Ativação dos receptores estrogênicos, sinalização intracelular e função em células de carcinoma embrionário testicular humano NT2/D1
Data
2022-09-15
Autores
Lima, Carla Macheroni 
Orientadores
Porto, Catarina Segreti
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os mecanismos envolvidos no desenvolvimento e na progressão do tumor de células germinativas testiculares (TGCTs) não estão totalmente esclarecidos. Estudos recentes do nosso laboratório mostraram a expressão dos receptores estrogênicos ESR1 e ESR2 em células de carcinoma embrionário testicular humano (NT2/D1). É possível que a ativação dos receptores estrogênicos e a modulação de vias de sinalização intracelular estejam envolvidos nestes processos. Assim, o objetivo deste estudo foi investigar os efeitos da ativação dos receptores estrogênicos na expressão e na fosforilação de proteínas envolvidas com sinalização intracelular SRC, ERK1/2 e AKT e suas funções na proliferação, invasão, migração e formação de colônias em linhagem de células de carcinoma embrionário testicular humano NT2/D1. Além disso, foi determinado o perfil proteico destas células tratadas com 17β-estradiol. Foi observado que:
1. 17β-estradiol (E2), agonista seletivo do ESR1 (PPT) e agonista seletivo do ESR2 (DPN) levaram ao rápido aumento no estado de fosforilação de SRC e ERK1/2 nas células NT2/D1. Esses efeitos foram bloqueados pelos antagonistas seletivos do ESR1 (MPP) e do ESR2 (PHTPP).
2. A forma fosforilada da AKT foi praticamente não detectável tanto nas células NT2/D1 controle (ausência de tratamento) como nas células tratadas com E2 por diferentes tempos quando comparada com o controle positivo.
3. E2, agonista seletivo do ESR1 (PPT) e agonista seletivo do ESR2 (DPN) levaram ao aumento do número de células NT2/D1. O pré-tratamento dessas células com os inibidores de SRC (PP2) e MEK1/2 (U0126) bloquearam estes efeitos. Estes resultados indicam o envolvimento destas vias de sinalização na proliferação das células NT2/D1.
4. E2 e o agonista seletivo do ESR2 (DPN) causaram um aumento na migração das células NT2/D1. O pré-tratamento com o antagonista seletivo do ESR2 (PHTPP) bloqueou o efeito estimulado pelo E2 e do DPN na migração das células NT2/D1, indicando que o complexo E2-ESR2 está envolvido neste processo. O tratamento com o agonista seletivo do ESR1 (PPT) não alterou a migração destas células.
5. E2, o agonista seletivo do ESR1 (PPT) e o agonista seletivo do ESR2 (DPN) levaram ao aumento na invasão das células NT2/D1. O pré-tratamento com os antagonistas seletivos do ESR1 (MPP) e do ESR2 (PHTPP) bloquearam estes efeitos, indicando que o complexo E2-ESR1 e E2-ESR2 estão envolvidos neste processo.
6. E2, o agonista seletivo do ESR1 (PPT) e o agonista seletivo do ESR2 (DPN) levaram a formação de colônias das células NT2/D1. O pré-tratamento com os antagonistas seletivos do ESR1 (MPP) e do ESR2 (PHTPP) bloquearam estes efeitos, indicando que o complexo E2-ESR1 e E2-ESR2 estão envolvidos neste processo.
7. Os pré-tratamentos com inibidores de SRC (PP2) e MEK1/2 (U0126) bloquearam os efeitos da ativação dos receptores estrogênicos na migração e na invasão celular e na formação de colônias das células NT2/D1. Estes resultados indicam o envolvimento destas vias de sinalização nestes processos.
8. Foi investigado o papel das metaloproteínases na invasão das células NT2/D1 ativadas pelos ERs. Na presença do inibidor das metaloproteínases (GM6001) os efeitos do E2, do agonista seletivo do ESR1 (PPT) e do agonista seletivo do ESR2 (DPN) foram bloqueados.
