RESUMO
Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.
Assuntos
Epigênese Genética , Terapia Genética , Células Supressoras Mieloides/fisiologia , Neoplasias/terapia , Microambiente Tumoral , Animais , Azacitidina/farmacologia , Benzamidas/farmacologia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Camundongos , Células Supressoras Mieloides/citologia , Metástase Neoplásica/terapia , Neoplasias/cirurgia , Piridinas/farmacologia , Receptores CCR2/genética , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
PURPOSE: Differentiated thyroid cancer (DTC) responds to VEGF receptor inhibitors. VEGF signals through RAS/RAF/MEK signaling. We evaluated the safety and efficacy of the VEGF receptor inhibitor pazopanib and MEK inhibitor trametinib in advanced solid tumors and DTC. PATIENTS AND METHODS: Patients with advanced solid tumors were enrolled in a phase I, multicenter trial with a DTC expansion cohort. Patients received pazopanib 400-800 mg and trametinib 1-2 mg daily. Efficacy in the expansion cohort was assessed with objective response (OR) at 6 months of treatment. RESULTS: Twenty-six patients were enrolled in five dose levels. MTD was not reached; the recommended phase II dose was pazopanib 800 mg orally and trametinib 2 mg orally every day. There was one dose-limiting toxicity on dose level 1 with grade 3 fatigue and muscle weakness. Common grade 3 adverse events were elevated transaminases (19%), diarrhea (15%), hypertension (12%), and fatigue (8%). Thirteen patients were enrolled in the DTC cohort; OR was 33% (95% confidence interval, 9.9, 65.1%) and median progression-free survival was 10.7 months. The cohort was terminated after planned interim analysis suggested insufficiently increased activity against the historical control of pazopanib alone. Reduction in tumor diameter negatively correlated with p-ERK change in tumor (Spearman ρ = -0.71; P = 0.05). NRAS mutation was associated with response (Fisher exact P = 0.008). CONCLUSIONS: Pazopanib + trametinib was tolerable at full single-agent doses with clinical activity in DTC but did not achieve the prespecified response rate target.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais , Feminino , Humanos , Indazóis , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirimidinonas/administração & dosagem , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/mortalidade , Neoplasias da Glândula Tireoide/patologia , Resultado do TratamentoRESUMO
We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.
Assuntos
Movimento Celular/fisiologia , Quinase 5 Dependente de Ciclina/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/biossíntese , Quinase 5 Dependente de Ciclina/genética , Citoesqueleto/enzimologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , RNA Interferente Pequeno/genéticaRESUMO
Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked ß-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/enzimologia , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células A549 , Acilação , Substituição de Aminoácidos , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Glucose/genética , Glucose/metabolismo , Células HEK293 , Hexosaminas/genética , Hexosaminas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
CONTEXT: Activating mutations in the BRAF gene, primarily at V600E, are associated with poorer outcomes in patients with papillary thyroid cancer. MAPK kinase (MEK), immediately downstream of BRAF, is a promising target for ras-raf-MEK-ERK pathway inhibition. OBJECTIVE: The objective of the investigation was to study the efficacy of a MEK1/2 inhibitor in thyroid cancer preclinical models with defined BRAF mutation status. EXPERIMENTAL DESIGN: After treatment with the potent MEK 1/2 inhibitor AZD6244, MEK inhibition and cell growth were examined in four BRAF mutant (V600E) and two BRAF wild-type thyroid cancer cell lines and in xenografts from a BRAF mutant cell line. RESULTS: AZD6244 potently inhibited MEK 1/2 activity in thyroid cancer cell lines regardless of BRAF mutation status, as evidenced by reduced ERK phosphorylation. Four BRAF mutant lines exhibited growth inhibition at low doses of the drug, with GI50 concentrations ranging from 14 to 50 nm, predominantly via a G0/G1 arrest, comparable with findings in a sensitive BRAF mutant melanoma cell line. In contrast, two BRAF wild-type lines were significantly less sensitive, with GI50 values greater than 200 nm. Nude mouse xenograft tumors derived from the BRAF mutant line ARO exhibited dose-dependent growth inhibition by AZD6244, with effective treatment at 10 mg/kg by oral gavage. This effect was primarily cytostatic and associated with marked inhibition of ERK phosphorylation. CONCLUSION: AZD6244 inhibits the MEK-ERK pathway across a spectrum of thyroid cancer cells. MEK inhibition is cytostatic in papillary thyroid cancer and anaplastic thyroid cancer cells bearing a BRAF mutation and may have less impact on thyroid cancer cells lacking this mutation.
