RESUMO
The clinical benefit conferred by vascular endothelial growth factors (VEGF)-targeted therapies is variable, and tumors from treated patients eventually reinitiate growth. Here, we identify a glycosylation-dependent pathway that compensates for the absence of cognate ligand and preserves angiogenesis in response to VEGF blockade. Remodeling of the endothelial cell (EC) surface glycome selectively regulated binding of galectin-1 (Gal1), which upon recognition of complex N-glycans on VEGFR2, activated VEGF-like signaling. Vessels within anti-VEGF-sensitive tumors exhibited high levels of α2-6-linked sialic acid, which prevented Gal1 binding. In contrast, anti-VEGF refractory tumors secreted increased Gal1 and their associated vasculature displayed glycosylation patterns that facilitated Gal1-EC interactions. Interruption of ß1-6GlcNAc branching in ECs or silencing of tumor-derived Gal1 converted refractory into anti-VEGF-sensitive tumors, whereas elimination of α2-6-linked sialic acid conferred resistance to anti-VEGF. Disruption of the Gal1-N-glycan axis promoted vascular remodeling, immune cell influx and tumor growth inhibition. Thus, targeting glycosylation-dependent lectin-receptor interactions may increase the efficacy of anti-VEGF treatment.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Células Endoteliais/metabolismo , Galectina 1/genética , Galectina 1/metabolismo , Glicosilação , Humanos , Hipóxia , Camundongos , Receptores Mitogênicos/metabolismoRESUMO
Kaposi's sarcoma (KS) may derive from Kaposi's sarcoma herpesvirus (KSHV)-infected human mesenchymal stem cells (hMSCs) that migrate to sites characterized by inflammation and angiogenesis, promoting the initiation of KS. By analyzing the RNA sequences of KSHV-infected primary hMSCs, we have identified specific cell subpopulations, mechanisms, and conditions involved in the initial stages of KSHV-induced transformation and reprogramming of hMSCs into KS progenitor cells. Under proangiogenic environmental conditions, KSHV can reprogram hMSCs to exhibit gene expression profiles more similar to KS tumors, activating cell cycle progression, cytokine signaling pathways, endothelial differentiation, and upregulating KSHV oncogenes indicating the involvement of KSHV infection in inducing the mesenchymal-to-endothelial (MEndT) transition of hMSCs. This finding underscores the significance of this condition in facilitating KSHV-induced proliferation and reprogramming of hMSCs towards MEndT and closer to KS gene expression profiles, providing further evidence of these cell subpopulations as precursors of KS cells that thrive in a proangiogenic environment.
Assuntos
Herpesvirus Humano 8 , Células-Tronco Mesenquimais , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virologia , Células-Tronco Mesenquimais/virologia , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Proliferação de CélulasRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS), an HIV/AIDS-associated malignancy. Effective treatments against KS remain to be developed. The sugar analog 2-deoxy- d-glucose (2-DG) is an anticancer agent that is well-tolerated and safe in patients and was recently demonstrated to be a potent antiviral, including KSHV and severe acute respiratory syndrome coronavirus 2. Because 2-DG inhibits glycolysis and N-glycosylation, identifying its molecular targets is challenging. Here we compare the antiviral effect of 2-DG with 2-fluoro-deoxy- d-glucose, a glycolysis inhibitor, and 2-deoxy-fluoro- d-mannose (2-DFM), a specific N-glycosylation inhibitor. At doses similar to those clinically achievable with 2-DG, the three drugs impair KSHV replication and virion production in iSLK.219 cells via downregulation of viral structural glycoprotein expression (K8.1 and gB), being 2-DFM the most potent KSHV inhibitor. Consistently with the higher potency of 2-DFM, we found that d-mannose rescues KSHV glycoprotein synthesis and virus production, indicating that inhibition of N-glycosylation is the main antiviral target using d-mannose competition experiments. Suppression of N-glycosylation by the sugar drugs triggers ER stress. It activates the host unfolded protein response (UPR), counteracting KSHV-induced inhibition of the protein kinase R-like endoplasmic reticulum kinase branch, particularly activating transcription factor 4 and C/EBP homologous protein expression. Finally, we demonstrate that sugar analogs induce autophagy (a prosurvival mechanism) and, thus, inhibit viral replication playing a protective role against KSHV-induced cell death, further supporting their direct antiviral effect and potential therapeutic use. Our work identifies inhibition of N-glycosylation leading to ER stress and UPR as an antienveloped virus target and sugar analogs such as 2-DG and the newly identified 2-DFM as antiviral drugs.
