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Arginase1 (ARG1) is a metabolic enzyme that is highly expressed in tumor-associated myeloid-derived suppressor cells (MDSCs) and causes the dysfunction of tumor-reactive T cells. Here, we present a protocol for detecting ARG1 expression in tumor MDSCs from a murine model of colon cancer using flow cytometry. We describe steps for tumor tissue processing, antibody staining, and data acquisition. We then detail procedures for identifying MDSC subsets and detecting ARG1 expression using a precise gating strategy. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.
RESUMEN
Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.
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Antígenos B7 , Linfocitos T CD8-positivos , Glicoproteínas de Membrana , Microambiente Tumoral , Animales , Humanos , Ratones , Antígenos B7/inmunología , Antígenos B7/genética , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/genética , Proteínas de la Membrana , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso , Microambiente Tumoral/inmunologíaRESUMEN
Myeloid-derived suppressor cells (MDSCs) impair antitumor immune responses. Identifying regulatory circuits during MDSC development may bring new opportunities for therapeutic interventions. We report that the V-domain suppressor of T cell activation (VISTA) functions as a key enabler of MDSC differentiation. VISTA deficiency reduced STAT3 activation and STAT3-dependent production of polyamines, which causally impaired mitochondrial respiration and MDSC expansion. In both mixed bone marrow (BM) chimera mice and myeloid-specific VISTA conditional knockout mice, VISTA deficiency significantly reduced tumor-associated MDSCs but expanded monocyte-derived dendritic cells (DCs) and enhanced T cell-mediated tumor control. Correlated expression of VISTA and arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival. Taken together, these findings unveil the VISTA/polyamine axis as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy.
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Células Supresoras de Origen Mieloide , Neoplasias , Animales , Humanos , Ratones , Ratones Noqueados , Células Mieloides/metabolismo , Neoplasias/patología , Poliaminas/metabolismo , Factor de Transcripción STAT3/metabolismo , Linfocitos TRESUMEN
The V-domain Ig suppressor of T-cell activation (VISTA) has been recognized as a critical negative regulator of antitumor immune response and is gaining growing interest as a potential pharmacological target in immunotherapy. This molecule is highly expressed in hematopoietic stem cells and myeloid compartment, and it has been found upmodulated in acute myeloid leukemia (AML). However, VISTA-associated immune features are relatively unexplored in myeloid malignancies. Herein, we aimed to explore whether this immune checkpoint regulator could play a role in the generation of an immune escape environment in AML patients. We characterized VISTA mRNA expression levels in leukemia cell lines and in large publicly available cohorts of specimens from bone marrow of healthy individuals and AML patients at diagnosis by deploying bulk and single-cell RNA sequencing. We also defined the correlations with leukemia-associated burden using results of whole-exome sequencing of AML samples at disease onset. We showed that VISTA expression linearly increased across the myeloid differentiation tree in normal hematopoiesis. Accordingly, its transcript was highly enriched in AML cell lines as well as in AML patients at diagnosis presenting with myelomonocytic and monocytic differentiation. A strong correlation was seen with NPM1 mutations regardless of the presence of FLT3 lesions. Furthermore, VISTA expression levels at baseline correlated with disease recurrence in patients with normal karyotype and NPM1 mutations, a subgroup traditionally considered as favorable according to current diagnostic schemes. Indeed, when compared to patients with long-term remission (>5 years after standard chemotherapy regimens), cases relapsing within 2 years from diagnosis had increased VISTA expression in both leukemia and T cells. Our results suggest a rationale for developing VISTA-targeted therapeutic strategies to treat molecularly defined subgroups of AML patients to prevent disease recurrence and treatment resistance.
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Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Pronóstico , Mutación , Nucleofosmina , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.
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Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/genética , Antígeno de Maduración de Linfocitos B/genética , Linfoma de Células del Manto/terapia , Mieloma Múltiple/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/inmunología , Antígeno de Maduración de Linfocitos B/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Activación de Linfocitos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/inmunología , Linfoma de Células del Manto/patología , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/inmunología , Masculino , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Unión Proteica , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/trasplante , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinß1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinß1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.
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Replicación del ADN , Matriz Extracelular/metabolismo , Lamina Tipo B/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Western Blotting , Línea Celular , Ensayo Cometa , Reparación del ADN , Matriz Extracelular/enzimología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.
