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BACKGROUND: The absence of prominent, actionable genetic alternations in osteosarcomas (OS) implies that transcriptional and epigenetic mechanisms significantly contribute to the progression of this life-threatening form of cancer. Therefore, the identification of potential transcriptional events that promote the survival of OS cells could be key in devising targeted therapeutic approaches for OS. We have previously shown that RUNX2 is a transcription factor (TF) essential for OS cell survival. Unfortunately, the transcriptional network or circuitry regulated by RUNX2 in OS cells is still largely unknown. METHODS: The TFs that are in the RUNX2 transcriptional circuitry were identified by analyzing RNAseq and ChIPseq datasets of RUNX2. To evaluate the effect of SOX9 knockdown on the survival of osteosarcoma cells in vitro, we employed cleaved caspase-3 immunoblotting and propidium iodide staining techniques. The impact of SOX9 and JMJD1C depletion on OS tumor growth was examined in vivo using xenografts and immunohistochemistry. Downstream targets of SOX9 were identified and dissected using RNAseq, pathway analysis, and gene set enrichment analysis. Furthermore, the interactome of SOX9 was identified using BioID and validated by PLA. RESULT: Our findings demonstrate that SOX9 is a critical TF that is induced by RUNX2. Both in vitro and in vivo experiments revealed that SOX9 plays a pivotal role in the survival of OS. RNAseq analysis revealed that SOX9 activates the transcription of MYC, a downstream target of RUNX2. Mechanistically, our results suggest a transcriptional network involving SOX9, RUNX2, and MYC, with SOX9 binding to RUNX2. Moreover, we discovered that JMJD1C, a chromatin factor, is a novel binding partner of SOX9, and depletion of JMJD1C impairs OS tumor growth. CONCLUSION: The findings of this study represent a significant advancement in our understanding of the transcriptional network present in OS cells, providing valuable insights that may contribute to the development of targeted therapies for OS.
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Understanding functional interactions between cancer mutations is an attractive strategy for discovering unappreciated cancer pathways and developing new combination therapies to improve personalized treatment. However, distinguishing driver gene pairs from passenger pairs remains challenging. Here, we designed an integrated omics approach to identify driver gene pairs by leveraging genetic interaction analyses of top mutated breast cancer genes and the proteomics interactome data of their encoded proteins. This approach identified that PIK3CA oncogenic gain-of-function (GOF) and CBFB loss-of-function (LOF) mutations cooperate to promote breast tumor progression in both mice and humans. The transcription factor CBFB localized to mitochondria and moonlighted in translating the mitochondrial genome. Mechanistically, CBFB enhanced the binding of mitochondrial mRNAs to TUFM, a mitochondrial translation elongation factor. Independent of mutant PI3K, mitochondrial translation defects caused by CBFB LOF led to multiple metabolic reprogramming events, including defective oxidative phosphorylation, the Warburg effect, and autophagy/mitophagy addiction. Furthermore, autophagy and PI3K inhibitors synergistically killed breast cancer cells and impaired the growth of breast tumors, including patient-derived xenografts carrying CBFB LOF and PIK3CA GOF mutations. Thus, our study offers mechanistic insights into the functional interaction between mutant PI3K and mitochondrial translation dysregulation in breast cancer progression and provides a strong preclinical rationale for combining autophagy and PI3K inhibitors in precision medicine for breast cancer. SIGNIFICANCE: CBFB-regulated mitochondrial translation is a regulatory step in breast cancer metabolism and synergizes with mutant PI3K in breast cancer progression.
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Neoplasias de la Mama , Fosfatidilinositol 3-Quinasa Clase I , Subunidad beta del Factor de Unión al Sitio Principal , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/farmacología , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Transducción de Señal/genéticaRESUMEN
Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.
