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INTRODUCTION: Increasingly, early-stage non-small cell lung cancer (NSCLC) is treated with stereotactic body radiation therapy (SBRT). Although treatment is generally effective, a small subset of tumors will recur because of radioresistance. Preclinical studies suggested PI3K-AKT-mTOR activation mediates radioresistance. This study sought to validate this finding in tumor samples from patients who underwent SBRT for NSCLC. METHODS: Patients with T1-3N0 NSCLC treated with SBRT at our institution were included. Total RNA of formalin-fixed paraffin-embedded tumor biopsy specimens (pretherapy) was isolated and analyzed using the Clariom D assay. Risk scores from a PI3K activity signature and four published NSCLC signatures were generated and dichotomized by the median. Kaplan-Meier curves and Cox regressions were used to analyze their association with recurrence and overall survival (OS). The PI3K signature was also tested in a data set of resected NSCLC for additional validation. RESULTS: A total of 92 patients were included, with a median follow-up of 18.3 months for living patients. There was no association of any of the four published gene expression signatures with recurrence or OS. However, high PI3K risk score was associated with higher local recurrence (hazard ratio [HR], 11.72; 95% CI, 1.40-98.0; p = .023) and worse disease-free survival (DFS) (HR, 3.98; 95% CI, 1.57-10.09; p = .0035), but not OS (p = .49), regional recurrence (p = .15), or distant recurrence (p = .85). In the resected NSCLC data set (n = 361), high PI3K risk score was associated with decreased OS (log-rank p = .013) but not DFS (p = 0.54). CONCLUSIONS: This study validates that higher PI3K activity, measured by gene expression, is associated with local recurrence and worse DFS in early-stage NSCLC patients treated with SBRT. This may be useful in prognostication and/or tailoring treatment, and merits further validation.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Radiocirugia , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Estadificación de Neoplasias , Fosfatidilinositol 3-Quinasas/genética , Estudios Retrospectivos , Carcinoma Pulmonar de Células Pequeñas/patología , Transcriptoma , Resultado del TratamientoRESUMEN
KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.
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Neoplasias del Colon/genética , Reparación del ADN por Unión de Extremidades , Factor 2 Relacionado con NF-E2/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Tolerancia a Radiación/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias del Colon/radioterapia , Roturas del ADN de Doble Cadena , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Rayos gamma , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Mutación , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.
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Carcinoma/genética , Mutación Missense/genética , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal , Animales , Carcinoma/enzimología , Carcinoma/fisiopatología , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Mamíferos , Activación Enzimática , Femenino , Técnicas de Sustitución del Gen , Ratones , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Estabilidad ProteicaRESUMEN
Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.
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Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Proteínas Angiogénicas/antagonistas & inhibidores , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Proteínas de Unión al Calcio , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Supervivencia sin Enfermedad , Familia de Proteínas EGF , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Femenino , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Pronóstico , Proteínas/metabolismo , Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factores de Riesgo , Regulación hacia Arriba , Adulto JovenRESUMEN
The ataxia telangiectasia mutated (ATM) checkpoint is the central surveillance system that maintains genome integrity. We found that in the context of childhood sarcoma, mammalian target of rapamycin (mTOR) signaling suppresses ATM by up-regulating miRNAs targeting ATM. Pharmacological inhibition or genetic down-regulation of the mTOR pathway resulted in increase of ATM mRNA and protein both in mouse sarcoma xenografts and cultured cells. mTOR Complex 1 (mTORC1) suppresses ATM via S6K1/2 signaling pathways. microRNA-18a and microRNA-421, both of which target ATM, are positively controlled by mTOR signaling. Our findings have identified a negative feedback loop for the signaling between ATM and mTOR pathways and suggest that oncogenic growth signals may promote tumorigenesis by dampening the ATM checkpoint.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Sarcoma/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Células Cultivadas , Retroalimentación Fisiológica/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Ratones , MicroARNs/metabolismo , MorfolinasRESUMEN
PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.