Assuntos
COVID-19 , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Manose/farmacologia , Glucose , Glicosilação , Resposta a Proteínas não Dobradas , Replicação Viral , Antivirais/farmacologiaRESUMO
Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-induced sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is found in all KS-lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of KS-spindle cells that have lost KSHV and proliferate autonomously or via paracrine mechanisms. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although infected cells that lose the KSHV-episome prior to tumor formation lose their tumorigenicity, explanted tumor cells that lost the KSHV-episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV tumorigenesis supporting the possibility of hit and run viral-sarcomagenesis. RNA-sequencing and CpG-methylation analysis were performed on KSHV-positive and KSHV-negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV-positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors occurring en exactly the same position, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV-negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V and other proposed mechanism, could be responsible for driving tumorigenesis in the absence of KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of "hit and run" KSHV-sarcomagenesis and point to the existence of selectable KSHV-induced host mutations that may impact AIDS-KS treatment.
Assuntos
Transformação Celular Viral , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8 , Neoplasias Experimentais , Plasmídeos , Sarcoma de Kaposi , Animais , Linhagem Celular , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Plasmídeos/genética , Plasmídeos/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologiaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi's sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades. We show that vGPCR expression triggers signaling pathways that upregulate COX-2 levels due to a dual effect upon both its gene promoter region and, in mature mRNA, the 3'UTR region that control mRNA stability. Both events are mediated by signaling through ERK1/2 MAPK pathway. Inhibition of COX-2 in vGPCR-transformed cells impairs vGPCR-driven angiogenesis and treatment with the COX-2-selective inhibitory drug Celecoxib produces a significant decrease in tumor growth, pointing to COX-2 activity as critical for vGPCR oncogenicity in vivo and indicating that COX-2-mediated angiogenesis could play a role in KS tumorigenesis. These results, along with the overexpression of COX-2 in KS lesions, define COX-2 as a potential target for the prevention and treatment of KSHV-oncogenesis.
Assuntos
Herpesvirus Humano 8/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Receptores Acoplados a Proteínas G/metabolismo , Sarcoma de Kaposi/irrigação sanguínea , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Células Endoteliais/metabolismo , Proteínas de Ligação ao GTP/genética , Herpesvirus Humano 8/genética , Sistema de Sinalização das MAP Quinases , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Células NIH 3T3 , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , Oncogenes , Receptores Acoplados a Proteínas G/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Transdução de Sinais , Ativação TranscricionalRESUMO
The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Camundongos , Rhadinovirus/metabolismoRESUMO
Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.
Assuntos
Células-Tronco Mesenquimais/virologia , Neovascularização Patológica/virologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sarcoma de Kaposi/virologia , Animais , Carcinogênese/metabolismo , Expressão Gênica/fisiologia , Herpesvirus Humano 8/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Mechanisms discovered to drive increased glucose metabolism in cancer cells are found to be similar to those in viral-infected cells. In this mini review, we summarize the major pathways by which the sugar analog, 2-Deoxy-d-glucose, has been shown to exploit increased glucose metabolism in cancer and how this information applies to viral-infected cells. Moreover, we highlight the relevance of these findings to the emergency approval of 2-Deoxy-d-glucose in India to be used against SARS-CoV-2, the virus responsible for COVID-19.