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Células Madre Embrionarias , Metabolismo de los Lípidos , Ácidos Grasos , Oxidación-Reducción , Estrés FisiológicoRESUMEN
The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Subunidad beta del Factor de Unión al Sitio Principal/genética , Regulación Neoplásica de la Expresión Génica , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Femenino , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Ratones , Mutación , Receptor Notch3/genética , Receptor Notch3/metabolismo , Transcripción Genética , Proteína Tumoral p73/deficiencia , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
No approved therapy exists for cancer-associated cachexia. The colon-26 mouse model of cancer cachexia mimics recent late-stage clinical failures of anabolic anti-cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx-024. The histone deacetylase inhibitor (HDACi) AR-42 exhibited anti-cachectic activity in this model. We explored combined SARM/AR-42 therapy as an improved anti-cachectic treatment paradigm. A reduced dose of AR-42 provided limited anti-cachectic benefits, but, in combination with GTx-024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR-42 suppressed the IL-6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx-024-mediated ß-catenin target gene regulation was apparent in cachectic mice only when combined with AR-42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx-024 therapy and a blockade of GTx-024-mediated anabolic signaling. AR-42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx-024. Combining GTx-024, a clinically established anabolic therapy, with AR-42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.
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Andrógenos/uso terapéutico , Caquexia/tratamiento farmacológico , Resistencia a Antineoplásicos , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.
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Neoplasias de la Mama/metabolismo , Interleucina-6/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , HumanosRESUMEN
BACKGROUND: Cancer cachexia is a debilitating condition that impacts patient morbidity, mortality, and quality of life and for which effective therapies are lacking. The anticachectic activity of the novel HDAC inhibitor AR-42 was investigated in murine models of cancer cachexia. METHODS: The effects of AR-42 on classic features of cachexia were evaluated in the C-26 colon adenocarcinoma and Lewis lung carcinoma (LLC) models. Effects on survival in comparison with approved HDAC inhibitors (vorinostat, romidepsin) were determined. The muscle metabolome and transcriptome (by RNA-seq), as well as serum cytokine profile, were evaluated. Data were analyzed using mixed effects models, analysis of variance, or log-rank tests. All statistical tests were two-sided. RESULTS: In the C-26 model, orally administered AR-42 preserved body weight (23.9±2.6 grams, AR-42-treated; 20.8±1.3 grams, vehicle-treated; P = .005), prolonged survival (P < .001), prevented reductions in muscle and adipose tissue mass, muscle fiber size, and muscle strength and restored intramuscular mRNA expression of the E3 ligases MuRF1 and Atrogin-1 to basal levels (n = 8). This anticachectic effect, confirmed in the LLC model, was not observed after treatment with vorinostat and romidepsin. AR-42 suppressed tumor-induced changes in inflammatory cytokine production and multiple procachexia drivers (IL-6, IL-6Rα, leukemia inhibitory factor, Foxo1, Atrogin-1, MuRF1, adipose triglyceride lipase, uncoupling protein 3, and myocyte enhancer factor 2c). Metabolomic analysis revealed cachexia-associated changes in glycolysis, glycogen synthesis, and protein degradation in muscle, which were restored by AR-42 to a state characteristic of tumor-free mice. CONCLUSIONS: These findings support further investigation of AR-42 as part of a comprehensive therapeutic strategy for cancer cachexia.
