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Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.
Assuntos
Alérgenos , Cryptomeria , Animais , Camundongos , Cryptomeria/química , Antígenos de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/análise , Proteínas de Plantas/química , Pólen , Peptídeos , Receptores de Antígenos de Linfócitos TRESUMO
Specific MHC class II alleles are strongly associated with susceptibility to various autoimmune diseases. Although the primary function of MHC class II molecules is to present peptides to helper T cells, MHC class II molecules also function like a chaperone to transport misfolded intracellular proteins to the cell surface. In this study, we found that autoantibodies in patients with Graves' disease preferentially recognize thyroid-stimulating hormone receptor (TSHR) complexed with MHC class II molecules of Graves' disease risk alleles, suggesting that the aberrant TSHR transported by MHC class II molecules is the target of autoantibodies produced in Graves' disease. Mice injected with cells expressing mouse TSHR complexed with MHC class II molecules, but not TSHR alone, produced anti-TSHR autoantibodies. These findings suggested that aberrant self-antigens transported by MHC class II molecules exhibit antigenic properties that differ from normal self-antigens and abrogate self-tolerance, providing a novel mechanism for autoimmunity.
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Genomic deletion of donor-patient-mismatched HLA alleles in leukemic cells is a major cause of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Mismatched HLA is frequently lost as an individual allele or a whole region in HLA-class I, however, it is downregulated in HLA-class II. We hypothesized that there might be a difference in T cell recognition capacity against epitopes associated with HLA-class I and HLA-class II and consequently such allogeneic immune pressure induced HLA alterations in leukemic cells. To investigate this, we conducted in vitro experiments with T cell receptor-transduced T (TCR-T) cells. The cytotoxic activity of NY-ESO-1-specific TCR-T cells exhibited similarly against K562 cells with low HLA-A*02:01 expression. However, we demonstrated that the cytokine production against low HLA-DPB1*05:01 expression line decreased gradually from the HLA expression level approximately 2-log lower than normal expressors. Using sort-purified leukemia cells before and after HSCT, we applied the next-generation sequencing, and revealed that there were several marked downregulations of HLA-class II alleles which demonstrated consistently low expression from pre-transplantation. The marked downregulation of HLA-class II may lead to decreased antigen recognition ability of antigen-specific T cells and may be one of immune evasion mechanism associated with HLA-class II downregulation.
Assuntos
Regulação para Baixo , Epitopos/imunologia , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Leucemia/genética , Leucemia/imunologia , Linfócitos T/imunologia , Transplante Homólogo , Alelos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Humanos , Células K562 , Leucemia/terapia , RecidivaRESUMO
Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.
Assuntos
Aspartato-Amônia Ligase/genética , Neoplasias Colorretais/terapia , Pinocitose/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Asparagina/genética , Asparagina/metabolismo , Aspartato-Amônia Ligase/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Técnicas de Silenciamento de Genes , Humanos , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms - a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition - apart from its radiosensitizing potential - obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.
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HLA-DPB1 T-cell epitope (TCE) mismatching algorithm and rs9277534 SNP at the 3' untranslated region (3'UTR) in the HLA-DPB1 gene are key factors for transplant-related events in unrelated hematopoietic cell transplantation (UR-HCT). However, the association of these 2 mechanisms has not been elucidated. We analyzed 19 frequent HLA-DPB1 alleles derived from Japanese healthy subjects by next-generation sequencing of the entire HLA-DPB1 gene region and multi-SNP data of the HLA region in 1589 UR-HCT pairs. The risk of acute graft-versus-host disease (aGVHD) was analyzed in 1286 patients with single HLA-DPB1 mismatch UR-HCT. The phylogenetic tree constructed using the entire gene region demonstrated that HLA-DPB1 alleles were divided into 2 groups, HLA-DP2 and HLA-DP5. Although a phylogenetic relationship in the genomic region from exon 3 to 3'UTR (Ex3-3'UTR) obviously supported the division of HLA-DP2 and HLA-DP5 groups, which in exon 2 showed intermingling of HLA-DPB1 alleles in a non-HLA-DP2 and non-HLA-DP5-group manner. Multi-SNP data also showed 2 discriminative HLA-DPB1 groups according to Ex3-3'UTR. Risk of grade 2-4 aGVHD was significantly higher in patient HLA-DP5 group mismatch than patient HLA-DP2 group mismatch (hazard ratio, 1.28; P = .005), regardless of donor mismatch HLA-DP group. Regarding TCE mismatch, increasing risk of aGVHD in patient HLA-DP5 group mismatch and TCE-nonpermissive mismatch were observed only in patients with TCE-permissive mismatch and patient HLA-DP2 group mismatch, respectively. Evolutionary analysis revealed that rs9277534 represented a highly conserved HLA-DPB1 Ex3-3'UTR region and may provoke aGVHD differently to TCE mismatching algorithm, reflecting exon 2 polymorphisms. These findings enrich our understanding of the mechanism of aGVHD in HLA-DPB1 mismatch UR-HCT.