Assuntos
Benzimidazóis/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Mutação/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Inibidores do Crescimento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transativadores/metabolismo , Proteínas ras/metabolismo , Adenoviridae/genética , Antígenos CD/metabolismo , Northern Blotting , Western Blotting , Calcitonina/metabolismo , Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Receptor gp130 de Citocina , DNA/biossíntese , Ativação Enzimática , Genes Reporter , Humanos , Immunoblotting , Fator Inibidor de Leucemia , Luciferases/metabolismo , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Espectrometria de Massas , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3 , Transdução de Sinais , Frações Subcelulares , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Regulação Neoplásica da Expressão Gênica , Meia-Vida , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/química , Complexos Multienzimáticos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1 , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ubiquitina/metabolismoRESUMO
CONTEXT: Medullary thyroid cancer (MTC) is a cancer of the parafollicular C cells that commonly presents with an inherited or acquired RET gene mutation. There is currently no effective systemic treatment for MTC. OBJECTIVE: The objective of this study was to investigate a systemic therapeutic approach to treat MTC. We studied the sensitivity of an MTC cell line and xenograft to irinotecan, alone and in combination with the tyrosine kinase inhibitor, CEP-751. RESULTS: In TT cell culture and xenografts, irinotecan treatment was highly effective. This effect was augmented by treatment with CEP-751. Treatment of TT cell xenografts resulted in durable complete remission in 100% of the mice, with median time to recurrence of 70 d for irinotecan alone and more than 130 d for irinotecan plus CEP-751. Although irinotecan induced an S phase checkpoint arrest in TT cells, CEP-751 in combination with irinotecan resulted in a loss of this arrest. CEP-751 induced a loss in the induction of the DNA repair program marked by phospho-H2AX and the checkpoint pathway marked by the activated Chk1 pathway. CONCLUSIONS: Irinotecan treatment was highly effective in a preclinical model of human MTC, resulting in complete remission in 100% of the xenografts treated. The duration of remission was further enhanced by combination with the kinase inhibitor, CEP-751. These results suggest that irinotecan, alone or in combination, may be useful for the treatment of MTC.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Carbazóis/uso terapêutico , Carcinoma Medular/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Western Blotting , Camptotecina/uso terapêutico , Carcinoma Medular/genética , Carcinoma Medular/patologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Histonas/biossíntese , Histonas/genética , Humanos , Irinotecano , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Fase S/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Transplante Heterólogo , Fosfatases cdc25/biossíntese , Fosfatases cdc25/genéticaRESUMO
The aberrant growth of head and neck squamous cell carcinoma (HNSCC) is often associated with the constitutive activation of signal-transducer-and-activator-of-transcription-3 (STAT3), which is believed to result from the persistent stimulation of EGF receptors that are highly expressed in squamous cell carcinoma (SCC) cells. To investigate the mechanism underlying STAT3 deregulation in HNSCC, we examined the interplay of the STAT3 and epidermal growth factor receptor (EGFR) signaling pathways using a panel of HNSCC cell lines. Although STAT3 was active in most HNSCC cell lines, only 3 of 10 HNSCC cell lines were moderately to strongly positive for activated EGFR. Even in the EGFR-positive cell lines, STAT3 activation was not dependent on EGFR activation, as STAT3 tyrosine phosphorylation levels persisted after treatment with AG1478, a chemical inhibitor of EGFR activity. Furthermore, we found that conditioned medium harvested from HNSCC cells could induce STAT3 tyrosine phosphorylation in immortalized keratinocytes regardless of the status of EGFR signaling. In contrast, blocking the cytokine gp130 coreceptor abolished STAT3 tyrosine phosphorylation in HNSCC cells and that induced by the conditioned medium. Immunodepletion studies suggested interleukin 6 (IL6) as the major autocrine/paracrine factor for STAT3 activation, which coincided with high levels of secretion of IL6 into the culture medium by these cancer cells. Treatment with a specific inhibitor of Janus kinase, AG490, in HNSCC cells led to a reduction of active STAT3 and caused significant growth retardation and apoptosis. Thus, constitutive activation of STAT3 in HNSCC may use an autocrine/paracrine-activating loop mediated by IL6 and other cytokines acting through the gp130 receptor family, which may confer both proliferative and survival potential in this malignancy.