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Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Células Cultivadas , Citocinas/biosíntesis , Citotoxicidad Inmunológica/genética , Granzimas/metabolismo , Humanos , Melanoma Experimental , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Proto-Oncogénicas c-rel/genéticaRESUMEN
Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin's lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Receptor del Factor Activador de Células B , Linfoma de Células del Manto , Animales , Apoptosis , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/genética , Humanos , Linfoma de Células del Manto/tratamiento farmacológico , Ratones , Recurrencia Local de NeoplasiaRESUMEN
Drug-resistant acute lymphoblastic leukemia (ALL) patients do not respond to standard chemotherapy, and an urgent need exists to develop new treatment strategies. Our study exploited the presence of B-cell activating factor receptor (BAFF-R) on the surface of drug-resistant B-ALL cells as a therapeutic target. We used anti-BAFF-R (VAY736), optimized for natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), to kill drug-resistant ALL cells. VAY736 antibody and NK cell treatments significantly decreased ALL disease burden and provided survival benefit in vivo However, if the disease was advanced, the ADCC efficacy of NK cells was inhibited by microenvironmental transforming growth factor-beta (TGFß). Inhibiting TGFß signaling in NK cells using the TGFß receptor 1 (R1) inhibitor (EW-7197) significantly enhanced VAY736-induced NK cell-mediated ALL killing. Our results highlight the potential of using a combination of VAY736 antibody with EW-7197 to treat advance-stage, drug-resistant B-ALL patients.
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Compuestos de Anilina/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Triazoles/farmacología , Adulto , Animales , Apoptosis , Receptor del Factor Activador de Células B/antagonistas & inhibidores , Proliferación Celular , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dietary lectins are carbohydrate-binding proteins found in food sources. We used a panel of seven dietary lectins to analyze cytotoxicity against hematological cancers. Wheat germ agglutinin (WGA), even at low doses, demonstrated maximum toxicity toward acute myeloid leukemia (AML) cells. Using AML cell lines, we show time- and dose-dependent killing by WGA. We also show that low doses of WGA kills primary patient AML cells, irrespective of subtype, with no significant toxicity to normal cells. WGA caused AML cell agglutination, but failed to agglutinate RBC's at this dose. WGA, primarily, binds to N-acetyl-D-glucosamine (GlcNAc) and is also reported to interact with sialic-acid-containing glycoconjugates and oligosaccharides. After neuraminidase pre-treatment, which catalyzes the hydrolysis of terminal sialic acid residues, AML cells were less sensitive to WGA-induced cell death. AML cells were also not sensitive to succinyl-WGA, which does not react with sialic acid. Incubation with LEL lectin, which recognizes GlcNAc or SNA, which binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree α-2,3 linkage, did not alter AML cell viability. These data indicate that WGA-induced AML cell death is dependent on both GlcNAc binding and interaction with sialic acids. We did not observe any in vitro or in vivo toxicity of WGA toward normal cells at the concentrations tested. Finally, low doses of WGA injection demonstrated significant in vivo toxicity toward AML cells, using xenograft mouse model. Thus, WGA is a potential candidate for leukemia therapy.
RESUMEN
Breast cancer is the second leading cause of death among women in the US. Targeted therapies exist, however resistance is common and patients resort to chemotherapy. Chemotherapy is also a main treatment for triple negative breast cancer (TNBC) patients; while radiation is delivered to patients with advanced disease to counteract metastasis. Yet, resistance to both chemo- and radiotherapy is still frequent, highlighting a need to provide novel sensitizers. We discovered that MT1-MMP modulates DNA damage responses (DDR) in breast cancer. MT1-MMP expression inversely correlates to chemotherapy response of breast cancer patients. Inhibition of MT1-MMP sensitizes TNBC cells to IR and doxorubicin in vitro, and in vivo in an orthotopic breast cancer model. Specifically, depletion of MT1-MMP causes stalling of replication forks and Double Strand Breaks (DBSs), leading to increased sensitivity to additional genotoxic stresses. These effects are mediated by integrinß1, as a constitutive active integrinß1 reverts replication defects and protects cells depleted of MT1-MMP from IR and chemotherapy. These data highlight a novel DNA damage response triggered by MT1-MMP-integrinß1 and provide a new point of therapeutic targeting that may improve breast cancer patient outcomes.
