This thesis investigates estrogens' modulation of three aspects of social cognition (aggression and agonistic behaviour, social learning, and social recognition). Sex-typical agonistic behaviour (males: overt attacks, females: more subtle dominance behaviours) was increased in gonadectomized mice by estrogen receptor alpha (ERα) agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), while non-overt agonistic behaviour was increased in male and female gonadally intact mice by ERβ agonist 7-Bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY-200070). Estrogens also affected the social transmission of food preferences (STFP). Acute estrogen and ERβ agonists WAY-200070 and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) prolonged the preference for the demonstrated food when administered pre-acquisition, likely by affecting motivation or the nature of the social interaction, while acute PPT blocked the STFP. All mice receiving any of the three treatments chronically showed a prolonged demonstrated food preference, suggesting a loss of ER specificity. Individual differences in social recognition may relate to increased oxytocin (OT) and vasopressin (AVP) mRNA, and ERα and ERβ gene activation, in the medial preoptic area, and decreased mRNA for ERs, OT receptor (OTR), AVP and AVP receptors 1a and 1b in the lateral amygdala. Additionally, dorsolateral septum ERs, progesterone receptor, and OTR may relate to social interest without affecting social recognition. Our and others' results suggest that estrogens, OT and AVP are all involved in social behaviours and mediate social recognition, social learning, social interactions, and aggression. ERs differently modulate the two types of social learning investigated here: ERα is critical for social recognition, but impairs social learning, while ERβ is less important in social recognition, and prolongs the demonstrated food preference in the STFP. This may be due to differences in receptor brain distributions or in downstream neurochemical systems that mediate these behaviours. The results of this thesis suggest that estrogens, through the various systems they modulate, have a key role to play in social behaviour. Further investigations of how estrogens effect change in these systems at the molecular and cellular level, as well as the critical brain areas and downstream effectors involved in these complex behaviours, are needed, and could contribute to therapeutic interventions in socially-based, sexually dimorphic disorders, like the autism spectrum disorders, and women receiving hormone replacement therapy for negative peri- or post-menopausal symptoms. / National Science and Engineering Research Council (PGS-D, CGS-M)

Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.

Introdução: Estimativas mostram que mais de dois terços das mulheres com câncer de mama possuem receptores hormonais positivos, e recebem terapia endócrina como tratamento, sendo tamoxifeno (TAM) o tratamento padrão (EBCTCG 2005; Davies et al., 2012). Porém, muitas pacientes se tornam resistentes com o passar do tempo. Estudos prévios mostraram que a expressão do receptor de estrogênio β (REβ) aumenta a resposta ao tratamento com TAM em células de câncer de mama, assim como a coexpressão de REα e REβ esta associada com maior ação proliferativa de TAM (Treeck et al., 2010; Sun et al., 2014). Também foi observada a existência de “cross-talk” entre os RE e a família do receptor do fator de crescimento epidérmico (HER) na resposta ao tratamento com TAM (Lindberg et al., 2011; Blows et al., 2010). Objetivo: Verificar a expressão do REβ, e suas interações com REα e receptores HER, durante o tratamento com TAM e em células resistentes ao TAM. Métodos: A expressão do REβ foi analisada em dois bancos de dados contendo informações de pacientes com câncer de mama. A expressão de RNAm dos RE, receptores HER e vias de sinalização PTEN, Akt e MAPK foram avaliadas após tratamento com TAM, em células resistentes ao TAM e em células