RESUMO
BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Células Dendríticas/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Cabeça e Pescoço/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Hipofracionamento da Dose de Radiação , Tolerância a Radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Subunidade alfa de Receptor de Interleucina-2/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Depleção Linfocítica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Carga Tumoral , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.
Assuntos
Melanoma Experimental/imunologia , Células Supressoras Mieloides/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.
Assuntos
Citoesqueleto de Actina/metabolismo , Burkholderia cenocepacia/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fagossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Células da Medula Óssea/citologia , Feminino , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Células RAW 264.7 , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidoresRESUMO
Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-ß, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.
Assuntos
Interleucina-1/metabolismo , Melanoma/metabolismo , Linfócitos T Reguladores/metabolismo , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/fisiologiaRESUMO
: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.
Assuntos
Proteína Quinase CDC2/metabolismo , Transformação Celular Neoplásica/metabolismo , Melanoma/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação , Ligação Proteica , Transporte Proteico , Transdução de SinaisRESUMO
As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.
Assuntos
Citocinas/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Estabilidade de RNA , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Benzilaminas , Ciclamos , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Células Jurkat , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Linfoma Cutâneo de Células T/patologia , Modelos Biológicos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome de Sézary/patologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus.
Assuntos
Proteínas dos Microfilamentos/metabolismo , NF-kappa B/genética , Neoplasias Pancreáticas/genética , Proteínas/genética , Fator de Transcrição RelA/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Proteínas dos Microfilamentos/genética , NF-kappa B/metabolismo , Sinais de Localização Nuclear/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a Fosfato , Ligação Proteica/genética , Fator de Transcrição RelA/genética , GencitabinaRESUMO
COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.
Assuntos
Citoesqueleto de Actina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Movimento Celular , Cobre/metabolismo , ATPases Transportadoras de Cobre , Citoplasma/metabolismo , Endossomos/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte VesicularRESUMO
CD4(+) T cell recognition of MHC:peptide complexes in the context of a costimulatory signal results in the large-scale redistribution of molecules at the T cell-APC interface to form the immunological synapse. The immunological synapse is the location of sustained TCR signaling and delivery of a subset of effector functions. T cells activated in the absence of costimulation are rendered anergic and are hyporesponsive when presented with Ag in the presence of optimal costimulation. Several previous studies have looked at aspects of immunological synapses formed by anergic T cells, but it remains unclear whether there are differences in the formation or composition of anergic immunological synapses. In this study, we energized primary murine CD4(+) T cells by incubation of costimulation-deficient, transfected fibroblast APCs. Using a combination of TCR, MHC:peptide, and ICAM-1 staining, we found that anergic T cells make mature immunological synapses with characteristic central and peripheral supramolecular activation cluster domains that were indistinguishable from control synapses. There were small increases in total phosphotyrosine at the anergic synapse along with significant decreases in phosphorylated ERK 1/2 accumulation. Most striking, there was specific accumulation of c-Cbl and Cbl-b to the anergic synapses. Cbl-b, previously shown to be essential in anergy induction, was found in both the central and the peripheral supramolecular activation clusters of the anergic synapse. This Cbl-b (and c-Cbl) accumulation at the anergic synapse may play an important role in anergy maintenance, induction, or both.