RESUMEN
Peptides with dual binding specificity for classical HLA class I and non-classical HLA-E molecules have been identified in virus-encoded proteins, but not in cellular proteins from normal or neoplastic cells. Expression screening of a melanoma cDNA library with a CTL clone recognizing an HLA-A2-restricted tumor-specific epitope encoded by mutant peroxiredoxin 5 (Prdx5), a stress-inducible peroxidase, led to the identification of two alternatively spliced isoforms of the same gene. These isoforms, which lack the catalytic cysteine fundamental for enzymatic activity, showed widespread expression in neoplastic and normal tissues but were unstable at the protein level, being detectable, following transient transfection, only after lactacystin treatment to inhibit proteasomal degradation. Isoform-specific sequences which formed, respectively, as result of exon 1 splicing to either exon 3 or 4, encoded two distinct nonapeptides (AMAPIKTHL and AMAPIKVRL, not present in the full-length protein) with anchor residues for HLA-A2 and HLA-E molecules and able to stabilize HLA-A2 and HLA-E cell surface expression. HLA-E+ targets, loaded with these peptides, were not recognized by NK cells expressing CD94/NKG2A inhibitory or CD94/NKG2C activatory receptors. However, both peptides were recognized, although with low avidity, by HLA-E-restricted CD8+ CTL. The nonapeptide AMAPIKVRL was used to elicit HLA-A2-restricted CTL clones that killed peptide-pulsed lymphoblastoid cell lines and melanoma cells expressing the corresponding Prdx5 isoform. Our results suggest that alternatively spliced isoforms of Prdx5, through the generation of HLA-E- and HLA-A2-restricted peptides may be part of immune-mediated stress response contributing to the detection and elimination of damaged normal or neoplastic cells.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos HLA/metabolismo , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Peroxirredoxinas/metabolismo , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antioxidantes/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Células Clonales , Cisteína/deficiencia , Humanos , Inmunidad Celular , Células Asesinas Naturales/metabolismo , Melanoma/genética , Melanoma/metabolismo , Estrés Oxidativo/inmunología , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/inmunología , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estabilidad Proteica/efectos de los fármacos , Eliminación de Secuencia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Antígenos HLA-ERESUMEN
Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front.
RESUMEN
Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+ EZH2+ melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Transición Epitelial-Mesenquimal/genética , Melanoma/genética , Factores de Transcripción NFATC/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Femenino , Proteína Forkhead Box M1/genética , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen/fisiología , Humanos , Melanoma/patología , Ratones , Ratones SCID , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-myc/genética , Regulación hacia Arriba/genéticaRESUMEN
Human melanomas can express unique tumor antigens, resulting from mutated proteins, and shared epitopes encoded for by normal genes, but these two classes of antigens have not been previously compared for immunogenicity and retention in metastatic cells. Here, we identified a new unique antigen generated by a point mutation in the peroxiredoxin 5 (Prdx5) gene in an HLA-A*0201(+) human metastatic melanoma lacking the wild-type allele. An antioxidant assay, with recombinant Prdx5 proteins, and evaluation of peroxide accumulation in transiently transfected cells, indicated that the mutant protein retained its enzymatic activity. The mutation in the Prdx5 protein did not generate a new HLA agretope but yielded an HLA-A*0201-restricted T cell epitope (Prdx5(110-119)). By HLA-tetramer analysis, in a tumor-invaded lymph node, >50% of mutant Prdx5-specific CD8(+) T cells (frequency 0.37%/CD8(+)) showed a CCR7(+/-) CD45RA(-) "T(CM)" or "T(EM)" phenotype, as found in Melan-A/MART-1-specific T cells (frequency 0.68%/CD8(+)) in the same tissue. In agreement with their memory phenotype, the Prdx5-specific T cells readily expanded in vitro in mixed lymphocyte-tumor culture, as did the Melan-/MART-1-specific T cells. By immunohistochemistry of the invaded lymph node, the mutant Prdx5 protein was expressed in all neoplastic cells, in contrast with the heterogeneous expression of shared antigens as Melan-A/MART-1, gp100 and tyrosinase. Thus, a unique tumor antigen can be as immunogenic as the melanoma differentiation antigens but, in contrast to the latter, may be retained in all metastatic cells possibly as result of the relevant cellular function exerted by the mutated protein.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/enzimología , Melanoma/inmunología , Peroxidasas/inmunología , Alelos , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antioxidantes/metabolismo , Células COS , Chlorocebus aethiops , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos HLA-A/inmunología , Antígeno HLA-A2/inmunología , Humanos , Inmunohistoquímica , Memoria Inmunológica/inmunología , Melanoma/genética , Melanoma/secundario , Datos de Secuencia Molecular , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas , Mutación Puntual , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin beta receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin beta receptor expression on the stromal cells was significantly associated with a "brisk" T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT(+) melanoma-derived microvesicles or even with LIGHT(+) melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3(+)CD8(+) T-cell proliferation. However, lymphocyte coculture with LIGHT(+) microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3(+)CD8(+) T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.
