate (R-2HG) production; however, functional mechanisms of R-2HG in regulating cholangiocarcinoma (CCA) development remain to be further investigated. We first applied the CRISPR-Cas9 gene-editing system to create IDH1R132H-mutated CCA cells. Interestingly, our data showed that R-2HG could function through downregulating estrogen receptor alpha (ERα) and Yes-associated protein 1 (YAP1) pathways to decrease CCA growth. Detailed mechanistic studies revealed that R-2HG could target and degrade the fat mass and obesity-associated protein (FTO), the first identified mRNA demethylase. This reduced FTO can increase the N6-methyladenosine (m6A) to methylate the mRNA of ERα, and consequently decrease protein translation of the ERα. Further mechanistic studies revealed that ERα could transcriptionally suppress miR-16-5p expression, which could then increase YAP1 expression due to the reduced miR-16-5p binding to the 3′ UTR of YAP1. Furthermore, data from the pre-clinical animal model with implantation of IDH1R132H QBC939 cells demonstrated that R-2HG generated by the IDH1 mutation could downregulate ERα and YAP1 to suppress CCA tumor growth. Taken together, our new findings suggested that IDH1 mutation-induced R-2HG could suppress CCA growth via regulating the FTO/m6A-methylated ERα/miR16-5p/YAP1 signaling pathway. Upregulating R-2HG or downregulating the ERα signal by short hairpin RNA ERα (shERα) or antiestrogen could be effective strategies to inhibit CCA., Graphical abstract, Our study demonstrates that (R)-2-hydroxyglutarate (R-2HG), produced by IDH1/2 mutations, could suppress tumor growth in cholangiocarcinoma through regulating the FTO/m6A-methylated ERα/miR16-5p/YAP1 pathway.

Estrogen receptor alpha (ERα) expression in ~2/3 breast tumors selects patients for hormonal therapies. Tumors negative for ERα but positive for the progesterone receptor (PR, encoded by PGR) have also been candidates for ER-targeting therapies, as PR expression may reflect undetected ER activity. Conversely, PR− status in ER+ tumors predicts a worse therapeutic response. Our analysis of breast tumor transcriptome datasets, however, revealed that in tumors with lower PGR expression, the clinical PR status does not correlate accurately with the expression of ESR1 or of ER target genes, including PGR itself. We identified carbonic anhydrase 12 (CA12) as an estrogen target gene better correlated with ESR1 than PGR, reflecting CA12 regulation by both ERα and the luminal factor and upstream ESR1 regulator GATA3. Immunostaining supported strong positive correlations at the protein level with ERα and GATA3 in a cohort of 118 tumors. Most ER+PR− tumors expressed CAXII at levels similar to those of ER+PR+ tumors, consistent with observations in tumor transcriptome datasets and with active estrogenic signaling in some ER+PR− breast cancer cell lines. The few ER−PR+ tumors did not express CAXII or the other luminal markers FOXA1 and GATA3. Overall, CAXII is a luminal marker that can help interpret ER status in single ER/PR positive tumors.

The identification of novel prognostic biomarkers for breast cancer is an unmet clinical need. Cullin-associated and neddylation-dissociated 1 (CAND1) has been implicated in mediating carcinogenesis in prostate and lung cancers. In addition, CAND1 is an established prognostic biomarker for worse prognosis in liver cancer. However, the prognostic significance of CAND1 in breast cancer has not yet been explored. In this study, Breast Cancer Gene-Expression Miner (Bc-GenExMiner) and TIMER2.0 were utilized to explore the mRNA expression of CAND1 in ERα-positive breast cancer patients. The Kaplan–Meier plotter was used to explore the relationship between CAND1 expression and several prognostic indicators. The Gene Set Cancer Analysis (GSCA) web server was then used to explore the pathways of the genes that correlate with CAND1 in ERα-positive breast cancer. Immune infiltration was investigated using Bc-GenExMiner. Our bioinformatics analysis illustrates that breast cancer patients have higher CAND1 compared to normal breast tissue and that ERα-positive breast cancer patients with a high expression of CAND1 have poor overall survival (OS), distant metastasis-free survival (DMFS), and relapse-free survival (RFS) outcomes. Higher CAND1 expression was observed in histologic grade 3 compared to grades 2 and 1. Our results revealed that CAND1 positively correlates with lymph nodes and negatively correlates with the infiltration of immune cells, which is in agreement with published reports. Our findings suggest that CAND1 might mediate invasion and metastasis in ERα-positive breast cancer, possibly through the activation of estrogen and androgen signaling pathways; however, experiments should be carried out to further explore the role of CAND1 in activating the androgen and estrogen signaling pathways. In conclusion, the results suggest that CAND1 could be used as a potential novel biomarker for worse prognosis in ERα-positive breast cancer.

Purpose: The stimulator of interferon genes (STING) pathway plays a crucial role in antitumor immunity, and it is strictly regulated by many types of post-translational modifications. However, the contribution of acetylation involved in the regulation of STING to endometrial tumorigenesis remains unclear. Methods: We attempted to identify the key role of STING in endometrial carcinoma (EC) tissue and cell lines and explore its epigenetic regulation mechanism by HDACs that are critically involved in EC. We used IHC and qRT-PCR to detect the protein level and mRNA level of STING expression in endometrial carcinoma tissues, then explored the potential role of STING in tumor proliferation and apoptosis by CCK8 and flow cytometry, and identified the STING effect in the tumorigenicity by a mouse xenograft assay. We explored the possible relationship of acetylation alteration in STING regulation by ChIP analysis and Co-IP, and we knocked out STING in ECC1 and Ishikawa cells using CRISPR-Cas9 to further confirm the critical role of STING restoration induced by HDAC3 inhibitor RGFP-966 in the proliferation and apoptosis. Results: We found that STING expression was largely decreased and worked as an important regulator of cell proliferation and apoptosis; either activated or overexpressed STING, with both pharmacological and genetic approaches, largely blocked cell proliferation and induced apoptosis in EC. Moreover, STING expression was deregulated by both β-estradiol and HDAC3. Mechanically, we determined that HDAC3 can interact with β-estradiol-ERα and induce deacetylation of histone 3 lysine 4 at the STING promoter, thereby decreasing STING expression. Inhibition of HDAC3 increased STING expression, thereby inhibiting tumorigenesis. Conclusion: This study reveals a novel molecular mechanism by which HDAC3 inhibits STING transcription via β-estradiol-ERα and provides a promising therapy (a combination of HDAC and STING) for combating endometrial cancer.

Tubulin β class I gene (TUBB) is highly expressed in various cancers and plays several roles in carcinogenesis. However, the prognostic value of TUBB in breast cancer remains to be investigated. GEPIA and Breast Cancer Gene-Expression Miner were used to explore TUBB expression in breast cancer patients. Kaplan–Meier Plotter was used to assess the relationship between TUBB expression and several prognostic indicators including overall, distant metastasis-free, and relapse-free survival in ERα-positive and ERα-negative breast cancer. The genes that correlate with TUBB in ERα-positive and ERα-negative breast cancer were explored and the pathways were investigated using GSCA. The correlation between TUBB and several gene markers of immune cells was explored using GEPIA. ERα-positive breast cancer patients with increased TUBB showed worse prognosis, possibly through the activation of the TSC/mTOR pathway, whereas ERα-negative breast cancer patients with increased TUBB mRNA showed better prognosis. Significant positive correlations were observed between TUBB and gene markers of immune cells in ERα-positive breast cancer patients, whereas significant negative correlations were observed in ERα-negative breast cancer patients. The analysis revealed that TUBB might be considered as a predictive biomarker for worse prognosis in ERα-positive and better prognosis in ERα-negative breast cancer.

