RESUMEN
Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted 89Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of 89Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting 89Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, 89Zr-DFOPODS-DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to 89Zr-DFOMal-DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a 89Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited â¼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.
Asunto(s)
Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Línea Celular Tumoral , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Circonio/químicaRESUMEN
PURPOSE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. EXPERIMENTAL DESIGN: Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the "other tumor" cohort of NCT02709889 and assessed for response. RESULTS: The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. CONCLUSIONS: DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. IMPLICATIONS FOR PRACTICE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.
Asunto(s)
Carcinoma de Células de Merkel , Neoplasias Cutáneas , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genéticaRESUMEN
Rovalpituzumab tesirine (Rova-T) offers a targeted therapy for ~85% of SCLC patients whose tumors express DLL3, but clinical dosing is limited due to off-target toxicities. We hypothesized that a sub-efficacious dose of Rova-T combined with anti-PD1, which alone shows a clinical benefit to ~15% of SCLC patients, might elicit a novel mechanism of action and extend clinical utility. Using a pre-clinical murine SCLC tumor model that expresses Dll3 and has an intact murine immune system, we found that sub-efficacious doses of Rova-T with anti-PD1 resulted in enhanced anti-tumor activity, compared to either monotherapy. Multiplex immunohistochemistry (IHC) showed CD4 and CD8 T-cells primarily in normal tissue immediately adjacent to the tumor. Combination treatment, but not anti-PD1 alone, increased Ki67+/CD8 T-cells and Granzyme B+/CD8 in tumors by flow cytometry and IHC. Antibody depletion of T-cell populations showed CD8+ T-cells are required for in vivo anti-tumor efficacy. Whole transcriptome analysis as well as flow cytometry and IHC showed that Rova-T activates dendritic cells and increases Ccl5, Il-12, and Icam more than anti-PD1 alone. Increased tumor expression of PDL1 and MHC1 following Rova-T treatment also supports combination with anti-PD1. Mice previously treated with Rova-Tâ¯+â¯anti-PD1 withstood tumor re-challenge, demonstrating sustained anti-tumor immunity. Collectively our pre-clinical data support clinical combination of sub-efficacious Rova-T with anti-PD1 to extend the benefit of immune checkpoint inhibitors to more SCLC patients.
RESUMEN
In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas ELAV/genética , Histona Desacetilasas/genética , Estabilidad del ARN , Receptor ErbB-2/genética , Secuencia de Bases , Línea Celular Tumoral , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 6 , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Datos de Secuencia Molecular , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/farmacología , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-2/metabolismoRESUMEN
PURPOSE: Transcriptomic profiling can shed light on the biology of small-cell bladder cancer (SCBC), nominating biomarkers, and novel therapeutic targets. EXPERIMENTAL DESIGN: Sixty-three patients with SCBC had small-cell histology confirmed and quantified by a genitourinary pathologist. Gene expression profiling was performed for 39 primary tumor samples, 1 metastatic sample, and 6 adjacent normal urothelium samples (46 total) from the same cohort. Protein levels of differentially expressed therapeutic targets, DLL3 and PDL1, and also CD56 and ASCL1, were confirmed by IHC. A SCBC PDX model was utilized to assess in vivo efficacy of DLL3-targeting antibody-drug conjugate (ADC). RESULTS: Unsupervised hierarchical clustering of 46 samples produced 4 clusters that correlated with clinical phenotypes. Patients whose tumors had the most "normal-like" pattern of gene expression had longer overall survival (OS) compared with the other 3 clusters while patients with the most "metastasis-like" pattern had the shortest OS (P = 0.047). Expression of DLL3, PDL1, ASCL1, and CD56 was confirmed by IHC in 68%, 30%, 52%, and 81% of tissue samples, respectively. In a multivariate analysis, DLL3 protein expression on >10% and CD56 expression on >30% of tumor cells were both prognostic of shorter OS (P = 0.03 each). A DLL3-targeting ADC showed durable antitumor efficacy in a SCBC PDX model. CONCLUSIONS: Gene expression patterns in SCBC are associated with distinct clinical phenotypes ranging from more indolent to aggressive disease. Overexpression of DLL3 mRNA and protein is common in SCBC and correlates with shorter OS. A DLL3-targeted ADC demonstrated in vivo efficacy superior to chemotherapy in a PDX model of SCBC.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Pronóstico , Transcriptoma/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Inmunoconjugados/inmunología , Masculino , Ratones , Persona de Mediana Edad , Proteoma/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
PURPOSE: Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. EXPERIMENTAL DESIGN: We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. RESULTS: Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10-15) and protein by IHC (P = 0.0014 and P < 4.3 × 10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. CONCLUSIONS: DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.