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Astrocitoma/genética , Astrocitoma/radioterapia , Bencimidazoles/efectos adversos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Radioterapia/efectos adversos , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Complejos Multiproteicos/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/radioterapia , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic RAS and RAF signaling has been implicated in contributing to radioresistance in pancreatic and thyroid cancers. In this study, we sought to better clarify molecular mechanisms contributing to this effect. We discovered that miRNA 296-3p (miR-296-3p) is significantly correlated with radiosensitivity in a panel of pancreatic cancer cells, and miR-296-3p is highly expressed in normal cells, but low in cancer cell lines. Elevated expression of miR-296-3p increases radiosensitization while decreasing the expression of the DNA repair enzyme RAD18 in both pancreatic and thyroid cancer cells. RAD18 is overexpressed in both pancreatic and thyroid tumors compared to matched normal controls, and high expression of RAD18 in tumors is associated with poor prognostic features. Modulating the expression of mutant KRAS in pancreatic cancer cells or mutant BRAF in thyroid cancer cells demonstrates a tight regulation of RAD18 expression in both cancer types. Depletion of RAD18 results in DNA damage and radiation-induced cell death. Importantly, RAD18 depletion in combination with radiotherapy results in marked and sustained tumor regression in KRAS mutant pancreatic cancer orthotopic tumors and BRAF mutant thyroid heterotopic tumors. Overall, our findings identify a novel coordinated RAS/RAF-miR-296-3p-RAD18 signaling network in pancreatic and thyroid cancer cells, which leads to enhanced radioresistance.
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Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , MicroARNs , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Tolerancia a Radiación , Transducción de Señal , Neoplasias de la Tiroides , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Tolerancia a Radiación/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/radioterapia , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Ratones Desnudos , Mutación , Daño del ADN , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismo , TransfecciónRESUMEN
Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo, triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM.
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Glioblastoma , Animales , Ratones , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/radioterapia , Piridinas/farmacología , Mutaciones Letales SintéticasRESUMEN
PURPOSE: Caveolin-1 and -2 (CAV1/2) dysregulation are implicated in driving cancer progression and may predict response to nab-paclitaxel. We explored the prognostic and predictive potential of CAV1/2 expression for patients with early-stage HER2-negative breast cancer receiving neoadjuvant paclitaxel-based chemotherapy regimens, followed by epirubicin and cyclophosphamide. EXPERIMENTAL DESIGN: We correlated tumor CAV1/2 RNA expression with pathologic complete response (pCR), disease-free survival (DFS), and overall survival (OS) in the GeparSepto trial, which randomized patients to neoadjuvant paclitaxel- versus nab-paclitaxel-based chemotherapy. RESULTS: RNA sequencing data were available for 279 patients, of which 74 (26.5%) were hormone receptor (HR)-negative, thus triple-negative breast cancer (TNBC). Patients treated with nab-paclitaxel with high CAV1/2 had higher probability of obtaining a pCR [CAV1 OR, 4.92; 95% confidence interval (CI), 1.70-14.22; P = 0.003; CAV2 OR, 5.39; 95% CI, 1.76-16.47; P = 0.003] as compared with patients with high CAV1/2 treated with solvent-based paclitaxel (CAV1 OR, 0.33; 95% CI, 0.11-0.95; P = 0.040; CAV2 OR, 0.37; 95% CI, 0.12-1.13; P = 0.082). High CAV1 expression was significantly associated with worse DFS and OS in paclitaxel-treated patients (DFS HR, 2.29; 95% CI, 1.08-4.87; P = 0.030; OS HR, 4.97; 95% CI, 1.73-14.31; P = 0.003). High CAV2 was associated with worse DFS and OS in all patients (DFS HR, 2.12; 95% CI, 1.23-3.63; P = 0.006; OS HR, 2.51; 95% CI, 1.22-5.17; P = 0.013), in paclitaxel-treated patients (DFS HR, 2.47; 95% CI, 1.12-5.43; P = 0.025; OS HR, 4.24; 95% CI, 1.48-12.09; P = 0.007) and in patients with TNBC (DFS HR, 4.68; 95% CI, 1.48-14.85; P = 0.009; OS HR, 10.43; 95% CI, 1.22-89.28; P = 0.032). CONCLUSIONS: Our findings indicate high CAV1/2 expression is associated with worse DFS and OS in paclitaxel-treated patients. Conversely, in nab-paclitaxel-treated patients, high CAV1/2 expression is associated with increased pCR and no significant detriment to DFS or OS compared with low CAV1/2 expression.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Paclitaxel , Expresión Génica , Terapia Neoadyuvante , Receptor ErbB-2/metabolismoRESUMEN