silenciadas para os genes dos RE. Também foi avaliada a viabilidade celular após tratamento com TAM e nas células silenciadas para os genes dos RE. Resultados: Pacientes com câncer de mama apresentaram expressão reduzida do REβ, e os subtipos de câncer de mama REα positivos apresentaram baixa expressão do REβ quando comparados aos subtipos REα negativos. Células expressando níveis moderados de REβ apresentaram melhor resposta ao tratamento com TAM. Diminuição nos níveis dos RE é acompanhada por aumento nos níveis dos receptores ErbB2 e ErbB3, aumento de PTEN e diminuição de Akt e MAPK3 após tratamento com TAM. ERβ modula a ação antiproliferativa do TAM através da via de MAPK3. Células resistentes ao TAM apresentaram baixos níveis dos RE e altos níveis dos receptores EGFR, ErbB3 e ErbB4. Conclusão: Estes resultados demonstram que o REβ, e suas interações com REα e receptores HER, possuem papel importante na resposta ao tratamento com TAM. / Introduction: Approximately two-thirds of all breast cancer patients overexpress hormonal receptors, and are treated with endocrine therapy, being tamoxifen (TAM) the standard treatment. However many of initial responders to TAM as first-line experience relapse. Several mechanisms have been proposed to explain the occurrence of acquired TAM resistance. Previous studies showed that estrogen receptor β (ERβ) expression is associated with better response to tamoxifen treatment, as the co-expression of ERα and ERβ is associated with TAM antiproliferative effects. Moreover, there is growing interest about the cross-talk between ERs and ErbB family in response to endocrine therapy. Suggesting that TAM can acts through ERβ and/or ErbB family as compensatory pathways. Objective: To evaluate the expression of ERβ and the relation of ERβ with ERα and ErbB family in response to TAM treatment and in TAM resistant cells. Methods: ERβ expression was analyzed in two different databases of breast cancer patients. The mRNA levels of ER, HER receptors and PTEN, Akt and MAPK signal pathways were measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. The cellular viability was also measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. Results: Breast cancer patients presented reduced ERβ expression and the ERα-positive breast cancer subtypes presented lower ERβ levels when compared to ERα-negative breast cancer subtypes. Cells expressing moderates levels of ERβ presented better response to TAM treatment. Down-regulation of ERs induced by TAM treatment are accompanied with an increase in ErbB2 and ErbB3, reduced AKT and MAPK3 mRNA levels and increased PTEN levels. ERβ modulates TAM anti-proliferative effects through MAPK3 pathway. TAM– resistant cells expressed decreased ER mRNA levels and increased EGFR, ErbB3 and ErbB4 levels. Demonstrating that the cross-talk between ERs and HER family influence the response to TAM treatment. Conclusion: These results provide additional data indicating the importance of ERβ, and the relation with ERα and HER receptors, to predict TAM responsiveness.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer de mama é a neoplasia que mais acomete mulheres no mundo. Dentre os fatores prognósticos levados em consideração estão os receptores hormonais. O receptor de estrógeno beta (ERβ) é um dos receptores hormonais que pode ser encontrado na mama, mas que até então não é utilizado como um marcador preditivo de prognóstico tumoral por conta da sua ação paradoxal. Células tumorais de mama da linhagem MCF-7 foram utilizadas neste trabalho para averiguar a função dicotômica do ERβ no processo tumoral. De tal forma que foi avaliado alguns dos processos que são observados nas etapas do desenvolvimento do câncer de mama, como proliferação, transição epitélio-mesênquima (EMT) e investigação das células -tronco cancerosas (CSCs) . Para isso, foram utilizados o agonista de ERβ, Diarilpropionitrilo (DPN), o agonista ambíguo (ERα e ERβ) estradiol (E2) e o fator de crescimento transformante beta (TGF-β), indutor