RESUMO
PURPOSE: Endocrine-based therapy is the initial primary treatment option for hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (mBC). However, patients eventually experience disease progression due to resistance to endocrine therapy. Molibresib (GSK525762) is a small-molecule inhibitor of bromodomain and extraterminal (BET) family proteins (BRD2, BRD3, BRD4, and BRDT). Preclinical data suggested that the combination of molibresib with endocrine therapy might overcome endocrine resistance. This study aimed to investigate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy [objective response rate (ORR)] of molibresib combined with fulvestrant in women with HR+/HER2- mBC. PATIENTS AND METHODS: In this phase I/II dose-escalation and dose-expansion study, patients received oral molibresib 60 or 80 mg once daily in combination with intramuscular fulvestrant. Patients enrolled had relapsed/refractory, advanced/metastatic HR+/HER2- breast cancer with disease progression on prior treatment with an aromatase inhibitor, with or without a cyclin-dependent kinase 4/6 inhibitor. RESULTS: The study included 123 patients. The most common treatment-related adverse events (AE) were nausea (52%), dysgeusia (49%), and fatigue (45%). At a 60-mg dosage of molibresib, >90% of patients experienced treatment-related AE. Grade 3 or 4 treatment-related AE were observed in 47% and 48% of patients treated with molibresib 60 mg and molibresib 80 mg, respectively. The ORR was 13% [95% confidence interval (CI), 8-20], not meeting the 25% threshold for proceeding to phase II. Among 82 patients with detected circulating tumor DNA and clinical outcome at study enrollment, a strong association was observed between the detection of copy-number amplification and poor progression-free survival (HR, 2.89; 95% CI, 1.73-4.83; P < 0.0001). CONCLUSIONS: Molibresib in combination with fulvestrant did not demonstrate clinically meaningful activity in this study.
Assuntos
Benzodiazepinas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fulvestranto , Proteínas Nucleares , Receptor ErbB-2/metabolismo , Fatores de Transcrição , Progressão da Doença , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo CelularRESUMO
PURPOSE: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). PATIENTS AND METHODS: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non-Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). RESULTS: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8-18.7], 25% (95% CI, 7.3-52.4), and 13% (95% CI, 6.9-20.6), respectively. CONCLUSIONS: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.
Assuntos
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Linfoma não Hodgkin , Trombocitopenia , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
Molibresib is an orally bioavailable, selective, small molecule BET protein inhibitor. Results from a first time in human study in solid tumors resulted in the selection of a 75 mg once daily dose of the besylate formulation of molibresib as the recommended Phase 2 dose (RP2D). Here we present the results of Part 2 of our study, investigating safety, pharmacokinetics, pharmacodynamics and clinical activity of molibresib at the RP2D for nuclear protein in testis carcinoma (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer and gastrointestinal stromal tumor. The primary safety endpoints were incidence of adverse events (AEs) and serious AEs; the primary efficacy endpoint was overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). Molibresib 75 mg once daily demonstrated no unexpected toxicities. The most common treatment-related AEs (any grade) were thrombocytopenia (64%), nausea (43%) and decreased appetite (37%); 83% of patients required dose interruptions and 29% required dose reductions due to AEs. Antitumor activity was observed in NC and CRPC (one confirmed partial response each, with observed reductions in tumor size), although predefined clinically meaningful response rates were not met for any tumor type. Total active moiety median plasma concentrations after single and repeated administration were similar across tumor cohorts. GSEA revealed that gene expression changes with molibresib varied by patient, response status and tumor type. Investigations into combinatorial approaches that use BET inhibition to eliminate resistance to other targeted therapies are warranted.
