RESUMO
Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.
Assuntos
Tolerância Imunológica , Neoplasias/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Vacinas Anticâncer/imunologia , Deleção de Genes , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The transcriptional co-activators YAP and TAZ are key regulators of organ size and tissue homeostasis, and their dysregulation contributes to human cancer. Here, we discover YAP/TAZ as bona fide downstream effectors of the alternative Wnt signaling pathway. Wnt5a/b and Wnt3a induce YAP/TAZ activation independent of canonical Wnt/ß-catenin signaling. Mechanistically, we delineate the "alternative Wnt-YAP/TAZ signaling axis" that consists of Wnt-FZD/ROR-Gα12/13-Rho GTPases-Lats1/2 to promote YAP/TAZ activation and TEAD-mediated transcription. YAP/TAZ mediate the biological functions of alternative Wnt signaling, including gene expression, osteogenic differentiation, cell migration, and antagonism of Wnt/ß-catenin signaling. Together, our work establishes YAP/TAZ as critical mediators of alternative Wnt signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Receptores Frizzled/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transativadores , Fatores de Transcrição , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein homologous to angiopoietins. Previous studies suggest that tumor cell-derived ANGPTL2 has tumor-promoting function. Here, we conducted mechanistic analysis comparing ANGPTL2 function in cancer progression in a murine syngeneic model of melanoma and a mouse model of translocation renal cell carcinoma (tRCC). ANGPTL2 deficiency in tumor cells slowed tRCC progression, supporting a tumor-promoting role. However, systemic ablation of ANGPTL2 accelerated tRCC progression, supporting a tumor-suppressing role. The syngeneic model also demonstrated a tumor-suppressing role of ANGPTL2 in host tumor microenvironmental cells. Furthermore, the syngeneic model showed that PDGFRα+ fibroblasts in the tumor microenvironment express abundant ANGPTL2 and contribute to tumor suppression. Moreover, host ANGPTL2 facilitates CD8+ T-cell cross-priming and enhances anti-tumor immune responses. Importantly, ANGPTL2 activates dendritic cells through PIR-B-NOTCH signaling and enhances tumor vaccine efficacy. Our study provides strong evidence that ANGPTL2 can function in either tumor promotion or suppression, depending on what cell type it is expressed in.
Assuntos
Proteínas Semelhantes a Angiopoietina/genética , Proteínas Semelhantes a Angiopoietina/metabolismo , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Renais/fisiopatologia , Progressão da Doença , Melanoma/fisiopatologia , Transdução de Sinais , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/deficiência , Proteínas Semelhantes a Angiopoietina/imunologia , Animais , Vacinas Anticâncer/imunologia , Carcinoma de Células Renais/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Melanoma/imunologia , Camundongos , Transdução de Sinais/genética , Células Estromais/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Although cells are frequently maintained at cold temperatures during experiments, the effects of cold stress on cell viability and subsequent cellular conditions remain elusive. In this study, we investigated the effects of cold stress on cancer cells under various culture conditions. We showed that cold stress induces ferroptosis, a form of cell death characterized by lipid peroxidation, in sensitive cancer cell lines. High cell density and serum starvation activate the Hippo pathway and suppress cold-induced cell death. Genetic deletion of Hippo pathway components enhances cold stress susceptibility. Furthermore, the cell attachment status influences the response to cold stress, with suspended cells showing greater resistance and faster recovery than attached cells. This study highlights the importance of cellular conditions and the Hippo pathway in the handling and storage of cancer cells at cold temperatures, thereby offering insights into experimental and clinical contexts.
Assuntos
Adesão Celular , Resposta ao Choque Frio , Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases , Humanos , Resposta ao Choque Frio/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Linhagem Celular Tumoral , Animais , Ferroptose , Camundongos , Sobrevivência Celular , Temperatura BaixaRESUMO
Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.