Lung squamous cell carcinoma (LUSC) accounts for one of three of non-small cell lung carcinoma (NSCLC) and 30% of LUSC patients present with locally advanced, unresectable/medically inoperable disease, who are commonly treated with definitive chemoradiation. However, disease relapse in the radiation fields occurs in one of three cases. We aim to explore the underlying molecular mechanisms of chemoradiation resistance of LUSC. Patient-derived xenograft (PDX) models of LUSC were established in immunodeficient mice, followed by treatment with cisplatin in combination with clinically relevant courses of ionizing radiation (20, 30, and 40 Gy). The recurrent tumors were extracted for functional proteomics using reverse phase protein analysis (RPPA). We found that phospho-AKT-S473, phospho-AKT-T308, phospho-S6-S235/6, and phospho-GSK3ß-S9 were upregulated in the chemoradiation-resistant 20 Gy + cisplatin and 40 Gy + cisplatin tumors compared with those in the control tumors. Ingenuity pathway analysis of the RPPA data revealed that AKT-mTOR signaling was the most activated signaling pathway in the chemoradiation-resistant tumors. Similarly, elevated AKT-mTOR signaling was observed in stable 40 Gy and 60 Gy resistant HARA cell lines compared with the parental cell line. Accordingly, pharmacologic inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. In conclusion, using chemoradiation-resistant PDX models coupled with RPPA proteomics analysis, we revealed that deregulation of AKT-mTOR signaling may contribute to the chemoradiation resistance of LUSC. IMPLICATIONS: Clonal selection of subpopulations with high AKT-mTOR signaling in heterogeneous tumors may contribute to relapse of LUSC after chemoradiation. mTOR kinase inhibitors may be promising radiosensitizing agents in upfront treatment to prevent acquired resistance.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Ratones , Recurrencia Local de Neoplasia , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
OBJECTIVES: In line with the cardiac fast track, the 'green pathway for patients with heart attack' policy in China is implemented to reduce door-to-balloon time in patients with ST-segment elevation myocardial infarction (STEMI). However, the difference in prehospital delay between urban and rural areas of China and its impact on prognosis is unclear. DESIGN: Prospective observational study. SETTING: This study was conducted in a tertiary hospital, the only nationally accredited chest pain centre with percutaneous coronary intervention (PCI) capacity in Pizhou, China. PARTICIPANTS: 394 patients with STEMI without patients with in-hospital STEMI or patients lost to follow-up were included. PRIMARY OUTCOME MEASURES: Primary outcome was major adverse cardiovascular events (MACEs), including cardiac death, non-fatal myocardial infarction and heart failure. RESULTS: Among 394 patients enrolled, 261 (66.2%) were men, the median age was 69 years (interquartile range: 61-77 years), and 269 (68.3%) were from rural areas. Symptom-to-door (S2D) time was significantly longer for rural patients than for urban patients (p<0.001). Cox regression analyses revealed living in rural areas was independently associated with prolonged S2D time (adjusted HR 0.59; 95% CI 0.43 to 0.81; p=0.001). HR of <1 indicates that the S2D time is longer for patients in the rural group (group of interest). During 1-year follow-up, the incidence of MACEs was higher in rural patients (p=0.008). The unadjusted OR for MACEs between rural and urban patients was 2.22 (95% CI 1.22 to 4.01). Adjusting for sex did not attenuate the association (OR 2.06; 95% CI 1.13 to 3.76), but after further adjusting for age, cardiac function classification, S2D time and performance of primary PCI, we found that odds were similar for rural and urban patients (OR 1.19; 95% CI 0.59 to 2.38). CONCLUSIONS: Rural patients with STEMI had a longer S2D time, which led to a higher incidence of MACEs. This study provides rationales for taking all the measures to avoid prehospital delay.