de EMT. As células que receberam tratamento com DPN obtiveram maior número de CSCs comparadas às que não foram cultivadas com esse agonista, resultado encontrado pela técnica de citometria de fluxo. As células que tiveram um tratamento prévio com TGF-β demonstraram menor taxa de proliferação e aumento na expressão de p21, uma proteína com ação bloqueadora de ciclina D1, cuja expressão ficou inalterada nos tratamentos com TGF-β e agonista de ERβ. A respeito dos genes relacionados a EMT (SLUG, SNAIL,VIMENTINA, ZEB1,TWIST1,RBFOX2,CICLINAD1 e p21), ERβ inibiu a expressão dos mesmos, sugerindo que esse receptor induza o fenômeno denominado transição mesenquimal epitelial (MET). Nesse cenário, DPN causou a diminuição da expressão de SLUG, SNAIL, TWIST, contrastando com a expressão obtida no tratamento com TGF-β que, além desses genes, também demonstrou aumento na expressão de ZEB1, RBFOX2 e vimentina. O efeito do TGF-β na EMT foi revertido ao associá-lo com DPN. Os dados corroboram com a literatura acerca do possível papel pró tumoral de ERβ no aumento da proliferação celular e da geração das células-tronco cancerígenas de mama (BSCs), além de ir ao encontro do fenótipo relacionado a MET nos tratamentos com DPN sozinho ou associado ao TGF-β. Com esses resultados, torna-se claro no nosso trabalho que dependendo o que é avaliado em relação a ação do ERβ, previamente tratado ou não com TGF-β, o seu efeito dicotômico ainda é observado. / Breast cancer is the most common neoplasm of women in the world. Among the prognostic factors taken into consideration are the hormonal receptors. The estrogen receptor beta (ERβ) is one of the hormone receptors that can be found in the breast, but is not used as a predictive marker of tumor prognosis due to its paradoxical action. Tumor cells of the MCF-7 lineage were used in this work to ascertain the dichotomic function of ERβ in the tumor process. Thus, we evaluated some of the processes that are observed in the stages of breast cancer development, such as proliferation, epithelial-mesenchymal transition (EMT) and cancer stem cell research (CSCs). For this, ERβ agonist Diarilpropionitrile (DPN), ambiguous agonist (ERα and ERβ) estradiol (E2) and transforming growth factor beta (TGF-β), inducer of EMT, were used. Cells receiving TGF- β treatment obtained a higher number of CSCs compared to those that were not cultured with this agonist, a result found by the flow cytometry technique. Cells that were pretreated with TGF-β demonstrated a lower rate of proliferation and increased expression of p21, a protein with cyclin D1 blocking action, whose expression was unchanged in TGF-β and ERβ agonist treatments. Regarding the EMT-related genes (SLUG, SNAIL, VIMENTINA, ZEB1, TWIST1, RBFOX2, CYCLINAD1 and p21), ERβ inhibited their expression, suggesting that this receptor induces the phenomenon called epithelial mesenchymal transition (MET). In this scenario, DPN caused a decrease in the expression of SLUG, SNAIL, TWIST, in contrast to TGF-β expression, which, in addition to these genes, also showed increased expression of ZEB1, RBFOX2 and VIMENTIN. The effect of TGF-β on EMT was reversed by associating it with DPN. The data corroborate with the literature about the possible ERβ pro-tumor role in increasing cell proliferation and the generation of breast cancer stem cells (BSCs), in addition to the MET related phenotype in treatments with DPN alone or associated to TGF-β. With these results, it becomes clear in our work that depending on what is evaluated in relation to the action of ERβ, previously treated or not with TGF-β, its dichotomous effect is still observed. / Dissertação (Mestrado)