[ES] Con la menopausia se inicia el climaterio, periodo fisiológico en el que se produce el cese de la fertilidad debido a que los ovarios de una mujer dejan de liberar óvulos. Durante esta etapa, las mujeres pueden experimentar signos y síntomas que se atribuyen a la disminución de hormonas sexuales, principalmente estrógenos y progesterona. La menopausia es una característica casi exclusiva del ser humano, por lo que para el estudio experimental de posibles alteraciones metabólicas y fisiológicas es necesario contar con un modelo experimental. En este sentido, las ratonas SAM (Senescence Accelerated Mouse) resistentes y propensas a desarrollar senescencia (SAMR1 y SAMP8, respectivamente) sometidas a un procedimiento quirúrgico de extirpación de ovarios (ovariectomía), permiten estudiar el efecto de la ausencia de hormonas sexuales y, además, determinar la contribución del envejecimiento, proceso también asociado a la menopausia. Dentro del grupo de los estrógenos, es el 17β-estradiol (estradiol) el que ejerce los efectos estrogénicos más potentes. Las funciones fisiológicas del estradiol están reguladas por los receptores de estrógenos (ER, de sus siglas en inglés), siendo los principales el ERα, el ERβ y el receptor de estrógenos asociado a proteína G, GPER. ERα y ERβ tienen funciones reguladoras en la transcripción de genes diana, se expresan en gran variedad de células y tejidos, y modulan distintas funciones fisiológicas además de las reproductivas, principalmente a través de acciones a nivel cardiovascular, esquelético y nervioso. El GPER, también tiene un papel importante en eventos rápidos de señalización no genómica debido a la activación de numerosas vías de señalización intracelular como la PI3/AKT o MAPK ERK1/2. Todos los receptores de estrógenos tienen algunas funciones comunes pero su papel es más o menos destacado dependiendo del tejido en el que se expresen. En este contexto, el presente trabajo tiene como objetivo estudiar si la ausencia de hormonas ováricas inducida por una menopausia experimental produce una alteración en la expresión de receptores de estrógenos en diferentes tejidos. Para ello, nos hemos centrado en estudiar la expresión por qRT-PCR de los principales receptores de estrógenos ERα, ERβ y GPER en diferentes tejidos procedentes del modelo murino de menopausia experimental SAMR1/SAMP8, concretamente en corazón, bazo, pulmón, hígado y riñón., [EN] Menopause is the beginning of the climacteric, a physiological period in which fertility ceases because a woman's ovaries stop releasing eggs. During this stage, women may experience signs and symptoms that are attributed to the decrease in sex hormones, mainly estrogen and progesterone. Menopause is an almost exclusive characteristic of human beings, so for the experimental study of possible metabolic and physiological alterations it is necessary to have an experimental model. In this sense, SAM (Senescence Accelerated Mouse) resistant and senescence-prone mice (SAMR1 and SAMP8, respectively) subjected to a surgical procedure of ovarian removal (ovariectomy), allow us to study the effect of the absence of sex hormones and, in addition, to determine the contribution of aging, a process also associated with menopause. Within the estrogen group, it is 17β-estradiol (estradiol) that exerts the most potent estrogenic effects. The physiological functions of estradiol are regulated by estrogen receptors (ER), the main ones being ERα, ERβ and the G protein-associated estrogen receptor, GPER. ERα and ERβ have regulatory functions in the transcription of target genes, are expressed in a wide variety of cells and tissues, and modulate different physiological functions in addition to reproductive ones, mainly through actions at the cardiovascular, skeletal and nervous levels. GPER also plays an important role in rapid non-genomic signaling events due to the activation of numerous intracellular signaling pathways such as PI3/AKT or MAPK ERK1/2. All estrogen receptors have some common functions but their role is more or less prominent depending on the tissue in which they are expressed. In this context, the present work aims to study whether the absence of ovarian hormones induced by an experimental menopause produces an alteration in the expression of estrogen receptors in different tissues. To this end, we have focused on studying the expression by qRT-PCR of the main estrogen receptors ERα, ERβ and GPER in different tissues from the murine model of experimental menopause SAMR1/SAMP8, specifically in heart, spleen, lung, liver and kidney.

En 2020, le cancer du sein est le type de cancer qui affecte le plus de personnes dans le monde mais est une maladie hétérogène. Le type luminal, représentant plus de 70 % des tumeurs, est identifiable par l'expression du récepteur des œstrogènes alpha (ERα). Certaines tumeurs comprennent cependant un mélange de cellules ERα-positives/négatives (ERα+/–). Les raisons de cette hétérogénéité restant inconnues, nous avons cherché à identifier une protéine membranaire positivement corrélée à ERα, dans le but d'isoler et caractériser des populations de cellules tumorales viables ERα+/– par la technique de Fluorescence Activated Cell Sorting (FACS). Nos résultats démontrent la régulation de CA12 par ERα et par le facteur de transcription luminal GATA3 dans des lignées cellulaires tumorales mammaires et son expression protéique prépondérante dans les tumeurs mammaires luminales. Nous avons développé une approche de purification cellulaire par FACS en utilisant un anticorps contre la partie extracellulaire de CAXII afin de trier des cellules tumorales ERα+/–. Nous avons finalement examiné la fonction de CAXII dans les tumeurs mammaires luminales et ainsi démontré une contribution significative à l’acidification du pH extracellulaire et à la migration cellulaire. Cette étude indique que CAXII représente un bon marqueur de l'activité de ERα et GATA3 dans les tumeurs luminales et pourrait être utilisé pour séparer les cellules ERα+ des cellules ERα– à partir de tumeurs hétérogènes et ainsi déterminer par profilage génique la relation évolutive entre les cellules tumorales ERα+/– et identifier les régulateurs potentiels de la différenciation luminale dans le cancer du sein., In 2020, breast cancer is the type of cancer that affects the most people in the world but is a heterogeneous disease. The luminal type, representing more than 70% of tumors, is identifiable by the expression of the estrogen receptor alpha (ERα). However, some tumors include a mixture of ERα-positive/negative cells (ERα+/–). As the reasons for this heterogeneity remain unknown, we aimed to identify a membrane protein positively correlated with ERα, in order to isolate and characterize populations of viable ERα+/- tumor cells by the Fluorescence Activated Cell Sorting (FACS) technique. Our results demonstrate the regulation of CA12 by ERα and by the luminal transcription factor GATA3 in mammary tumor cell lines and its predominant protein expression in luminal mammary tumors. We have developed a cellular purification approach by FACS using an anticoprs against the extracellular part of CAXII in order to sort ERα+/– tumor cells. We finally examined the function of CAXII in luminal mammary tumors and thus demonstrated a significant contribution to extracellular pH acidification and cell migration. This study indicates that CAXII represents a good marker of the activity of ERα and GATA3 in luminal tumors and could be used to separate ERα+ cells from ERα– cells from heterogeneous tumors and thus determine by gene profiling the evolutionary relationship between ERα+/– tumor cells and identify potential regulators of luminal differentiation in breast cancer.