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Servicios Médicos de Urgencia , Infarto del Miocardio , Intervención Coronaria Percutánea , Infarto del Miocardio con Elevación del ST , Masculino , Humanos , Anciano , Femenino , Infarto del Miocardio con Elevación del ST/terapia , Factores de Tiempo , Infarto del Miocardio/epidemiología , Infarto del Miocardio/terapiaRESUMEN
Accurate and complete DNA replication and separation are essential for genetic information inheritance and organism maintenance. Errors in DNA duplication are the main source of genetic instability. Understanding DNA duplication regulation is the key to elucidate the mechanisms and find treatment strategies for human genetic disorders, especially cancer. The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and proliferation by integrating and processing extracellular and intracellular signals to monitor the well-being of cell physiology. mTOR signaling dysregulation is associated with many human diseases including cancer and diabetes. Emerging evidence has demonstrated that mTOR signaling plays a key role in DNA duplication. We herein review the current knowledge of mTOR signaling in the regulation of DNA replication origin licensing, replication fork progression, and stabilization.
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Replicación del ADN/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular/fisiología , Humanos , Serina-Treonina Quinasas TOR/genéticaRESUMEN
OBJECTIVE: To summarize the roles of AKT-mTOR signaling in the regulation of the DNA damage response and PD-L1 expression in cancer cells, and propose a novel strategy of targeting AKT-mTOR signaling in combination with radioimmunotherapy in the era of cancer immunotherapy. BACKGROUND: Immunotherapy has greatly improved the clinical outcomes of many cancer patients and has changed the landscape of cancer patient management. However, only a small subgroup of cancer patients (~20-30%) benefit from immune checkpoint blockade-based immunotherapy. The current challenge is to find biomarkers to predict the response of patients to immunotherapy and strategies to sensitize patients to immunotherapy. METHODS: Search and review the literature which were published in PUBMED from 2000-2021 with the key words mTOR, AKT, drug resistance, DNA damage response, immunotherapy, PD-L1, DNA repair, radioimmunotherapy. CONCLUSIONS: More than 50% of cancer patients receive radiotherapy during their course of treatment. Radiotherapy has been shown to reduce the growth of locally irradiated tumors as well as metastatic non-irradiated tumors (abscopal effects) by affecting systemic immunity. Consistently, immunotherapy has been demonstrated to enhance radiotherapy with more than one hundred clinical trials of radiation in combination with immunotherapy (radioimmunotherapy) across cancer types. Nevertheless, current available data have shown limited efficacy of trials testing radioimmunotherapy. AKT-mTOR signaling is a major tumor growth-promoting pathway and is upregulated in most cancers. AKT-mTOR signaling is activated by growth factors as well as genotoxic stresses including radiotherapy. Importantly, recent advances have shown that AKT-mTOR is one of the main signaling pathways that regulate DNA damage repair as well as PD-L1 levels in cancers. These recent advances clearly suggest a novel cancer therapy strategy by targeting AKT-mTOR signaling in combination with radioimmunotherapy.
