RESUMEN
Circadian misalignment due to night work has been associated with an elevated risk for chronic diseases. We investigated the effects of circadian misalignment using shotgun protein profiling of peripheral blood mononuclear cells taken from healthy humans during a constant routine protocol, which was conducted immediately after participants had been subjected to a 3-day simulated night shift schedule or a 3-day simulated day shift schedule. By comparing proteomic profiles between the simulated shift conditions, we identified proteins and pathways that are associated with the effects of circadian misalignment and observed that insulin regulation pathways and inflammation-related proteins displayed markedly different temporal patterns after simulated night shift. Further, by integrating the proteomic profiles with previously assessed metabolomic profiles in a network-based approach, we found key associations between circadian dysregulation of protein-level pathways and metabolites of interest in the context of chronic metabolic diseases. Endogenous circadian rhythms in circulating glucose and insulin differed between the simulated shift conditions. Overall, our results suggest that circadian misalignment is associated with a tug of war between central clock mechanisms controlling insulin secretion and peripheral clock mechanisms regulating insulin sensitivity, which may lead to adverse long-term outcomes such as diabetes and obesity. Our study provides a molecular-level mechanism linking circadian misalignment and adverse long-term health consequences of night work.
Asunto(s)
Ritmo Circadiano , Inflamación , Insulina , Leucocitos Mononucleares , Humanos , Leucocitos Mononucleares/metabolismo , Insulina/metabolismo , Insulina/sangre , Inflamación/metabolismo , Inflamación/sangre , Masculino , Adulto , Horario de Trabajo por Turnos , Femenino , Proteómica/métodos , Glucemia/metabolismo , Transducción de Señal , Resistencia a la Insulina , Adulto JovenRESUMEN
Solar ultraviolet B radiation (UVB) is one of the leading causes of various skin conditions, including photoaging, sunburn erythema, and melanoma. As a protective response, the skin has inbuilt defense mechanisms, including DNA repair, cell cycle, apoptosis, and melanin synthesis. Though DNA repair, cell cycle, and apoptosis are clock controlled, the circadian mechanisms associated with melanin synthesis are not well understood. Using human melanocytes and melanoma cells under synchronized clock conditions, we observed that the microphthalmia-associated transcription factor (MITF), a rate-limiting protein in melanin synthesis, is expressed rhythmically with 24-hr periodicity in the presence of circadian clock protein, BMAL1. Furthermore, we demonstrated that BMAL1 binds to the promoter region of MITF and transcriptionally regulates its expression, which positively influences melanin synthesis. Finally, we report that an increase in melanin levels due to BMAL1 overexpression protects human melanoma cells from UVB. In conclusion, our studies provide novel insights into the mechanistic role of the circadian clock in melanin synthesis and protection against UVB-mediated DNA damage and genomic instability.
Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas de Neoplasias/genéticaRESUMEN
Circadian disruption has been identified as a risk factor for health disorders such as obesity, cardiovascular disease, and cancer. Although epidemiological studies suggest an increased risk of various cancers associated with circadian misalignment due to night shift work, the underlying mechanisms have yet to be elucidated. We sought to investigate the potential mechanistic role that circadian disruption of cancer hallmark pathway genes may play in the increased cancer risk in shift workers. In a controlled laboratory study, we investigated the circadian transcriptome of cancer hallmark pathway genes and associated biological pathways in circulating leukocytes obtained from healthy young adults during a 24-hour constant routine protocol following 3 days of simulated day shift or night shift. The simulated night shift schedule significantly altered the normal circadian rhythmicity of genes involved in cancer hallmark pathways. A DNA repair pathway showed significant enrichment of rhythmic genes following the simulated day shift schedule, but not following the simulated night shift schedule. In functional assessments, we demonstrated that there was an increased sensitivity to both endogenous and exogenous sources of DNA damage after exposure to simulated night shift. Our results suggest that circadian dysregulation of DNA repair may increase DNA damage and potentiate elevated cancer risk in night shift workers.
