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1.
Cancer Lett ; 591: 216873, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38604313

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Tolerância a Radiação , Transdução de Sinais , Neoplasias da Glândula Tireoide , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Tolerância a Radiação/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/radioterapia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Linhagem Celular Tumoral , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Camundongos Nus , Mutação , Dano ao DNA , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Proteínas ras/metabolismo , Transfecção
2.
Cancer ; 129(24): 3971-3977, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-37560930

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Radiocirurgia , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Estudos Retrospectivos , Carcinoma de Pequenas Células do Pulmão/patologia , Transcriptoma , Resultado do Tratamento
3.
Cancer Lett ; 570: 216308, 2023 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-37482342

RESUMO

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.


Assuntos
Glioblastoma , Animais , Camundongos , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/radioterapia , Piridinas/farmacologia , Mutações Sintéticas Letais
4.
Clin Cancer Res ; 29(17): 3384-3394, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37432976

RESUMO

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.


Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Paclitaxel , Expressão Gênica , Terapia Neoadjuvante , Receptor ErbB-2/metabolismo
5.
BMJ Open ; 12(11): e063795, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396304

RESUMO

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.


Assuntos
Serviços Médicos de Emergência , Infarto do Miocárdio , Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST , Masculino , Humanos , Idoso , Feminino , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Fatores de Tempo , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/terapia
6.
Mol Cancer Res ; 20(3): 425-433, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34810212

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Camundongos , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
7.
Ann Transl Med ; 9(20): 1596, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34790802

RESUMO

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.

8.
Nucleic Acids Res ; 49(19): 11067-11082, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34606602

RESUMO

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.


Assuntos
Neoplasias do Colo/genética , Reparo do DNA por Junção de Extremidades , Fator 2 Relacionado a NF-E2/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Tolerância a Radiação/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Neoplasias do Colo/radioterapia , Quebras de DNA de Cadeia Dupla , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Raios gama , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Mutação , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
9.
Cell Cycle ; 20(8): 742-751, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33691584

RESUMO

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.


Assuntos
Replicação do DNA/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Proliferação de Células/fisiologia , Humanos , Serina-Treonina Quinases TOR/genética
10.
Clin Cancer Res ; 26(14): 3740-3750, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32220892

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Quimiorradioterapia/métodos , Neoplasias Esofágicas/terapia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Radiossensibilizantes/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Neoplasias Esofágicas/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Pirazóis/uso terapêutico , Pirimidinonas/uso terapêutico , Radiossensibilizantes/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Clin Cancer Res ; 26(3): 669-678, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672772

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Família de Proteínas EGF/metabolismo , Leucemia Mieloide Aguda/patologia , Receptores Notch/antagonistas & inibidores , Animais , Apoptose , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Família de Proteínas EGF/genética , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores Notch/metabolismo , Transdução de Sinais
12.
Sci Rep ; 9(1): 9805, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285446

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Rabdomiossarcoma/genética , Transdução de Sinais , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rabdomiossarcoma/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima
13.
Clin Cancer Res ; 25(15): 4749-4760, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31097454

RESUMO

PURPOSE: Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. EXPERIMENTAL DESIGN: Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. RESULTS: BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. CONCLUSIONS: BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Neoplasias da Glândula Tireoide/radioterapia , Vemurafenib/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Reparo do DNA , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Tolerância a Radiação/genética , Radioterapia/métodos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Cancer Ther ; 18(6): 1025-1035, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31015310

RESUMO

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.


Assuntos
Proteínas Reguladoras de Apoptose/química , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Proteínas Mitocondriais/química , Papillomaviridae/isolamento & purificação , Radiossensibilizantes/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Tiazóis/uso terapêutico , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mimetismo Molecular , Infecções por Papillomavirus/virologia , Prognóstico , Radiossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Tiazóis/farmacologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Oncotarget ; 8(25): 41422-41431, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28507282