Assuntos
Benzodiazepinas/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Neoplasias Testiculares/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzodiazepinas/administração & dosagem , Benzodiazepinas/efeitos adversos , Benzodiazepinas/farmacocinética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Adulto JovemRESUMO
Molibresib (GSK525762) is an investigational orally bioavailable small-molecule bromodomain and extraterminal (BET) protein inhibitor for the treatment of advanced solid tumors. In the first-time-in-human BET115521 study of molibresib in patients with solid tumors, thrombocytopenia was the most frequent treatment-related adverse event (AE), QT prolongation was an AE of special interest based on preclinical signals, and gastrointestinal (GI) AEs (nausea, vomiting, diarrhea, and dysgeusia) were often observed. The aims of this analysis were the following: (i) develop a population pharmacokinetic (PK)/pharmacodynamic (PD) model capable of predicting platelet time courses in individual patients after administration of molibresib and identify covariates of clinical interest; (ii) evaluate the effects of molibresib (and/or its two active metabolites [GSK3529246]) exposure on cardiac repolarization by applying a systematic modeling approach using high-quality, intensive, PK time-matched 12-lead electrocardiogram measurements; (iii) evaluate the exposure-response (ER) relationship between molibresib and/or GSK3529246 exposures and the occurrence of Grade 2 or higher GI AEs. Overall, the PK/PD model (including a maximal drug effect model and molibresib concentration) adequately described platelet counts following molibresib treatment and was used to simulate the impact of molibresib dosing on thrombocytopenia at different doses and regimens. ER analyses showed no clinically meaningful QT interval prolongation with molibresib at up to 100 mg q.d., and no strong correlation between molibresib exposure and the occurrence of Grade 2 or higher GI AEs. The models described here can aid dosing/schedule and drug combination strategies and may support a thorough QT study waiver request for molibresib.
Assuntos
Síndrome do QT Longo , Neoplasias , Trombocitopenia , Benzodiazepinas/farmacocinética , Benzodiazepinas/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Síndrome do QT Longo/induzido quimicamente , Neoplasias/tratamento farmacológico , Trombocitopenia/induzido quimicamenteRESUMO
Molibresib (GSK525762) is an investigational, orally bioavailable, small-molecule bromodomain and extraterminal (BET) protein inhibitor for the treatment of advanced solid tumors. Molibresib was initially evaluated in a first-time-in-human (FTIH) study BET115521 consisting of two parts: Part 1 of the study (dose escalation) was conducted in 94 patients with nuclear protein in testis midline carcinoma and other solid tumors, and Part 2 (expansion cohort) was conducted in 99 patients with different solid tumor types. Molibresib is metabolized by cytochrome P450 3A4 enzymes to produce two major active metabolites that are equipotent to the parent molecule. The metabolites are measured together after full conversion of one to the other and reported as an active metabolite composite (GSK3529246). The molibresib pharmacokinetic (PK) profile has been characterized by a decrease in exposure over time, with the decrease more pronounced at higher doses, and accompanied by a slight increase of the metabolite concentrations. Autoinduction of molibresib metabolism was suspected and confirmed in vitro. Here we report the development of a semimechanistic liver-compartment population PK model using PK data from the FTIH study, which adequately describes the autoinduction of molibresib clearance and the PK of both molibresib and GSK3529246. Covariate analysis indicated body weight had a significant effect on the volume of distribution of molibresib and GSK3529246, and higher levels of aspartate aminotransferase resulted in the lower clearance of GSK3529246. This model was used to simulate individual patient exposures based on covariate information for use in future alternative dosing strategies and exposure-response analyses.
