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1.
J Neurooncol ; 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358642

RESUMO

OBJECTIVE: Dose escalation via stereotactic radiation therapy techniques has been necessary for hepatobiliary malignancies in the primary and oligometastatic setting, but such dose escalation is challenging for spine metastases due to spinal cord proximity. Here, we investigate the role of spine stereotactic radiosurgery (SSRS) in the management of such metastases. METHODS: We retrospectively reviewed patients treated with SSRS to spinal metastases from hepatobiliary malignancies between 2004 and 2017 at our Institution. We used the Kaplan-Meier method to calculate overall survival (OS) and local control (LC) and Cox regression analysis to identify factors associated with disease-related outcomes. RESULTS: We identified 28 patients treated to 43 spinal metastases with SSRS for either HCC or cholangiocarcinoma. The 1-year LC and OS were 85% and 23%, respectively. The median time to death was 6.2 months, while median time to local failure was not reached. Tumor volume > 60 cc (SHR 6.65, p = 0.03) and Bilsky ≥ 1c (SHR 4.73, p = 0.05) predicted for poorer LC, while BED10 > 81 Gy trended towards better local control (SHR 4.35, p = 0.08). Child-Pugh Class (HR 3.02, p = 0.003), higher PRISM Group (HR 3.49, p = 0.001), and systemic disease progression (HR 3.65, p = 0.001) were associated with worse mortality based on univariate modeling in patients treated with SSRS; on multivariate analysis, PRISM Group (HR 2.28, p = 0.03) and systemic disease progression (HR 2.67, p = 0.03) remained significant. Four patients (10%) developed compression deformity and one patient (2%) developed radiation neuritis. CONCLUSION: SSRS provides durable local control in patients with metastatic hepatobiliary malignancies, with higher BED necessary to ensure excellent LC. PRISM scoring is a promising prognostic tool to aid SSRS patient selection.

2.
JAMA Netw Open ; 3(4): e201929, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32236532

RESUMO

Importance: Historical data suggest that there is an overall survival benefit associated with prophylactic cranial irradiation (PCI) in patients with small cell lung cancer (SCLC). However, as the fidelity of magnetic resonance imaging (MRI) of the brain continues to improve, this idea is now being questioned, with recent research showing no survival benefit associated with PCI in extensive-stage SCLC; however, the role for PCI is not clear in patients with limited-stage SCLC (LS-SCLC). Objective: To report the overall survival and rates of intracranial control for patients with LS-SCLC, all staged with MRI, who either did or did not undergo PCI. Design, Setting, and Participants: This cohort study included 297 patients with LS-SCLC at a large US academic cancer center. Patients were treated with thoracic radiation; 205 also underwent PCI and 92 did not. All patients underwent at least baseline MRI, with restaging brain MRI and/or computed tomography; they did not have disease progression after thoracic radiation treatment. A propensity score-matching analysis was undertaken in an attempt to adjust for potential bias. Of the 297 patients who met the inclusion criteria, the propensity score was calculated for 295 patients, using patient, tumor, and treatment characteristics. Data were analyzed in October 2019. Intervention: Prophylactic cranial irradiation in patients with LS-SCLC. Main Outcomes and Measures: The rate of overall survival and intracranial control. Results: Of the 297 patients, 162 (54.5%) were men. The median age was 62.2 years (range, 27.0-85.0 years) for patients who underwent PCI and 68.6 years (range, 40.0-86.0 years) for those who did not undergo PCI. The 3-year cumulative incidence rate of brain metastases was higher in the no-PCI group vs the PCI group, when counting death as a competing risk, but the difference was not statistically significant (20.40% [95% CI, 12.45%-29.67%] vs 11.20% [95% CI, 5.40%-19.20%]; P = .10). The use of PCI was not associated with a difference in overall survival between the patient groups (hazard ratio, 0.844; 95% CI, 0.604-1.180; P = .32). Conclusions and Relevance: These findings suggest that patients with LS-SCLC staged with MRI who undergo PCI after thoracic radiation treatment were not associated with a decreased risk of developing new brain metastases compared with patients who do not undergo PCI. The use of PCI was not associated with an overall survival benefit for such patients.

