RESUMO
TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinogênese/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Quinases Semelhantes a Duplacortina , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prognóstico , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/metabolismo , Infecções por Papillomavirus/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Papillomavirus Humano , Carcinogênese , Papillomaviridae/genética , Papillomaviridae/metabolismoRESUMO
Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth in vivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.
Assuntos
Carcinoma de Células Renais , Proteínas de Homeodomínio , Neoplasias Renais , Fatores de Transcrição , Proteases Específicas de Ubiquitina , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.
Assuntos
Aminoacil-tRNA Sintetases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Código Genético , Aminoácidos/química , Peptídeos/genética , RNA de Transferência/metabolismoRESUMO
Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.
Assuntos
Proteínas Culina , Infecções por Citomegalovirus , Proteínas de Ligação a DNA , Proteínas Virais , Proteínas Culina/metabolismo , Infecções por Citomegalovirus/imunologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Fator de Transcrição STAT2/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas Virais/metabolismoRESUMO
Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/patologia , Endopeptidases/metabolismo , Neoplasias Renais/patologia , Animais , Carcinogênese , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Regulação para Baixo , Endopeptidases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Camundongos , Estabilidade Proteica , RNA Interferente Pequeno/metabolismo , RNA-Seq , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability to regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.
Assuntos
Quinase I-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inanição/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Linhagem Celular , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Sequência Conservada , Retroalimentação Fisiológica , Fibroblastos/metabolismo , Humanos , Quinase I-kappa B/genética , Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfotirosina/metabolismo , Domínios de Homologia de srcRESUMO
Thioridazine is an antipsychotic that has been shown to induce cell death and inhibit self-renewal in a broad spectrum of cancer cells. The mechanisms by which these effects are mediated are currently unknown but are presumed to result from the inhibition of dopamine receptor 2 (DRD2). Here we show that the self-renewal of several, but not all, triple-negative breast cancer cell lines is inhibited by thioridazine. The inhibition of self-renewal by thioridazine in these cells is mediated by DRD2 inhibition. Further, we demonstrate that DRD2 promotes self-renewal in these cells via a STAT3- and IL-6-dependent mechanism. We also show that thioridazine induces a G1 arrest and a loss in cell viability in all tested cell lines. However, the reduction in proliferation and cell viability is independent of DRD2 and STAT3. Our results indicate that although there are cell types in which DRD2 inhibition results in inhibition of STAT3 and self-renewal, the dramatic block in cancer cell proliferation across many cell lines caused by thioridazine treatment is independent of DRD2 inhibition.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Autorrenovação Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Tioridazina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/farmacologia , Antagonistas dos Receptores de Dopamina D2/farmacologia , Humanos , Fator de Transcrição STAT3/metabolismoRESUMO
Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Fator de Transcrição STAT4/metabolismo , Proteínas com Domínio T/metabolismo , Células Th1/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Elementos Reguladores de Transcrição/genética , Fator de Transcrição STAT4/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Células Th1/imunologia , Células Th1/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Ativação TranscricionalRESUMO
Cytosolic DNA can elicit beneficial as well as undesirable immune responses. For example, viral or microbial DNA triggers cell-intrinsic immune responses to defend against infections, whereas aberrant cytosolic accumulation of self-DNA results in pathological conditions, such as autoimmunity. Given the importance of these DNA-provoked responses, a better understanding of their molecular mechanisms is needed. Cytosolic DNA engages stimulator of interferon genes (STING) to activate TANK-binding kinase 1 (TBK1), which subsequently phosphorylates the transcription factor interferon regulatory factor 3 (IRF3) to promote interferon expression. Recent studies have reported that additional transcription factors, including nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6), are also activated by cytosolic DNA, suggesting that cytosolic DNA-induced gene expression is orchestrated by multiple factors. Here we show that cytosolic DNA activates STAT3, another member of the STAT family, via an autocrine mechanism involving interferon ß (IFNß) and IL-6. Additionally, we observed a novel cytosolic DNA-induced phosphorylation at serine 754 in the transactivation domain of STAT3. Upon cytosolic DNA stimulation, Ser754 is directly phosphorylated by TBK1 in a STING-dependent manner. Moreover, Ser754 phosphorylation inhibits cytosolic DNA-induced STAT3 transcriptional activity and selectively reduces STAT3 target genes that are up-regulated in response to cytosolic DNA. Taken together, our results suggest that cytosolic DNA-induced STAT3 activation via IFNß and IL-6 is restrained by Ser754 phosphorylation of STAT3. Our findings reveal a new signaling axis downstream of the cytosolic DNA pathway and suggest potential interactions between innate immune responses and STAT3-driven oncogenic pathways.