Assuntos
Antineoplásicos/farmacocinética , Benzodiazepinas/farmacocinética , Modelos Biológicos , Neoplasias/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Benzodiazepinas/administração & dosagem , Peso Corporal , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Distribuição Tecidual , Adulto JovemRESUMO
BACKGROUND: Bromodomain and extra-terminal domain proteins are promising epigenetic anticancer drug targets. This first-in-human study evaluated the safety, recommended phase II dose, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the bromodomain and extra-terminal domain inhibitor molibresib (GSK525762) in patients with nuclear protein in testis (NUT) carcinoma (NC) and other solid tumors. METHODS: This was a phase I and II, open-label, dose-escalation study. Molibresib was administered orally once daily. Single-patient dose escalation (from 2 mg/d) was conducted until the first instance of grade 2 or higher drug-related toxicity, followed by a 3 + 3 design. Pharmacokinetic parameters were obtained during weeks 1 and 3. Circulating monocyte chemoattractant protein-1 levels were measured as a pharmacodynamic biomarker. RESULTS: Sixty-five patients received molibresib. During dose escalation, 11% experienced dose-limiting toxicities, including six instances of grade 4 thrombocytopenia, all with molibresib 60-100 mg. The most frequent treatment-related adverse events of any grade were thrombocytopenia (51%) and gastrointestinal events, including nausea, vomiting, diarrhea, decreased appetite, and dysgeusia (22%-42%), anemia (22%), and fatigue (20%). Molibresib demonstrated an acceptable safety profile up to 100 mg; 80 mg once daily was selected as the recommended phase II dose. Following single and repeat dosing, molibresib showed rapid absorption and elimination (maximum plasma concentration: 2 hours; t1/2: 3-7 hours). Dose-dependent reductions in circulating monocyte chemoattractant protein-1 levels were observed. Among 19 patients with NC, four achieved either confirmed or unconfirmed partial response, eight had stable disease as best response, and four were progression-free for more than 6 months. CONCLUSIONS: Once-daily molibresib was tolerated at doses demonstrating target engagement. Preliminary data indicate proof-of-concept in NC.
RESUMO
BACKGROUND: NUT midline carcinoma, renamed NUT carcinoma (NC), is an aggressive squamous cancer defined by rearrangement of the NUTM1 gene. Although a subset of patients can be cured, for the majority of patients the prognosis is grim. We sought to classify patients into risk groups based on molecular and clinicopathologic factors at the time of diagnosis. METHODS: Clinicopathologic variables and survival outcomes were extracted for a total of 141 NC patients from the NUT midline carcinoma Registry using questionnaires and medical records. Translocation type was identified by molecular analyses. Survival tree regression analysis was performed to determine risk factors associated with overall survival (OS). RESULTS: For 141 patients, the median age at diagnosis was 23.6 years. Fifty-one percent had thoracic origin compared with 49% nonthoracic sites (41% head and neck, 6% bone or soft tissue, 1% other). The median OS was 6.5 months (95% confidence interval [CI] = 5.8 to 9.1 months). Most patients had the BRD4-NUTM1 fusion (78%), followed by BRD3-NUTM1 (15%) and NSD3-NUTM1 (6%). Survival tree regression identified three statistically distinct risk groups among 124 patients classified by anatomical site and genetics: group A is nonthoracic primary, BRD3-, or NSD3-NUT (n = 12, median OS = 36.5 months, 95% CI = 12.5 to not reported months); group B is nonthoracic primary, BRD4-NUT (n = 45, median OS = 10 months, 95% CI = 7 to 14.6 months); and group C is thoracic primary (n = 67, median OS = 4.4 months, 95% CI = 3.5 to 5.6 months). Only groups A and B had long-term (≥3 years, n = 12) survivors. CONCLUSIONS: We identify three risk groups defined by anatomic site and NUT fusion type. Nonthoracic primary with non-BRD4-NUT fusion confers the best prognosis, followed by nonthoracic primary with BRD4-NUT. Thoracic NC patients, regardless of the NUT fusion, have the worst survival.