Deregulated metabolism is one of the characteristics of hepatocellular carcinoma. Sex hormone receptor signalling has been involved in the marked gender dimorphism of hepatocellular carcinoma pathogenesis. Oestrogen receptor (ER) has been reported to reduce the incidence of liver cancer. However, it remains unclear how oestrogen and ER regulate metabolic alterations in liver tumour cells. Our previous work revealed that ERα interacted with carbohydrate responsive element binding protein (ChREBP), which is a transcription factor promoting aerobic glycolysis and proliferation of hepatoma cells. Here, the data showed that ERα overexpression with E2 treatment reduced aerobic glycolysis and cell proliferation of hepatoma cells. In addition to modestly down‐regulating ChREBP transcription, ERα promoted ChREBP degradation. ERα co‐immunoprecipitated with both ChREBP‐α and ChREBP‐β, the two known subtypes of ChREBP. Although E2 promoted ERα to translocate to the nucleus, it did not change subcellular localization of ChREBP. In addition to interacting with ChREBP‐β and promoting its degradation, ERα decreased ChREBP‐α–induced ChREBP‐β transcription. Taken together, we confirmed an original role of ERα in suppressing aerobic glycolysis in liver cancer cells and elucidated the mechanism by which ERα and ChREBP‐α together regulated ChREBP‐β expression.

Protein arginine methyltransferase 5 (PRMT5) has previously been reported to be upregulated in many malignant tumors. This study investigated the significance of PRMT5 in endometrial carcinoma (EC) and explored its function in tumorigenesis. Immunohistochemistry was performed to evaluate PRMT5 expression in 62 EC and 66 endometrial hyperplasia samples. The functions of PRMT5 were investigated by cell counting kit‐8, plate colony formation, wound healing, and transwell and flow cytometry assays. Quantitative reverse transcription‐polymerase chain reaction and western blotting were used to measure the expression of PRMT5, changes in estrogen receptor α (ERα), and related functional proteins. Coimmunoprecipitation was performed to examine the interaction of PRMT5 with ERα and its coactivator steroid receptor coactivator‐1 (SRC1). Compared to endometrial hyperplasia tissue, PRMT5 was overexpressed in endometrioid adenocarcinoma (EAC) but not overexpressed in mucinous EC. The main expression pattern of PRMT5 in EAC was cytoplasmic. However, the positive cases of endometrial hyperplasia showed both cytoplasmic and nuclear positivity in the endometrial glands or were mainly positive in stromal cells. Knockdown of PRMT5 significantly inhibited the growth and migration ability of EAC cells and promoted their apoptosis by regulating cyclin D1, c‐myc, p53, and Bcl2 proteins. Furthermore, PRMT5 could form a complex with ERα and SRC1 to promote the expression of ERα. In conclusion, PRMT5 plays a significant role in the progression of EAC by interacting with ERα and impacting the cell cycle signaling pathways.

Although different types of drugs are available for postmenopausal osteoporosis, the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents. Here, we defined that norlichexanthone (NOR), a natural product, is a ligand of estrogen receptor-alpha (ERα) and revealed its therapeutic potential for postmenopausal osteoporosis. We used mammalian-one hybrid assay to screen for ERα modulators from crude extracts of several plant endophytes. As a result, NOR purified from the extract of endophyte ARL-13 was identified as a selective ERα modulator. NOR directly bound to ERα with an affinity in nanomolar range, revealing that it is a natural ligand of ERα. NOR induced osteoblast formation in MC3T3-E1 precursor cells. Conversely, NOR inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation in both RAW264.7 macrophages and mouse primary monocytes. Mechanistically, NOR inhibited RANKL-induced association of ERα and TRAF6 to prevent ERα-mediated TRAF6 activation via Lys63-linked ubiquitination. Importantly, NOR exhibited potent anti-osteoporosis efficacy in an ovariectomized mouse model. Comparing to estrogen, NOR was of much less capability in stimulating endometrial hyperplasia and promoting mammalian cancer cell proliferation. Taken together, our study identified NOR as a natural and high affinity ligand of ERα with substantial anti-osteoporosis but less estrogenic activity.

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

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

Yanfang Liu,* Hong Ma,* Jing Yao Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing YaoCancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1277, Wuhan, Hubei Province 430022, People’s Republic of ChinaTel +86-189 8627 1157Email 2007XH0839@hust.edu.cnAbstract: Estrogen receptor α (ERα) is closely associated with both hormone-dependent and hormone-independent tumors, and it is also essential for the development of these cancers. The functions of ERα are bi-faceted; it can contribute to cancer progression as well as cancer inhibition. Therefore, understanding ERα is vital for the treatment of those cancers that are closely associated with its expression. Here, we will elaborate on ERα based on its structure, localization, activation, modification, and mutation. Also, we will look at co-activators of ERα, elucidate the signaling pathway activated by ERα, and identify cancers related to its activation. A comprehensive understanding of ERα could help us to find new ways to treat cancers.Keywords: ERα, estrogen receptors, estradiol, signaling pathway, cancer

Estrogen receptor α (ERα) is closely associated with both hormone-dependent and hormone-independent tumors, and it is also essential for the development of these cancers. The functions of ERα are bi-faceted; it can contribute to cancer progression as well as cancer inhibition. Therefore, understanding ERα is vital for the treatment of those cancers that are closely associated with its expression. Here, we will elaborate on ERα based on its structure, localization, activation, modification, and mutation. Also, we will look at co-activators of ERα, elucidate the signaling pathway activated by ERα, and identify cancers related to its activation. A comprehensive understanding of ERα could help us to find new ways to treat cancers.

We report on a stabilizer of the interaction between 14‐3‐3ζ and the Estrogen Receptor alpha (ERα). ERα is a driver in the majority of breast cancers and 14‐3‐3 proteins are negative regulators of this nuclear receptor, making the stabilization of this protein‐protein interaction (PPI) an interesting strategy. The stabilizer (1) consists of three symmetric peptidic arms containing an arginine mimetic, previously described as the GCP motif. 1 stabilizes the 14‐3‐3ζ/ERα interaction synergistically with the natural product Fusicoccin‐A and was thus hypothesized to bind to a different site. This is supported by computational analysis of 1 binding to the binary complex of 14‐3‐3 and an ERα‐derived phosphopeptide. Furthermore, 1 shows selectivity towards 14‐3‐3ζ/ERα interaction over other 14‐3‐3 client‐derived phosphomotifs. These data provide a solid support of a new binding mode for a supramolecular 14‐3‐3ζ/ERα PPI stabilizer., A stabilizer of the interaction between the Estrogen Receptor alpha (ERα), a driver in the majority of breast cancers, and 14‐3‐3ζ, a negative regulator of ERα, is developed as anti‐cancer strategy. The stabilizer of this protein‐protein interaction (PPI) consists of three symmetric peptidic arms and stabilizes the 14‐3‐3ζ/ERα interaction synergistically with the natural product Fusicoccin‐A.

