RESUMO
Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERalpha. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIalpha, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIalpha converts tamoxifen from an ERalpha inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).
Assuntos
Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Receptores de Estrogênio/química , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Sítios de Ligação , Western Blotting , Neoplasias da Mama/enzimologia , Divisão Celular , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio , Feminino , Transferência Ressonante de Energia de Fluorescência , Fulvestranto , Humanos , Luciferases/metabolismo , Microscopia Confocal , Modelos Biológicos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Estrogênio/metabolismo , Serina/química , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
Estrogen receptor-mediated transcription is enhanced by overexpression of G1/S cyclins D1, E or A in the presence as well in the absence of estradiol. Excess of G1/S cyclins also prevents the inhibition of transactivation of estrogen receptor (ER) by the pure antiestrogen ICI 182780. Cyclin D1 mediates this transactivation independent of complex formation to its CDK4/6 partner. This raises the possibility that overexpression of G1/S cyclins renders growth of ER-positive breast cancer hormone-independent and resistant to treatment with antiestrogens. Transient transfection of ER-positive breast cancer cell lines T47D and MCF7 with G1/S cyclins could overcome the growth arrest induced by ICI 182780 treatment. The ability of various cyclin D1 mutants to overcome the ICI 182780 mediated growth arrest corresponded with their ability to stimulate cyclin A- and E2F- promoter based reporter activities in the presence of ICI 182780. Transfection of a mutant cyclin D1 (cyclin D1-KE) that was unable to bind CDK4 and was reported to transactivate ER in the presence of ICI 182780, could not stimulate proliferation in ICI 182780 treated cells. On the other hand, cyclin D1-LALA, which is unable to stimulate ERE transactivation, could overcome the ICI 182780 cell cycle arrest. Furthermore, transient transfection of T47D cells using cyclin D1 together with a catalytic inactive mutant of CDK4 (CDK4-DN) indicated that the observed effect is due to binding to CDK inhibitors. However, a moderate, sixfold overexpression of cyclin D1 in stably transfected MCF7 cells did not overcome the ICI 182780 mediated growth arrest. These results indicate that CDK-independent transactivation of the estrogen receptor by cyclin D1 is by itself, not sufficient to result in estradiol-independent growth of breast cancer cells, whereas a vast overexpression of G1/S cyclins is able to do so, most likely by capturing of CDK inhibitors.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Ciclina A/fisiologia , Ciclina D1/fisiologia , Ciclina E/fisiologia , Proteínas de Ligação a DNA , Estradiol/análogos & derivados , Estrogênios , Fase G1/fisiologia , Proteínas de Neoplasias/análise , Neoplasias Hormônio-Dependentes/patologia , Proteínas Proto-Oncogênicas , Receptores de Estrogênio/análise , Fase S/fisiologia , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Ciclina D1/genética , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Fulvestranto , Humanos , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Treatment of MCF7 human breast cancer cells with taxol induces G2/M arrest followed by mitotic death. A moderate overexpression of ectopic cyclin D1 accelerated these G2/M associated events and resulted in a reduced clonogenic survival upon taxol treatment. Taxol treatment resulted in elevated expression of p53 and of p21, which was more pronounced and persistent in cyclin D1 overexpressing cells. Overexpression of cyclin D1 altered sensitivity to taxol by modulating exit from mitosis, which is controlled by p21. These results indicate that overexpression of cyclin D1 sensitizes MCF7 cells to treatment with taxol.