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1.
Int J Cancer ; 132(3): E149-57, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22948716

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 , Temozolomida
2.
Clin Exp Metastasis ; 28(8): 899-908, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21953073

RESUMO

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-Like
3.
Clin Cancer Res ; 17(10): 3420-30, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21459796

RESUMO

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-Like
4.
Oncotarget ; 2(12): 1254-64, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22248814

RESUMO

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-Like
5.
Cancer Res ; 70(9): 3677-86, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406975

RESUMO

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 Testes
6.
Curr Opin Genet Dev ; 18(1): 68-72, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18339543

RESUMO

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 Genética
7.
Nat Cell Biol ; 8(8): 855-62, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16845383

RESUMO

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 Ubiquitina
8.
Proc Natl Acad Sci U S A ; 102(44): 15901-6, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16247015

RESUMO

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/metabolismo
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