Assuntos
COVID-19/metabolismo , Desoxiglucose/farmacologia , Glucose/metabolismo , Neoplasias/metabolismo , SARS-CoV-2/efeitos dos fármacos , COVID-19/virologia , Humanos , Índia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidadeRESUMO
Kaposi's sarcoma (KS) herpesvirus (KSHV) causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Tyrosine kinase receptor (RTK) proteomic arrays, identified PDGF receptor-alpha (PDGFRA) as the predominantly-activated RTK in KSHV-induced mouse KS-tumors. We show that: 1) KSHV lytic replication and the vGPCR can activate PDGFRA through upregulation of its ligands PDGFA/B, which increase c-myc, VEGF and KSHV gene expression in infected cells 2) KSHV infected spindle cells of most AIDS-KS lesions display robust phospho-PDGFRA staining 3) blocking PDGFRA-signaling with N-acetyl-cysteine, RTK-inhibitors Imatinib and Sunitinib, or dominant-negative PDGFRA inhibits tumorigenesis 4) PDGFRA D842V activating-mutation confers resistance to Imatinib in mouse-KS tumorigenesis. Our data show that KSHV usurps sarcomagenic PDGFRA signaling to drive KS. This and the fact that PDGFRA drives non-viral sarcomas highlights the importance for KSHV-induced ligand-mediated activation of PDGFRA in KS sarcomagenesis and shows that this oncogenic axis could be successfully blocked to impede KS tumor growth.
Assuntos
Carcinogênese/metabolismo , Herpesvirus Humano 8/patogenicidade , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sarcoma de Kaposi/virologia , Animais , Humanos , Camundongos , Camundongos Nus , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Sarcoma de Kaposi/metabolismo , Transdução de SinaisRESUMO
Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent, monomethyl auristatin E. Brentuximab vedotin is an effective treatment of relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest, and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunoconjugados/farmacologia , Antígeno Ki-1/imunologia , Linfoma de Efusão Primária/patologia , Animais , Western Blotting , Brentuximab Vedotin , Citometria de Fluxo , Humanos , Linfoma de Efusão Primária/imunologia , Linfoma de Efusão Primária/prevenção & controle , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Transfection of a Kaposi's sarcoma (KS) herpesvirus (KSHV) Bacterial Artificial Chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells generates a cell (mECK36) that forms KS-like tumors in mice. mECK36 expressed most KSHV genes and were angiogenic, but they didn't form colonies in soft agar. In nude mice, mECK36 formed KSHV-harboring vascularized spindle cell sarcomas that were LANA+/podoplanin+, overexpressed VEGF and Angiopoietin ligands and receptors, and displayed KSHV and host transcriptomes reminiscent of KS. mECK36 that lost the KSHV episome reverted to nontumorigenicity. siRNA suppression of KSHV vGPCR, an angiogenic gene upregulated in mECK36 tumors, inhibited angiogenicity and tumorigenicity. These results show that KSHV malignancy is in vivo growth restricted and reversible, defining mECK36 as a biologically sensitive animal model of KSHV-dependent KS.
Assuntos
Modelos Animais de Doenças , Herpesvirus Humano 8 , Sarcoma de Kaposi/patologia , Angiopoietinas/metabolismo , Animais , Antígenos Virais/metabolismo , Células da Medula Óssea/patologia , Linhagem da Célula , Transformação Celular Neoplásica , Transformação Celular Viral , Células Cultivadas , Cromossomos Artificiais Bacterianos , Células Endoteliais/patologia , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Neovascularização Patológica , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-kappaB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.
Assuntos
Ácidos Borônicos/uso terapêutico , Linfoma de Efusão Primária/tratamento farmacológico , Pirazinas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Fator de Transcrição E2F3/metabolismo , Evolução Fatal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/fisiologia , Humanos , Linfoma de Efusão Primária/virologia , Masculino , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazinas/farmacologia , Análise de Sobrevida , Resultado do Tratamento , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Células Vero , Vírion/efeitos dos fármacos , Vírion/metabolismoRESUMO
Endothelial-to-mesenchymal transition has been described in tumors as a source of mesenchymal stroma, while the reverse process has been proposed in tumor vasculogenesis and angiogenesis. A human oncogenic virus, Kaposi's sarcoma herpes virus (KSHV), can regulate both processes in order to transit through this transition 'boulevard' when infecting KS oncogenic progenitor cells. Endothelial or mesenchymal circulating progenitor cells can serve as KS oncogenic progenitors recruited by inflammatory cytokines because KSHV can reprogram one into the other through endothelial-to-mesenchymal and mesenchymal-to-endothelial transitions. Through these novel insights, the identity of the potential oncogenic progenitor of KS is revealed while gaining knowledge of the biology of the mesenchymal-endothelial differentiation axis and pointing to this axis as a therapeutic target in KS.
Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/patologia , Herpesvirus Humano 8/fisiologia , Diferenciação CelularRESUMO
Evidence indicates that the microbiome plays a significant role in HIV immunopathogenesis and associated complications. This study aimed to characterize the oral and anal microbiome of Men who have Sex with Men (MSM) and Transgender Women (TGW), with and without HIV. One hundred and thirty oral and anal DNA-derived samples were obtained from 78 participants and subjected to shotgun metagenomics sequencing for further microbiome analysis. Significant differences in the microbiome composition were found among subjects associated with HIV infection, gender, sex behavior, CD4+ T-cell counts, antiretroviral therapy (ART), and the presence of HPV-associated precancerous anal lesions. Results confirm the occurrence of oncogenic viromes in this high HIV-risk population. The oral microbiome in HIV-associated cases exhibited an enrichment of bacteria associated with periodontal disease pathogenesis. Conversely, anal bacteria showed a significant decrease in HIV-infected subjects (Coprococcus comes, Finegoldia magna, Blautia obeum, Catenibacterium mitsuokai). TGW showed enrichment in species related to sexual transmission, which concurs that most recruited TGW are or have been sex workers. Prevotella bivia and Fusobacterium gonidiaformans were positively associated with anal precancerous lesions among HIV-infected subjects. The enrichment of Holdemanella biformis and C. comes was associated with detectable viral load and ART-untreated patients. Metabolic pathways were distinctly affected by predominant factors linked to sexual behavior or HIV pathogenesis. Gene family analysis identified bacterial gene signatures as potential prognostic and predictive biomarkers for HIV/AIDS-associated malignancies. Conclusions: Identified microbial features at accessible sites are potential biomarkers for predicting precancerous anal lesions and therapeutic targets for HIV immunopathogenesis.
Assuntos
Infecções por HIV , Microbiota , Minorias Sexuais e de Gênero , Masculino , Humanos , Feminino , Infecções por HIV/complicações , Homossexualidade Masculina , Redes e Vias MetabólicasRESUMO
Lytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.
Assuntos
Antivirais/farmacologia , Desoxiglucose/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Herpesvirus Humano 8/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/virologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Fosforilação , Ativação Transcricional/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Ensaio de Placa Viral , Proteínas Virais/biossíntese , Proteínas Virais/genética , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The G protein-coupled receptor oncogene (vGPCR) of the Kaposi's sarcoma (KS) associated herpesvirus (KSHV), an oncovirus implicated in angioproliferative neoplasms, induces angiogenesis by VEGF secretion. Accordingly, we found that expression of vGPCR in human umbilical vein endothelial cells (HUVEC) leads to immortalization with constitutive VEGF receptor-2/ KDR expression and activation. vGPCR immortalization was associated with anti-senescence mediated by alternative lengthening of telomeres and an anti-apoptotic response mediated by vGPCR constitutive signaling and KDR autocrine signaling leading to activation of the PI3K/AKT pathway. In the presence of the KS growth factor VEGF, this mechanism can sustain suppression of signaling by the immortalizing gene. We conclude that vGPCR can cause an oncogenic immortalizing event and recapitulate aspects of the KS angiogenic phenotype in human endothelial cells, pointing to this gene as a pathogenic determinant of KSHV.
Assuntos
Endotélio Vascular/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Quimiocinas/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Virais/fisiologia , Apoptose , Transformação Celular Neoplásica , Células Cultivadas , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Herpesvirus Humano 8/patogenicidade , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Retroviridae/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Transdução de Sinais , Telomerase/metabolismo , Telômero/metabolismo , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Kaposi's sarcoma (KS) is the major AIDS-associated malignancy. It is characterized by the proliferation of spindle cells, inflammatory infiltrate, and aberrant angiogenesis caused by Kaposi's sarcoma herpesvirus (KSHV) infection. Small GTPase Rac1, an inflammatory signaling mediator triggering reactive oxygen species (ROS) production by NADPH-oxidases, is implicated in carcinogenesis and tumor angiogenesis. Here, we show that expression of a constitutively active Rac1 (RacCA) driven by the alpha-smooth muscle actin promoter in transgenic mice is sufficient to cause KS-like tumors through mechanisms involving ROS-driven proliferation, up-regulation of AKT signaling, and hypoxia-inducible factor 1-alpha-related angiogenesis. RacCA-induced tumors expressed KS phenotypic markers; displayed remarkable transcriptome overlap with KS lesions; and were, like KS, associated with male gender. The ROS scavenging agent N-acetyl-cysteine inhibited angiogenesis and completely abrogated transgenic RacCA tumor formation, indicating a causal role of ROS in tumorigenesis. Consistent with a pathogenic role in KS, immunohistochemical analysis revealed that Rac1 is overexpressed in KSHV(+) spindle cells of AIDS-KS biopsies. Our results demonstrate the direct oncogenicity of Rac1 and ROS and their contribution to a KS-like malignant phenotype, further underscoring the carcinogenic potential of oxidative stress in the context of chronic infection and inflammation. They define the RacCA transgenic mouse as a model suitable for studying the role of oxidative stress in the pathogenesis and therapy of KS, with relevance to other inflammation-related malignancies. Our findings suggest host and viral genes triggering Rac1 or ROS production as key determinants of KS onset and potential KS chemopreventive or therapeutic targets.
