RESUMO
Inhibition of centromere-associated protein-E (CENP-E) has demonstrated preclinical anti-tumor activity in a number of tumor types including neuroblastoma. A potent small molecule inhibitor of the kinesin motor activity of CENP-E has recently been developed (GSK923295). To identify an effective drug combination strategy for GSK923295 in neuroblastoma, we performed a screen of siRNAs targeting a prioritized set of genes that function in therapeutically tractable signaling pathways. We found that siRNAs targeted to extracellular signal-related kinase 1 (ERK1) significantly sensitized neuroblastoma cells to GSK923295-induced growth inhibition (p = 0.01). Inhibition of ERK1 activity using pharmacologic inhibitors of mitogen-activated ERK kinase (MEK1/2) showed significant synergistic growth inhibitory activity when combined with GSK923295 in neuroblastoma, lung, pancreatic and colon carcinoma cell lines. Synergistic growth inhibitory activity of combined MEK/ERK and CENP-E inhibition was a result of increased mitotic arrest and apoptosis. There was a significant correlation between ERK1/2 phosphorylation status in neuroblastoma cell lines and GSK923295 growth inhibitory activity (r = 0.823, p = 0.0006). Consistent with this result we found that lung cancer cell lines harboring RAS mutations, which leads to oncogenic activation of MEK/ERK signaling, were significantly more resistant than cell lines with wild-type RAS to GSK923295-induced growth inhibition (p = 0.047). Here we have identified (MEK/ERK) activity as a potential biomarker of relative GSK923295 sensitivity and have shown the synergistic effect of combinatorial MEK/ERK pathway and CENP-E inhibition across different cancer cell types including neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Sarcosina/análogos & derivados , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Biomarcadores Tumorais , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Irinotecano , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuroblastoma/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Interferência de RNA , RNA Interferente Pequeno , Sarcosina/farmacologia , TemozolomidaRESUMO
WAY-255348 is a potent nonsteroidal progesterone receptor (PR) antagonist previously characterized in rodents and nonhuman primates. This report describes the novel mechanism by which WAY-255348 inhibits the activity of progesterone. Most PR antagonists bind to and block PR action by inducing a unique "antagonist" conformation of the PR. However, WAY-255348 lacks the bulky side chains or chemical groups that have been associated with the conformation changes of helix 12 that lead to functional antagonism. We show that WAY-255348 achieves antagonist activity by binding to and subsequently preventing progesterone-induced nuclear accumulation, phosphorylation and promoter interactions of the PR. This effect was concentration dependent, as high concentrations of WAY-255348 alone are able to induce nuclear translocation, phosphorylation and subsequent promoter interactions resulting in partial agonist activity at these concentrations. However, at lower concentrations where nuclear accumulation and phosphorylation are prevented, the progesterone-induced DNA binding is blocked along with PR-dependent gene expression. Analysis of the PR conformation induced by WAY-255348 using a limited protease digestion assay, suggested that the WAY-255348 bound PR conformation was similar to that of a progesterone agonist-bound PR and distinct from steroidal antagonist-bound PR conformations. Furthermore, the recruitment and binding of peptides derived from nuclear receptor co-activators is consistent with WAY-255348 inducing an agonist-like conformation. Taken together, these data suggest that WAY-255348 inhibits PR action through a novel molecular mechanism that is distinct from previously studied PR modulators and may be a useful tool to further understanding of PR signaling pathways. Development of therapeutic molecules with this 'passive' antagonism mechanism may provide distinct advantages for patients with reproductive disorders or PR positive breast cancers.
Assuntos
Indóis/farmacologia , Pirróis/farmacologia , Receptores de Progesterona/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Ligação Competitiva , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas Correpressoras/metabolismo , Agonismo Parcial de Drogas , Humanos , Modelos Moleculares , Coativadores de Receptor Nuclear/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Conformação Proteica , Ensaio Radioligante , Receptores de Progesterona/agonistas , Receptores de Progesterona/genéticaRESUMO
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.