Introdução: Estimativas mostram que mais de dois terços das mulheres com câncer de mama possuem receptores hormonais positivos, e recebem terapia endócrina como tratamento, sendo tamoxifeno (TAM) o tratamento padrão (EBCTCG 2005; Davies et al., 2012). Porém, muitas pacientes se tornam resistentes com o passar do tempo. Estudos prévios mostraram que a expressão do receptor de estrogênio β (REβ) aumenta a resposta ao tratamento com TAM em células de câncer de mama, assim como a coexpressão de REα e REβ esta associada com maior ação proliferativa de TAM (Treeck et al., 2010; Sun et al., 2014). Também foi observada a existência de “cross-talk” entre os RE e a família do receptor do fator de crescimento epidérmico (HER) na resposta ao tratamento com TAM (Lindberg et al., 2011; Blows et al., 2010). Objetivo: Verificar a expressão do REβ, e suas interações com REα e receptores HER, durante o tratamento com TAM e em células resistentes ao TAM. Métodos: A expressão do REβ foi analisada em dois bancos de dados contendo informações de pacientes com câncer de mama. A expressão de RNAm dos RE, receptores HER e vias de sinalização PTEN, Akt e MAPK foram avaliadas após tratamento com TAM, em células resistentes ao TAM e em células silenciadas para os genes dos RE. Também foi avaliada a viabilidade celular após tratamento com TAM e nas células silenciadas para os genes dos RE. Resultados: Pacientes com câncer de mama apresentaram expressão reduzida do REβ, e os subtipos de câncer de mama REα positivos apresentaram baixa expressão do REβ quando comparados aos subtipos REα negativos. Células expressando níveis moderados de REβ apresentaram melhor resposta ao tratamento com TAM. Diminuição nos níveis dos RE é acompanhada por aumento nos níveis dos receptores ErbB2 e ErbB3, aumento de PTEN e diminuição de Akt e MAPK3 após tratamento com TAM. ERβ modula a ação antiproliferativa do TAM através da via de MAPK3. Células resistentes ao TAM apresentaram baixos níveis dos RE e altos níveis dos receptores EGFR, ErbB3 e ErbB4. Conclusão: Estes resultados demonstram que o REβ, e suas interações com REα e receptores HER, possuem papel importante na resposta ao tratamento com TAM. / Introduction: Approximately two-thirds of all breast cancer patients overexpress hormonal receptors, and are treated with endocrine therapy, being tamoxifen (TAM) the standard treatment. However many of initial responders to TAM as first-line experience relapse. Several mechanisms have been proposed to explain the occurrence of acquired TAM resistance. Previous studies showed that estrogen receptor β (ERβ) expression is associated with better response to tamoxifen treatment, as the co-expression of ERα and ERβ is associated with TAM antiproliferative effects. Moreover, there is growing interest about the cross-talk between ERs and ErbB family in response to endocrine therapy. Suggesting that TAM can acts through ERβ and/or ErbB family as compensatory pathways. Objective: To evaluate the expression of ERβ and the relation of ERβ with ERα and ErbB family in response to TAM treatment and in TAM resistant cells. Methods: ERβ expression was analyzed in two different databases of breast cancer patients. The mRNA levels of ER, HER receptors and PTEN, Akt and MAPK signal pathways were measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. The cellular viability was also measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. Results: Breast cancer patients presented reduced ERβ expression and the ERα-positive breast cancer subtypes presented lower ERβ levels when compared to ERα-negative breast cancer subtypes. Cells expressing moderates levels of ERβ presented better response to TAM treatment. Down-regulation of ERs induced by TAM treatment are accompanied with an increase in ErbB2 and ErbB3, reduced AKT and MAPK3 mRNA levels and increased PTEN levels. ERβ modulates TAM anti-proliferative effects through MAPK3 pathway. TAM– resistant cells expressed decreased ER mRNA levels and increased EGFR, ErbB3 and ErbB4 levels. Demonstrating that the cross-talk between ERs and HER family influence the response to TAM treatment. Conclusion: These results provide additional data indicating the importance of ERβ, and the relation with ERα and HER receptors, to predict TAM responsiveness.

Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.

