RESUMO
Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.
Assuntos
Antineoplásicos/administração & dosagem , Benzoatos/administração & dosagem , Ciclopropanos/administração & dosagem , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Histona Desmetilases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/farmacologia , Benzoatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Dados de Sequência Molecular , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.
Assuntos
Benzodiazepinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors.
Assuntos
Biomarcadores Tumorais/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Piridonas/farmacologia , Pirimidinonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Estrutura Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Piridonas/química , Pirimidinonas/química , Transcriptoma , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Few therapeutic strategies exist for the treatment of metastatic tumor cells in the brain because the blood-brain barrier (BBB) limits drug access. Thus the identification of molecular targets and accompanying BBB permeable drugs will significantly benefit brain metastasis patients. Polo-like kinase 1 (Plk1) is an attractive molecular target because it is only expressed in dividing cells and its expression is upregulated in many tumors. Analysis of a publicly available database of human breast cancer metastases revealed Plk1 mRNA expression was significantly increased in brain metastases compared to systemic metastases (P = 0.0018). The selective Plk1 inhibitor, GSK461364A, showed substantial uptake in normal rodent brain. Using a breast cancer brain metastatic xenograft model (231-BR), we tested the efficacy of GSK461364A to prevent brain metastatic colonization. When treatment was started 3 days post-injection, GSK461364A at 50 mg/kg inhibited the development of large brain metastases 62% (P = 0.0001) and prolonged survival by 17%. GSK461364A sensitized tumor cells to radiation induced cell death in vitro. Previously, it was reported that mutations in p53 might render tumor cells more sensitive to Plk1 inhibition; however, p53 mutations are uncommon in breast cancer. In a cohort of 41 primary breast tumors and matched brain metastases, p53 immunostaining was increased in 61% of metastases; 44% of which were associated with primary tumors with low p53. The data suggest that p53 overexpression occurs frequently in brain metastases and may facilitate sensitivity to Plk1 inhibition. These data indicate Plk1 may be a new druggable target for the prevention of breast cancer brain metastases.
Assuntos
Neoplasias Encefálicas/prevenção & controle , Neoplasias da Mama/prevenção & controle , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Radiação Ionizante , Taxa de Sobrevida , Tiofenos/farmacologia , Análise Serial de Tecidos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53 , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
BACKGROUND: Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916. METHODS: 59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response. RESULTS: 20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test). CONCLUSIONS: High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.
Assuntos
Compostos Aza/farmacologia , Cromossomos Humanos/genética , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Indóis/farmacologia , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinase B , Aurora Quinases , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Diploide , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hematológicas/patologia , Humanos , Mutação/genética , Fenótipo , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/genéticaRESUMO
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
Assuntos
Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Apoptose/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Hibridização Genômica Comparativa/métodos , Feminino , Dosagem de Genes , Humanos , Imuno-Histoquímica/métodos , Mutação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
Identification of biomarkers for positive and negative predictors of response to cancer therapeutics can help direct clinical strategies. However, challenges with tissue availability and costs are significant limiting factors for diagnostic assays. To address these challenges, we have customized a high-throughput single nucleotide polymorphism genotyping assay with the objective of simultaneously surveying known somatic mutations and copy number alterations for translational studies in cancer. As constructed, this assay can interrogate 376 known somatic mutations and quantify copy number alterations of genes commonly implicated in tumorigenesis or progression. Validation of this assay on a panel of 321 cell lines demonstrates sensitivity to accurately detect mutations, robust accuracy in the presence of infiltrating normal tissue, and the ability to detect both DNA copy number amplifications and deletions. This technology, with its high sensitivity, small DNA requirements, and low costs is an attractive platform for biomarker exploration in cancer.