Asunto(s)
Biomarcadores de Tumor/genética , Trastornos Cronobiológicos/etiología , Ritmo Circadiano , Daño del ADN , Reparación del ADN , Neoplasias/etiología , Horario de Trabajo por Turnos/efectos adversos , Transcriptoma , Ciclos de Actividad , Adulto , Trastornos Cronobiológicos/genética , Trastornos Cronobiológicos/fisiopatología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias/genética , Neoplasias/patología , Medición de Riesgo , Factores de Riesgo , Sueño , Factores de Tiempo , Adulto JovenRESUMEN
Radiation therapy (RT) is commonly used to treat solid tumors of the breast, lung, and esophagus; however, the heart is an unintentional target of ionizing radiation (IR). IR exposure to the heart results in chronic toxicities including heart failure. We hypothesize that the circadian system plays regulatory roles in minimizing the IR-induced cardiotoxicity. We treated mice in control (Day Shift), environmentally disrupted (Rotating Shift), and genetically disrupted (Per 1/2 mutant) circadian conditions with 18 Gy of IR to the heart. Compared to control mice, circadian clock disruption significantly exacerbated post-IR systolic dysfunction (by ultrasound echocardiography) and increased fibrosis in mice. At the cellular level, Bmal1 protein bound to Atm, Brca1, and Brca2 promoter regions and its expression level was inversely correlated with the DNA damage levels based on the state of the clock. Further studies with circadian synchronized cardiomyocytes revealed that Bmal1 depletion increased the IR-induced DNA damage and apoptosis. Collectively, these findings suggest that the circadian clock protects from IR-induced toxicity and potentially impacts RT treatment outcome in cancer patients through IR-induced DNA damage responses.
Asunto(s)
Miocitos Cardíacos/metabolismo , Proteínas Circadianas Period/genética , Traumatismos Experimentales por Radiación/genética , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Línea Celular , Daño del ADN , Ratones , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/efectos de la radiación , Regiones Promotoras Genéticas , Traumatismos Experimentales por Radiación/metabolismo , Radiación Ionizante , Ratas , SístoleRESUMEN
Misalignment between internal circadian rhythmicity and externally imposed behavioral schedules, such as occurs in shift workers, has been implicated in elevated risk of metabolic disorders. To determine underlying mechanisms, it is essential to assess whether and how peripheral clocks are disturbed during shift work and to what extent this is linked to the central suprachiasmatic nuclei (SCN) pacemaker and/or misaligned behavioral time cues. Investigating rhythms in circulating metabolites as biomarkers of peripheral clock disturbances may offer new insights. We evaluated the impact of misaligned sleep/wake and feeding/fasting cycles on circulating metabolites using a targeted metabolomics approach. Sequential plasma samples obtained during a 24-h constant routine that followed a 3-d simulated night-shift schedule, compared with a simulated day-shift schedule, were analyzed for 132 circulating metabolites. Nearly half of these metabolites showed a 24-h rhythmicity under constant routine following either or both simulated shift schedules. However, while traditional markers of the circadian clock in the SCN-melatonin, cortisol, and PER3 expression-maintained a stable phase alignment after both schedules, only a few metabolites did the same. Many showed reversed rhythms, lost their rhythms, or showed rhythmicity only under constant routine following the night-shift schedule. Here, 95% of the metabolites with a 24-h rhythmicity showed rhythms that were driven by behavioral time cues externally imposed during the preceding simulated shift schedule rather than being driven by the central SCN circadian clock. Characterization of these metabolite rhythms will provide insight into the underlying mechanisms linking shift work and metabolic disorders.