Assuntos
Adjuvantes Imunológicos , Ouro , Nanopartículas Metálicas , Oligodesoxirribonucleotídeos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Animais , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Camundongos , Açúcares/química , Humanos , Camundongos Endogâmicos C57BLRESUMO
Mammalian cells are surrounded by neighbouring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes1. The Hippo pathway effectors YAP (also known as YAP1) and TAZ (also known as WWTR1) are regulated by mechanical cues and mediate cellular responses to ECM stiffness2,3. Here we identified the Ras-related GTPase RAP2 as a key intracellular signal transducer that relays ECM rigidity signals to control mechanosensitive cellular activities through YAP and TAZ. RAP2 is activated by low ECM stiffness, and deletion of RAP2 blocks the regulation of YAP and TAZ by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, which activates RAP2 through PDZGEF1 and PDZGEF2 (also known as RAPGEF2 and RAPGEF6). At low stiffness, active RAP2 binds to and stimulates MAP4K4, MAP4K6, MAP4K7 and ARHGAP29, resulting in activation of LATS1 and LATS2 and inhibition of YAP and TAZ. RAP2, YAP and TAZ have pivotal roles in mechanoregulated transcription, as deletion of YAP and TAZ abolishes the ECM stiffness-responsive transcriptome. Our findings show that RAP2 is a molecular switch in mechanotransduction, thereby defining a mechanosignalling pathway from ECM stiffness to the nucleus.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transformação Celular Neoplásica , Matriz Extracelular/química , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Quinases do Centro Germinativo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Transcriptoma , Proteínas de Sinalização YAP , Proteínas rap de Ligação ao GTP/genéticaRESUMO
The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell-cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Drosophila melanogaster/enzimologia , Humanos , Fatores de Transcrição/genéticaRESUMO
Accumulating evidence suggests an association between iron metabolism and lung cancer progression. In biological systems, iron is present in either reduced (Fe2+ ; ferrous) or oxidized (Fe3+ ; ferric) states. However, ferrous and ferric iron exhibit distinct chemical and biological properties, the role of ferrous and ferric iron in lung cancer cell growth has not been clearly distinguished. In this study, we manipulated the balance between cellular ferrous and ferric iron status by inducing gene mutations involving the FBXL5-IRP2 axis, a ubiquitin-dependent regulatory system for cellular iron homeostasis, and determined its effects on lung cancer cell growth. FBXL5 depletion (ferrous iron accumulation) was found to suppress lung cancer cell growth, whereas IRP2 depletion (ferric iron accumulation) did not suppress such growth, suggesting that ferrous iron but not ferric iron plays a suppressive role in cell growth. Mechanistically, the depletion of FBXL5 impaired the degradation of the cyclin-dependent kinase inhibitor, p27, resulting in a delay in the cell cycle at the G1/S phase. FBXL5 depletion in lung cancer cells also improved the survival of tumor-bearing mice. Overall, this study highlights the important function of ferrous iron in cell cycle progression and lung cancer cell growth.
Assuntos
Proteínas F-Box , Neoplasias Pulmonares , Animais , Camundongos , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Neoplasias Pulmonares/genética , Ferro/metabolismo , Ubiquitina/metabolismo , Compostos Férricos , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMO
YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivation impairs tissue development and regeneration. Dynamic and precise control of YAP/TAZ activity is thus important to ensure proper physiological regulation and homeostasis of the cells. Here, we show that YAP/TAZ activation results in activation of their negative regulators, LATS1/2 (large tumor suppressor 1/2) kinases, to constitute a negative feedback loop of the Hippo pathway in both cultured cells and mouse tissues. YAP/TAZ in complex with the transcription factor TEAD (TEA domain family member) directly induce LATS2 expression. Furthermore, YAP/TAZ also stimulate the kinase activity of LATS1/2 through inducing NF2 (neurofibromin 2). This feedback regulation is responsible for the transient activation of YAP upon lysophosphatidic acid (LPA) stimulation and the inhibition of YAP-induced cell migration. Thus, this LATS-mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP/TAZ regulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retroalimentação Fisiológica/fisiologia , Homeostase/fisiologia , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Movimento Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/fisiologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Via de Sinalização Hippo , Homeostase/genética , Humanos , Fígado/metabolismo , Masculino , Camundongos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E2 (PGE2). Although inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is reported to lead to PGE2 accumulation, the role of 15-PGDH expression in the tumor microenvironment remains unclear. We utilized Panc02 murine pancreatic cancer cells for orthotopic transplantation into wild-type and 15-pgdh+/- mice and found that 15-pgdh depletion in the tumor microenvironment leads to enhanced tumorigenesis accompanied by an increase in cancer-associated fibroblasts (CAFs) and the promotion of fibrosis. The fibrotic tumor microenvironment is widely considered to be hypovascular; however, we found that the angiogenesis level is maintained in 15-pgdh+/- mice, and these changes were also observed in a genetically engineered PDAC mouse model. Further confirmation revealed that fibroblast growth factor 1 (FGF1) is secreted by pancreatic cancer cells after PGE2 stimulation, consequently promoting CAF proliferation and vascular endothelial growth factor A (VEGFA) expression in the tumor microenvironment. Finally, in 15-pgdh+/- Acta2-TK mice, depletion of fibroblasts inhibited angiogenesis and cancer cell viability in orthotopically transplanted tumors. These findings highlighted the role of 15-pgdh downregulation in enhancing PGE2 accumulation in the pancreatic tumor microenvironment and in subsequently maintaining the angiogenesis level in fibrotic tumors along with CAF expansion.
