RESUMO
Inhibitor of apoptosis (IAP) proteins constitute a family of conserved molecules that regulate both apoptosis and receptor signaling. They are often deregulated in cancer cells and represent potential targets for therapy. In our work, we investigated the effect of IAP inhibition in vivo to identify novel downstream genes expressed in an IAP-dependent manner that could contribute to cancer aggressiveness. To this end, immunocompromised mice engrafted subcutaneously with the triple-negative breast cancer MDA-MB231 cell line were treated with SM83, a Smac mimetic that acts as a pan-IAP inhibitor, and tumor nodules were profiled for gene expression. SM83 reduced the expression of Snai2, an epithelial-to-mesenchymal transition factor often associated with increased stem-like properties and metastatic potential especially in breast cancer cells. By testing several breast cancer cell lines, we demonstrated that Snai2 downregulation prevents cell motility and that its expression is promoted by cIAP1. In fact, the chemical or genetic inhibition of cIAP1 blocked epidermal growth factor receptor (EGFR)-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and caused the reduction of Snai2 transcription levels. In a number of breast cancer cell lines, cIAP1 depletion also resulted in a reduction of EGFR protein levels which derived from the decrease of its gene transcription, though, paradoxically, the silencing of cIAP1 promoted EGFR protein stability rather than its degradation. Finally, we provided evidence that IAP inhibition displays an anti-tumor and anti-metastasis effect in vivo. In conclusion, our work indicates that IAP-targeted therapy could contribute to EGFR inhibition and to the reduction of its downstream mediators. This approach could be particularly effective in tumors characterized by high levels of EGFR and Snai2, such as triple-negative breast cancer.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Administração Intravenosa , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/deficiência , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células Tumorais CultivadasRESUMO
Using a high-throughput approach, we identified lemur tyrosine kinase 2 (LMTK2) as a novel determinant of cell sensitivity to TRAIL. LMTK2 is a poorly characterized serine/threonine kinase believed to play a role in endosomal membrane trafficking and neuronal physiology, and recently found to be mutated in diverse tumor types. We show that LMTK2 silencing sensitizes immortalized epithelial cells and cancer cells to TRAIL, and this phenomenon is accompanied by changes in the expression of BCL2 family members. In epithelial cells, LMTK2 targeting causes the down-regulation of the BCL2 and BCL-xL anti-apoptotic proteins and the reciprocal up-regulation of the pro-apoptotic protein BIM, while, in cancer cells, LMTK2 knock-down reduces BCL2 without increasing BIM levels. We provide evidence that both BIM and BCL2 proteins are regulated by LMTK2 in a GSK3ß- and PP1A-dependent manner and that their perturbation, together with BCL-xL reduction, determines an increased sensitivity not only to TRAIL, but also to other compounds. Overall, our findings suggest a broad function of LMTK2 in the regulation of the apoptotic pathway and highlight LMTK2 as a novel candidate target to increase the cytotoxic activity of chemotherapeutic compounds.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteína bcl-X/análise , Proteína 11 Semelhante a Bcl-2/análise , Linhagem Celular Tumoral , Receptores ErbB/análise , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteína Fosfatase 1/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Interferente Pequeno/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The oncogenic transcription factor Myc is required for the progression and maintenance of diverse tumors. This has led to the concept that Myc itself, Myc-activated gene products, or associated biological processes might constitute prime targets for cancer therapy. Here, we present an in vivo reverse-genetic screen targeting a set of 241 Myc-activated mRNAs in mouse B-cell lymphomas, unraveling a critical role for the mitochondrial ribosomal protein (MRP) Ptcd3 in tumor maintenance. Other MRP-coding genes were also up regulated in Myc-induced lymphoma, pointing to a coordinate activation of the mitochondrial translation machinery. Inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired respiratory activity and tumor cell survival in vitro, and significantly extended lifespan in lymphoma-bearing mice. We have thus identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-overexpressing tumors.
Assuntos
Linfoma de Burkitt/genética , Mitocôndrias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Transgênicos , Minociclina/análogos & derivados , Minociclina/farmacologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tigeciclina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Colorretais/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Glândulas Mamárias Humanas/citologia , Mutação/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Animais , Apoptose/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Modelos Biológicos , Invasividade Neoplásica/genética , Prognóstico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Non-covalent (NP-1/3) and covalent (NP-A-1/3) pro-apoptotic SPION-Smac mimetic nano-conjugates antitumor agents are reported. The solution synthesis of key Smac mimetics, their support onto SPIONs through non-covalent adsorption (NP-1/3) or APTES-mediated covalent binding (NP-A-1/3), the analytical characterization of SPION-Smac mimetic conjugates, their target affinity in cell-free assays, and their cytotoxicity against tumor cells are thoroughly described.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Dextranos/química , Dextranos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Nanopartículas de Magnetita/química , Proteínas Mitocondriais/química , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação ProteicaRESUMO
Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.