Background: Some stemness-associated transcription factors consistently play essential roles in the maintenance of pluripotency or induce the differentiation of cancer stem cells (CSCs). However, the regulatory mechanism of CSC stemness mediated by transcription factors has not been extensively explored. Here, we show that two transcription factors (YB-1 and ERα), which are simultaneously highly expressed in estrogen receptor (ER)-positive CSCs, interact with each other to regulate the stemness and differentiation of ER-positive CSCs. Methods: The expression of YB-1 was examined in ER-positive CSCs and patient specimens. Western blot, real-time PCR, cell viability analysis, tumorsphere formation assay and subcutaneous tumorigenesis assays were used to study the stemness functions of YB-1 and ERα in CSCs. The relationship between YB-1 and ERα in cells was studied by promoter activity analysis, the electrophoretic mobility shift assay (EMSA) and the Co-IP assay. The mechanisms and functional significance of YB-1 in the sensitivity of CSCs to tamoxifen were further investigated with both in vitro and in vivo models. Results: YB-1 was aberrantly upregulated in the cancerous tissue of ER-positive breast cancer patients and in CSCs. Knockdown of YB-1 in ER-positive CSCs significantly inhibited cell stemness and induced differentiation, and the expression of YB-1 could be regulated by estrogen signaling and ERα in ER-positive breast CSCs. The Co-IP results showed that YB-1 interacted directly with ERα specifically in ER-positive non-CSCs and that YB-1 induced ERα degradation by ubiquitination via direct interaction in differentiated cells. Cell differentiation induced by FBS could inhibit YB-1 phosphorylation and promote YB-1 protein transfer from the nucleus to the cytoplasm. Moreover, cell differentiation induced by targeting inhibited the expression of YB-1 in ER-positive CSCs, which increased the sensitivity of cells to tamoxifen in vitro and in vivo. Conclusion: The ERα/YB-1 axis has an important role in the regulation of ER-positive breast cancer stemness. The dephosphorylation of YB-1 and the interaction between YB-1 and ERα may be the switch that initiates the differentiation of ER-positive CSCs. Targeting YB-1 to sensitize ER-positive CSCs to antiestrogen therapy might represent a new therapeutic strategy that warrants further exploration.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

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

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

The incidence and mortality of breast cancer (BCa) are the highest among female cancers. There are approximate 70% BCa that are classified as estrogen receptor alpha (ERα) positive. Therefore, targeting ERα is the most significantly therapeutic schedule. However, patients with breast cancer develop resistance to ERα or estrogen (E2) antagonists such as fulvestrant and tamoxifen. In the present study, we found that L-Tetrahydropalmatine (L-THP) significantly suppressed cell proliferation in ERα+ BCa cells via inducing cell cycle arrest rather than apoptosis. Additionally, L-THP enhanced the sensitivity of ERα+ BCa cells to tamoxifen and fulvestrant. Mechanically, the application of L-THP promotes ERα degradation through accumulating ubiquitin chains on ERα. Overexpressing ERα abrogates L-THP induced-antiproliferation in ERα+ BCa cells. Collectively, our work indicates that L-THP may represent a potentially novel therapeutic medicine for ERα+ breast cancer patient.

Background Cystatin SN (CST1) has been reported to act as an oncogene in cancers, but its underlying mechanism remains unclear. Methods We performed Western blotting analyses to observe protein expression and conducted transwell invasion, wound healing, and colony formation assays to assess cell invasion, migration, and proliferation, respectively. We also performed cell cycle analyses by flow cytometry to determine the role of CST1 in the cell cycle. In vivo experiments used subcutaneous tumor models in BALB/c-nu athymic female mice to evaluate the effect of CST1 on tumor growth. Results Western blotting analyses showed that CST1 was upregulated in ER+ breast cancer cells such as MCF7, T47D, and BT474. CST1 knockdown led to slower cell growth and inhibited the G1 to S phase transition in ER+ breast cancer cells. In vivo experiments showed that CST1 deletion inhibited tumor growth, and led to decreased expression of estrogen receptor α (ERα) and p-AKT. In vitro experiments showed that the over-expression of CST1 led to the upregulation of ERα, and inhibition of CST1 inhibited the expression of ERα. Western blotting analyses showed that CST1 regulated the activity of the PI3K/AKT signaling pathway in breast cancer cells. We confirmed that CST1 acted as an oncogene in ER+ breast cancer by regulating the ERα/PI3K/AKT/ERα loopback pathway. Conclusion CST1 acts as an oncogene in ER+ breast cancer, and CST1 contributes to cancer development by regulating the ERα/PI3K/AKT/ERα loopback pathway in ER+ breast cancer. Our findings indicate that CST1 could be a significant therapeutic target for ER+ breast cancer patients. Our discovery should inspire further studies on the role of CST1 in cancers.

Yanfang Liu,* Hong Ma,* Ye Wang, Xinyang Du, Jing Yao Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing YaoCancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1277, Wuhan 430022, Hubei Province, People’s Republic of ChinaTel +86-18986271157Email 2007XH0839@hust.edu.cnBackground: Cystatin SN (CST1) has been reported to act as an oncogene in cancers, but its underlying mechanism remains unclear.Methods: We performed Western blotting analyses to observe protein expression and conducted transwell invasion, wound healing, and colony formation assays to assess cell invasion, migration, and proliferation, respectively. We also performed cell cycle analyses by flow cytometry to determine the role of CST1 in the cell cycle. In vivo experiments used subcutaneous tumor models in BALB/c-nu athymic female mice to evaluate the effect of CST1 on tumor growth.Results: Western blotting analyses showed that CST1 was upregulated in ER+ breast cancer cells such as MCF7, T47D, and BT474. CST1 knockdown led to slower cell growth and inhibited the G1 to S phase transition in ER+ breast cancer cells. In vivo experiments showed that CST1 deletion inhibited tumor growth, and led to decreased expression of estrogen receptor α (ERα) and p-AKT. In vitro experiments showed that the over-expression of CST1 led to the upregulation of ERα, and inhibition of CST1 inhibited the expression of ERα. Western blotting analyses showed that CST1 regulated the activity of the PI3K/AKT signaling pathway in breast cancer cells. We confirmed that CST1 acted as an oncogene in ER+ breast cancer by regulating the ERα/PI3K/AKT/ERα loopback pathway.Conclusion: CST1 acts as an oncogene in ER+ breast cancer, and CST1 contributes to cancer development by regulating the ERα/PI3K/AKT/ERα loopback pathway in ER+ breast cancer. Our findings indicate that CST1 could be a significant therapeutic target for ER+ breast cancer patients. Our discovery should inspire further studies on the role of CST1 in cancers.Keywords: ERα, CST1, breast cancer, cancer, PI3K/AKT signaling pathway