Asunto(s)
Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Ayuno/sangre , Regulación de la Expresión Génica/fisiología , Hidrocortisona/sangre , Melatonina/sangre , Proteínas Circadianas Period/biosíntesis , Adulto , Femenino , Humanos , MasculinoRESUMEN
Cisplatin is one of the most commonly used chemotherapeutic drugs; however, toxicity and tumor resistance limit its use. Studies using murine models and human subjects have shown that the time of day of cisplatin treatment influences renal and blood toxicities. We hypothesized that the mechanisms responsible for these outcomes are driven by the circadian clock. We conducted experiments using wild-type and circadian disrupted Per1/2-/- mice treated with cisplatin at selected morning (AM) and evening (PM) times. Wild-type mice treated in the evening showed an enhanced rate of removal of cisplatin-DNA adducts and less toxicity than the morning-treated mice. This temporal variation in toxicity was lost in the Per1/2-/- clock-disrupted mice, suggesting that the time-of-day effect is linked to the circadian clock. Observations in blood cells from humans subjected to simulated day and night shift schedules corroborated this view. Per1/2-/- mice also exhibited a more robust immune response and slower tumor growth rate, indicating that the circadian clock also influences the immune response to melanoma tumors. Our findings indicate that cisplatin chronopharmacology involves the circadian clock control of DNA repair as well as immune responses, and thus affects both cisplatin toxicity and tumor growth. This has important implications for chronochemotherapy in cancer patients, and also suggests that influencing the circadian clock (e.g., through bright light treatment) may be explored as a tool to improve patient outcomes.
RESUMEN
TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/metabolismo , Invasividad Neoplásica/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/patología , ARN Largo no Codificante/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Cromatina , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Humanos , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Invasividad Neoplásica/patología , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , Proteína 1 Relacionada con Twist/genéticaRESUMEN
The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína 1 Relacionada con Twist/metabolismo , Secuencias de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Análisis por Conglomerados , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Masculino , Mutación , Metástasis de la Neoplasia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Proteína 1 Relacionada con Twist/química , Proteína 1 Relacionada con Twist/genéticaRESUMEN
UNLABELLED: Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial-mesenchymal transition (EMT) that promotes cancer metastasis. Structure-function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. IMPLICATIONS: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential.
Asunto(s)
Metástasis de la Neoplasia/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Estructura Terciaria de Proteína/genética , Proteína 1 Relacionada con Twist/metabolismo , Sustitución de Aminoácidos , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia/patología , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Neoplasias de la Próstata/patología , Activación Transcripcional , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G1-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/terapia , Terapia Combinada/métodos , Rayos gamma/uso terapéutico , Histonas/genética , Neoplasias Hepáticas/terapia , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Miembro Posterior/patología , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Neovascularización Patológica/prevención & control , Niacinamida/uso terapéutico , Tolerancia a Radiación , Sorafenib , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK-driven NSCLCs, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene-driven NSCLCs. We recently showed that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene-driven human NSCLCs. Silencing of TWIST1 in KRAS-mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or, in some cases, apoptosis. Similar effects were observed in EGFR mutation-driven and c-Met-amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS-mutant, EGFR-mutant, and c-Met-amplified NSCLCs. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS-mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver-dependent NSCLCs represents a novel and promising therapeutic strategy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Oncogenes , Proteína 1 Relacionada con Twist/antagonistas & inhibidores , Proteína 1 Relacionada con Twist/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Senescencia Celular/genética , Silenciador del Gen , Células HEK293 , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the "non-oncogene" addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded "client" proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4-1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G 2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Resorcinoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de la radiación , Línea Celular Tumoral , Terapia Combinada , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fase G2/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Transgénicos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Daño del ADN , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Reparación del ADN/genética , Células HeLa , Humanos , Mutación Missense , Fosforilación/genética , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution, and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavir's effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p < 0.05). There is increased apoptotic cell death, as determined by annexin-V expression and cleaved caspase-3 levels. Nelfinavir does not affect prolactin secretion or cell cycle distribution. In vivo, untreated tumors reached 4-fold volume in 12 days, 17 days with nelfinavir treatment, 27 days with radiation 6 Gy, and 41 days with nelfinavir plus radiation (one-way ANOVA p < 0.001). Decreased phospho-S6 on Western blotting in vitro and immunohistochemistry in vivo demonstrated nelfinavir inhibition of the PI3K-AKT-mTOR pathway. Our data suggests a promising combination therapy with nelfinavir plus radiation in pituitary adenomas, which should be investigated in clinical studies.