Invasiv äggstockscancer anses vara en av de mest dödligaste gynekologiska maligniteterna. Granulosacelltumör i äggstocken är en sällsynt subtyp av äggstockstumör, som utgör 1–5% av alla äggstockstumörer. De kan kännetecknas av den långsamma tillväxthastigheten och produktionen av höga östrogennivåer. Återfallsfrekvensen efter den första behandlingen är låg, men dödligheten på ett återfall är så hög som 80%. Det är därför av intresse att undersöka nya terapeutiska mål som kan användas för att behandla granulosacelltumör i framtiden. Granulosacelltumörer tros uppstå från de sena preovulatoriska granulosacellerna eftersom de delar liknande egenskaper; de uttrycker follikelstimulerande hormonreceptorer och producerar östrogen som respons på follikelstimulerandehormoner. Östrogen och dess motsvarande kärnreceptorer, östrogenreceptor α och β spelar en avgörande roll i utvecklingen av äggstocksfolliklarna under äggstockscykeln. Uttrycket av östrogenreceptorn α är ganska lågt i granulosacellerna, istället tros östrogenreceptorn β (ERβ/ESR2) spela den övervägande rollen att aktivera intracellulära signalvägar som främjar cellproliferation och överlevnad i äggstocken. Det finns också många varianter av östrogenreceptorn β, främst ERβ1, ERβ2 ibland kallad för ERβcx, ERβ3, ERβ4, och ERβ5. Deras roll i äggstocken är fortfarande okända och har varit svårt att studera eftersom de saknar ligand-bindningsdomänen. DNA-bindningsdomänen är fortfarande bevarad, så de kan fortfarande vara viktiga transkriptionsfaktorer i äggstocken. Nya ChIP-sekvenseringsresultat från en studie visade att ERβ och leverreceptorn homolog 1 (LRH-1/NR5A2) delar många av kromatinbindningsställena i en normal musäggstock. Detta resultat tyder på att dessa två transkriptionsfaktorer troligtvis interagerar med varandra fysiskt eller indirekt. ChIP-qPCR användes för att bekräfta ERβ-varianternas kromatinbindning i den mänskliga granulosatumörcellinjen COV434. Dubbel luciferasanalys användes för att undersöka om ERβ påverkar LRH-1s transaktivering aktivitet. Co-IP utfördes för att undersöka om ERβ och LRH-1 fysiskt interagerar med varandra. Vi kunde bekräfta att ERβ-varianterna ERβcx, ERβ5 och ERβ4 binder till LRP6 genen och att varianterna kan binda direkt till DNA. För att ytterligare bekräfta flera målgener för ERβ-varianterna måste DNA-proverna skickas för sekvensering. Resultaten från denna studie indikerar att ERβ undertrycker LRH-1s transaktiverings aktivitet med eller utan liganden östradiol, hur ERβ fungerar som repressor är fortfarande okänt. Vi kunde inte visa om ERβ fysiskt interagerar med LRH-1 i denna studie. / Invasive ovarian cancer is considered to be one of the most fatal gynecological malignancies. Granulosa cell tumor in the ovary is a rare subtype of ovarian tumor that makes up almost 1-5% of all ovarian tumors. It can be characterized by the slow growth rate and production of high estrogen levels. The recurrence rate after the first treatment is fairly low, but the mortality rate for the recurrence is as high as 80%. Therefore, it is interesting to investigate new therapeutic targets that can be used to treat granulosa cell tumors in the future. Granulosa cell tumors are thought to originate from the late preovulatory granulosa cells because they share similar features; they express follicle-stimulating hormone receptors and produce estrogen in response to follicle-stimulating hormone. Estrogen and its corresponding nuclear receptors, estrogen receptors α and β, play a crucial role in the development of the ovarian follicles during the ovarian cycle. The expression of the estrogen receptor α is relatively low in the nucleus of the granulosa cells; instead, estrogen receptor β (ERβ/ESR2) is thought to play the predominant role of activating intracellular signaling pathways that promote cell proliferation and survival in the ovary. There are also many splice variants of the estrogen receptor β, mainly ERβ1, ERβ2 or also sometimes called ERβcx, ERβ3, ERβ4, and ERβ5. Their role in the ovary is still unknown. Most of the splice variants lack the ligand-binding domain. However, the DNA-binding domain is still preserved, so they could be crucial transcriptional factors of different pathways in the ovary. Recent ChIP-sequencing results from a study showed that two nuclear receptors, ERβ and the liver receptor homolog 1 (LRH-1/NR5A2), share many chromatin binding sites in normal mouse ovary. This finding suggests that these two transcriptional factors may interact with each other physically or indirectly. ChIP-qPCR was used to confirm chromatin binding of ERβ’s splice variants in the human granulosa tumor cell line COV434. Dual luciferase assay was used to investigate if ERβ affects the transactivation activity of LRH-1. Co-IP was performed to investigate if ERβ and LRH-1 physically interact. We could confirm that LRP6 is a target gene of ERβ splice variants ERβcx, ERβ4 and ERβ5. We also demonstrated that these ERβ splice variants bind directly to DNA which has not been shown before. The DNA samples must be sent for further sequencing to confirm more target genes of the ERβ splice variants. The results from this study indicate that ERβ represses the transactivation activity of LRH-1, how ERβ acts as a repressor is still unknown. We could not show whether ERβ and LRH-1 physically interact in this study.