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

ObjectiveThe curative effect of high-efficiency progesterone and other therapeutic drugs for endometrioid adenocarcinoma patients with preservation of reproductive capacity has not been satisfactory so far. Novel therapeutic drugs need to be explored.MethodsWe investigated the cytoplastic and nuclear expression levels of LMTK3 between endometrioid adenocarcinoma tissues and adjacent endometrial tissues by immunohistochemistry. We detected the effects of LMTK3 on cell viability of Ishikawa cells by CCK-8. We detected the effects of LMTK3 on cell cycle and apoptosis of Ishikawa cells by flow cytometry. We also detected the effects of LMTK3 knockdown on mRNA and protein levels of ERα by qRT-PCR and western blotting, respectively. We also used the cBioPortal online database to analyze the coexpression of LMTK3 and ESR1 in 1647 UCEC samples.ResultsWe used TMAs to identify that LMTK3 was mainly detected in the cytoplasm of endometrioid tissues, and cytoplasmic LMTK3 expression in endometrioid tissues was higher than that in adjacent endometrial tissues (P < 0.05). LMTK3 knockdown decreased the proliferation of Ishikawa cells through decreasing cell viability (P < 0.01), increasing G1 (P < 0.001) arrest, and promoting apoptosis (P < 0.01). There was a positive correlation between the mRNA expression levels of LMTK3 and ESR1 (Spearman: P=2.011e-5, R=0.13; Pearson: P=7.18e-8, R=0.17). Knockdown of LMTK3 also reduced the mRNA (P < 0.001) and protein (P < 0.001) levels of ERα.ConclusionsInhibitors of LMTK3 may be a possible future treatment for ERα and LMTK3 highly expressed endometrioid adenocarcinoma following appropriate studies.

Frontiers in Pediatrics 9, 1-9 (2021). doi:10.3389/fped.2021.729198, Published by Frontiers Media, Lausanne

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

TP53 est l'un des gènes les plus fréquemment mutés dans le cancer du sein (~ 30% de tumeurs) et code pour le suppresseur de tumeur p53. La majorité des mutations TP53 observées dans le cancer du sein sont exclusives à chaque sous-type, avec une fréquence de mutation élevée (75-80%) dans les sous-types de récepteurs aux œstrogènes négatifs (statut ER-), qu’ils soient de type HER2 + ou basal, alors que cette fréquence est plus faible (15-30%) dans les tumeurs ER + de sous-types de luminal A ou B. Les tumeurs mammaires ER + dépendent des œstrogènes pour leur croissance et peuvent être traitées avec des anti-œstrogènes, alors que les tumeurs ER- sont résistantes à un tel traitement. Il a été publié que p53 régule positivement l'expression de ER, suggérant que des mutations pourraient contribuer à la perte de ER. Nous avons donc émis l'hypothèse que les mutations TP53 trouvées dans les sous-types ER- pourraient provoquer la perte d'expression de ER, de la dépendance aux œstrogènes pour la prolifération, et de la sensibilité aux anti-œstrogènes. L'expression de TP53, qui n’est pas muté dans la lignée cellulaire de cancer du sein ER + MCF-7, a été supprimée par une approche CRISPR-Cas9. Un panel de mutations de TP53 a été sélectionné en fonction de leur distribution dans les différents sous-type, des niveaux ER associés à la présence de ces mutations et de leur fréquence dans des collections de tumeurs à grande échelle. Ces mutations ont ensuite été introduites dans une lignée cellulaire clonale TP53 KO par rétrovirus. Ni la suppression de TP53 ni l'introduction de mutations en combinaison avec la stimulation Nutlin-3a n'ont affecté l'expression de ER dans les cellules MCF7. En outre, alors que la sensibilité à Nutlin-3a est abolie dans les lignées cellulaires KO ou porteuses de mutations de p53, aucune capacité proliférative supplémentaire n'a été observée en présence d'estradiol. Enfin, l'arrêt du cycle cellulaire par les anti-œstrogènes n'a pas été aboli par le KO de TP53. Cependant, le TP53 KO a démontré, outre une résistance à la doxorubicine et aux rayonnements ionisants, une capacité accrue de croissance clonale en l'absence d'oestrogènes. Cela suggère que la mutation TP53 n'est pas impliquée dans les phénotypes liés à ER+, mais pourrait constituer un médiateur de transition vers une croissance indépendante des œstrogènes., TP53 is one of the most commonly mutated genes in breast cancer (~30% tumors) and encodes the tumour suppressor p53. The majority of TP53 mutations observed in breast cancer are enriched in different subtypes, with an overall higher frequency of mutation found in the estrogen receptor negative (ER-) subtypes of HER2+ and basal-like (75-80%) compared to the ER+ subtypes of luminal A and B (15-30%). ER+ breast tumours are dependent on estrogens for growth and may be treated with endocrine therapies, whereas ER- tumours are resistant to such treatments. p53 has been reported to regulate ER expression, suggesting that mutations could contribute to loss of ER. We therefore hypothesized that the TP53 mutations found in ER- subtypes may directly or indirectly lead to loss of ER expression levels and of estrogen-dependent proliferation and antiestrogen sensitivity. Wildtype TP53 expression in the ER+ breast cancer cell line MCF-7 was suppressed by CRISPR-Cas9. A panel of TP53 mutations was selected based on subtype bias, associated ER levels, and mutation frequency. This panel was then stably expressed into the TP53 KO clonal cell lines using retroviral expression vectors. Neither suppression of TP53 or introduction of mutations in combination with Nutlin-3a stimulation suppressed estrogen receptor expression in MCF7 cells. Furthermore, while sensitivity to Nutlin-3a was abolished in the mutant p53 bearing cell lines in proliferation assays in the presence of estradiol, no estrogen-independent proliferative capacity was observed. Finally, antiestrogen-mediated cell cycle arrest was not relieved upon TP53 KO. However, TP53 KO demonstrated an increased capacity for clonal growth in the absence of estrogens, and resistance to doxorubicin and ionizing radiation. Our results suggest that TP53 mutations are not involved in loss of ER+ related phenotypes but may act as mediators of transition to estrogen-independent growth.

Background The heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in their biological features, invasiveness, and response to chemotherapy, but the transcriptional regulators causing their differences remain nebulous. Methods In this study, we compared high-grade serous ovarian cancers (HGSOCs) to low malignant potential or serous borderline tumors (SBTs). Our aim was to discover new regulatory factors causing distinct biological properties of HGSOCs and SBTs. Results In a discovery dataset, we identified 11 differentially expressed genes (DEGs) between SBTs and HGSOCs. Their expression correctly classified 95% of 267 validation samples. Two of the DEGs, TMEM30B and TSPAN1, were significantly associated with worse overall survival in patients with HGSOC. We also identified 17 DEGs that distinguished stage II vs. III HGSOC. In these two DEG promoter sets, we identified significant enrichment of predicted transcription factor binding sites, including those of RARA, FOXF1, BHLHE41, and PITX1. Using published ChIP-seq data acquired from multiple non-ovarian cell types, we showed additional regulatory factors, including AP2-gamma/TFAP2C, FOXA1, and BHLHE40, bound at the majority of DEG promoters. Several of the factors are known to cooperate with and predict the presence of nuclear hormone receptor estrogen receptor alpha (ER-alpha). We experimentally confirmed ER-alpha and PITX1 presence at the DEGs by performing ChIP-seq analysis using the ovarian cancer cell line PEO4. Finally, RNA-seq analysis identified recurrent gene fusion events in our EOC tumor set. Some of these fusions were significantly associated with survival in HGSOC patients; however, the fusion genes are not regulated by the transcription factors identified for the DEGs. Conclusions These data implicate an estrogen-responsive regulatory network in the differential gene expression between ovarian cancer subtypes and stages, which includes PITX1. Importantly, the transcription factors associated with our DEG promoters are known to form the MegaTrans complex in breast cancer. This is the first study to implicate the MegaTrans complex in contributing to the distinct biological trajectories of malignant and indolent ovarian cancer subtypes.