Asunto(s)
Glioma/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/genética , Isocitrato Deshidrogenasa/genética , Proteínas de la Membrana/genética , Terapia Molecular Dirigida , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Benzodiazepinonas/uso terapéutico , Encéfalo/patología , Metilación de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genotipo , Glioma/genética , Glioma/patología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/uso terapéutico , Ligandos , Masculino , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , ARN/genética , Receptores Notch/genéticaRESUMEN
Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets.Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC.Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2-expressing SCLC tumors in vivo BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2-expressing SCLCs.Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2-expressing SCLCs. Clin Cancer Res; 24(2); 360-9. ©2017 AACR.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Expresión Génica , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Sulfonamidas/farmacología , Animales , Antineoplásicos/uso terapéutico , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Metilación de ADN , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Regiones Promotoras Genéticas , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patología , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibitors of histone deacetylases (HDAC) show significant promise as targeted anticancer agents against a variety of hematologic and solid tumors. HDAC inhibitors arrest the growth of primary cells, but they induce apoptosis or differentiation of tumor cells. Although the precise mechanism is unknown, differences in cell cycle checkpoints and chromatin structure may be responsible. Cellular polyamines regulate both cell cycle progression and chromatin structure. In tumors, polyamines are abundantly produced because of increased activity of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC). To determine if polyamines contribute to the cellular response to HDAC inhibitors, we inhibited ODC activity with alpha-difluoromethylornithine. Polyamine depletion increased resistance to apoptosis induced by HDAC inhibitors. In addition, we found that ODC activity levels correlated with sensitivity to HDAC inhibitors in a panel of tumor cell lines. We conclude that polyamines participate in the cellular response to HDAC inhibitors and that ODC activity correlates with sensitivity to HDAC inhibitor-induced apoptosis. Thus, elevated polyamine levels might be a biomarker for tumor sensitivity to HDAC inhibitor-induced apoptosis. These findings warrant clinical evaluation of tumor samples to determine if high ODC activity levels predict sensitivity to HDAC inhibitors.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Ornitina Descarboxilasa/metabolismo , Apoptosis , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cromatina/fisiología , Resistencia a Antineoplásicos , Eflornitina/farmacología , Femenino , Fase G1 , Células HCT116 , Células HeLa , Histona Desacetilasas/metabolismo , Humanos , Masculino , Inhibidores de la Ornitina Descarboxilasa , Poliaminas/metabolismoRESUMEN
The dsRNA binding proteins (DRBPs) comprise a growing family of eukaryotic, prokaryotic, and viral-encoded products that share a common evolutionarily conserved motif specifically facilitating interaction with dsRNA. Proteins harboring dsRNA binding domains (DRBDs) have been reported to interact with as little as 11 bp of dsRNA, an event that is independent of nucleotide sequence arrangement. More than 20 DRBPs have been identified and reportedly function in a diverse range of critically important roles in the cell. Examples include the dsRNA-dependent protein kinase PKR that functions in dsRNA signaling and host defense against virus infection and DICER, which is implicated in RNA interference (RNAi) -mediated gene silencing. Other DRBPs such as Staufen, adenosine deaminase acting on RNA (ADAR), and spermatid perinuclear RNA binding protein (SPNR) are known to play essential roles in development, translation, RNA editing, and stability. In many cases, homozygous and even heterozygous disruption of DRBPs in animal models results in embryonic lethality. These results implicate the recognition of dsRNA as an evolutionarily conserved mechanism important in the regulation of gene expression and in host defense and underscore the diversity of essential biological tasks performed by dsRNA-related processes in the cell.
Asunto(s)
ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Citoplasma/metabolismo , Proteínas de Escherichia coli/fisiología , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Edición de ARN , Interferencia de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/clasificación , Homología de Secuencia de Aminoácido , Proteínas Virales/fisiologíaRESUMEN
The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de la Membrana/inmunología , Tumores Neuroendocrinos/tratamiento farmacológico , Animales , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Tumores Neuroendocrinos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Concentrations of acetyl-coenzyme A and nicotinamide adenine dinucleotide (NAD(+)) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-ß-hydroxybutyrate (ßOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous ßOHB, or fasting or calorie restriction, two conditions associated with increased ßOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by ßOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with ßOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with ßOHB conferred substantial protection against oxidative stress.
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Ácido 3-Hidroxibutírico/metabolismo , Inhibidores de Histona Desacetilasas/metabolismo , Histona Desacetilasas/metabolismo , Riñón/metabolismo , Estrés Oxidativo , Ácido 3-Hidroxibutírico/sangre , Ácido 3-Hidroxibutírico/farmacología , Acetilación , Animales , Restricción Calórica , Catalasa/metabolismo , Ayuno , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Células HEK293 , Inhibidores de Histona Desacetilasas/sangre , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histonas/metabolismo , Humanos , Riñón/efectos de los fármacos , Peroxidación de Lípido , Metalotioneína/genética , Metalotioneína/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Superóxido Dismutasa/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD+ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.