3.
J Neurosurg Spine ; : 1-9, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059183

RESUMO

OBJECTIVE: Patients with metastatic thyroid cancer have prolonged survival compared to those with other primary tumors. The spine is the most common site of osseous involvement in cases of metastatic thyroid cancer. As a result, obtaining durable local control (LC) in the spine is crucial. This study aimed to evaluate the efficacy of spine stereotactic radiosurgery (SSRS) in patients with metastatic thyroid cancer. METHODS: Information on patients with metastatic thyroid cancer treated with SSRS for spinal metastases was retrospectively evaluated. SSRS was delivered with a simultaneous integrated boost technique using single- or multiple-fraction treatments. LC, defined as stable or reduced disease volume, was evaluated by examining posttreatment MRI, CT, and PET studies. RESULTS: A total of 133 lesions were treated in 67 patients. The median follow-up duration was 31 months. Dose regimens for SSRS included 18 Gy in 1 fraction, 27 Gy in 3 fractions, and 30 Gy in 5 fractions. The histology distribution was 36% follicular, 33% papillary, 15% medullary, 13% Hurthle cell, and 3% anaplastic. The 1-, 2-, and 5-year LC rates were 96%, 89%, and 82%, respectively. The median overall survival (OS) was 43 months, with 1-, 2-, and 5-year survival rates of 86%, 74%, and 44%, respectively. There was no correlation between the absolute biological equivalent dose (BED) and OS or LC. Patients with effective LC had a trend toward improved OS when compared to patients who had local failure: 68 versus 28 months (p = 0.07). In terms of toxicity, 5 vertebral compression fractures (2.8%) occurred, and only 1 case (0.6%) of greater than or equal to grade 3 toxicity (esophageal stenosis) was reported. CONCLUSIONS: SSRS is a safe and effective treatment option with excellent LC and minimal toxicity for patients with metastatic thyroid cancer. No association with increased radiation dose or BED was found, suggesting that such patients can be effectively treated with reduced dose regimens.

4.
Br J Radiol ; 93(1107): 20190378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31430188

RESUMO

Radiation therapy is an essential component of treatment for locally advanced non-small cell lung cancer (NSCLC) but can be technically challenging because of the proximity of lung tumors to nearby critical organs or structures. The most effective strategy for reducing radiation-induced toxicity is to reduce unnecessary exposure of normal tissues by using advanced technology; examples from photon (X-ray) therapy have included three-dimensional conformal radiation therapy versus its predecessor, two-dimensional radiation therapy, and intensity-modulated photon radiation therapy versus its predecessor, three-dimensional conformal therapy. Using particle-beam therapy rather than photons offers the potential for further advantages because of the unique depth-dose characteristics of the particles, which can be exploited to allow still higher dose escalation to tumors with greater sparing of normal tissues, with the ultimate goal of improving local tumor control and survival while preserving quality of life by reducing treatment-related toxicity. However, the costs associated with particle therapy with protons are considerably higher than the current state of the art in photon technology, and evidence of clinical benefit from protons is increasingly being demanded to justify the higher financial burden on the healthcare system. Some such evidence is available from preclinical studies, from retrospective, single-institution clinical series, from analyses of national databases, and from single-arm prospective studies in addition to several ongoing randomized comparative trials. This review summarizes the rationale for and challenges of using proton therapy to treat thoracic cancers, reviews the current clinical experience, and suggests topics for future research.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , Órgãos em Risco/efeitos da radiação , Terapia com Prótons/métodos , Lesões por Radiação/prevenção & controle , Radioterapia de Intensidade Modulada/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Previsões , Humanos , Neoplasias Pulmonares/patologia , Tratamentos com Preservação do Órgão/métodos , Estudos Prospectivos , Terapia com Prótons/economia , Terapia com Prótons/tendências , Qualidade de Vida , Radioterapia Conformacional/métodos , Radioterapia de Intensidade Modulada/economia , Radioterapia de Intensidade Modulada/tendências , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Resultado do Tratamento
5.
J Oncol Pract ; : JOP1900480, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31765268

RESUMO

PURPOSE: Several treatment options for spinal metastases exist, including multiple radiation therapy (RT) techniques: three-dimensional (3D) conventional RT (3D-RT), intensity-modulated RT (IMRT), and spine stereotactic radiosurgery (SSRS). Although data exist regarding reimbursement differences across regimens, differences in provider care delivery costs have yet to be evaluated. We quantified institutional costs associated with RT for spinal metastases, using a time-driven activity-based costing model. METHODS: Comparisons were made between (1) 10-fraction 3D-RT to 30 Gy, (2) 10-fraction IMRT to 30 Gy, (3) 3-fraction SSRS (SSRS-3) to 27 Gy, and (4) single-fraction SSRS (SSRS-1) to 18 Gy. Process maps were developed from consultation through follow-up 30 days post-treatment. Process times were determined through panel interviews, and personnel costs were extracted from institutional salary data. The capacity cost rate was determined for each resource, then multiplied by activity time to calculate costs, which were summed to determine total cost. RESULTS: Full-cycle costs of SSRS-1 were 17% lower and 17% higher compared with IMRT and 3D-RT, respectively. Full-cycle costs for SSRS-3 were only 1% greater than 10-fraction IMRT. Technical costs for IMRT were 50% and 77% more than SSRS-3 and SSRS-1. In contrast, personnel costs were 3% and 28% higher for SSRS-1 than IMRT and 3D-RT, respectively (P < .001). CONCLUSIONS: Resource utilization varies significantly among treatment options. By quantifying provider care delivery costs, this analysis supports the institutional resource efficiency of SSRS-1. Incorporating clinical outcomes with such resource and cost data will provide additional insight into the highest value modalities and may inform alternative payment models, operational workflows, and institutional resource allocation.