Assuntos
Antígenos CD/fisiologia , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Interleucina-6/fisiologia , Glicoproteínas de Membrana/fisiologia , Transativadores/metabolismo , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma de Células Escamosas/patologia , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Quinazolinas , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/antagonistas & inibidores , Transativadores/fisiologia , Células Tumorais Cultivadas , Tirfostinas/farmacologiaRESUMO
All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.
Assuntos
Carbazóis/farmacologia , Carcinoma Medular/tratamento farmacológico , Indóis , Proteínas Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Carcinoma Medular/enzimologia , Carcinoma Medular/patologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Furanos , Inibidores do Crescimento/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasia Endócrina Múltipla Tipo 2b/genética , Mutação , Proteínas Oncogênicas/metabolismo , Fosforilação/efeitos dos fármacos , Pró-Fármacos/farmacologia , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT: Gastrointestinal (GI) carcinoid tumors elaborate serotonin and other vasoactive substances, causing the carcinoid syndrome. Based on developmental biology data, we hypothesized that basic helix-loop-helix transcription factors, including achaete-scute complex homolog-like 1 (Ascl1)/hASH1, and the Notch signaling pathway might regulate the neuroendocrine phenotype in GI carcinoids. OBJECTIVE: The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in GI carcinoids and model their interaction in a relevant GI carcinoid cell line. DESIGN: Fourteen GI carcinoid tumor specimens, five paired adjacent normal tissues, fetal tissues, and tumor cell lines were analyzed by RT-PCR and immunoblot. BON carcinoid cells were further analyzed after Notch overexpression for neuroendocrine marker expression, serotonin production, and growth. SETTING: The study was conducted in an academic referral center. PATIENTS OR OTHER PARTICIPANTS: Deidentified archival pathology specimens were examined. RESULTS: Among a panel of six developmental transcription factors tested, only Ascl1 mRNA was overexpressed compared with surrounding normal tissue (seven of 10 GI carcinoid tumors and in BON cells, none of five normal tissues). Ascl1 protein was also expressed in four of four carcinoid tumors and BON cells). Notch pathway ligands, receptors, and downstream effectors were widely expressed in tumor and normal specimens. Overexpression of activated Notch1 in BON cells led to induction of the Notch effector hairy and enhancer of split 1 (Hes1), loss of Ascl1, reductions in neuron-specific enolase, synaptophysin, and chromogranin A, and most significantly, an 89% decrease in serotonin concentration and equivalent reductions in serotonin-reactive cells and repression of tryptophan hydroxylase 1 mRNA. CONCLUSIONS: The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.