Estrogen receptor α (ERα) is the major driver for breast tumor carcinogenesis and progression, while ERα positive breast cancer is the major subtype in breast malignancies, which account for 70% breast cancers in patients. The success of endocrine therapy such as tamoxifen is one of the biggest breakthroughs in breast cancer treatments. However, the endocrine therapy resistance is a headache problem in breast cancer. Further mechanisms need to be identified to the effect of ERα signaling in controlling breast cancer progression and drug resistance. HOIL-1 was firstly identified as the ERα transcriptional co-activator in modulating estrogen signaling in breast cancer. In our current study, we showed that HOIL-1, which was elevated in breast cancer, related to good prognosis in ERα positive breast cancer, but correlated with poor outcome in endocrine-treated patients. HOIL-1 was required for ERα positive breast cancer proliferation and clone formation, which effect could be rescued by further ERα overexpression. Further mechanism studies showed that HOIL-1 is required for ERα signaling activity in breast cancer cells. HOIL-1 could interact with ERα in the cytosol and modulate ERα stability via inhibiting ERα K48-linked poly-ubiquitination. Thus, our study demonstrated a novel post-translational modification in ERα signaling, which could provide novel strategy for ERα-driven breast cancer therapy.

XiaoDan Mao,1,* Binhua Dong,1,* Min Gao,2 GuanYu Ruan,1 MeiMei Huang,1 Elena Ioana Braicu,3 Jalid Sehouli,3 PengMing Sun11Laboratory of Gynecologic Oncology, Fujian Provincial Maternity and Children’s Hospital, Affiliated Hospital of Fujian Medical University, Fuzhou 350001, People’s Republic of China; 2Department of Gynecology Oncology, Peking University Cancer Hospital, Beijing 100142, People’s Republic of China; 3Department of Gynecology, Campus Virchow-Klinikum, Charité Universitätmedizin Berlin, Berlin D-13353, GermanyCorrespondence: PengMing SunLaboratory of Gynecologic Oncology, Fujian Provincial Maternity and Children’s Hospital, Affiliated Hospital of Fujian Medical University, No. 18 Daoshan Road, Fuzhou 350001, Fujian, People’s Republic of ChinaTel +86 1 378 887 3900Fax +86 5 918 755 1247Email sunfemy@hotmail.com*These authors contributed equally to this workBackground: Endometrial cancer (EC) is a hormone dependent carcinoma that may involve complex molecular mechanisms. Endocrine therapy by blocking the estrogen and estrogen receptor α (ERα) has been effective in breast cancer, while it is still controversial in EC. Recently, estrogen-related receptor α (ERRα) was proven to be another endocrine therapy target.Methods: The anti-tumor effect of selective estrogen receptor modulators (SERMs) and XCT790 (XCT) used alone or in combination were evaluated in both of ERα-positive (ERα+) and ERα-negative (ERα-) EC cells. ERα and ERRα mRNA were tested by qPCR, while the protein was detected by Western blot. The proliferation was tested by MTS and cell cycle, apoptosis rate were analyzed by flow cytometry.Results: A relatively high dose (10μM) of tamoxifen (TAM) suppressed the expression of ERα and ERRα in two types of EC cells. However, 10μM raloxifene (RAL) exhibited no effect on ERα and ERRα, while 10μM XCT down regulated ERRα specifically, but not ERα in all EC cells. When dual targeting on ERα and ERRα by combining TAM with XCT, the proliferation inhibitory effect and apoptosis reached the strongest in all EC cells (P

Background Endometrial cancer (EC) is a hormone dependent carcinoma that may involve complex molecular mechanisms. Endocrine therapy by blocking the estrogen and estrogen receptor α (ERα) has been effective in breast cancer, while it is still controversial in EC. Recently, estrogen-related receptor α (ERRα) was proven to be another endocrine therapy target. Methods The anti-tumor effect of selective estrogen receptor modulators (SERMs) and XCT790 (XCT) used alone or in combination were evaluated in both of ERα-positive (ERα+) and ERα-negative (ERα-) EC cells. ERα and ERRα mRNA were tested by qPCR, while the protein was detected by Western blot. The proliferation was tested by MTS and cell cycle, apoptosis rate were analyzed by flow cytometry. Results A relatively high dose (10 μM) of tamoxifen (TAM) suppressed the expression of ERα and ERRα in two types of EC cells. However, 10 μM raloxifene (RAL) exhibited no effect on ERα and ERRα, while 10 μM XCT down regulated ERRα specifically, but not ERα in all EC cells. When dual targeting on ERα and ERRα by combining TAM with XCT, the proliferation inhibitory effect and apoptosis reached the strongest in all EC cells (P

Background Adult stem cells and progenitors are responsible for breast tissue regeneration. Human breast epithelial progenitors are organized in a lineage hierarchy consisting of bipotent progenitors (BPs), myoepithelial- and luminal-restricted progenitors (LRPs) where the LRP differentiation into mature luminal cells requires estrogen receptor (ER) signaling. However, the experimental evidence exploring the relationship between the BPs and LRPs has remained elusive. In this study, we report the presence of a basal-like luminal progenitor (BLP) in human breast epithelial cells. Methods Breast reduction samples were used to obtain different subsets of human breast epithelial cell based on cell surface marker expression using flow cytometry. Loss of function and gain of function studies were employed to demonstrate the role of NOTCH3 (NR3)-FRIZZLED7 (FZD7) signaling in luminal cell fate commitment. Results Our results suggest that, NR3-FZD7 signaling axis was necessary for luminal cell fate commitment. Similar to LRPs, BLPs (NR3highFZD7highCD90+MUC1−ER−) differentiate to generate NR3medFZD7medCD90−MUC1+ER+ luminal cells. Unlike LRPs however, BLP’s proliferation and differentiation potentials depend on NR3 and regulated in part by FZD7 signaling. Lastly, we show that BLPs have a higher colony-forming potential than LRPs and that they are continuously generated from the NOTCH3−FZD7low subset of the bipotent progenitors. Conclusion Our data indicate that BPs differentiate to generate basal-like luminal progenitors that in turn differentiate into LRPs. These results provide new insights into the hierarchical organization of human breast epithelial cell and how cooperation between the Notch and Wnt signaling pathways define a new progenitor cell type. Electronic supplementary material The online version of this article (10.1186/s13287-019-1361-3) contains supplementary material, which is available to authorized users.

Background: We previously demonstrated that liver resection triggers estradiol production, which, in turn, induces the proliferation of hepatocytes to promote liver regeneration in mice. In this study, we demonstrated estradiol-induced estrogen receptor alpha (ERα) expression. Methods: To further explore the role of ERα in estradiol-mediated liver regeneration, in the present study, we confirmed impaired liver regeneration ability in ERα knockout mice. Results: Further analysis during liver regeneration revealed a role for ERα in hepatic steatosis, tumor necrosis factor-alpha and interleukin 6 expression, and nuclear factor-κB and signal transducer and activator of transcription 3 DNA-binding activities. Conclusion: Moreover, estradiol administration accelerated liver regeneration through ERα, indicating the feasibility of the estrogen-ERα axis as a target for accelerating the rate of liver regeneration.