Assuntos
Doença Enxerto-Hospedeiro/genética , Cadeias beta de HLA-DP/genética , Doença Aguda , Adolescente , Adulto , Idoso , Alelos , Criança , Pré-Escolar , Evolução Molecular , Feminino , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Teste de Histocompatibilidade , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Filogenia , Polimorfismo Genético , Doadores não Relacionados , Adulto JovemRESUMO
Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful due to insufficient understanding of the salient mechanisms of RAS signaling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and nonapoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.
Assuntos
Proteína 12 Relacionada à Autofagia/metabolismo , Autofagia , Neoplasias Colorretais/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Apoptose , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/patologia , Regulação para Baixo , Humanos , Mucosa Intestinal/patologia , MAP Quinase Quinase Quinases/metabolismo , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis.
Assuntos
Apoptose , Neoplasias Colorretais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Receptor fas/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Nitrilas , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fatores de Tempo , Transativadores , Fatores de Transcrição/genética , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC) can predict a lack of responses to anti-epidermal growth factor receptor-based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.
Assuntos
Adaptação Biológica , Aspartato-Amônia Ligase/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Glutamina/metabolismo , Mutação , Proteínas ras/genética , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Carga TumoralRESUMO
Associations of variants located in the HLA class II region with chronic hepatitis B (CHB) infection have been identified in Asian populations. Here, HLA imputation method was applied to determine HLA alleles using genome-wide SNP typing data of 1,975 Japanese individuals (1,033 HBV patients and 942 healthy controls). Together with data of an additional 1,481 Japanese healthy controls, association tests of six HLA loci including HLA-A, C, B, DRB1, DQB1, and DPB1, were performed. Although the strongest association was detected at a SNP located in the HLA-DP locus in a SNP-based GWAS using data from the 1,975 Japanese individuals, HLA genotyping-based analysis identified DQB1*06:01 as having the strongest association, showing a greater association with CHB susceptibility (OR = 1.76, P = 6.57 × 10(-18)) than any one of five HLA-DPB1 alleles that were previously reported as CHB susceptibility alleles. Moreover, HLA haplotype analysis showed that, among the five previously reported HLA-DPB1 susceptibility and protective alleles, the association of two DPB1 alleles (DPB1*09:01, and *04:01) had come from linkage disequilibrium with HLA-DR-DQ haplotypes, DRB1*15:02-DQB1*06:01 and DRB1*13:02-DQB1*06:04, respectively. The present study showed an example that SNP-based GWAS does not necessarily detect the primary susceptibility locus in the HLA region.
Assuntos
Antígenos HLA/imunologia , Hepatite B Crônica/imunologia , Suscetibilidade a Doenças , Genótipo , Antígenos HLA/genética , Haplótipos , Hepatite B Crônica/genética , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
HLA molecules play an important role for immunoreactivity in allogeneic hematopoietic stem cell transplantation. To elucidate the effect of specific HLA alleles on acute graft-versus-host disease, we conducted a retrospective analysis using 6967 Japanese patients transplanted with T-cell-replete marrow from an unrelated donor. Using unbiased searches of patient and donor HLA alleles, patient and/or donor HLA-B*51:01 (patient: HR, 1.37,P<0.001; donor: HR, 1.35,P<0.001) and patient HLA-C*14:02 (HR, 1.35,P<0.001) were significantly associated with an increased risk of severe acute graft-versus-host disease. The finding that donor HLA-C*14:02 was not associated with severe acute graft-versus-host disease prompted us to elucidate the relation of these high-risk HLA alleles with patient and donor HLA-C allele mismatches. In comparison to HLA-C allele match, patient mismatched HLA-C*14:02 showed the highest risk of severe acute graft-versus-host disease (HR, 3.61,P<0.001) and transplant-related mortality (HR, 2.53,P<0.001) among all patient mismatched HLA-C alleles. Although patient HLA-C*14:02 and donor HLA-C*15:02 mismatch was usually KIR2DL-ligand mismatch in the graft-versus-host direction, the risk of patient mismatched HLA-C*14:02 for severe acute graft-versus-host disease was obvious regardless of KIR2DL-ligand matching. The effect of patient and/or donor HLA-B*51:01 on acute graft-versus-host disease was attributed not only to strong linkage disequilibrium of HLA-C*14:02 and -B*51:01, but also to the effect of HLA-B*51:01 itself. With regard to clinical implications, patient mismatched HLA-C*14:02 proved to be a potent risk factor for severe acute graft-versus-host disease and mortality, and should be considered a non-permissive HLA-C mismatch in donor selection for unrelated donor hematopoietic stem cell transplantation.