RESUMO
PURPOSE: Enhancer of zeste homolog 2 (EZH2) activity is dysregulated in many cancers. PATIENTS AND METHODS: This phase I study determined the safety, maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of the intravenously administered, highly selective EZH2 inhibitor, GSK2816126, (NCT02082977). Doses of GSK2816126 ranged from 50 to 3,000 mg twice weekly, and GSK2816126 was given 3-weeks-on/1-week-off in 28-day cycles. Eligible patients had solid tumors or B-cell lymphomas with no available standard treatment regimen. RESULTS: Forty-one patients (21 solid tumors, 20 lymphoma) received treatment. All patients experienced ≥1 adverse event (AE). Fatigue [22 of 41 (53.7%)] and nausea [20 of 41 (48.8%)] were the most common toxicity. Twelve (32%) patients experienced a serious AE. Dose-limiting elevated liver transaminases occurred in 2 of 7 patients receiving 3,000 mg of GSK2816126; 2,400 mg was therefore established as the MTD. Following intravenous administration of 50 to 3,000 mg twice weekly, plasma GSK2816126 levels decreased biexponentially, with a mean terminal elimination half-life of approximately 27 hours. GSK2816126 exposure (maximum observed plasma concentration and area under the plasma-time curve) increased in a dose-proportional manner. No change from baseline in H3K27me3 was seen in peripheral blood mononuclear cells. Fourteen of 41 (34%) patients had radiological best response of stable disease, 1 patient with lymphoma achieved a partial response, 21 of 41 (51%) patients had progressive disease, and 5 patients were unevaluable for antitumor response. CONCLUSIONS: The MTD of GSK2816126 was established at 2,400 mg, but the dosing method and relatively short half-life limited effective exposure, and modest anticancer activity was observed at tolerable doses.
Assuntos
Antineoplásicos/administração & dosagem , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Indóis/administração & dosagem , Linfoma de Células B/tratamento farmacológico , Neoplasias/tratamento farmacológico , Piridonas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Indóis/efeitos adversos , Indóis/farmacocinética , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/patologia , Segurança do Paciente , Prognóstico , Piridonas/efeitos adversos , Piridonas/farmacocinética , Distribuição Tecidual , Adulto JovemRESUMO
INTRODUCTION: This first-time-in-humans study assessed the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of GSK2879552 in patients with relapsed or refractory SCLC. METHODS: This phase I, multicenter, open-label study (NCT02034123) enrolled patients (≥18 years old) with relapsed or refractory SCLC (after ≥1 platinum-containing chemotherapy or refusal of standard therapy). Part 1 was a dose-escalation study; Part 2 was a dose-expansion study. Dose escalations were based on safety, PK, and PD. The primary end point (Part 1) was to determine the safety, tolerability, and recommended dose and regimen of GSK2879552. Secondary end points were to characterize PK and PD parameters and measure disease control rate at week 16. Part 2 was not conducted. RESULTS: Between February 4, 2014, and April 18, 2017, a total of 29 patients were allocated to one of nine dose cohorts (0.25 mg-3 mg once daily and 3-mg or 4-mg intermittent dosing). In all, 22 patients completed the study; 7 withdrew, primarily owing to adverse events (AEs). Most patients (24 of 29 [83%]) had at least one treatment-related AE, most commonly thrombocytopenia (12 of 29 [41%]). Twelve serious AEs (SAEs) were reported by nine patients; six were considered treatment related, the most common of which was encephalopathy (four SAEs). Three patients died; one death was related to SAEs. PK was characterized by rapid absorption, slow elimination, and a dose-proportional increase in exposure. CONCLUSIONS: GSK2879552 is a potent, selective inhibitor of lysine demethylase 1A and has demonstrated favorable PK properties but provided poor disease control and a high AE rate in patients with SCLC. The study was terminated, as the risk-benefit profile did not favor continuation.