9. A análise proteômica nas células NT2/D1 controles (sem tratamento) e tratadas com E2, mostraram proteínas diferencialmente expressas após o tratamento com E2. Foi observado o aumento de algumas proteínas envolvidas com o processo de migração celular com o tratamento com o E2.
Em conclusão, a ativação do complexo E2-ESR1 aumenta a fosforilação de SRC e ERK1/2 e leva à proliferação e invasão celular e formação de colônias. A ativação do complexo E2-ESR2 aumenta a fosforilação de SRC e ERK1/2 e leva à proliferação, migração e invasão celular e formação de colônias. O tratamento com os agonistas seletivos do ESR1 e do ESR2 ativa metaloproteinases e estas proteínas estão também envolvidas na invasão das células NT2/D1
Nosso estudo fornece uma nova visão sobre mecanismos moleculares de ERs e vias de sinalização intracelular em células de carcinoma embrionário testicular humano NT2/D1. O mecanismo de ação dos ERs em tumor de células germinativas testiculares ainda precisa ser profundamente compreendido, mas representa um importante alvo terapêutico no futuro.
The mechanisms involved in the development and progression of testicular germ cell tumors (TGCTs) are not fully understood. Recent studies from our laboratory have shown the expression of estrogen receptors ESR1 and ESR2 in human embryonic testicular carcinoma (NT2/D1) cells. The activation of estrogen receptors and the modulation of intracellular signaling pathways may be involved in these processes. Thus, the aims of the present study were to investigate the effects of estrogen receptor activation on the expression and phosphorylation of proteins involved in intracellular signaling SRC, ERK1/2, and AKT and their roles in proliferation, invasion, migration, and colony formation in NT2/D1 human embryonic testicular carcinoma. Furthermore, the protein profile of these cells treated with 17β-estradiol was determined. It was noticed that: 1. 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN led to a rapid increase in the phosphorylation state of SRC and ERK1/2 in NT2/D1 cells. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP). 2. The phosphorylated form of AKT was practically undetectable both in NT2/D1 control cells (no treatment) and in cells treated with E2 for different times, compared to the positive control. 3. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to an increase of NT2/D1 cells number. Pretreatment of these cells with SRC inhibitors (PP2) and MEK1/2 (U0126) blocked these effects. These results indicate the involvement of these proteins in NT2/D1 cell proliferation. 4. E2 and ESR2-selective agonist DPN caused an increase in the migration of NT2/D1 cells. Pretreatment with ESR2-selective antagonists (PHTPP) blocked these effects, indicating that the E2-ESR2 complex is involved in this process. Treatment with ESR1-selective agonist PPT did not alter the migration of these cells. 5. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to the increase of NT2/D1 cells invasion. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP), indicating that the E2-ESR1 and E2-ESR2 complex are involved in this process. 6. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to the increase of NT2/D1 cells colony formation. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP), indicating that the E2-ESR1 and E2-ESR2 complex are involved in this process. 7. Pretreatments with SRC inhibitors (PP2) and MEK1/2 (U0126) blocked the effects of estrogen receptor activation on cell migration, invasion, and colony formation of NT2/D1 cells. These results indicate the involvement of these proteins in these processes. 8. The role of metalloproteinases in the invasion of NT2/D1 cells activated by ERs was investigated. In the presence of the metalloproteinase inhibitor (GM6001) the effects of E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN were blocked. 9. Proteomic analysis on NT2/D1 control (no treatment) and E2-treated cells showed differential expression of proteins with E2 treatment. An increase in some proteins involved in the cell migration process was observed with the treatment with E2. In conclusion, activation of the E2-ESR1 complex increases SRC and ERK1/2 phosphorylation and leads to cell proliferation and invasion, and colony formation. Activation of the E2-ESR2 complex increases SRC and ERK1/2 phosphorylation and leads to cell proliferation, migration and invasion, and colony formation. In the presence of the metalloproteinase inhibitor (GM6001) the effects of E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN on cell invasion were blocked. Our study provides novel insights into the signatures and molecular mechanisms of ERs in human testicular embryonal carcinoma NT2/D1 cells. The mechanism of action of ERs in testicular germ cell cancers remains to be deeply understood, but it could represent an important therapeutic target in the future.