Asunto(s)
Neoplasias de la Mama/terapia , Resistencia a Antineoplásicos , Metaloproteinasa 14 de la Matriz/metabolismo , Tolerancia a Radiación , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Daño del ADN , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina beta1/metabolismo , Células MCF-7 , Ratones , Trasplante de Neoplasias , Regulación hacia ArribaRESUMEN
Treatment for acute myeloid leukemia (AML) has remained unchanged for past 40 years. Targeting cell metabolism is a promising avenue for future cancer therapy. We found that enzymes involved in metabolic hexosamine biosynthetic pathway (HBP) are increased in patients with AML. Inhibiting GFAT (the rate-limiting enzyme of HBP) induced differentiation and apoptosis in AML cells, sparing normal cells. UDP-GlcNAc, the end product of HBP, is the substrate for O-GlcNAcylation, a posttranslational modification. O-GlcNAc transferase (OGT) is the enzyme which transfers GlcNAc from UDP-GlcNAc to target proteins. Inhibition of O-GlcNAcylation, using OGT inhibitors as well as genetic knockdown of OGT, also led to cell differentiation and apoptosis of AML cells. Finally, HBP inhibition in vivo reduced the tumor growth in a subcutaneous AML xenograft model and tumor cells showed signs of differentiation in vivo A circulating AML xenograft model also showed clearance of tumor load in bone marrow, spleen, and blood, after HBP inhibition, with no signs of general toxicity. This study reveals an important role of HBP/O-GlcNAcylation in keeping AML cells in an undifferentiated state and sheds light into a new area of potential AML therapy by HBP/O-GlcNAc inhibition. Mol Cancer Ther; 17(10); 2226-37. ©2018 AACR.
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Vías Biosintéticas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Hexosaminas/biosíntesis , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite significant advances in melanoma therapy, melanoma remains the deadliest form of skin cancer, with a 5-year survival rate of only 15%. Thus, novel treatments are required to address this disease. Notch and ERBB are evolutionarily conserved signaling cascades required for the maintenance of melanocyte precursors. We show that active Notch1 (Notch1(NIC)) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly and are similarly expressed in both mutated and wild-type BRAF melanomas, suggesting these receptors are co-reactivated in melanoma to promote survival. Whereas blocking either pathway triggers modest effects, combining a ?-secretase inhibitor to block Notch activation and a tyrosine kinase inhibitor to inhibit ERBB3/2 elicits synergistic effects, reducing cell viability by 90% and hampering melanoma tumor growth. Specific inhibition of Notch1 and ERBB3 mimics these results, suggesting these are the critical factors triggering melanoma tumor expansion. Notch and ERBB inhibition blunts AKT and NF?B signaling. Constitutive expression of NF?B partially rescues cell death. Blockade of both Notch and ERBB signaling inhibits the slow cycling JARID1B-positive cell population, which is critical for long-term maintenance of melanoma growth. We propose that blocking these pathways is an effective approach to treatment of melanoma patients regardless of whether they carry mutated or wild-type BRAF.
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Antineoplásicos/uso terapéutico , Melanoma/genética , Receptor ErbB-3/genética , Receptor Notch1/genética , Neoplasias Cutáneas/genética , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Terapia Molecular Dirigida/métodos , Fosforilación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Metastatic melanoma is the deadliest of all skin cancers. Despite progress in diagnostics and treatment of melanoma, the prognosis for metastatic patients remains poor. We previously showed that Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is one of the drivers of melanoma metastasis. Classically, MT1-MMP regulates a verity of cellular functions including cell-to-cell interaction and cell-to-matrix communication. Recently, MT1-MMP has been found to also modulate gene expression. To specifically assess MT1-MMP dependent gene regulation in melanoma, microarray gene expression analysis was performed in a melanoma cell line whose metastatic properties depend on the activity of MT1-MMP. We identified the tumor suppressor gene SPRY4 as a new transcriptional target of MT1-MMP that is negatively regulated by the protease. Knockdown of MT1-MMP enhances SPRY4 expression at the mRNA and protein level. SPRY4 expression inversely correlates with that of MT1-MMP in melanoma samples and importantly, correlates with melanoma patient survival. SPRY4 modulates MT1-MMP dependent cell migration such that inhibition of SPRY4 rescues cell migration that has been impaired by MT1-MMP knock down. MT1-MMP decreases SPRY4 in part through an MMP2/RAC1 axis we previously show promotes cell motility downstream of MT1-MMP. These results identify the tumor suppressor SPRY4 as a novel molecular effector of MT1-MMP affecting melanoma cell motility.