Assuntos
Neoplasias Pancreáticas , Fator A de Crescimento do Endotélio Vascular , Animais , Ácido Araquidônico , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Fator 1 de Crescimento de Fibroblastos , Fibrose , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Camundongos , Neoplasias Pancreáticas/genética , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias PancreáticasRESUMO
Coordination of growth and genomic stability is critical for normal cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player in maintenance of genomic stability, its role in growth signaling remains elusive. Here, we show that BRCA1 facilitates stabilization of YAP1 protein and turning "off" the Hippo pathway through ubiquitination of NF2. In BRCA1-deficient cells Hippo pathway is "turned On." Phosphorylation of YAP1 is crucial for this signaling process because a YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 kinase recognition sites not only resists degradation but also rescues YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, an ectopic expression of the active Mt-YAP5SA, but not inactive Mt-YAP6SA, promotes EGF-independent proliferation and tumorigenesis in BRCA1-/- mammary epithelial cells. These findings establish an important role of BRCA1 in regulating stability of YAP1 protein that correlates positively with cell proliferation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Via de Sinalização Hippo , Humanos , Mutação de Sentido Incorreto , Neurofibromina 2/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAPRESUMO
The Hippo intracellular signaling pathway plays a pivotal role in cell fate determination. Although previous studies have identified many components of the Hippo pathway, the whole picture of the Hippo network is just beginning to be delineated. Recent discoveries in the past decade have shed light on a newly discovered signaling network where the Hippo pathway interplays with several types of non-coding RNAs, including microRNAs, long non-coding RNAs and circular RNAs, to mediate diverse biological processes. Those non-coding RNAs communicate with each other to maintain cellular homeostasis. In this review article, we summarize the current and emerging understanding of the roles of non-coding RNAs in the regulation of and by the Hippo pathway.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , RNA não Traduzido/metabolismo , Animais , Via de Sinalização Hippo , Humanos , Transdução de SinaisRESUMO
Loss of tumor suppressor adenomatous polyposis coli (APC) activates ß-catenin to initiate colorectal tumorigenesis. However, ß-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Receptor gp130 de Citocina/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptor gp130 de Citocina/genética , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.
Assuntos
Adenina/análogos & derivados , Adjuvantes Imunológicos/farmacologia , Ouro/química , Manose/química , Nanopartículas Metálicas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia , Esplenomegalia/prevenção & controle , Receptor 7 Toll-Like/agonistas , Adenina/química , Adenina/farmacologia , Adjuvantes Imunológicos/química , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunização , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ligantes , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Bibliotecas de Moléculas Pequenas/química , Esplenomegalia/imunologia , Esplenomegalia/patologia , Receptor 7 Toll-Like/imunologiaRESUMO
FBXL5 (F-box and leucine-rich repeat protein 5) is the F-box protein subunit of, and therefore responsible for substrate recognition by, the SCF(FBXL5) ubiquitin-ligase complex, which targets iron regulatory protein 2 (IRP2) for proteasomal degradation. IRP2 plays a central role in the maintenance of cellular iron homeostasis in mammals through posttranscriptional regulation of proteins that contribute to control of the intracellular iron concentration. The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo, given that mice lacking FBXL5 die during early embryogenesis as a result of unrestrained IRP2 activity and oxidative stress attributable to excessive iron accumulation. Despite its pivotal role in the control of iron homeostasis, however, little is known of the upstream regulation of FBXL5 activity. We now show that FBXL5 undergoes constitutive ubiquitin-dependent degradation at the steady state. With the use of a proteomics approach to the discovery of proteins that regulate the stability of FBXL5, we identified the large HECT-type ubiquitin ligase HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) as an FBXL5-associated protein. Inhibition of the HERC2-FBXL5 interaction or depletion of endogenous HERC2 by RNA interference resulted in the stabilization of FBXL5 and a consequent increase in its abundance. Such accumulation of FBXL5 in turn led to a decrease in the intracellular content of ferrous iron. Our results thus suggest that HERC2 regulates the basal turnover of FBXL5, and that this ubiquitin-dependent degradation pathway contributes to the control of mammalian iron metabolism.