Assuntos
Tumor Carcinoide/patologia , Neoplasias Gastrointestinais/patologia , Proteínas de Membrana/fisiologia , Sistemas Neurossecretores/citologia , Transdução de Sinais/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proliferação de Células , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Humanos , RNA Mensageiro/análise , Receptores Notch , Serotonina/biossíntese , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
KRAS is activated by mutation in the vast majority of cases of pancreatic cancer; unfortunately, therapeutic attempts to inhibit KRAS directly have been unsuccessful. Our previous studies showed that inhibition of cyclin-dependent kinase 5 (CDK5) reduces pancreatic cancer growth and progression, through blockage of the centrally important RAL effector pathway, downstream of KRAS. In the current study, the therapeutic effects of combining the CDK inhibitor dinaciclib (SCH727965; MK-7965) with the pan-AKT inhibitor MK-2206 were evaluated using orthotopic and subcutaneous patient-derived human pancreatic cancer xenograft models. The combination of dinaciclib (20 mg/kg, i.p., three times a week) and MK-2206 (60 mg/kg, orally, three times a week) dramatically blocked tumor growth and metastasis in all eight pancreatic cancer models examined. Remarkably, several complete responses were induced by the combination treatment of dinaciclib and MK-2206. The striking results obtained in these models demonstrate that the combination of dinaciclib with the pan-AKT inhibitor MK-2206 is promising for therapeutic evaluation in pancreatic cancer, and strongly suggest that blocking RAL in combination with other effector pathways downstream from KRAS may provide increased efficacy in pancreatic cancer. Based on these data, an NCI-CTEP-approved multicenter phase I clinical trial for pancreatic cancer of the combination of dinaciclib and MK-2206 (NCT01783171) has now been opened.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Óxidos N-Cíclicos , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Esquema de Medicação , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imuno-Histoquímica , Indolizinas , Injeções Intraperitoneais , Camundongos Nus , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/farmacologia , Proteína do Retinoblastoma/metabolismo , Resultado do TratamentoRESUMO
Multikinase inhibitors (MKIs) targeting VEGF receptors and other receptor tyrosine kinases have shown considerable activity in clinical trials of thyroid cancer. Thyroid cancer frequently exhibits activation of the RAS/RAF/MEK/ERK pathway. In other types of cancer, paradoxical ERK activation has emerged as a potential resistance mechanism to RAF-inhibiting drugs including MKIs such as sorafenib and pazopanib. We therefore queried whether the MEK inhibitor trametinib, could augment the activity of pazopanib in thyroid cancer cell lines. Trametinib potently inhibited growth in vitro (GI50 1.1-4.8 nM), whereas pazopanib had more limited in vitro activity, as anticipated (GI50 1.4-7.1 µM). We observed progressive upregulation of ERK activity with pazopanib treatment, an effect abrogated by trametinib. For xenografts (bearing either KRASG12R or BRAFV600E mutations), the combination of trametinib and pazopanib led to sustained shrinkage in tumor volume by 50% or more, compared to pre-treatment baseline. Trametinib also was highly effective as a single agent, compared to pazopanib alone. These preclinical findings support the evaluation of trametinib, alone or in combination with pazopanib or other kinase inhibitors, in thyroid cancer clinical trials. We highlight the importance of pharmacodynamic assessment of the ERK pathway for patients enrolled in trials involving MKIs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Indazóis , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas/administração & dosagem , Sulfonamidas/administração & dosagem , Neoplasias da Glândula Tireoide/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epithelial ovarian cancer (EOC) is the most common cause of death from gynaecological malignancy. Resistance to platinum chemotherapy is a major reason for treatment failure and poor prognosis. The human homeobox gene BARX2 is located within a minimal region at 11q25 that is associated with frequent loss of heterozygosity (LOH) and adverse survival in EOC. BARX2 is a transcription factor known to regulate transcription of specific cell adhesion molecules in the mouse. We have previously shown that BARX2 expression is low in clear cell/endometrioid and high in serous adenocarcinomas of the ovary, histologic variants that are less and more sensitive, respectively to platinum chemotherapy. The aim of this study was to define whether BARX2 could modulate sensitivity to cisplatin in human epithelial ovarian cancer. In two cell line series sequentially derived from ovarian cancer patients pre- and post-cisplatin chemotherapy, BARX2 expression was downregulated in the cell lines established upon tumor recurrence after platinum therapy. Transfection of BARX2 into a platinum resistant cell line significantly reversed cisplatin resistance compared with its isogenic platinum sensitive parent, in both growth inhibition and clonogenic assays. Taken together, our data demonstrate that the homeobox gene BARX2 may be a biological factor involved in determining sensitivity or resistance to the cytotoxic effects of cisplatin.