Assuntos
DNA/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Comunicação Autócrina , Linhagem Celular , DNA/fisiologia , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Interferon beta , Interleucina-6 , Proteínas de Membrana/metabolismo , FosforilaçãoRESUMO
Idiopathic pneumonia syndrome (IPS) is a noninfectious inflammatory disorder of the lungs that occurs most often after fully myeloablative allogeneic hematopoietic stem cell transplantation (HSCT). IPS can be severe and is associated with high 1-year mortality rates despite existing therapies. The canonical nuclear factor-(NF) κB signaling pathway has previously been linked to several inflammatory disorders of the lung, including asthma and lung allograft rejection. It has never been specifically targeted as a novel IPS treatment approach, however. Here, we report that the IκB kinase 2 (IKK2) antagonist BAY 65-5811 or "compound A," a highly potent and specific inhibitor of the NF-κB pathway, was able to improve median survival times and recipient oxygenation in a well-described mouse model of IPS. Compound A impaired the production of the proinflammatory chemokines CCL2 and CCL5 within the host lung after transplantation. This resulted in significantly lower numbers of donor lung infiltrating CD4+ and CD8+ T cells and reduced pulmonary inflammatory cytokine production after allograft. Compound A's beneficial effects appeared to be specific for limiting pulmonary injury, as the drug was unable to improve outcomes in a B6 into B6D2 haplotype-matched murine HSCT model in which recipient mice succumb to lethal acute graft-versus-host disease of the gastrointestinal tract. Collectively, our data suggest that the targeting of the canonical NF-κB pathway with a small molecule IKK2 antagonist may represent an effective and novel therapy for the specific management of acute lung injury that can occur after allogeneic HSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Quinase I-kappa B/antagonistas & inibidores , Lesão Pulmonar/tratamento farmacológico , Terapia de Alvo Molecular/métodos , NF-kappa B/metabolismo , Pneumonia/tratamento farmacológico , Animais , Lesão Pulmonar/etiologia , Camundongos , Resultado do TratamentoRESUMO
The cellular response to survive or to undergo death is fundamental to the benefit of the organism, and errors in this process can lead to autoimmunity and cancer. The transcription factor nuclear factor κB (NF-κB) functions to block cell death through transcriptional induction of genes encoding anti-apoptotic and antioxidant proteins. This is essential for survival of activated cells of the immune system and for cells undergoing a DNA damage response. In Ras-transformed cells and tumors as well as other cancers, NF-κB functions to suppress apoptosis--a hallmark of cancer. Critical prosurvival roles for inhibitor of NF-κB kinase (IKK) family members, including IKKε and TBK1, have been reported, which are both NF-κB-dependent and -independent. While the roles of NF-κB in promoting cell survival in lymphocytes and in cancers is relatively clear, evidence has been presented that NF-κB can promote cell death in particular contexts. Recently, IKK was shown to play a critical role in the induction of autophagy, a metabolic response typically associated with cell survival but which can lead to cell death. This review provides an historical perspective, along with new findings, regarding the roles of the IKK and NF-κB pathways in regulating cell survival.
Assuntos
Quinase I-kappa B/metabolismo , Imunidade/fisiologia , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Linfócitos B/metabolismo , Morte Celular , Sobrevivência Celular , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Quinase I-kappa B/genética , NF-kappa B/genética , Neoplasias/genética , Transdução de Sinais , Linfócitos T/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.
Assuntos
Quinase I-kappa B/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Feminino , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/genética , Immunoblotting , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Complexos Multiproteicos/genética , Mutação , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Proteína Regulatória Associada a mTOR , Serina/genética , Serina/metabolismo , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Acute graft-versus-host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A(-/-)), an actin-associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins C-C chemokines receptor type 7 (CCR7) and sphingosine 1 phosphate receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF-κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A(-/-) mice were not able to initially traffic to SLT or exit SLT after BM transplantation. However, this alteration did not abrogate the graft-versus-leukemia response. Our data suggest that blocking T-cell migration into and out of SLT is a valid approach to prevent aGvHD.
