RESUMO
FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation, and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38 MAPK. Here, we studied the potential regulation of FOXO3a by p38 in response to doxorubicin. Co-immunoprecipitation studies in MCF-7 cells demonstrated a direct interaction between p38 and FOXO3a. We also showed that p38 can bind and phosphorylate a recombinant FOXO3a directly in vitro. HPLC-coupled phosphopeptide mapping and mass spectrometric analyses identified serine 7 as a major site for p38 phosphorylation. Using a phosphorylated Ser-7 FOXO3a antibody, we demonstrated that FOXO3a is phosphorylated on Ser-7 in response to doxorubicin. Immunofluorescence staining studies showed that upon doxorubicin treatment, the wild-type FOXO3a relocalized to the nucleus, whereas the phosphorylation-defective FOXO3a (Ala-7) mutant remained largely in the cytoplasm. Treatment with SB202190 also inhibits the doxorubicin-induced FOXO3a Ser-7 phosphorylation and nuclear accumulation in MCF-7 cells. In addition, doxorubicin caused the nuclear translocation of FOXO3a in wild-type but not p38-depleted mouse fibroblasts. Together, our results suggest that p38 phosphorylation of FOXO3a on Ser-7 is essential for its nuclear relocalization in response to doxorubicin.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Doxorrubicina/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Inibidores Enzimáticos/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, DeltaN-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinazolinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Estudos de Casos e Controles , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteína Forkhead Box M1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Gefitinibe , Humanos , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.
Assuntos
Neoplasias da Mama/enzimologia , Fatores de Transcrição Forkhead/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lapatinib , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Inclusão em Parafina , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
This chapter describes methods for studying downstream events of the PI3K/Akt signaling cascade, focusing on the FoxO transcription factors. These approaches also represent alternative means for gauging the phosphoinositide-3 kinase/Akt activity. We describe protocols for the fractionation of cytoplasmic and nuclear protein extracts and for studying transcription factor DNA-binding activity in vitro and in vivo.