RESUMEN
Objective: CR1447, a novel transdermal formulation of 4-hydroxytestosterone, has aromatase-inhibiting and androgen receptor (AR)-modulating properties (IC504.4 nM) with antitumor effects against AR-positive tumor cells in vitro. This trial investigated the efficacy and safety of CR1447 for patients with metastatic estrogen receptor-positive (A) and AR-positive triple-negative breast cancers (B). Design and methods: (A) included patients with at most one prior endocrine therapy line without progression ≥6 months, whereas (B) included patients with ≤2 prior chemotherapy lines, all displaying advanced signs of disease. The primary endpoint was disease control at week 24 (DC24). The null hypothesis was DC24 ≤30% (A) and ≤15% (B). Thirty-seven patients were recruited (29 in (A) and 8 in (B)); accrual was stopped following an interim analysis demonstrating futility in (A) and slow accrual in (B). Results: DC24 was attained in 5/21 (95% CI: 8.2-47.2) patients in (A) and none in (B). The median progression-free survival was 5.1 months (95% CI: 2.5-5.6) in (A) and 2.5 months (95% CI: 0.7-2.6) in (B). The median overall survival was 24.6 months (95% CI: 22.9-not applicable) in (A) and 10.8 months (95% CI: 3.3-10.9) in (B). CR1447 had a favorable safety profile without treatment-related grade 3-5 toxicities in (A). Especially no side effects linked to androgenic effects were observed. Conclusions: Despite this trial being negative, the 24% DC24 rate in a second-line setting, and the prolonged partial response experienced by a patient, indicate activity. Further evaluation of CR1447 in endocrine-sensitive patients or combination trials appears warranted.
RESUMEN
CR1447 (4-hydroxytestosterone, 4-OHT) binds to the androgen receptor and has antiproliferative activity in both ER-positive and ER-negative/AR-positive breast cancer cells in preclinical studies. The objective of this first-in man trial was to evaluate the safety and to determine the dose of CR1447, administered as an ointment, for Phase II. Escalating doses (100, 200, 400 mg) of CR1447 were administered topically on a daily basis to patients with ER-positive/AR-positive/HER2-negative advanced breast cancer pretreated with several lines of therapy. 14 patients have been treated for a total of 42 cycles. Two patients, one at dose level 100 mg and one at dose level 200 mg, showed early tumour progression and were replaced. Related adverse events were all ≤ grade 2 and included fatigue, bone and joint pain, stiffness, dry skin and mouth, nausea, sweating, urinary tract infection, rash, headache and distress. No drug-related dose-limiting toxicities (DLTs) were seen. Two patients (17%) achieved stable disease at 3 months. Pharmacokinetic analysis confirmed dose-dependent transdermal uptake of CR1447. 4-OH-androstenedione (4-OHA), a key metabolite of 4-OHT, was undetectable in most of the plasma samples. Urine metabolites of 4-OHT and 4-OHA indicate high exposure of 4-OHT after topical administration. Oestradiol serum concentrations did not increase, confirming preclinical data that CR1447 is not converted to estrogens in vivo In conclusion, CR1447 administered transdermally as an ointment is well tolerated and appears to have single-agent activity in heavily pretreated ER-positive/HER2-negative breast cancer patients. The recommended phase II dose is 400 mg/day.
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NF-κB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. In this study, we identified SIRT2 as a deacetylase of the transcription factor p65. SIRT2 is a member of the family of sirtuins, which are NAD(+)-dependent deacetylases involved in several cellular processes. SIRT2 interacts with p65 in the cytoplasm and deacetylates p65 in vitro and in vivo at Lys310. Moreover, p65 is hyperacetylated at Lys310 in Sirt2(-/-) cells after TNFα stimulation, which results in the increase in expression of a subset of p65 acetylation-dependent target genes. Our work provides evidence that p65 is deacetylated by SIRT2 in the cytoplasm to regulate the expression of specific NF-κB-dependent genes.
