RESUMO
Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Carcinoma/patologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinoma/tratamento farmacológico , Carcinoma/genética , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/fisiologia , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Camundongos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.