Asunto(s)
Melanoma/inmunología , Proteínas de la Membrana/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Apoptosis/inmunología , Complejo CD3/inmunología , Linfocitos T CD8-positivos/inmunología , Técnicas de Cocultivo , Humanos , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/genética , Melanoma/metabolismo , Melanoma/secundario , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Células Tumorales Cultivadas , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Antígenos CD/análisis , Antígeno CTLA-4/análisis , Factores de Transcripción Forkhead/análisis , Antígenos HLA-DR/análisis , Receptor 2 Celular del Virus de la Hepatitis A/análisis , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Receptor de Muerte Celular Programada 1/análisis , Receptores Inmunológicos/análisis , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1(-) cells) nor up-regulated (in APAF-1(+) cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1(+) and APAF-1(-) tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1(+) and APAF-1(-) tumor cells. In response to 1 to 100 micromol/L of cisplatin, camptothecin, or celecoxib, APAF-1(+) melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1(-) tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 micromol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/patología , Proteínas/metabolismo , Amidinas/farmacología , Apoptosis/fisiología , Factor Apoptótico 1 Activador de Proteasas , Bencilaminas/farmacología , Camptotecina/farmacología , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Celecoxib , Línea Celular Tumoral , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Melanoma/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas/genética , Pirazoles , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Sulfonamidas/farmacologíaRESUMEN
Activation of CTL-mediated antitumor immunity to self-epitopes expressed by neoplastic cells is thought to be prevented, at any stage of tumor progression, by tolerance mechanisms. In contrast, in 74 American Joint Committee on Cancer stages I-IV melanoma patients, we found that development of lymph node metastases is a key event triggering CD8(+) T-cell-mediated immunity to self-epitopes encoded by melanocyte differentiation antigens. This was shown by the increased peripheral precursor frequency to Melan-A/Mart-1, gp100, and tyrosinase epitopes in stage III and IV compared with stage I and II patients, and by accumulation of functional memory T cells directed to Melan-A/Mart-1(26-35) in tumor-invaded lymph nodes. However, in tumor-invaded lymph nodes of most patients, CD8(+) T cells directed to melanocyte differentiation antigens or to tumor-restricted antigens (MAGE-3 and NY-ESO-1 epitopes), showed a CCR7(+) CD45RA(+) CD27(+) CD28(+) perforin(-) "precursor" phenotype. Only in 7 of 23 cases antigen-specific CD8(+) T cells in invaded lymph nodes showed a predominant CCR7(-) CD45RA(-) CD27(+) CD28(-) perforin(+) "preterminally differentiated" phenotype. In the latter subset of patients, by immunohistochemistry in lymph node lesions, we found that CD8(+) T lymphocytes intermingling with the neoplastic tissue expressed a CCR7(-) CD45RO(+)/RA(-) phenotype, whereas CD4(+) lymphocytes did not infiltrate the tumor. Furthermore, perforin and granzyme B were expressed on a higher fraction of the CD8(+) cells surrounding the invading tumor compared with the lymphocytes infiltrating the neoplastic tissue. In addition, no evidence for tumor regression was found in such metastatic lesions, as documented by absence of neoplastic cell necrosis or apoptosis. These data indicate that neoplastic cells in the lymph nodes and/or increased tumor burden in metastatic disease activate CD8(+) T-cell-mediated antitumor immunity to self-epitopes. However, the paucity of terminally differentiated CD8(+) T cells at tumor site suggests that immunotherapy strategies may require not only the boosting of tumor immunity, but also effective means to promote CD8(+) T-cell differentiation in the neoplastic tissue.