Assuntos
Cisplatino/farmacologia , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/genética , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Northern Blotting , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Transfecção , Células Tumorais CultivadasRESUMO
Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.
Assuntos
Antineoplásicos/farmacologia , Quinase 5 Dependente de Ciclina/metabolismo , Invasividade Neoplásica/patologia , Neoplasias da Próstata/patologia , Tilorona/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
CONTEXT: Medullary thyroid cancer (MTC) is a rare thyroid cancer that can occur sporadically or as part of a hereditary syndrome. OBJECTIVE: To explore the genetic origin of MTC, we sequenced the protein coding exons of approximately 21,000 genes in 17 sporadic MTCs. PATIENTS AND DESIGN: We sequenced the exomes of 17 sporadic MTCs and validated the frequency of all recurrently mutated genes and other genes of interest in an independent cohort of 40 MTCs comprised of both sporadic and hereditary MTC. RESULTS: We discovered 305 high-confidence mutations in the 17 sporadic MTCs in the discovery phase, or approximately 17.9 somatic mutations per tumor. Mutations in RET, HRAS, and KRAS genes were identified as the principal driver mutations in MTC. All of the other additional somatic mutations, including mutations in spliceosome and DNA repair pathways, were not recurrent in additional tumors. Tumors without RET, HRAS, or KRAS mutations appeared to have significantly fewer mutations overall in protein coding exons. CONCLUSIONS: Approximately 90% of MTCs had mutually exclusive mutations in RET, HRAS, and KRAS, suggesting that RET and RAS are the predominant driver pathways in MTC. Relatively few mutations overall and no commonly recurrent driver mutations other than RET, HRAS, and KRAS were seen in the MTC exome.
Assuntos
Carcinoma Medular/genética , Exoma/genética , Mutação , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias da Glândula Tireoide/genética , HumanosRESUMO
PURPOSE: In thyroid cancer clinical trials, agents targeting VEGF receptors (VEGFR) and RET, among other kinases, have led to partial responses but few complete or durable responses. The RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are frequently activated in differentiated and medullary thyroid cancer (DTC and MTC) and may provide therapeutic targets for these diseases. We tested a novel drug combination targeting RAF, phosphoinositide 3-kinase (PI3K), and mTOR, plus VEGFR2 and RET, in thyroid cancer preclinical models with defined genetic backgrounds. EXPERIMENTAL DESIGN: RAF265, an ATP-competitive pan-RAF inhibitor active against VEGFR2, and BEZ-235, a PI3K inhibitor also active against Torc1 and Torc2, were tested alone and in combination in a panel of thyroid cancer lines. We tested RAF265 and BEZ-235 for kinase inhibition, growth inhibition and cell-cycle alterations, and inhibition of signaling targets and tumor growth in xenograft models. RESULTS: Both drugs potently inhibited their kinase targets in the extracellular signal-regulated kinase (ERK) and PI3K pathways. In addition, RAF265 had significant RET inhibitory activity (IC50 = 25-50 nmol/L for RET(C634W)). The combination strongly inhibited proliferation of DTC and MTC cell lines with mutations in RAS, BRAF, PTEN, and RET. Synergy was shown for B-CPAP (BRAF(V600E)) and TT cells (RET(C634W)). The combination of both drugs significantly inhibited growth of CAL62 (KRAS(G12R/G12R)) and TT xenografts, thoroughly inhibiting ERK and PI3K pathway signaling. CONCLUSIONS: Combined blockade of ERK and PI3K signaling potently inhibits growth in preclinical models representing the key genotypes seen in refractory thyroid cancer. These targets and therapies are promising for further development in both differentiated and medullary thyroid cancers.