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

Τα αστροκυττώματα αποτελούν το συχνότερο τύπο πρωτοπαθών όγκων του κεντρικού νευρικού συστήματος (ΚΝΣ) και παραδοσιακά θεωρούνται ότι σχετίζονται με ιδιαίτερα δυσμενή πρόγνωση. Η Συστημική προσέγγιση της καρκινογένεσης, εστιάζοντας στην αποκρυπτογράφηση του τρόπου λειτουργίας και δυναμικής αλληλεπίδρασης πολύπλοκων παθοβιολογικών δικτύων, προσφέρει σήμερα καινούριες ερευνητικές προοπτικές και πιθανές εναλλακτικές, περισσότερο αποτελεσματικές θεραπευτικές στρατηγικές. Οι οιστρογονικοί υποδοχείς και οι συν – ρυθμιστές της μεταγραφής συνιστούν κομβικά σημεία “συνομιλίας” (cross – talk) πολύπλοκων μοριακών οδών του κυττάρου, διαμεσολαβώντας πλήθος κυτταρικών λειτουργιών φυσιολογικά αλλά και σε παθολογικές καταστάσεις, ανάμεσα στις οποίες και ο καρκίνος. Οι παράγοντες EZH2 και SOX2 θεωρούνται μόρια κλειδιά του ρυθμιστικού μεταγραφικού κυκλώματος που χαρακτηρίζει το stemness. Η αποσαφήνιση της συμπεριφοράς του συγκεκριμένου αυτού δικτύου στα διάφορα νεοπλάσματα και ρόλος του σε σχέση με την απόκτηση ιδιότητας καρκινικού stem κυττάρου, θεωρείται καθοριστικής σημασίας στην προσπάθεια ερμηνείας του φαινομένου του καρκίνου ως πολύπλοκο προσαρμόσιμο σύστημα, που θα αναδείξει εναλλακτικούς θεραπευτικούς στόχους και θα επιτρέψει περισσότερο αποτελεσματικές σε σχέση με τις υπάρχουσες παρεμβάσεις. Σκοπός. Υπό το πρίσμα της Συστημικής προσέγγισης της κατανόησης της κακοήθους ανάπτυξης και εξέλιξης των αστροκυτταρικών όγκων, η παρούσα μελέτη διερεύνησε τα επίπεδα έκφρασης του Οιστρογονικού υποδοχέα β (ERβ), και των συν – ρυθμιστών AIB1, TIF2 and PELP1, όπως και την έκφραση των παραγόντων EZH2 και SOX2 σε αστροκυττώματα grade II ως IV και τη συσχέτιση μεταξύ του προφίλ έκφρασης των συγκεκριμένων παραγόντων, με κλινικοπαθολογικά δεδομένα. Υλικό και μέθοδος. Η έκφραση των πρωτεϊνών ERβ, AIB1, TIF2, PELP1, EZH2 και SOX2 εκτιμήθηκε σε 86 περιπτώσεις αστροκυτταρικών όγκων χρησιμοποιώντας τη μέθοδο της ανοσοϊστοχημείας. Είκοσι grade II αστροκυττώματα, 22 grade III αναπλαστικά αστροκυττώματα και 46 grade IV πλειόμορφα γλοιοβλαστώματα (GBM) συμπεριλήφθησαν στη συγκεκριμένη μελέτη. Η μέθοδος με χρήση συστήματος ανίχνευσης EnVision (Envision, Dako, CA, USA) ή MACH4 Universal HRP-Polymer Detection (Biocare Medical, CA, USA) και πρωτογενή αντισώματα έναντι των ERβ (Biogenex, CA, USA), AIB1 (BD Biosciences, Ca, USA), TIF2 (BD Biosciences, Ca, USA), PELP-1/MNAR (Novus Biologicals, CO, USA) EZH2 (Novocastra, UK) και SOX2 (R&D Systems, Inc.) χρησιμοποιήθηκαν στην παρούσα μελέτη. Σε κάθε περιστατικό και για κάθε δείκτη εκτιμήθηκε το ποσοστό των καρκινικών κυττάρων που εμφάνιζαν θετική ανοσοχρώση. Αντιπροσωπευτικές περιοχές επιλέχθηκαν κατόπιν σάρωσης του πλακιδίου σε οπτικό πεδίο μικρής μεγέθυνσης (Χ100), ενώ η καταμέτρηση των θετικών κυττάρων πραγματοποιήθηκε σε μεγάλης μεγέθυνσης πεδίο (400X). Η στατιστική ανάλυση έγινε με τη χρήση του SPSS στατιστικού πακέτου (SPSS©, Release 17.0, Chicago, IL, USA). Τιμές p<0.05 θεωρήθηκαν ως στατιστικά σημαντικές. Αποτελέσματα. Σημαντική μείωση των επιπέδων του ERβ παρατηρήθηκε παράλληλα με την αύξηση