Assuntos
Dosagem de Genes/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Oncogenes/genética , Mutação Puntual/genética , Linhagem Celular Tumoral , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Sensibilidade e EspecificidadeRESUMO
PURPOSE: GSK461364 is an ATP-competitive inhibitor of polo-like kinase 1 (Plk1). A phase I study of two schedules of intravenous GSK461364 was conducted. EXPERIMENTAL DESIGN: GSK461364 was administered in escalating doses to patients with solid malignancies by two schedules, either on days 1, 8, and 15 of 28-day cycles (schedule A) or on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (schedule B). Assessments included pharmacokinetic and pharmacodynamic profiles, as well as marker expression studies in pretreatment tumor biopsies. RESULTS: Forty patients received GSK461364: 23 patients in schedule A and 17 in schedule B. Dose-limiting toxicities (DLT) in schedule A at 300 mg (2 of 7 patients) and 225 mg (1 of 8 patients) cohorts included grade 4 neutropenia and/or grade 3-4 thrombocytopenia. In schedule B, DLTs of grade 4 pulmonary emboli and grade 4 neutropenia occurred at 7 or more days at 100 mg dose level. Venous thrombotic emboli (VTE) and myelosuppression were the most common grade 3-4, drug-related events. Pharmacokinetic data indicated that AUC (area under the curve) and C(max) (maximum concentration) were proportional across doses, with a half-life of 9 to 13 hours. Pharmacodynamic studies in circulating tumor cells revealed an increase in phosphorylated histone H3 (pHH3) following drug administration. A best response of prolonged stable disease of more than 16 weeks occurred in 6 (15%) patients, including 4 esophageal cancer patients. Those with prolonged stable disease had greater expression of Ki-67, pHH3, and Plk1 in archived tumor biopsies. CONCLUSIONS: The final recommended phase II dose for GSK461364 was 225 mg administered intravenously in schedule A. Because of the high incidence (20%) of VTE, for further clinical evaluation, GSK461364 should involve coadministration of prophylactic anticoagulation.
Assuntos
Benzimidazóis/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tiofenos/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Benzimidazóis/metabolismo , Benzimidazóis/farmacocinética , Ligação Competitiva , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Tiofenos/metabolismo , Tiofenos/farmacocinética , Quinase 1 Polo-LikeRESUMO
RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.
Assuntos
Benzimidazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Tiofenos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Farmacológicos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Análise em Microsséries , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Retinoides/farmacologia , Transdução de Sinais/genética , Quinase 1 Polo-LikeRESUMO
Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G(1) state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Instabilidade Cromossômica , Mutação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tiofenos/farmacologia , Proteína Supressora de Tumor p53/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Immunoblotting , Concentração Inibidora 50 , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Poliploidia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Quinase 1 Polo-LikeRESUMO
Preclinical cellular response profiling of tumor models has become a cornerstone in the development of novel cancer therapeutics. As efforts to predict clinical efficacy using cohorts of in vitro tumor models have been successful, expansive panels of tumor-derived cell lines can recapitulate an "all comers" efficacy trial, thereby identifying which tumors are most likely to benefit from treatment. The response profile of a therapy is most often studied in isolation; however, drug treatment effect patterns in tumor models across a diverse panel of compounds can help determine the value of unique molecular target classes in specific tumor cohorts. To this end, a panel of 19 compounds was evaluated against a diverse group of cancer cell lines (n = 311). The primary oncogenic targets were a key determinant of concentration-dependent proliferation response, as a total of five of six, four of four, and five of five phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, insulin-like growth factor-I receptor (IGF-IR), and mitotic inhibitors, respectively, clustered with others of that common target class. In addition, molecular target class was correlated with increased responsiveness in certain histologies. A cohort of PI3K/AKT/mTOR inhibitors was more efficacious in breast cancers compared with other tumor types, whereas IGF-IR inhibitors more selectively inhibited growth in colon cancer lines. Finally, specific phenotypes play an important role in cellular response profiles. For example, luminal breast cancer cells (nine of nine; 100%) segregated from basal cells (six of seven; 86%). The convergence of a common cellular response profile for different molecules targeting the same oncogenic pathway substantiates a rational clinical path for patient populations most likely to benefit from treatment. Cancer Res; 70(9); 3677-86. (c)2010 AACR.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral , Humanos , Valor Preditivo dos TestesRESUMO
Polo-like kinases (Plk) function in mitosis and maintaining DNA integrity. There are four family members, of which Plk1 represents a target for anticancer therapy. Plk1 is only expressed in dividing cells with peak expression during G2/M. Plk1 functions in multiple steps of mitosis, and is overexpressed in many tumor types. Mitotic arrest and inhibition of proliferation, apoptosis, and tumor growth inhibition have been observed in preclinical studies using small interfering RNAs (siRNA) or small molecules that inhibit Plk1. Preclinical studies also show that Plk1 inhibitors may be active against tumors with RAS mutations and that tumor cells with mutations in TP53 are more sensitive to inhibition of Plk1. Several Plk inhibitors are in phase I or II clinical studies. As expected, hematologic toxicity is the primary dose-limiting toxicity. Some patients have achieved clinical response, although in some studies only at doses above the maximum tolerated dose defined in the study. Further evaluation is necessary to discern the clinical utility of Plk1 inhibitors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Biológicos , Inibidores de Proteínas Quinases/administração & dosagem , Especificidade por Substrato , Quinase 1 Polo-LikeRESUMO
Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas.