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/inmunología , Proteínas de la Membrana , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Epítopos de Linfocito T/inmunología , Granzimas , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Humanos , Memoria Inmunológica , Antígenos Comunes de Leucocito/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Antígeno MART-1 , Melanoma/patología , Melanoma/secundario , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Monofenol Monooxigenasa/inmunología , Proteínas de Neoplasias/inmunología , Estadificación de Neoplasias , Fragmentos de Péptidos/inmunología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas/inmunología , Receptores CCR7 , Receptores de Quimiocina/inmunología , Serina Endopeptidasas/biosíntesis , Antígeno gp100 del MelanomaRESUMEN
Dysregulation of receptor tyrosine kinases (RTKs) contributes to several aspects of oncogenesis including drug resistance. In melanoma, distinct RTKs have been involved in BRAF inhibitors (BRAFi) resistance, yet the utility of RTKs expression pattern to identify intrinsically resistant tumors has not been assessed. Transcriptional profiling of RTKs and integration with a previous classification, reveals three robust subtypes in two independent datasets of melanoma cell lines and one cohort of melanoma samples. This classification was validated by Western blot in a panel of patient-derived melanoma cell lines. One of the subtypes identified here for the first time displayed the highest and lowest expression of EGFR and ERBB3, respectively, and included BRAF-mutant tumors all intrinsically resistant to BRAFi PLX4720, as assessed by analysis of the Cancer Cell Line Encyclopedia pharmacogenomic study and by in vitro growth inhibition assays. High levels of EGFR were detected, even before therapy, in tumor cells of one of three melanoma patients unresponsive to BRAFi. Use of different pharmacological inhibitors highlighted the relevance of PI3K/mTOR signaling for growth of this PLX4720-resistant subtype. Our results identify a specific molecular profile of melanomas intrinsically resistant to BRAFi and suggest the PI3K/mTOR pathway as a potential therapeutic target for these tumors.
Asunto(s)
Melanoma/clasificación , Melanoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas con Dominio LIM/metabolismo , Melanoma/enzimología , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Semaforinas/metabolismo , Neoplasias Cutáneas/enzimología , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Western Blotting , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Proteínas del Citoesqueleto/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Humanos , Inmunohistoquímica , Proteínas con Dominio LIM/genética , Masculino , Melanoma/genética , Melanoma/patología , Ratones Desnudos , Proteínas de Microfilamentos , Oxigenasas de Función Mixta , Invasividad Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Semaforinas/genética , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , TransfecciónRESUMEN
A phase I clinical trial using autologous, IL-7 gene-modified tumor cells in patients with disseminated melanoma has been recently completed. Although no major clinical responses were observed, increased antitumor cytotoxicity was measured in postvaccine peripheral blood lymphocytes in a subset of treated patients. To analyze the in situ immune response, the T cell receptor beta-chain variable region (BV) repertoire of T cells infiltrating postvaccine lesions was studied in two patients, and compared with that of T cells present in prevaccine ones, in peripheral blood lymphocytes, and, in one patient, in delayed type hypersensitivity (DTH) sites of autologous melanoma inoculum. A relative expansion of T cells expressing few BVs was observed in all postvaccine metastases, and their intratumoral presence was confirmed by immunohistochemistry. Length pattern analysis of the complementarity determining region 3 (CDR3) indicated that the repertoire of T cells expressing some of these BVs was heterogeneous. At difference, CDR3, beta-chain joining region usage, and sequence analysis enabled us to demonstrate, within a T-cell subpopulation commonly expanded at DTH sites and at the postvaccine lesion of patient 1, that both DTH sites contained identical dominant T-cell clonotypes. One of them was also expressed at increased relative frequency in the postvaccine lesion compared to prevaccine specimens. These results provide evidence for immunological changes, including in situ clonally expanded T cells, in metastases of patients vaccinated with IL-7 gene-transduced cells.