Assuntos
Benzoatos/uso terapêutico , Ciclopropanos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Terapia de Salvação , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Adolescente , Adulto , Idoso , Benzoatos/farmacocinética , Ciclopropanos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de Sobrevida , Distribuição Tecidual , Adulto JovemRESUMO
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transativadores/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Epigênese Genética/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brivanib alaninate is the orally available prodrug of brivanib, a dual inhibitor of fibroblast growth factor and vascular endothelial growth factor signaling pathways that is under therapeutic investigation for various malignancies. Brivanib alaninate inhibits CYP3A4 in vitro, and thus there is potential for drug-drug interaction with CYP3A4 substrates, such as midazolam. The present study evaluated pharmacokinetic parameters and safety/tolerability upon coadministration of brivanib alaninate and midazolam. Healthy participants received intravenous (IV) or oral midazolam with and without oral brivanib alaninate. Blood samples for pharmacokinetic analysis were collected up to 12 hours after midazolam and up to 48 hours after brivanib alaninate. Twenty-four participants were administered study drugs; 21 completed the trial. No clinically relevant effect of brivanib alaninate on the overall exposure to midazolam following IV or oral administration was observed. Orally administered brivanib alaninate was generally well tolerated in the presence of IV or oral midazolam. The lack of a pharmacokinetic interaction between brivanib and midazolam indicates that brivanib alaninate does not influence either intestinal or hepatic CYP3A4 and confirms that brivanib alaninate may be safely coadministered with midazolam and other CYP3A4 substrates.
Assuntos
Alanina/análogos & derivados , Antineoplásicos/farmacologia , Inibidores do Citocromo P-450 CYP3A , Midazolam/farmacocinética , Pró-Fármacos/farmacologia , Triazinas/farmacologia , Administração Oral , Adulto , Alanina/efeitos adversos , Alanina/sangue , Alanina/farmacocinética , Alanina/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Estudos Cross-Over , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Meia-Vida , Humanos , Injeções Intravenosas , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Desintoxicação Metabólica Fase I , Midazolam/administração & dosagem , Midazolam/efeitos adversos , Midazolam/sangue , Pessoa de Meia-Idade , Pacientes Desistentes do Tratamento , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacocinética , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Triazinas/efeitos adversos , Triazinas/sangue , Triazinas/farmacocinéticaRESUMO
Activation of the transcription factor AP-1 (activator protein-1) is required for tumor promotion and maintenance of malignant phenotype. A number of AP-1-regulated genes that play a role in tumor progression have been identified. However, AP-1-regulated genes driving tumor induction are yet to be defined. Previous studies have established that expression of a dominant-negative c-Jun (TAM67) inhibits phorbol 12-tetradecanoyl-13-acetate (TPA)-induced AP-1 transactivation as well as transformation in mouse epidermal JB6/P+ cells and tumor promotion in mouse skin carcinogenesis. In this study, we utilized the tumor promotion-sensitive JB6/P+ cells to identify AP-1-regulated TAM67 target genes and to establish causal significance in transformation for one target gene. A 2700 cDNA microarray was queried with RNA from TPA-treated P+ cells with or without TAM67 expression. Under conditions in which TAM expression inhibited TPA-induced transformation, microarray analysis identified a subset of six genes induced by TPA and suppressed by TAM67. One of the identified genes, the high-mobility group protein A1 (Hmga1) is induced by TPA in P+, but not in transformation-resistant P cells. We show that TPA induction of the architectural transcription factor HMGA1 is inhibited by TAM67, is extracellular-signal-regulated kinase (ERK)-activation dependent, and is mediated by AP-1. HMGA1 antisense construct transfected into P+ cells blocked HMGA1 protein expression and inhibited TPA-induced transformation indicating that HMGA1 is required for transformation. HMGA1 is not however sufficient as HMGA1a or HMGA1b overexpression did not confer transformation sensitivity on P- cells. Although HMGA1 expression is ERK dependent, it is not the only ERK-dependent event required for transformation because it does not suffice to rescue ERK-deficient P- cells. Our study shows (a) TAM 67 when it inhibits AP-1 and transformation, targets a relatively small number of genes; (b) HMGA1, a TAM67 target gene, is causally related to transformation and therefore a potentially important target for cancer prevention.