RESUMEN
The conserved TOR (target of rapamycin) kinase is part of a TORC1 complex that regulates cellular responses to environmental stress, such as amino acid starvation and hypoxia. Dysregulation of Akt-TOR signaling has also been linked to the genesis of cancer, and thus, this pathway presents potential targets for cancer chemotherapeutics. Here we report that rapamycin-sensitive TORC1 signaling is required for the S-phase progression and viability of yeast cells in response to genotoxic stress. In the presence of the DNA-damaging agent methyl methanesulfonate (MMS), TOR-dependent cell survival required a functional S-phase checkpoint. Rapamycin inhibition of TORC1 signaling suppressed the Rad53 checkpoint-mediated induction of ribonucleotide reductase subunits Rnr1 and Rnr3, thereby abrogating MMS-induced mutagenesis and enhancing cell lethality. Moreover, cells deleted for RNR3 were hypersensitive to rapamycin plus MMS, providing the first demonstration that Rnr3 contributes to the survival of cells exposed to DNA damage. Our findings support a model whereby TORC1 acts as a survival pathway in response to genotoxic stress by maintaining the deoxynucleoside triphosphate pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA-damaging agents coincides with increased mutation rates, which may contribute to the acquisition of chemotherapeutic drug resistance.
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Supervivencia Celular , Daño del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Adulto , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Cicloheximida/metabolismo , Humanos , Metilmetanosulfonato/metabolismo , Complejos Multiproteicos , Mutágenos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/metabolismo , Fase S/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Deregulated cell cycle progression is a hallmark of cancer. Accordingly, a major part of therapeutic drugs has been designed to inhibit cell proliferation and tumor growth. Metabolic imaging with positron emission tomography (PET) and the glucose analog 2'-[(18)F]fluoro-2'-deoxyglucose (FDG) has been demonstrated to sensitively detect malignant tumors and to identify responding tumors early in the course of anticancer treatment. However, tumoral uptake of FDG reflects proliferation only in part and is associated with false positive findings due to unspecific tracer retention in inflammatory processes. Most recent advances in cancer treatment have come from the development of disease specific, molecular agents, many of which induce cell cycle arrest (cytostatic effect) instead of tumor cell death (cytotoxic effect). Thus, evaluating alterations in DNA metabolism may reflect response to treatment better than alterations in glucose utilization. PET with the thymidine analog 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) enables non-invasive imaging and quantification of the proliferation fraction of tumors. Furthermore, FLT has been suggested as surrogate marker for assessment of response to treatment, especially when targeted drugs are utilized. This article reports on metabolic pathways of radionucleosides in proliferating cells. Methods for in vivo assessment of the proliferative activity in preclinical and clinical studies are described with a focus on early monitoring response to therapy.
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Proliferación Celular , Didesoxinucleósidos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Tomografía de Emisión de Positrones/métodos , Animales , Ensayos Clínicos como Asunto , Diagnóstico Diferencial , Modelos Animales de Enfermedad , Fluorodesoxiglucosa F18 , Humanos , Estadificación de Neoplasias/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Neoplasias/patología , RadiofármacosRESUMEN
PURPOSE: Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G2-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2-M checkpoint sensitizes esophageal cancer cells to radiotherapy. EXPERIMENTAL DESIGN: Cell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo. RESULTS: The IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G2-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts. CONCLUSIONS: Abrogation of G2-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Quimioradioterapia/métodos , Neoplasias Esofágicas/terapia , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Neoplasias Esofágicas/patología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Pirazoles/uso terapéutico , Pirimidinonas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. EXPERIMENTAL DESIGN: Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated in vitro with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. RESULTS: We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that in vivo treatment with anti-EGFL7 alone results in increased survival. CONCLUSIONS: Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.
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Anticuerpos Monoclonales Humanizados/farmacología , Proteínas de Unión al Calcio/metabolismo , Familia de Proteínas EGF/metabolismo , Leucemia Mieloide Aguda/patología , Receptores Notch/antagonistas & inhibidores , Animales , Apoptosis , Proteínas de Unión al Calcio/genética , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Familia de Proteínas EGF/genética , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Survivin is an attractive target for anti-cancer drug development; however targeting it by small molecules or antibodies is difficult, as survivin is neither a kinase nor a cell surface protein. Protein transduction domain (PTD)-mediated macromolecular therapeutics provides an alternative avenue for targeting survivin. MATERIALS AND METHODS: A plasmid expressing a dominant-negative survivin-T34A fused with the immunodeficiency virus protein transduction domain TAT was constructed. The fusion protein was expressed and purified from E. coli. The inhibition of proliferation and induction of apoptosis was tested in human lung carcinoma cell line A549 by directly adding survivin-T34A to the cell culture medium. RESULTS: Recombinant survivin-T34A was efficiently expressed and purified by affinity chromatography. It induced cell apoptosis as demonstrated by induction of caspase 3 activation and higher percentage of Annexin V staining, and inhibited cell proliferation as determined by cell number counting. CONCLUSION: This functional recombinant protein is promising for development of macromolecular therapeutics targeting survivin.