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Hipersensibilidad Tardía/inmunología , Interleucina-7/genética , Melanoma/terapia , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Neoplasias Cutáneas/terapia , Linfocitos T/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos CD/metabolismo , Cartilla de ADN/química , Femenino , Reordenamiento Génico de Linfocito T/inmunología , Humanos , Técnicas para Inmunoenzimas , Linfocitos Infiltrantes de Tumor/inmunología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Linfocitos T/citología , Transcripción Genética/inmunología , VacunaciónRESUMEN
BACKGROUND AND OBJECTIVES: Dendritic cells (DC) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Several experimental studies indicate that apoptotic bodies are an optimal source of tumor antigens for ex vivo priming of DC. However, the clinical use of killed tumor cells as a source of antigens will require an optimal methodology to induce effective tumor cell apoptosis. DESIGN AND METHODS: The goal of this study was to compare the efficiency of three agents for inducing neoplastic B lymphocyte apoptosis; staurosporine, infection by modified vaccinia (MVA) viral particles and ultraviolet C (UVC) radiation. RESULTS: The three methods were finely tuned to induce apoptosis in more than 90% of tumor cells after 24 hours of exposure. However, the viability of monocyte-derived DC, loaded with B-cell tumor apoptotic bodies induced by staurosporine or MVA viral particles, decreased dramatically within 48 hours after phagocytosis of the killed neoplastic cells. The persistence of the apoptosis-inducing agents in the apoptotic bodies and not in the tumor supernatant, was responsible for the observed damage to DC viability. In contrast, DC viability was not affected after uptake of tumor cells killed through UVC-irradiation. Furthermore, B-lymphoblastic cell line (LCL)-specific T cells were reactivated by DC loaded with apoptotic bodies induced by UVC-rays. INTERPRETATION AND CONCLUSIONS: Since the method used to induce tumor cell apoptosis might be detrimental to DC viability, these findings should be considered when designing anticancer vaccination programs.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Células Dendríticas/citología , Fagocitosis , Estaurosporina/farmacología , Rayos Ultravioleta , Virus Vaccinia/fisiología , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Apoptosis/efectos de la radiación , Linfocitos B/patología , Linfocitos B/efectos de la radiación , Vacunas contra el Cáncer , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Humanos , Interferón gamma/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma Folicular/patología , Monocitos/citología , Monocitos/efectos de los fármacos , Linfocitos T/inmunología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/efectos de la radiación , Células Tumorales Cultivadas/virología , VacunaciónRESUMEN
The identification of intracellular signaling pathways that promote cell proliferation and resistance to cell death may lead to the development of improved treatment for advanced melanoma. Here we show that the calcineurin/nuclear factor of activated T cells c2 (NFATc2) pathway has an antiapoptotic role in melanoma cells. Expression of NFATc2 was constitutive in vitro and in vivo in human melanoma, and cyclosporin A (CsA) treatment of melanoma cells led to downmodulation of NFATc2. Inhibition of the calcineurin/NFAT pathway by CsA, or by NFATc2 silencing, led to modulation of cell cycle inhibitors and apoptosis-related proteins such as Apollon, and promoted caspase-dependent apoptosis of neoplastic cells. Calcineurin/NFATc2 targeting significantly enhanced melanoma cell death induced by antitumor agents, such as MEK- or BRAF(V600E)-specific inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand, which trigger the intrinsic or extrinsic pathway of apoptosis, respectively. These findings identify NFATc2 as a potential therapeutic target in melanoma.