Assuntos
Transplante de Medula Óssea , Movimento Celular/imunologia , Doença Enxerto-Hospedeiro/imunologia , Proteínas dos Microfilamentos/imunologia , Linfócitos T/imunologia , Doença Aguda , Aloenxertos , Animais , Movimento Celular/genética , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas dos Microfilamentos/isolamento & purificação , NF-kappa B/genética , NF-kappa B/imunologia , Receptores CCR7/genética , Receptores CCR7/imunologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/imunologia , Receptores de Esfingosina-1-Fosfato , Linfócitos T/patologiaRESUMO
Hematopoiesis is a tightly regulated process resulting in the production of blood cells. Self-renewal and differentiation of hematopoietic stem cells (HSCs) are key processes in hematopoietic development. Disruption of these steps can lead to altered cell distribution and disease. To investigate the role of the nuclear factor-κB subunit RelA/p65 in the regulation of HSCs in vivo, we generated mice lacking RelA/p65 in the hematopoietic compartment. Using this model system, we show that loss of p65 severely impairs HSC function and occurs in conjunction with increased hematopoietic stem and progenitor cell cycling, extramedullary hematopoiesis, and differentiation defects. Gene array studies of phenotypic HSCs indicate the up-regulation of genes normally expressed in lineage restricted cells, as well as the down-regulation of genes involved in HSC maintenance and homeostasis. We hypothesize that changes in gene expression in p65-deficient cells lead to decreased self-renewal and differentiation efficiency of hematopoietic stem and progenitor cells. These studies demonstrate that p65 is an important regulator of hematopoiesis through the transcription of genes involved in HSC fate.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Fator de Transcrição RelA/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição RelA/genéticaRESUMO
LZAP has been reported to inhibit cellular proliferation and clonogenic growth. Here, we report that decreased LZAP expression promoted cellular transformation, xenograft tumor growth, and xenograft tumor vascularity. Loss of LZAP also increased cellular invasion, and MMP-9 expression dependent on NF-kappaB. LZAP directly bound to RelA, impaired serine 536 phosphorylation of RelA, increased HDAC association with RelA, inhibited basal and stimulated NF-kappaB transcriptional activity, and was found at the promoter of selective NF-kappaB-responsive genes. LZAP protein levels were markedly decreased in 32% of primary HNSCCs (n = 28) and decreased LZAP levels in primary HNSCC correlated with increased expression of the NF-kappaB-regulated genes IL-8 and IkappaBalpha. In aggregate, these data support a role of LZAP in NF-kappaB regulation and tumor suppression.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , NF-kappa B/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Regulação da Expressão Gênica , Células HeLa , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Histona Desacetilases/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Fator de Transcrição RelA/metabolismo , Transplante Heterólogo , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.
Assuntos
Apoptose , Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Doença Enxerto-Hospedeiro/etiologia , Neoplasias Hematológicas/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transdução de Sinais , Adulto , Idoso , Fator Ativador de Células B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Tamanho Celular , Células Cultivadas , Doença Crônica , Feminino , Citometria de Fluxo , Seguimentos , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Immunoblotting , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Transplante Homólogo , Adulto JovemRESUMO
Dihydroartemisinin (DHA) is an important derivative of the herb medicine Artemisia annua L., used in ancient China. DHA is currently used worldwide to treat malaria by killing malaria-causing parasites. In addition to this prominent effect, DHA is thought to regulate cellular functions, such as angiogenesis, tumor cell growth, and immunity. Nonetheless, how DHA affects T cell function remains poorly understood. We found that DHA potently suppressed Th cell differentiation in vitro. Unexpectedly, however, DHA greatly promoted regulatory T cell (Treg) generation in a manner dependent on the TGF-ßR:Smad signal. In addition, DHA treatment effectively reduced onset of experimental autoimmune encephalomyelitis (EAE) and ameliorated ongoing EAE in mice. Administration of DHA significantly decreased Th but increased Tregs in EAE-inflicted mice, without apparent global immune suppression. Moreover, DHA modulated the mammalian target of rapamycin (mTOR) pathway, because mTOR signal was attenuated in T cells upon DHA treatment. Importantly, enhanced Akt activity neutralized DHA-mediated effects on T cells in an mTOR-dependent fashion. This study therefore reveals a novel immune regulatory function of DHA in reciprocally regulating Th and Treg cell generation through the modulating mTOR pathway. It addresses how DHA regulates immune function and suggests a new type of drug for treating diseases in which mTOR activity is to be tempered.
Assuntos
Artemisininas/farmacologia , Inflamação/prevenção & controle , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/prevenção & controle , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/patologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.