RESUMO

Balanced deoxyribonucleotides pools are essential for cell survival and genome stability. Ribonucleotide reductase is the rate-limiting enzyme for the production of deoxyribonucleotides. We report here that p53 suppresses ribonucleotide reductase subunit 1 (RRM1) and 2 (RRM2) via inhibiting mammalian target of rapamycin complex 1 (mTORC1). In vitro, cancer cell lines and mouse embryonic fibroblast cells were treated with different concentrations of pharmacological inhibitors for different times. In vivo, rhabdomyosarcoma Rh30 cell tumor-bearing mice were treated with rapamycin or AZD8055. Protein levels and phosphorylation status were assessed by immunoblotting and mRNA levels were determined by real time RT-PCR. Pharmacological inhibition of mTORC1 with rapamycin, mTOR kinase with AZD8055 or protein kinase B with MK2206 resulted in decrease of RRM1 and RRM2 in Rh30 cells both in vitro and in mouse tumor xenografts. Moreover, eukaryotic translational initiation factor 4E-binding proteins 1 and 2 double knockout mouse embryonic fibroblast cells demonstrated an elevation of RRM1 and RRM2. Furthermore, down-regulation of mTOR-protein kinase B signaling or cyclin dependent kinase 4 led to decrease of RRM1 and RRM2 mRNAs. In addition, TP53 mutant cancer cells had elevation of RRM1 and RRM2, which was reduced by rapamycin. Importantly, human double minute 2 inhibitor nutlin-3 decreased RRM1 and RRM2 in TP53 wild type rhabdomyosarcoma Rh18 but not in TP53 mutated Rh30 cells. Our data demonstrated that mTOR enhances the cap-dependent protein translation and gene transcription of RRM1 and RRM2. Our findings might provide an additional mechanism by which p53 maintains genome stability.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Knockout , Camundongos SCID , Morfolinas/farmacologia , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Interferência de RNA , Ribonucleosídeo Difosfato Redutase/genética , Sirolimo/farmacologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Sci Rep ; 7(1): 1535, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28484242

RESUMO

Oncogenic transformation leads to dysregulated cell proliferation, nutrient deficiency, and hypoxia resulting in metabolic stress and increased DNA damage. In normal cells, such metabolic stress leads to inhibition of signaling through the mammalian Target of Rapamycin Complex 1 (mTORC1), reduction of protein translation, cell cycle arrest, and conservation of energy. In contrast, negative regulation of mTORC1 signaling by DNA damage is abrogated in many cancer cells, thus mTORC1 signaling remains active under microenvironmental conditions that potentially promote endogenous DNA damage. Here we report that mTORC1 signaling suppresses endogenous DNA damage and replication stress. Pharmacological inhibition of mTOR signaling resulted in phosphorylation of H2AX concomitant with the decrease of CHK1 levels both in cell culture and mouse rhadomyosarcoma xenografts. Further results demonstrated that mTORC1-S6K1 signaling controls transcription of CHK1 via Rb-E2F by upregulating cyclin D and E. Consistent with these results, downregulation of CHK1 by inhibition of mTOR kinase resulted in defects in the slow S phase progression following DNA damage. These results indicate that, under stressful conditions, maintained mTORC1 signaling in cancer cells promotes survival by suppressing endogenous DNA damage, and may control cell fate through the regulation of CHK1.


Assuntos
Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Serina-Treonina Quinases TOR/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Dano ao DNA/genética , Replicação do DNA , Feminino , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos SCID , Modelos Biológicos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fase S , Transdução de Sinais , Transcrição Gênica
17.
Proc Natl Acad Sci U S A ; 114(23): E4641-E4647, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533390

RESUMO

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.


Assuntos
Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Proteínas Angiogênicas/antagonistas & inibidores , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Proteínas de Ligação ao Cálcio , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Intervalo Livre de Doença , Família de Proteínas EGF , Fatores de Crescimento Endotelial/antagonistas & inibidores , Feminino , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Proteínas/metabolismo , Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Risco , Regulação para Cima , Adulto Jovem
18.
Genes Dev ; 29(16): 1707-20, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26302789

RESUMO

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.


Assuntos
Carcinoma/genética , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Carcinoma/enzimologia , Carcinoma/fisiopatologia , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática , Feminino , Técnicas de Introdução de Genes , Camundongos , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Estabilidade Proteica
19.
Pediatr Blood Cancer ; 62(10): 1768-74, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25981859

RESUMO

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.


Assuntos
Astrocitoma/genética , Astrocitoma/radioterapia , Benzimidazóis/efeitos adversos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Radioterapia/efeitos adversos , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos SCID , Complexos Multiproteicos/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/radioterapia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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