Assuntos
Regulação Neoplásica da Expressão Gênica , Complexo Principal de Histocompatibilidade/genética , Melanoma/genética , Antígenos de Diferenciação de Linfócitos B/análise , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Primers do DNA , Progressão da Doença , Regulação para Baixo , Amplificação de Genes , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Melanócitos/citologia , Melanócitos/imunologia , Melanoma/imunologia , Melanoma/patologia , Metástase Neoplásica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Valores de Referência , Transcrição GênicaRESUMO
New technologies as well as concerted brute-force approaches have increased the content (number of genes) that can be characterized for genomic DNA alterations. Recent advances include the detection of activating point mutations in key kinase genes (BRAF, EGFR, and PIK3CA) in multiple cancer types: preliminary insight into the entire repertoire of genes that can be mutated in cancer; the discovery of new oncogenes by high-resolution profiling of DNA copy number alterations; and the bioinformatic-driven discovery of oncogenic gene fusions. High-content promoter methylation detection systems have been used to discover additional methylated genes and have provided evidence for a stem cell origin for certain tumors. Some of these advances have had significant impact on the development and clinical testing of new therapeutics.
Assuntos
Genes Neoplásicos , Neoplasias/genética , Análise Mutacional de DNA , DNA de Neoplasias/química , Epigênese Genética , Dosagem de Genes , Genoma Humano , Humanos , Proteínas de Fusão Oncogênica/genética , Translocação GenéticaRESUMO
The tumour suppressor p53 induces apoptosis or cell-cycle arrest in response to genotoxic and other stresses. In unstressed cells, the anti-proliferative effects of p53 are restrained by mouse double minute 2 (Mdm2), a ubiquitin ligase (E3) that promotes p53 ubiquitination and degradation. Mdm2 also mediates its own degradation through auto-ubiquitination. It is unclear how the cis- and trans-E3 activities of Mdm2, which have opposing effects on cell fate, are differentially regulated. Here, we show that death domain-associated protein (Daxx) is required for Mdm2 stability. Downregulation of Daxx decreases Mdm2 levels, whereas overexpression of Daxx strongly stabilizes Mdm2. Daxx simultaneously binds to Mdm2 and the deubiquitinase Hausp, and it mediates the stabilizing effect of Hausp on Mdm2. In addition, Daxx enhances the intrinsic E3 activity of Mdm2 towards p53. On DNA damage, Daxx dissociates from Mdm2, which correlates with Mdm2 self-degradation. These findings reveal that Daxx modulates the function of Mdm2 at multiple levels and suggest that the disruption of the Mdm2-Daxx interaction may be important for p53 activation in response to DNA damage.
Assuntos
Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Correpressoras , Dano ao DNA , Endopeptidases/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase , Peptidase 7 Específica de UbiquitinaRESUMO
Metastasis of primary tumors leads to a very poor prognosis for patients suffering from cancer. Although it is well established that not every tumor will eventually metastasize, it is less clear whether primary tumors acquire genetic alterations in a stochastic process at a late stage, which make them invasive, or whether genetic alterations acquired early in the process of tumor development drive primary tumor growth and determine whether this tumor is going to be metastatic. To address this issue, we tested genes identified in a large-scale comparative genomic hybridization analysis of primary tumor for their ability to confer metastatic properties on a cancer cell. We identified amplification of the ACK1 gene in primary tumors, which correlates with poor prognosis. We further show that overexpression of Ack1 in cancer cell lines can increase the invasive phenotype of these cells both in vitro and in vivo and leads to increased mortality in a mouse model of metastasis. Biochemical studies show that Ack1 is involved in extracellular matrix-induced integrin signaling, ultimately activating signaling processes like the activation of the small GTPase Rac. Taken together, this study supports a theory from Bernards and Weinberg [Bernards, R. & Weinberg, R. A. (2002) Nature 418, 823], which postulates that the tendency to metastasize is largely predetermined.
Assuntos
Amplificação de Genes , Metástase Neoplásica/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Animais , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa3beta1/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Transplante de Neoplasias , Prognóstico , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Transplante Heterólogo , Células Tumorais Cultivadas , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
PPM1D encodes WIP1, a serine-threonine phosphatase that had previously been shown to be the driver oncogene of a 17q23 amplicon that is present in approximately 15% of human breast tumors. However, it is unknown whether it has any role in the remaining 85% of breast tumors. A recent study using Wip1-deficient mice revealed that blocking its function significantly impaired RAS and ERBB2-induced breast tumor formation, suggesting that the inhibition of Wip1 could be a broad-spectrum treatment for breast cancer. However, because of the structure of Wip1, the development of small molecule inhibitors is a significant challenge.