Background Sexually dimorphic growth has been attributed to the growth hormone (GH)/insulin-like growth factor 1 (IGF1) axis, particularly GH-induced activation of the intracellular signal transducer and activator of transcription 5B (STAT5B), because deletion of STAT5B reduces body mass and the mass of skeletal muscles in male mice to that in female mice. However, it remains unclear why these effects are sex- and species-specific, because the loss of STAT5B retards growth in girls, but not in male mice. Our objectives were to determine whether sexually dimorphic growth of skeletal muscle persisted in STAT5B−/− mice and investigate the mechanisms by which STAT5B regulates sexually dimorphic growth. Methods Blood and skeletal muscle were harvested from male and female STAT5B−/− mice and their wild-type littermates from the onset of puberty to adulthood. Results Growth of the skeleton and skeletal muscles was retarded in both sexes of STAT5B−/− mice, but more so in males. Although reduced, sexually dimorphic growth of skeletal muscle persisted in STAT5B−/− mice with an oxidative shift in the composition of myofibres in both sexes. Concentrations of IGF1 in blood and skeletal muscle were reduced in male STAT5B−/− mice at all ages, but only in female STAT5B−/− mice at the onset of puberty. Expression of androgen receptor (AR) and oestrogen receptor alpha (ERα) mRNA and protein was reduced in skeletal muscles of male and female STAT5B−/− mice, respectively. Loss of STAT5B abolished the sexually dimorphic expression of myostatin protein and Igf1, Ar, Erα, suppressor of cytokine signalling 2 (Socs2), and cytokine-inducible SH2-containing protein (Cis) mRNA in skeletal muscle. Conclusions STAT5B appears to mediate GH signalling in skeletal muscles of male mice at all ages, but only until puberty in female mice. STAT5B also appears to mediate the actions of androgens and oestrogens in both male and female mice, but sexually dimorphic growth persists in STAT5B−/− mice. Electronic supplementary material The online version of this article (10.1186/s13395-019-0204-3) contains supplementary material, which is available to authorized users.

Background Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG’s mechanism of action. Methods RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. Results MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. Conclusions Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-019-1130-3) contains supplementary material, which is available to authorized users.

Objective The endogenous estrogen 17β-estradiol (E2) promotes metabolic homeostasis in premenopausal women. In a mouse model of post-menopausal metabolic syndrome, we reported that estrogens increased energy expenditure, thus preventing estrogen deficiency-induced adiposity. Estrogens' prevention of fat accumulation was associated with increased serum concentrations of fibroblast growth factor 21 (FGF21), suggesting that FGF21 participates in estrogens' promotion of energy expenditure. Methods We studied the effect of E2 on FGF21 production and the role of FGF21 in E2 stimulation of energy expenditure and prevention of adiposity, using female estrogen receptor (ER)- and FGF21-deficient mice fed a normal chow and a cohort of ovariectomized women from the French E3N prospective cohort study. Results E2 acting on the hepatocyte ERα increases hepatic expression and production of FGF21 in female mice. In vivo activation of ERα increases the transcription of Fgf21 via an estrogen response element outside the promoter of Fgf21. Treatment with E2 increases oxygen consumption and energy expenditure and prevents whole body fat accumulation in ovariectomized female WT mice. The effect of E2 on energy expenditure is not observed in FGF21-deficient mice. While E2 treatment still prevents fat accumulation in FGF21-deficient mice, this effect is decreased compared to WT mice. In an observational cohort of ovariectomized women, E2 treatment was associated with lower serum FGF21 concentrations, which may reflect a healthier metabolic profile. Conclusions In female mice, E2 action on the hepatocyte ERα increases Fgf21 transcription and FGF21 production, thus promoting energy expenditure and partially decreasing fat accumulation., Highlights • Activation of the hepatocyte ERα increases FGF21 production in female mice. • ERα increases Fgf21 transcription in vivo via an estrogen response element. • Estrogen stimulation of energy expenditure is lost in female FGF21-deficient mice. • In women, estrogen treatment is associated with lower serum FGF21 concentrations.

Background Breast cancer is the most common cancer among women worldwide, and approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. ERα has been identified to promote the growth of primary breast cancer, however, it can also antagonize signaling pathways that lead to epithelial-mesenchymal transition (EMT), including transforming growth factor-β (TGF-β) signaling. miRNA alteration or dysfunction is involved in cancer development and progression. Although miR-1271 has identified as a tumor suppressor in various cancers, the role of miR-1271 in breast cancer is still limited. Methods The effect of miR-1271 on breast cancer progression was investigated both in vitro and in vivo. The EMT-related protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Chromatin immunoprecipitation and dual-luciferase reporter assays were used to validate the regulation of ERα-miR-1271-SNAI2 feedback loop. Results miR-1271 suppresses breast cancer progression and EMT phenotype both in vitro and in vivo by targeting SNAI2. Estrogen reverses TGF-β-induced EMT in a miR-1271 dependent manner. Furthermore, ERα transactivates the miR-1271 expression and is also transcriptionally repressed by SNAI2. Conclusions Our data uncover the ERα-miR-1271-SNAI2 feedback loop and provide a mechanism to explain the TGF-β network in breast cancer progression. Electronic supplementary material The online version of this article (10.1186/s13046-019-1112-4) contains supplementary material, which is available to authorized users.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70‐75% of primary breast cancers belong to estrogen receptor (ER)‐positive breast cancer. In the development of ER‐positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti‐ER‐positive breast cancer, which also provides an important reference for the study of CTX anti‐ER‐related tumors.

Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically. Methods: Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information (RNA-seq, ATAC-seq, Gene microarray, 450k methylation chip, GRO-seq), forming a multi-dimension network to illuminate ERα associated transcription. Results: We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers. Conclusions: The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors (NR2F2, FOXA1 and GATA3).The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription.

The effect of endogenous estrogens on the male reproductive tissues and development of gametes is an essential for the reproductive success. Estrogens affect the target cells via estrogen receptors (ERs) by both genomic and non-genomic pathways. The ER localization in the testis, epididymis, and sperm cells is a key to understanding the effect of estrogens on the sperm development, maturation, and function. The ER detection in male reproductive tissues and sperm cells at different development stages is described in representative mammalian species (human, mouse, rat, horse, and pig), in which the ER localization has been most described. According to various authors the ER occurrence in the male reproductive tissues and spermatozoa is quite distinct. Discrepancy in the published results is probably caused either by the application of different tissue preparation methods, or the choice of specific antibodies. Inconsistent findings should be subjected to further investigation to better understand the role of ERs in the male gamete development and mammalian reproduction

Loss of ovarian function is closely related to estrogen (E2) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E2 to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E2 injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E2 decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E2/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E2 exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E2 exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p–Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E2/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E2-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.