7.
World J Surg Oncol ; 16(1): 143, 2018 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-30007404

RESUMO

BACKGROUND: Gastric cancer is the fifth most prevalent and the third most lethal cancer worldwide, causing approximately 720,000 deaths annually. Although most cases of gastric cancers are sporadic, one of its inherited forms, hereditary diffuse gastric cancer (HDGC), constitutes about 1-3% of cases. Interestingly, females in families with HDGC are also predisposed to developing lobular breast cancer (LBC). Recent analyses have identified loss-of-function germline mutations in cadherein-1 (CDH1) as a culprit in HDGC and LBC. This discovery fueled several sequencing analyses and case series reports analyzing the pattern of inheritance of CDH1 and its propensity to induce HDGC. In 2015, a multinational and multidisciplinary task force updated the guidelines and criteria for screening, diagnosing, and managing HDGC. CASE PRESENTATION: Here, we present a case series of three siblings with family history of HDGC who tested positive for the CDH1 mutation and describe their surgical treatment course, post-operative management, and follow-up as they pertain to the updated guidelines. CONCLUSIONS: Despite recent updates in guidelines in the diagnosis and management of HDGC, the disease remains challenging to address with patients given the high level of uncertainty and the comorbidities associated with prophylactic intervention. We strongly recommend that an interdisciplinary team inclusive of clinical and surgical oncologists, along with geneticists, social work, and psychological support, should follow the patients in a longitudinal and comprehensive manner in order to achieve full recovery and return to normalcy, as with our patients.


Assuntos
Antígenos CD/genética , Caderinas/genética , Carcinoma de Células em Anel de Sinete/genética , Carcinoma de Células em Anel de Sinete/cirurgia , Gastrectomia/métodos , Neoplasias Gástricas/genética , Neoplasias Gástricas/cirurgia , Adulto , Carcinoma de Células em Anel de Sinete/diagnóstico , Carcinoma de Células em Anel de Sinete/prevenção & controle , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Mutação com Perda de Função , Masculino , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/prevenção & controle , Síndromes Neoplásicas Hereditárias/cirurgia , Prognóstico , Procedimentos Cirúrgicos Profiláticos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/prevenção & controle , Adulto Jovem
9.
J Virol ; 90(19): 8822-41, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27466416

RESUMO

UNLABELLED: IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation. IMPORTANCE: Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.


Assuntos
Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Latência Viral , Replicação Viral , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Proteólise
10.
J Virol ; 90(8): 3860-3872, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26819309

RESUMO

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. IMPORTANCE: Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endotélio Vascular/virologia , Herpesvirus Humano 8/fisiologia , Fosfoproteínas/fisiologia , Pinocitose , Internalização do Vírus , Actinina/metabolismo , Linhagem Celular , Membrana Celular/virologia , Derme/irrigação sanguínea , Derme/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Microvasos/citologia , Microvasos/virologia , Fosfoproteínas/genética , Quinases Associadas a rho/metabolismo
11.
J Virol ; 89(15): 7874-92, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25995248

RESUMO

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies. IMPORTANCE: KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Other than ganciclovir and alpha interferon (IFN-α) prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation, and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.


Assuntos
Antígenos Virais/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Linfoma de Efusão Primária/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sarcoma de Kaposi/metabolismo , Antígenos Virais/genética , Regulação para Baixo , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , Fator 2 Relacionado a NF-E2/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Repressoras/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Proteína 28 com Motivo Tripartido
12.
J Virol ; 89(4): 2268-86, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25505069

RESUMO

UNLABELLED: Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase Cζ (PKCζ) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (γGCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63-ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1ß-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis. IMPORTANCE: The transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/virologia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/biossíntese , Processamento de Proteína Pós-Traducional , Latência Viral , Antígenos de Neoplasias , Células Cultivadas , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Fosforilação , Ligação Proteica , Proteína Sequestossoma-1
13.
PLoS Pathog ; 10(10): e1004389, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25299066