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Apoptosis , Proliferación Celular , Genes Dominantes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Western Blotting , Cromatografía de Afinidad , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Survivin , Células Tumorales CultivadasRESUMEN
Targeting inhibitor of apoptosis proteins (IAP) with second mitochondria-derived activator of caspase (SMAC) mimetics may promote cancer cell death. We tested whether cIAP1 predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC) and whether a novel Smac-mimetic, LCL161, could radiosensitize human papillomavirus-positive (HPV+) and -negative (HPV-) HNSCC. The association of BIRC2 (encoding cIAP1) mRNA level with HPV status in HNSCC was analyzed using The Cancer Genome Atlas (TCGA) database. cIAP1 was assessed by IHC on an HNSCC tissue microarray (TMA, n = 84) followed by correlation analysis with HPV status and patient outcomes. Human cell culture and animal models of HNSCC were used to analyze the outcome and molecular characteristics following radiotherapy in combination with LCL161. cIAP1 expression is increased in HPV- compared with HPV+HNSCC tumors in the TCGA database. In our TMA, cIAP1 was overexpressed in HNSCC compared with normal tissues (P = 0.0003) and associated with a poor overall survival (P = 0.0402). cIAP1 levels were higher in HPV- than that in HPV+HNSCC tumors (P = 0.004) and patients with cIAP1+/HPV- HNSCC had the worst survival. LCL161 effectively radiosensitized HPV- HNSCC cells, which was accompanied with enhanced apoptosis, but not HPV+ HNSCC cells. Importantly, LCL161 in combination with radiotherapy led to dramatic tumor regression of HPV- HNSCC tumor xenografts, accompanied by cIAP1 degradation and apoptosis activation. These results reveal that cIAP1 is a prognostic and a potential therapeutic biomarker for HNSCC, and targeting cIAP1 with LCL161 preferentially radiosensitizes HPV- HNSCC, providing justification for clinical testing of LCL161 in combination with radiation for patients with HPV- HNSCC.
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Proteínas Reguladoras de la Apoptosis/química , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/radioterapia , Proteínas Mitocondriales/química , Papillomaviridae/aislamiento & purificación , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Tiazoles/uso terapéutico , Anciano , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/virología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Imitación Molecular , Infecciones por Papillomavirus/virología , Pronóstico , Fármacos Sensibilizantes a Radiaciones/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Tiazoles/farmacología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
mTOR signaling pathway is deregulated in most cancers and uncontrolled cell cycle progression is a hallmark of cancer cell. However, the precise molecular mechanisms of the regulation of DNA replication and chromatin metabolism by mTOR signaling are largely unknown. We herein report that mTOR signaling promotes the loading of MCM2-7 helicase onto chromatin and upregulates DNA replication licensing factor CDC6. Pharmacological inhibition of mTOR kinase resulted in CHK1 checkpoint activation and decreased MCM2-7 replication helicase and PCNA associated with chromatins. Further pharmacological and genetic studies demonstrated CDC6 is positively controlled by mTORC1-S6K1 and mTORC2 signaling. miRNA screening revealed mTOR signaling suppresses miR-3178 thereby upregulating CDC6. Analysis of TCGA data found that CDC6 is overexpressed in most cancers and associates with the poor survival of cancer patients. Our findings suggest that mTOR signaling may control DNA replication origin licensing and replisome stability thereby cell cycle progression through CDC6 regulation.