BackgroundThe development of new biomarkers with diagnostic, prognostic and therapeutic prominence will greatly enhance the management of breast cancer (BC). Several reports suggest the involvement of the histone acetyltransferases CREB-binding protein (CBP) and general control non-depressible 5 (GCN5) in tumor formation; however, their clinical significance in BC remains poorly understood. This study aims to investigate the value of CBP and GCN5 as markers and/or targets for BC prognosis and therapy. Expression of CBP, GCN5, estrogen receptor α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in BC was analyzed in cell lines by western blot and in patients’ tissues by immunohistochemistry. The gene amplification data were also analyzed for CBP and GCN5 using the publicly available data from BC patients.ResultsElevated expression of CBP and GCN5 was detected in BC tissues from patients and cell lines more than normal ones. In particular, CBP was more expressed in luminal A and B subtypes. Using chemical and biological inhibitors for CBP, ERα and HER2 showed a strong association between CBP and the expression of ERα and HER2. Moreover, analysis of theCREBBP(for CBP) andKAT2A(for GCN5) genes in a larger number of patients in publicly available databases showed amplification of both genes in BC patients. Amplification ofCREBBPgene was observed in luminal A, luminal B and triple-negative but not in HER2 overexpressing subtypes. Furthermore, patients with highCREBBPorKAT2Agene expression had better 5-year disease-free survival than the low gene expression group (p = 0.0018 andp ConclusionsWe conclude that the persistent amplification and overexpression of CBP in ERα- and PR-positive BC highlights the significance of CBP as a new diagnostic marker and therapeutic target in hormone-positive BC.

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

The molecular mechanisms by which uterine leiomyoma (UL) cells proliferate are unclear. Long noncoding RNA (lncRNA) is reported to participate in the occurrence and development of gynecological cancers. We investigated the molecular mechanisms that lncRNA uses in UL. We found that lncRNA Alu-mediated p21 transcriptional regulator (APTR) showed higher expression in UL tumor tissues compared with that in normal uterine tissues. APTR induced cell proliferation and colony formation both in vitro and in vivo. The JASPAR database showed that APTR was likely interacted with ERα, and these molecules were identified via laser scanning confocal microscopy and RNA immunoprecipitation analysis. To verify the correlation between APTR and ERα, we overexpressed and underexpressed APTR and simultaneously expressed ERα. The results showed that APTR function was suppressed. APTR increased the expressions of the proteins in the Wnt pathway, and inhibiting ERα eliminated these responses. In conclusion, our data suggest that APTR promoted leiomyoma cell proliferation through the Wnt pathway by targeting ERα, suggesting a new role of APTR in the Wnt signaling pathway in UL.

Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of which are transcription factors regulated by ligand binding. SRs regulate various human physiological functions essential for maintenance of vital biological pathways, including development, reproduction, and metabolic homeostasis. In addition, aberrant expression of SRs or dysregulation of their signaling has been observed in a wide variety of pathologies. SR activity is tightly and finely controlled by post-translational modifications (PTMs) targeting the receptors and/or their coregulators. Whereas major attention has been focused on phosphorylation, growing evidence shows that methylation is also an important regulator of SRs. Interestingly, the protein methyltransferases depositing methyl marks are involved in many functions, from development to adult life. They have also been associated with pathologies such as inflammation, as well as cardiovascular and neuronal disorders, and cancer. This article provides an overview of SR methylation/demethylation events, along with their functional effects and biological consequences. An in-depth understanding of the landscape of these methylation events could provide new information on SR regulation in physiology, as well as promising perspectives for the development of new therapeutic strategies, illustrated by the specific inhibitors of protein methyltransferases that are currently available., Graphical Abstract Graphical Abstract

Distinct from ovarian estradiol, the steroid hormone 17ß-estradiol (E2) is produced in the brain and is involved in numerous functions, particularly acting as a neurosteroid. However, the physiological role of E2 and the mechanism of its effects are not well known. In hippocampal slices, 17ß-estradiol has been found to cause a modest increase in fast glutamatergic transmission, because some of these effects are rapid and acute, they might be mediated by membrane-associated receptors via nongenomic action. Moreover, activation of membrane estrogen receptors can rapidly modulate neuron function in a sex-specific manner. To further investigate the neurological role of E2, we examined the effect of E2, as an estrogen receptor (ER) agonist, on synaptic transmission in slices of the prefrontal cortex (PFC) and hippocampus in both male and female mice. Whole-cell recordings of spontaneous excitatory postsynaptic currents (sEPSC) in the PFC showed that E2 acts as a neuromodulator in glutamatergic transmission in the PFC in both sexes, but often in a cell-specific manner. The sEPSC amplitude and/or frequency responded to E2 in three ways, namely by significantly increasing, decreasing or having no response. Additional experiments using an agonist selective for ERß, diarylpropionitrile (DPN) showed that in males the sEPSC and spontaneous inhibitory postsynaptic currents sIPSC responses were similar to their E2 responses, but in females the estrogen receptor ß (ERß) agonist DPN did not influence excitatory transmission in the PFC. In contrast, in the hippocampus of both sexes E2 potentiated the gluatmatergic synaptic transmission in a subset of hippocampal cells. These data indicate that activation of E2 targeting probably a estrogen subtypes or different downstream signaling affect synaptic transmission in the brain PFC and hippocampus between males versus females mice.

Cataloged from PDF version of article. Thesis (M.S.): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2021. Includes bibliographical references (leaves 58-62). ATAD2 is overexpressed in many distinct cancer types including breast cancer. Its elevated expression is an indicator of poor prognosis. High ATAD2 expression correlates with short overall survival, disease-free survival as well as shorter recurrence-free survival. Moreover, ATAD2 is associated with migration and invasion in some cancer types such as hepatocellular carcinoma and cervical cancer. In breast cancer, ATAD2 expression is higher in invasive tumors. ATAD2 is coactivator of steroid hormone receptor ERα. It directly interacts with ERα and enhances its target genes expressions. ERα is also a regulator of EMT in breast carcinomas. Based on our current knowledge it is proposed that ATAD2 may have a role in EMT and migration capacity of breast cancer cells and this role may be ER dependent as both ATAD2 and EMT are associated with ERα. Bioinformatics analysis revealed that siATAD2 silencing decreased mesenchymal gene expressions significantly in MCF7 and T47D cells. To investigate the possible mesenchymal inducing role of ATAD2, ATAD2 was silenced with shRNA transfection in ER+ MCF7 and T47D cells and ER- mda-mb-231 and sk-br-3 cells. ATAD2 silencing decreased mesenchymal markers expression at both the mRNA and protein level in ER- cells. In ER+ cells, no change in EMT marker proteins and mRNAs were observed with ATAD2 silencing. ER was silenced in ER+ cells and ER silencing introduced a mesenchymal phenotype to them. In this case, ATAD2 silencing reduced this mesenchymal phenotype introduced with ER loss. The EMT effect of the ATAD2 silencing on migration capacity of breast cancer cells was assessed with a scratch assay. Consistent with changes in the epithelial and mesenchymal markers, ATAD2 silencing reduced the migration capacity of mda-mb-231 cells. On the other hand, sk-br-3 migration did not change significantly. In ER+ cells ATAD2 silencing alone had no influence on migratory capacity. ER silencing increased their migration significantly while ATAD2 downregulation in ER-silenced cells suppressed this migration. Over all, this study suggests a possible involvement of ATAD2 in EMT and migration regulation in ER- cells. Targeting ATAD2 in ER- mesenchymal breast cancer cells could be a strategy to reduce their migration capacity. by Zeynep Ilgım Özerk M.S.