RESUMO

Kaposi's sarcoma associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) and B-cell proliferative primary effusion lymphoma (PEL), common malignancies seen in immunocompromised HIV-1 infected patients. The progression of these cancers occurs by the proliferation of cells latently infected with KSHV, which is highly dependent on autocrine and paracrine factors secreted from the infected cells. Glutamate and glutamate receptors have emerged as key regulators of intracellular signaling pathways and cell proliferation. However, whether they play any role in the pathological changes associated with virus induced oncogenesis is not known. Here, we report the first systematic study of the role of glutamate and its metabotropic glutamate receptor 1 (mGluR1) in KSHV infected cell proliferation. Our studies show increased glutamate secretion and glutaminase expression during de novo KSHV infection of endothelial cells as well as in KSHV latently infected endothelial and B-cells. Increased mGluR1 expression was detected in KSHV infected KS and PEL tissue sections. Increased c-Myc and glutaminase expression in the infected cells was mediated by KSHV latency associated nuclear antigen 1 (LANA-1). In addition, mGluR1 expression regulating host RE-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) was retained in the cytoplasm of infected cells. KSHV latent protein Kaposin A was also involved in the over expression of mGluR1 by interacting with REST in the cytoplasm of infected cells and by regulating the phosphorylation of REST and interaction with ß-TRCP for ubiquitination. Colocalization of Kaposin A with REST was also observed in KS and PEL tissue samples. KSHV infected cell proliferation was significantly inhibited by glutamate release inhibitor and mGluR1 antagonists. These studies demonstrated that elevated glutamate secretion and mGluR1 expression play a role in KSHV induced cell proliferation and suggest that targeting glutamate and mGluR1 is an attractive therapeutic strategy to effectively control the KSHV associated malignancies.


Assuntos
Proliferação de Células , Glutamatos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sarcoma de Kaposi/virologia , Linfócitos B/virologia , Linhagem Celular , Proliferação de Células/fisiologia , Células Endoteliais/metabolismo , Infecções por Herpesviridae/virologia , Humanos , Receptores de Glutamato Metabotrópico/imunologia , Sarcoma de Kaposi/metabolismo , Latência Viral/imunologia
14.
PLoS Pathog ; 10(10): e1004460, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340789

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.


Assuntos
Células Endoteliais/virologia , Herpesvirus Humano 8 , Fator 2 Relacionado a NF-E2/metabolismo , Sarcoma de Kaposi/virologia , Internalização do Vírus , Ciclo-Oxigenase 2/metabolismo , Humanos , Transporte Proteico/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Elife ; 3: e02057, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24771768

RESUMO

Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-α (ERα) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ERα ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNFα genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ERα and several ERα coregulators, suggesting that ERα functions as a primary conduit for resveratrol activity.DOI: http://dx.doi.org/10.7554/eLife.02057.001.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Inflamação/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Adenilato Quinase/metabolismo , AMP Cíclico/metabolismo , Receptor alfa de Estrogênio/química , Feminino , Humanos , Interleucina-6/genética , Ligantes , Células MCF-7 , Regiões Promotoras Genéticas , Conformação Proteica , Resveratrol , Fator de Necrose Tumoral alfa/metabolismo
16.
Cancer Res ; 73(13): 3913-26, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23636127

RESUMO

miRNAs (miR) have been shown to modulate critical gene transcripts involved in tumorigenesis, but their role in tumor-mediated immunosuppression is largely unknown. On the basis of miRNA gene expression in gliomas using tissue microarrays, in situ hybridization, and molecular modeling, miR-124 was identified as a lead candidate for modulating STAT3 signaling, a key pathway mediating immunosuppression in the tumor microenvironment. miR-124 is absent in all grades and pathologic types of gliomas. Upon upregulating miR-124 in glioma cancer stem cells (gCSC), the STAT3 pathway was inhibited, and miR-124 reversed gCSC-mediated immunosuppression of T-cell proliferation and induction of forkhead box P3 (Foxp3)(+) regulatory T cells (Treg). Treatment of T cells from immunosuppressed glioblastoma patients with miR-124 induced marked effector response including upregulation of interleukin (IL)-2, IFN-γ, and TNF-α. Both systemic administration of miR-124 or adoptive miR-124-transfected T-cell transfers exerted potent anti-glioma therapeutic effects in clonotypic and genetically engineered murine models of glioblastoma and enhanced effector responses in the local tumor microenvironment. These therapeutic effects were ablated in both CD4(+)- and CD8(+)-depleted mice and nude mouse systems, indicating that the therapeutic effect of miR-124 depends on the presence of a T-cell-mediated antitumor immune response. Our findings highlight the potential application of miR-124 as a novel immunotherapeutic agent for neoplasms and serve as a model for identifying miRNAs that can be exploited as immunotherapeutics.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/genética , Fator de Transcrição STAT3/genética , Linfócitos T/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/imunologia , Glioblastoma/terapia , Humanos , Tolerância Imunológica , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Análise Serial de Tecidos , Células Tumorais Cultivadas , Evasão Tumoral/genética
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