Assuntos
Imidazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Piridinas/farmacologia , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Transplante HeterólogoRESUMO
Pancreatic cancer is one of the most lethal of human malignancies, and potent therapeutic options are lacking. Inhibition of cell cycle progression through pharmacological blockade of cyclin-dependent kinases (CDK) has been suggested as a potential treatment option for human cancers with deregulated cell cycle control. Dinaciclib (SCH727965) is a novel small molecule multi-CDK inhibitor with low nanomolar potency against CDK1, CDK2, CDK5 and CDK9 that has shown favorable toxicity and efficacy in preliminary mouse experiments, and has been well tolerated in Phase I clinical trials. In the current study, the therapeutic efficacy of SCH727965 on human pancreatic cancer cells was tested using in vitro and in vivo model systems. Treatment with SCH727965 significantly reduced in vitro cell growth, motility and colony formation in soft agar of MIAPaCa-2 and Pa20C cells. These phenotypic changes were accompanied by marked reduction of phosphorylation of Retinoblastoma (Rb) and reduced activation of RalA. Single agent therapy with SCH727965 (40 mg/kg i.p. twice weekly) for 4 weeks significantly reduced subcutaneous tumor growth in 10/10 (100%) of tested low-passage human pancreatic cancer xenografts. Treatment of low passage pancreatic cancer xenografts with a combination of SCH727965 and gemcitabine was significantly more effective than either agent alone. Gene Set Enrichment Analysis identified overrepresentation of the Notch and Transforming Growth Factor-ß (TGF-ß) signaling pathways in the xenografts least responsive to SCH727965 treatment. Treatment with the cyclin-dependent kinase inhibitor SCH727965 alone or in combination is a highly promising novel experimental therapeutic strategy against pancreatic cancer.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Compostos de Piridínio/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Óxidos N-Cíclicos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indolizinas , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/embriologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Compostos de Piridínio/administração & dosagem , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ral de Ligação ao GTP/metabolismo , GencitabinaRESUMO
Cyclin-dependent kinase 5 (CDK5), a neuronal kinase that functions in migration, has been found to be activated in some human cancers in which it has been implicated in promoting metastasis. In this study, we investigated the role of CDK5 in pancreatic cancers in which metastatic disease is most common at diagnosis. CDK5 was widely active in pancreatic cancer cells. Functional ablation significantly inhibited invasion, migration, and anchorage-independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in the profound inhibition of Ras signaling through its critical effectors RalA and RalB. Conversely, restoring Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Our findings identify CDK5 as a pharmacologically tractable target to degrade Ras signaling in pancreatic cancer.
Assuntos
Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/terapia , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Proteínas de Ciclo Celular/biossíntese , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Quinase 5 Dependente de Ciclina/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ras/antagonistas & inibidoresRESUMO
Esophageal adenocarcinoma (EAC) arises in the backdrop of reflux-induced metaplastic phenomenon known as Barrett esophagus. The prognosis of advanced EAC is dismal, and there is an urgent need for identifying molecular targets for therapy. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to EAC during endoscopic surveillance. SAGE confirmed significant upregulation of Axl "tags" during the multistep progression of Barrett esophagus to EAC. In a cohort of 92 surgically resected EACs, Axl overexpression was associated with shortened median survival on both univariate (p < 0.004) and multivariate (p < 0.036) analysis. Genetic knockdown of Axl receptor tyrosine kinase (RTK) function was enabled in two EAC lines (OE33 and JH-EsoAd1) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of Axl in EAC cell lines inhibited invasion, migration, and in vivo engraftment, which was accompanied by downregulation in the activity of the Ral GTPase proteins (RalA and RalB). Restoration of Ral activation rescued the transformed phenotype of EAC cell lines, suggesting a novel effector mechanism for Axl in cancer cells. Pharmacological inhibition of Axl was enabled using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of Axl with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of Axl function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. Axl RTK is an adverse prognostic factor in EAC. The availability of small molecule inhibitors of Axl function provides a tractable strategy for molecular therapy of established EAC.