Assuntos
Transformação Celular Neoplásica/genética , Células Epidérmicas , Genes jun/genética , Proteína HMGA1a/fisiologia , Proteína HMGA1b/fisiologia , MAP Quinase Quinase Quinase 1 , Proteínas Proto-Oncogênicas c-jun/fisiologia , Transcrição Gênica/genética , Animais , Butadienos/farmacologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Ciclina D1/biossíntese , Ciclina D1/genética , DNA Complementar/genética , Suscetibilidade a Doenças , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Perfilação da Expressão Gênica , MAP Quinase Quinase Quinases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Nitrilas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , Osteopontina , Proteínas Proto-Oncogênicas c-jun/deficiência , Sialoglicoproteínas/biossíntese , Sialoglicoproteínas/genética , Acetato de Tetradecanoilforbol/toxicidade , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
UNLABELLED: Although inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are implicated in tumor pathology, their role in the early stages of carcinogenesis is not well defined. Tumor necrosis factor alpha (TNFalpha) induces iNOS and NO production in transformation-sensitive JB6 P+, but not in transformation-resistant JB6 P-, mouse epidermal cells. We tested the hypothesis that iNOS, by generating NO and reactive nitrogen species, mediates tumor promoter-induced transformation. Specific [N-[3-(aminomethyl)benzyl]acetamidine (1400W)] and non-specific (N(omega)-methyl-L-arginine) iNOS inhibitors significantly reduced TNFalpha-induced NO production in P+ cells but both iNOS inhibitors enhanced TNFalpha-induced anchorage-independent transformation, thus ruling out a mediator role and suggesting an inhibitor role for NO. Independent support for an inhibitor role came from the observation that the NO donor [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO)] inhibited TNFalpha- and 12-O-tetradecanoylphorbol-13-acetate-induced transformation. DETA/NO treatment also suppressed tumor phenotype in tumorigenic JB6 RT101 (Tx) cells. Higher concentrations of DETA/NO induced apoptosis. The transformation inhibitory effect of lower DETA/NO concentrations may be attributable in part to inhibition by NO of NF-kappaB-dependent but not of AP-1-dependent transcription. IN CONCLUSION: (a) induction of iNOS and NO production does not mediate but actually prevents tumor promotion; (b) iNOS inhibitors enhance the transformation response, and therefore appear not to be appropriate as chemoprevention agents; and (c) NO has both chemopreventive and tumoricidal effects, suggesting promise in cancer chemoprevention and therapy.
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Transformação Celular Neoplásica , Óxido Nítrico/fisiologia , Animais , Apoptose/fisiologia , Sequência de Bases , Linhagem Celular , Primers do DNA , Camundongos , NF-kappa B/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fenótipo , Espécies Reativas de Oxigênio , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Generation of reactive oxygen species (ROS) stimulates transcription by activating transcription factors activator protein 1 (AP-1) and nuclear factor kappaB (NF-KB). The mouse epidermal JB6 cells constitute a model system that has significantly contributed to the understanding of these events. Clonal variants of JB6 cells are differentially responsive to transformation induced by tumor promoters such as phorbol esters (TPA), epidermal growth factor (EGF) and tumor necrosis factor alpha (TNF-alpha), as well as oxidative stress. TPA and EGF, acting through the MAP kinase pathway, activate AP-1 and subsequently NF-kappaB proteins and downstream transcription processes that are involved in the transformation response in transformation-sensitive (P+) JB6 cells. The effect of TNF-alpha is primarily on the NF-kappaB pathway. ROS and other free radicals can activate AP-1 and NF-KB transcription coordinately. In JB6 cells, both ERK/Fra-1 and NF-kappaB activity is essential for the transformation response. Inhibition of NF-kappaB and AP-1 activity abrogates transformation in JB6 cells as well as in transgenic mice and human keratinocytes. A similar effect is seen with antioxidants, which inhibit NF-kappaB and AP-1 activity as well as transformation in JB6 cells. The JB6 model is therefore valuable for monitoring early events in oxidative stress related signaling leading to carcinogenesis, and for identifying molecular targets for cancer chemoprevention.