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Movimiento Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Melanoma/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Cutáneas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/enzimología , Melanoma/genética , Melanoma/metabolismo , Invasividad Neoplásica , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
Notch1 is an evolutionarily conserved transmembrane receptor involved in melanoma growth. Notch1 is first cleaved by furin in the Golgi apparatus to produce the biologically active heterodimer. Following ligand binding, Notch1 is cleaved at the cell membrane by proteases such as ADAM10 and -17 and membrane type 1 matrix metalloproteinase (MT1-MMP), the latter of which we recently identified as a novel protease involved in Notch1 processing. The final cleavage is γ-secretase dependent and releases the active Notch intracellular domain (NIC). We now demonstrate that Notch1 directly regulates furin expression. Aside from activating Notch1, furin cleaves and activates several proteases, including MT1-MMP, ADAM10, and ADAM17. By chromatin immunoprecipitation and a reporter assay, we demonstrate that Notch1 binds at position -1236 of the furin promoter and drives furin expression. The Notch1-dependent enhancement of furin expression increases the activities of MT1-MMP and ADAM10 but not that of ADAM17, as demonstrated by short hairpin RNA (shRNA) knockdown of furin, and promotes the cleavage of Notch1 itself. These data highlight a novel positive-feedback loop whereby Notch1-dependent furin expression can induce Notch1 signaling by increasing Notch1 processing and by potentiating the activity of the proteases responsible for Notch1 activation. This leads to Notch1 signal amplification, which can promote melanoma tumor growth and progression, as demonstrated by the inhibition of cell migration and invasion upon furin inhibition downstream of Notch1. Disruption of such feedback signaling might represent an avenue for the treatment of melanoma.
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Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Furina/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Activación Transcripcional , Proteína ADAM10 , Proteína ADAM17 , Línea Celular Tumoral , Movimiento Celular , Activación Enzimática , Furina/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Regiones Promotoras GenéticasRESUMEN
Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1-MMP is a membrane-associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We show that deregulation of MT1-MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1-MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. We demonstrate that MT1-MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1-MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1-MMP is required to sustain RAC1 activity and promote MT1-MMP-dependent cell motility. These data highlight a novel MT1-MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.
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Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Melanoma/enzimología , Melanoma/patología , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Proteína de Unión al GTP rac1/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Activación Enzimática , Humanos , Masculino , Ratones , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Transducción de Señal , Resultado del TratamientoRESUMEN
We recently identified neuregulin-1 (NRG1) as a novel target of Notch1 required in Notch-dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1-dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1-ERBB3-ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.
Asunto(s)
Melanoma/metabolismo , Melanoma/patología , Neurregulina-1/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transducción de Señal , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Fosforilación , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-4RESUMEN
BACKGROUND: Adenoviruses force quiescent cells to re-enter the cell cycle to replicate their DNA, and for the most part, this is accomplished after they express the E1A protein immediately after infection. In this context, E1A is believed to inactivate cellular proteins (e.g., p130) that are known to be involved in the silencing of E2F-dependent genes that are required for cell cycle entry. However, the potential perturbation of these types of genes by E1A relative to their functions in regulatory networks and canonical pathways remains poorly understood. FINDINGS: We have used DNA microarrays analyzed with Bayesian ANOVA for microarray (BAM) to assess changes in gene expression after E1A alone was introduced into quiescent cells from a regulated promoter. Approximately 2,401 genes were significantly modulated by E1A, and of these, 385 and 1033 met the criteria for generating networks and functional and canonical pathway analysis respectively, as determined by using Ingenuity Pathway Analysis software. After focusing on the highest-ranking cellular processes and regulatory networks that were responsive to E1A in quiescent cells, we observed that many of the up-regulated genes were associated with DNA replication, the cell cycle and cellular compromise. We also identified a cadre of up regulated genes with no previous connection to E1A; including genes that encode components of global DNA repair systems and DNA damage checkpoints. Among the down-regulated genes, we found that many were involved in cell signalling, cell movement, and cellular proliferation. Remarkably, a subset of these was also associated with p53-independent apoptosis, and the putative suppression of this pathway may be necessary in the viral life cycle until sufficient progeny have been produced. CONCLUSIONS: These studies have identified for the first time a large number of genes that are relevant to E1A's activities in promoting quiescent cells to re-enter the cell cycle in order to create an optimum environment for adenoviral replication.
Asunto(s)
Proteína MioD/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Ratones , Mioblastos Esqueléticos/citología , Factor 5 Regulador Miogénico/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
MyoD is a transcriptional factor that is required for the differentiation of muscle stem cells (satellite cells). In this study, we describe a previously unknown function for MyoD in regulating a gene (Cdc6) that is vital to endowing chromatin with the capability of replicating DNA. In C2C12 and primary mouse myoblasts, we show that MyoD can occupy an E-box within the promoter of Cdc6 and that this association, along with E2F3a, is required for its activity. MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6. Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase. Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.