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Caquexia/tratamiento farmacológico , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Experimentales/complicaciones , Fenilbutiratos/farmacología , Pérdida de Peso/efectos de los fármacos , Adenocarcinoma/complicaciones , Tejido Adiposo/efectos de los fármacos , Administración Oral , Animales , Caquexia/etiología , Caquexia/metabolismo , Caquexia/prevención & control , Carcinoma Pulmonar de Lewis/complicaciones , Neoplasias del Colon/complicaciones , Citocinas/biosíntesis , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Interleucina-6/metabolismo , Canales Iónicos/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Lipasa/metabolismo , Factores de Transcripción MEF2/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Fenilbutiratos/administración & dosificación , Receptores de Interleucina-6/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Análisis de Supervivencia , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Desacopladora 3RESUMEN
Reinstatement represents a phenomenon that may be used to model the effects of retraumatization observed in patients with posttraumatic stress disorder (PTSD). In this study, we found intraperitoneal injection of the ß-adrenergic receptor antagonist propranolol (10 mg/kg) 1 h before reinstatement training attenuated reinstatement of fear memory in rats. Conversely, reinstatement was facilitated by intra-amygdalar administration of ß-adrenergic receptor agonist isoproterenol (Iso; 2 µg per side) 30 min before reinstatement training. The frequency and amplitude of the miniature IPSC (mIPSC) and the surface expression of the ß3 and γ2 subunits of the GABA(A) receptor (GABA(A)R) were significantly lower in reinstated than in extinction rats, whereas the AMPA/NMDA ratio and the surface expression of GluR1 and GluR2 in the amygdala did not differ between groups. In amygdala slices, Iso-induced decrease in the surface ß3 subunit of GABA(A) receptor was blocked by a Tat-conjugated dynamin function-blocking peptide (Tat-P4) pretreatment (10 µm for 30 min). By contrast, Tat-scramble peptide had no effect. Intravenous injection (3 µmol/kg) or intra-amygdalar infusion (30 pmol per side) of Tat-P4 interfered with reinstatement. Reinstatement increased the association between protein phosphatase 2A (PP2A) and the ß3 subunit of the GABA(A)R, which was abolished by PP1/PP2A inhibitors okadaic acid and calyculin A. These results suggest the involvement of ß-adrenergic receptor activation and GABA(A) receptor endocytosis in the amygdala for the reinstatement in fear memory.
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Amígdala del Cerebelo/fisiología , Endocitosis/fisiología , Miedo , Memoria/fisiología , Receptores de GABA-A/metabolismo , Recompensa , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Agonistas Adrenérgicos beta/farmacología , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Biotinilación , Condicionamiento Clásico/efectos de los fármacos , Dinaminas/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Extinción Psicológica/efectos de los fármacos , Inmunoprecipitación , Isoproterenol/farmacología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Fosfohidrolasa PTEN/farmacología , Técnicas de Placa-Clamp , Propranolol/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/farmacología , Transmisión Sináptica/efectos de los fármacos , Valina/análogos & derivados , Valina/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
BACKGROUND: The nucleus tractus solitarius (NTS) is the primary integrative center for baroreflex. Adenosine has been shown to play an important modulatory role in blood pressure control in the NTS. Our previous results demonstrated that adenosine decreases blood pressure, heart rate, and renal sympathetic nerve activity and modulates baroreflex responses in the NTS. We also demonstrated that a nitric oxide synthase (NOS) inhibitor may block the cardiovascular effects of adenosine in the NTS, which suggests interaction between the adenosine receptor and NOS. However, the signaling mechanisms of adenosine that induce nitric oxide release in the NTS remain uncertain. The aim of the present study was to investigate the possible signal pathways involved in the cardiovascular regulation of adenosine in the NTS. METHODS AND RESULTS: Adenosine was microinjected into the NTS of urethane-anesthetized male Sprague-Dawley rats. Blood pressure and heart rate decreased significantly after microinjection. The cardiovascular effects of adenosine were attenuated by prior administration of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059 (6 nmol/60 nL) or an endothelial NOS-selective inhibitor, L-NIO (6 nmol/60 nL); however, the neuronal NOS-specific inhibitor vinyl-L-NIO (600 pmol/60 nL) did not attenuate the cardiovascular effects of adenosine. Western blot and immunohistochemistry studies demonstrated that adenosine induced extracellular signal-regulated kinases 1 and 2 and endothelial NOS phosphorylation in the NTS. Pretreatment with PD98059 diminished the endothelial NOS phosphorylation evoked by adenosine. CONCLUSIONS: These results represent a novel finding that extracellular signal-regulated kinases 1 and 2 is involved in cardiovascular regulation in the NTS. They also indicate that the cardiovascular modulatory effects of adenosine in the NTS are accomplished by activation of mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2 and then endothelial NOS.