Asunto(s)
Regulación de la Expresión Génica , Lisina/metabolismo , Sirtuina 2/metabolismo , Factor de Transcripción ReIA/metabolismo , Acetilación/efectos de los fármacos , Animales , Especificidad de Anticuerpos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Cinética , Ratones , Células 3T3 NIH , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Reproducibilidad de los Resultados , Sirtuina 1/deficiencia , Sirtuina 1/metabolismo , Sirtuina 2/deficiencia , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIMS: Endothelial activation, macrophage infiltration, and foam cell formation are pivotal steps in atherogenesis. Our aim in this study was to analyse the role of SIRT1, a class III deacetylase with important metabolic functions, in plaque macrophages and atherogenesis. METHODS AND RESULTS: Using partial SIRT1 deletion in atherosclerotic mice, we demonstrate that SIRT1 protects against atherosclerosis by reducing macrophage foam cell formation. Peritoneal macrophages from heterozygous SIRT1 mice accumulate more oxidized low-density lipoprotein (oxLDL), thereby promoting foam cell formation. Bone marrow-restricted SIRT1 deletion confirmed that SIRT1 function in macrophages is sufficient to decrease atherogenesis. Moreover, we show that SIRT1 reduces the uptake of oxLDL by diminishing the expression of lectin-like oxLDL receptor-1 (Lox-1) via suppression of the NF-κB signalling pathway. CONCLUSION: Our findings demonstrate protective effects of SIRT1 in atherogenesis and suggest pharmacological SIRT1 activation as a novel anti-atherosclerotic strategy by reducing macrophage foam cell formation.
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Aterosclerosis/etiología , Células Espumosas/patología , Receptores Depuradores de Clase E/fisiología , Sirtuina 1/fisiología , Animales , Aterosclerosis/prevención & control , Trasplante de Médula Ósea , Diferenciación Celular/fisiología , Células Cultivadas , Colesterol/metabolismo , Eliminación de Gen , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sirtuina 1/genéticaRESUMEN
BACKGROUND: NF-kappaB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. We have earlier reported that p65, a subunit of NF-kappaB, is acetylated in vitro and in vivo at three different lysines (K310, K314 and K315) by the histone acetyltransferase p300. RESULTS: In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-kappaB target genes including Mmp10 and Mmp13. Increased gene expression was mainly observed three hours after TNFalpha stimulation. Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314. CONCLUSIONS: Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-kappaB-dependent gene expression.
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Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Factor de Transcripción ReIA/genética , Acetilación , Animales , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Humanos , Lisina/genética , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/genética , Ratones , Mutación , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Recombinantes/genética , Factor de Necrosis Tumoral alfaRESUMEN
Coactivator-associated arginine methyltransferase 1 (CARM1) belongs to the protein arginine methyltransferase family. It was reported to methylate histone as well as non-histone proteins and thus to be involved in transcriptional activation and mRNA degradation/stability. Here we report the genetic complementation of carm1-/- cells with wild-type CARM1 or an enzymatic inactive mutant of CARM1 to investigate the requirement of CARM1 and its enzymatic activity for nuclear factor kappaB (NF-kappaB)-dependent gene expression. Using custom microarray and quantitative reverse transcription PCR, we could define a subset of NF-kappaB target genes that required CARM1 for their proper expression. Although several tumor necrosis factor-alpha- and phorbol-12-myristate-13-acetate/ionomycin-induced NF-kappaB target genes are CARM1 dependent, CARM1 enzymatic activity was dispensable for gene expression. Interestingly, CARM1 was not required for the stimulus-dependent recruitment of RelA/p65 to chromatin, suggesting that CARM1 is rather contributing in protein complex stabilization. Together, our results confirm the importance of CARM1 as transcriptional cofactor without the involvement of its catalytic activity.
Asunto(s)
FN-kappa B/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Prueba de Complementación Genética , Ionomicina/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteína-Arginina N-Metiltransferasas/deficiencia , Proteína-Arginina N-Metiltransferasas/genética , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65-/- cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor alpha was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFalpha identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.