Assuntos
Proteínas F-Box/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ferro/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Bases , Primers do DNA , Células HEK293 , Células HeLa , Humanos , Hidrólise , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína LigaseRESUMO
The Hippo pathway is a central regulator of tissue growth that has been widely studied in mammalian organ development, regeneration, and cancer biology. Although previous studies have convincingly revealed its cell-autonomous functions in controlling cell fate, such as cell proliferation, survival, and differentiation, accumulating evidence in recent years has revealed its non-cell-autonomous functions. This pathway regulates cell-cell communication through direct interactions, soluble factors, extracellular vesicles, and the extracellular matrix, providing a range of options for controlling diverse biological processes. Consequently, the Hippo pathway not only dictates the fate of individual cells but also triggers multicellular responses involving both tissue-resident cells and infiltrating immune cells. Here, we have highlighted the recent understanding of the molecular mechanisms by which the Hippo pathway controls cell-cell communication and discuss its importance in tissue homeostasis, especially in development and regeneration.
RESUMO
Use of immune checkpoint inhibitors (ICIs) as cancer immunotherapy has advanced rapidly in the clinic. We recently reported that tumor stroma-derived angiopoietin-like protein 2 (ANGPTL2) has tumor suppressive activity by enhancing dendritic cell-mediated CD8+ T cell anti-tumor immune responses. However, a direct impact of ANGPTL2 on ICI anti-tumor effect remains unclear. Here, we use a murine syngeneic model to show that host ANGPTL2 facilitates CD8+ T cell cross-priming and contributes to anti-tumor responses to ICIs in this context. Importantly, our analysis of public datasets indicated that ANGPTL2 expression is associated with positive responses to ICI therapy by human melanoma patients. We conclude that ANGPTL2-mediated stromal cell crosstalk facilitates anti-tumor immunity and ICI responsiveness. These findings overall provide novel insight into ANGPTL2 anti-tumor function and regulation of ICI-induced anti-tumor immunity.
Assuntos
Proteína 2 Semelhante a Angiopoietina , Inibidores de Checkpoint Imunológico , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Humanos , Proteínas Semelhantes a Angiopoietina/metabolismo , Proteínas Semelhantes a Angiopoietina/genética , Células Estromais/metabolismo , Células Estromais/imunologia , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Microambiente Tumoral/imunologia , Linhagem Celular Tumoral , Feminino , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/genéticaRESUMO
Although elucidation of enzyme-substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases.
Assuntos
Proteínas F-Box/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ubiquitinadas/metabolismo , Animais , Proteínas F-Box/isolamento & purificação , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Ligação Proteica , Proteólise , Proteômica , Proteínas Ubiquitinadas/isolamento & purificação , UbiquitinaçãoRESUMO
Fusobacterium nucleatum, found in the oral cavity, influences the progression of gastrointestinal cancers. Additionally, our previous results suggested that F. nucleatum is associated with poor patient prognosis in esophageal squamous cell carcinoma (ESCC). However, the mechanism by which F. nucleatum affects aggressive tumor behavior has yet to be elucidated. We have conducted this clinical, in vitro, and in vivo study to clarify the mechanism of ESCC progression induced by F. nucleatum. Transmission electron microscopy revealed that F. nucleatum invaded and occupied ESCC cells and impacted gene and protein expression. Comprehensive mRNA expression and pathway enrichment analyses of F. nucleatum-treated ESCC cells identified the "NF-κB" and "NOD-like receptor" signaling pathways as enriched. We confirmed the relationship between the presence of F. nucleatum and NF-κB activation in resected ESCC tissues. Furthermore, F. nucleatum-treated ESCC cells demonstrated enhanced growth ability, and NF-κB activation, as well as overexpression of NOD1 and phosphorylated RIPK2. Furthermore, treated cells showed accelerated tumor growth, with NF-κB activation in xenograft models. F. nucleatum invaded ESCC cells and induced the NF-κB pathway through the NOD1/RIPK2 pathway, leading to tumor progression.