Assuntos
Antioxidantes/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/enzimologia , Síndrome da Imunodeficiência Adquirida/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/etiologia , Transcrição Gênica/genética , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Kaposi's sarcoma (KS) is the most common tumor in AIDS patients. The highly vascularized patient's skin lesions are composed of cells derived from the endothelial tissue transformed by the KSHV virus. Heme oxygenase-1 (HO-1) is an enzyme upregulated by the Kaposi´s sarcoma-associated herpesvirus (KSHV) and highly expressed in human Kaposi Sarcoma (KS) lesions. The oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR) is expressed by the viral genome in infected cells. It is involved in KS development, HO-1 expression, and vascular endothelial growth factor (VEGF) expression. vGPCR induces HO-1 expression and HO-1 dependent transformation through the Ga13 subunit of heterotrimeric G proteins and the small GTPase RhoA. We have found several lines of evidence supporting a role for Nrf2 transcription factors and family members in the vGPCR-Ga13-RhoA signaling pathway that converges on the HO-1 gene promoter. Our current information assigns a major role to ERK1/2MAPK pathways as intermediates in signaling from vGPCR to Nrf2, influencing Nrf2 translocation to the cell nucleus, Nrf2 transactivation activity, and consequently HO-1 expression. Experiments in nude mice show that the tumorigenic effect of vGPCR is dependent on Nrf2. In the context of a complete KSHV genome, we show that the lack of vGPCR increased cytoplasmic localization of Nrf2 correlated with a downregulation of HO-1 expression. Moreover, we also found an increase in phospho-Nrf2 nuclear localization in mouse KS-like KSHV (positive) tumors compared to KSHV (negative) mouse KS-like tumors. Our data highlights the fundamental role of Nrf2 linking vGPCR signaling to the HO-1 promoter, acting upon not only HO-1 gene expression regulation but also in the tumorigenesis induced by vGPCR. Overall, these data pinpoint this transcription factor or its associated proteins as putative pharmacological or therapeutic targets in KS.
Assuntos
Síndrome da Imunodeficiência Adquirida , Transformação Celular Viral/genética , Herpesvirus Humano 8 , Sarcoma de Kaposi , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/metabolismo , Síndrome da Imunodeficiência Adquirida/patologia , Síndrome da Imunodeficiência Adquirida/virologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologiaRESUMO
Regulatory pathways involving non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long non-coding RNAs (lncRNA), have gained great relevance due to their role in the control of gene expression modulation. Using RNA sequencing of KSHV Bac36 transfected mouse endothelial cells (mECK36) and tumors, we have analyzed the host and viral transcriptome to uncover the role lncRNA-miRNA-mRNA driven networks in KSHV tumorigenesis. The integration of the differentially expressed ncRNAs, with an exhaustive computational analysis of their experimentally supported targets, led us to dissect complex networks integrated by the cancer-related lncRNAs Malat1, Neat1, H19, Meg3, and their associated miRNA-target pairs. These networks would modulate pathways related to KSHV pathogenesis, such as viral carcinogenesis, p53 signaling, RNA surveillance, and cell cycle control. Finally, the ncRNA-mRNA analysis allowed us to develop signatures that can be used to an appropriate identification of druggable gene or networks defining relevant AIDS-KS therapeutic targets.