The mechanisms involved in the development and progression of testicular germ cell tumors (TGCTs) are not fully understood. Recent studies from our laboratory have shown the expression of estrogen receptors ESR1 and ESR2 in human embryonic testicular carcinoma (NT2/D1) cells. The activation of estrogen receptors and the modulation of intracellular signaling pathways may be involved in these processes. Thus, the aims of the present study were to investigate the effects of estrogen receptor activation on the expression and phosphorylation of proteins involved in intracellular signaling SRC, ERK1/2, and AKT and their roles in proliferation, invasion, migration, and colony formation in NT2/D1 human embryonic testicular carcinoma. Furthermore, the protein profile of these cells treated with 17β-estradiol was determined. It was noticed that: 1. 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN led to a rapid increase in the phosphorylation state of SRC and ERK1/2 in NT2/D1 cells. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP). 2. The phosphorylated form of AKT was practically undetectable both in NT2/D1 control cells (no treatment) and in cells treated with E2 for different times, compared to the positive control. 3. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to an increase of NT2/D1 cells number. Pretreatment of these cells with SRC inhibitors (PP2) and MEK1/2 (U0126) blocked these effects. These results indicate the involvement of these proteins in NT2/D1 cell proliferation. 4. E2 and ESR2-selective agonist DPN caused an increase in the migration of NT2/D1 cells. Pretreatment with ESR2-selective antagonists (PHTPP) blocked these effects, indicating that the E2-ESR2 complex is involved in this process. Treatment with ESR1-selective agonist PPT did not alter the migration of these cells. 5. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to the increase of NT2/D1 cells invasion. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP), indicating that the E2-ESR1 and E2-ESR2 complex are involved in this process. 6. E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN led to the increase of NT2/D1 cells colony formation. These effects were blocked by pretreatment with the respective ESR1 or ESR2-selective antagonists (MPP and PHTPP), indicating that the E2-ESR1 and E2-ESR2 complex are involved in this process. 7. Pretreatments with SRC inhibitors (PP2) and MEK1/2 (U0126) blocked the effects of estrogen receptor activation on cell migration, invasion, and colony formation of NT2/D1 cells. These results indicate the involvement of these proteins in these processes. 8. The role of metalloproteinases in the invasion of NT2/D1 cells activated by ERs was investigated. In the presence of the metalloproteinase inhibitor (GM6001) the effects of E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN were blocked. 9. Proteomic analysis on NT2/D1 control (no treatment) and E2-treated cells showed differential expression of proteins with E2 treatment. An increase in some proteins involved in the cell migration process was observed with the treatment with E2. In conclusion, activation of the E2-ESR1 complex increases SRC and ERK1/2 phosphorylation and leads to cell proliferation and invasion, and colony formation. Activation of the E2-ESR2 complex increases SRC and ERK1/2 phosphorylation and leads to cell proliferation, migration and invasion, and colony formation. In the presence of the metalloproteinase inhibitor (GM6001) the effects of E2, ESR1-selective agonist PPT and ESR2-selective agonist DPN on cell invasion were blocked. Our study provides novel insights into the signatures and molecular mechanisms of ERs in human testicular embryonal carcinoma NT2/D1 cells. The mechanism of action of ERs in testicular germ cell cancers remains to be deeply understood, but it could represent an important therapeutic target in the future.
Descrição
Citação
MACHERONI, C. Ativação dos receptores estrogênicos, sinalização intracelular e função em células de carcinoma embrionário testicular humano NT2/D1. São Paulo, 2022. 149 f. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFSP). São Paulo, 2022.