του grade. Επιπλέον, η υψηλή ERβ έκφραση αναδείχθηκε ως ανεξάρτητος θετικός προγνωστικός παράγοντας της συνολικής επιβίωσης κατά την πολυπαραγοντική ανάλυση. Η έκφραση των AIB1, TIF2 και PELP1, δε συσχετίσθηκε με αυτή του ERβ, και ακολούθησε αντιστρόφως ανοδική τάση, παράλληλα με την αύξηση του grade. Η στατιστική ανάλυση περαιτέρω, ανέδειξε μία σημαντική αύξηση τόσο των επιπέδων EZH2 όσο και SOX2 στα grade III και IV σε σχέση με τα grade II αστροκυττώματα. Ισχυρή συσχέτιση παρατηρήθηκε ως προς την έκφραση των δύο δεικτών σε όλες τις κατά grade υποομάδες. Η Kaplan-Meier ανάλυση έδειξε ότι, η υψηλή EZH2 και SOX2 πρωτεϊνική έκφραση συνιστούν αρνητικό παράγοντα πρόγνωσης τόσο στο σύνολο των ασθενών όσο και κατόπιν διαστρωμάτωσης κατά grade. Τέλος, η πολυπαραγοντική Cox ανάλυση συνυπολογίζοντας την ηλικία, το φύλο, το grade και την έκφραση των δύο πρωτεϊνών έδειξε ότι μόνο η υψηλή EZH2 έκφραση μαζί με το υψηλό grade, αποτελούν ανεξάρτητους παράγοντες δυσμενούς πρόγνωσης. Συμπεράσματα. Οι παράγοντες ERβ, AIB1, TIF2 και PELP1 ενέχονται στους παθογενετικούς μηχανισμούς ανάπτυξης και εξέλιξης των αστροκυτταρικών όγκων, με τον ERβ να διαδραματίζει προστατευτικό ρόλο και τους AIB1, TIF2 και PELP1 να εμφανίζουν ογκο – προαγωγό δράση. Το ογκογενετικό δυναμικό των παραγόντων AIB1, TIF2 και PELP1 φαίνεται πως διαμεσολαβείται μέσω ανεξάρτητων του οιστρογονικού υποδοχέα μηχανισμών. Η έκφραση του ERβ, διαχωρίζοντας κλινικές εκβάσεις σε ασθενείς ιδίου grade, θα μπορούσε να αποτελέσει ένα χρήσιμο εργαλείο κατά τη λήψη εξατομικευμένων κλινικών αποφάσεων. Οι παράγοντες EZH2 και SOX2, θα μπορούσαν να χρησιμοποιηθούν ως εν δυνάμει δείκτες καρκινικών stem κυττάρων σε αστροκυτταρικούς όγκους, να βοηθήσουν τη βελτιστοποίηση τόσο διαγνωστικών όσο και προγνωστικών διαδικασιών στην κλινική πράξη, και να κατευθύνουν την ανάπτυξη εξατομικευμένων στρατηγικών θεραπείας. / Astrocytic tumors are the most common primary neoplasms of the central nervous system (CNS) and have traditionally been associated with disappointing clinical outcomes. The current challenge is to develop more efficacious and targeted therapeutic paradigms, exploiting the knowledge derived from the systems approach of understanding the complex networks underlying tumor formation and progression. Estrogen receptor beta (ERβ) and co-regulators of transcription AIB1, TIF2 and PELP1, are key components of complex cellular networks and integrate diverse signaling afferents with transcription programs controlling various physiological cellular processes and a variety of disease states including cancer. SOX2 and EZH2 represent crucial components of the reciprocal regulatory circuit that controls stemness. Elucidating the behavior of this particular network in cancer and its role in the formation of putative cancer stem cells is considered essential for the understanding of cancer as an adaptive complex system and subsequently allowing the discovery of more successful therapeutic designs. Purpose. In the context of the systems approach of comprehending tumorigenesis in astrocytomas, we sought to investigate the expression of ERβ and co – regulatory proteins AIB1, TIF1 and PELP1, as well as parallel expression of SOX2 and EZH2 in astrocytomas of various grades, and correlate the protein expression profiles with clinicopathological parameters and patients’ prognosis. Materials and methods. Expression of ERβ, AIB1, TIF2, PELP1, EZH2 and SOX2 was evaluated in 86 cases of astrocytic tumors, using Immunohistochemistry, on formalin-fixed paraffin-embedded tissue sections. Twenty grade II astrocytomas, 22 grade III anaplastic astrocytomas and 46 grade IV glioblastomas multiforme (GBM) were included in this study. Polymer based technique (Envision, Dako, CA, USA) or MACH4 Universal HRP-Polymer Detection (Biocare Medical, CA, USA) and primary antibodies against ERβ1 (Biogenex, CA, USA), AIB1 (BD Biosciences, Ca, USA), TIF2 (BD Biosciences, Ca, USA), PELP-1/MNAR (Novus Biologicals, CO, USA) EZH2 (Novocastra, UK) and SOX2 (R&D Systems, Inc.) were used. In each case, the percentage of cells exhibiting positive staining was determined. Representative areas were selected at low power (x100) magnification. Cell counts were performed at a 400X magnification. Data were analyzed using the SPSS statistical package (SPSS©, Release 17.0, Chicago, IL, USA). The level of significance was set at p-value <0.05. Results. ERβ levels were significantly decreased with the progression of tumors’ grade. High expression of ERβ was an independent favorable prognostic factor on multivariate analysis. Expression of AIB1, TIF2 and PELP1, was not correlated to ERβ expression and followed an opposite trend, with increasing levels in grade III and IV relative to grade II tumors. Univariate survival analysis revealed that high AIB1, TIF2 and PELP1 expression was associated with worse prognosis. Statistical analysis further revealed significantly higher expression of EZH2 and SOX2 in high grade III and IV astrocytomas, compared to low grade II astrocytomas. Strong correlation between EZH2 and SOX2 was also detected within all subgroups according to grade. Kaplan-Meier showed that EZH2 and SOX2 high expression was predictive of worse overall survival in the whole cohort as well as after subgroup analysis by grade. Finally, multivariate Cox analysis that included age, gender, grade, and expression of both proteins, revealed that high EZH2 together with higher grade were strong negative prognostic factors. Conclusions. ERβ, AIB1, TIF2 and PELP1 appear to play an important role in the pathogenesis of astrocytic tumors, with ERβ exhibiting a protective effect, whereas AIB1, TIF2 and PELP1 facilitate malignant progression. AIB1, TIF2 και PELP1 contribution in tumor progression is speculated to be achieved through ERβ independent pathways. Moreover, the expression status of ERβ, by distinguish patient subpopulations with different prognosis within the same grade, could be a useful tool accommodating personalized clinical decision-making. EZH2 and SOX2 may serve as potential cancer stem cell markers in astrocytomas and as such help optimizing diagnostic and prognostic assessments and devising novel individually tailored treatment strategies.