Asunto(s)
Apoptosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Melanoma/metabolismo , Factores de Transcripción NFATC/metabolismo , Neoplasias Cutáneas/metabolismo , Apoptosis/efectos de los fármacos , Calcineurina/metabolismo , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclosporina/farmacología , Cisteína Endopeptidasas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker ß-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Activadores de Enzimas/farmacología , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/genética , Secuencia de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Inmunohistoquímica , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Fenformina/farmacología , Fosforilación , Ribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genéticaRESUMEN
PURPOSE: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. EXPERIMENTAL DESIGN: Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents. RESULTS: Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors. CONCLUSIONS: Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Melanoma/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , Caspasa 2/metabolismo , Caspasa 3/biosíntesis , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Transformada , Polaridad Celular , Regulación hacia Abajo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Perfilación de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Melanoma/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Axl, a member of the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases, displays an increasingly important role in carcinogenesis. Analysis of 58 cutaneous melanoma lines indicated that Axl was expressed in 38% of them, with significant overrepresentation in NRAS- compared with BRAF-mutated tumors. Axl activation could be induced by autocrine production of its ligand, Gas6, in a significant fraction of Axl-positive tumors. Pearson's correlation analysis on expression data from five data sets of melanoma lines identified several transcripts correlating positively or negatively with Axl. By functionally grouping genes, those inversely correlated were involved in melanocyte development and pigmentation, whereas those positively correlated were involved in motility, invasion, and microenvironment interactions. Accordingly, Axl-positive melanomas did not express microphthalmia transcription factor (MITF) and melanocyte differentiation antigens (MDAs) such as MART-1 and gp100 and possessed a greater in vitro invasive potential compared with Axl-negative ones. Motility, invasivity, and ability to heal a wound or to migrate across an endothelial barrier were inhibited in vitro by Axl knockdown. Pharmacological inhibition of Axl using the selective inhibitor R428 had comparable effects in reducing migration and invasion. These results suggest that targeted inhibition of Axl signaling in the subset of melanomas lacking MITF and MDAs may represent a novel therapeutic strategy.
Asunto(s)
Antígenos Específicos del Melanoma/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias Cutáneas/metabolismo , Antígenos de Diferenciación/metabolismo , Benzocicloheptenos/farmacología , Línea Celular Tumoral , Movimiento Celular/genética , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/genética , Melanoma/patología , Antígenos Específicos del Melanoma/genética , Mutación , Invasividad Neoplásica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Triazoles/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
CD8(+) T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8(+) T cells expressing FOXP3. CD8(+) FOXP3(+) CD25(+) T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8(+) FOXP3(+) T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-ß1], exhausted (PD-1), or senescent (CD57) CD8(+) T lymphocytes. Instead, this subset showed an antigen-experienced "EM1" phenotype (CCR7(-) CD45RA(-) CD28(+) CD27(+)) and exhibited a CD127(-), KLRG1(-), HLA-DR(+), CD38(+), T-bet(+), perforin(+) "early effector" profile predicted by current models. CD8(+) FOXP3(+) T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8(+) FOXP3(+) T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8(+) FOXP3(+) "early effector" subset may be an invaluable tool for monitoring immunity at tumor site.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Western Blotting , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Técnicas para Inmunoenzimas , Memoria Inmunológica/inmunología , Ganglios Linfáticos/inmunología , Metástasis Linfática , Activación de Linfocitos/fisiología , Melanoma/secundario , Fenotipo , Neoplasias Cutáneas/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Activación de Linfocitos , Melanoma/tratamiento farmacológico , Linfocitos T/inmunología , Adulto , Antígeno CTLA-4/antagonistas & inhibidores , Humanos , Ipilimumab , Masculino , Melanoma/inmunología , Melanoma/mortalidad , Melanoma/secundario , Tomografía Computarizada por Rayos XRESUMEN
The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage given with GM-CSF and IFN-alpha in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days -1, 0 and +1, while IFN-alpha (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-alpha is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.