Assuntos
Anemia Aplástica/terapia , Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA-B/imunologia , Antígenos HLA-C/imunologia , Leucemia/terapia , Síndromes Mielodisplásicas/terapia , Adolescente , Adulto , Idoso , Alelos , Anemia Aplástica/genética , Anemia Aplástica/imunologia , Anemia Aplástica/mortalidade , Criança , Pré-Escolar , Contraindicações , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Teste de Histocompatibilidade , Humanos , Lactente , Recém-Nascido , Leucemia/genética , Leucemia/imunologia , Leucemia/mortalidade , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/mortalidade , Receptores KIR2DL1/genética , Receptores KIR2DL1/imunologia , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Transplante Homólogo , Doadores não RelacionadosRESUMO
The human leukocyte antigen (HLA) genomic region spanning about 4 Mb is the most gene dense and the polymorphic stretches in the human genome. A total of the 269 loci were identified, including 145 protein coding genes mostly important for immunity and 50 noncoding RNAs (ncRNAs). Biological function of these ncRNAs remains unknown, becoming hot spot in the studies of HLA-associated diseases. The genomic diversity analysis in the HLA region facilitated by next-generation sequencing will pave the way to molecular understanding of linkage disequilibrium structure, population diversity, histocompatibility in transplantation, and associations with autoimmune diseases. The 4-digit DNA genotyping of HLA for six HLA loci, HLA-A through DP, in the patients with Graves' disease (GD) and Hashimoto thyroiditis (HT) identified six susceptible and three resistant HLA alleles. Their epistatic interactions in controlling the development of these diseases are shown. Four susceptible and one resistant HLA alleles are shared by GD and HT. Two HLA alleles associated with GD or HT control the titers of autoantibodies to thyroid antigens. All these observations led us to propose a new model for the development of GD and HT. Hematopoietic stem cell transplantation from unrelated donor (UR-HSCT) provides a natural experiment to elucidate the role of allogenic HLA molecules in immune response. Large cohort studies using HLA allele and clinical outcome data have elucidated that (1) HLA locus, allele, and haplotype mismatches between donor and patient, (2) specific amino acid substitution at specific positions of HLA molecules, and (3) ethnic background are all responsible for the immunological events related to UR-HSCT including acute graft-versus-host disease (GVHD), chronic GVHD, graft-versus-leukemia (GvL) effect, and graft failure.
Assuntos
Mapeamento Cromossômico , Doença de Graves/genética , Doença de Hashimoto/genética , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Alelos , Autoanticorpos/sangue , Autoanticorpos/genética , Autoanticorpos/imunologia , Epistasia Genética , Predisposição Genética para Doença , Variação Genética , Doença Enxerto-Hospedeiro/genética , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.
Assuntos
Antineoplásicos/farmacologia , Autofagia/fisiologia , Neoplasias Colorretais/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Células HCT116 , Células HT29 , Humanos , Indóis/farmacologia , Macrolídeos/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína Sequestossoma-1/metabolismo , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
PURPOSE: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. EXPERIMENTAL DESIGN: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. RESULTS: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. CONCLUSIONS: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Expressão Gênica , Receptor EphA2/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Estimativa de Kaplan-Meier , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor EphA2/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Proteínas ral de Ligação ao GTP/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Proteínas ras/metabolismoRESUMO
Acute graft-versus-host disease (aGVHD) represents one of the major complications in allogeneic stem cell transplantation and is primarily caused by genetic disparity between the donor and recipient. In HLA-matched transplants, the disparity is thought to be determined by loci encoding minor histocompatibility antigens (minor H antigens), which are presented by specific HLA molecules. We performed a genome-wide association study (GWAS) to identify minor H antigen loci associated with aGVHD. A total of 500 568 single nucleotide polymorphisms (SNPs) were genotyped for donors and recipients from 1589 unrelated bone marrow transplants matched for HLA-A, -B, -C, -DRB1, and -DQB1, followed by the imputation of unobserved SNPs. We interrogated SNPs whose disparity between the donor and recipient was significantly associated with aGVHD development. Without assuming HLA unrestriction, we successfully captured a known association between HLA-DPB1 disparity (P = 4.50 × 10(-9)) and grade II-IV aGVHD development, providing proof of concept for the GWAS design aimed at discovering genetic disparity associated with aGVHD. In HLA-restricted analyses, whereby association tests were confined to major subgroups sharing common HLA alleles to identify putative minor H antigen loci, we identified 3 novel loci significantly associated with grade III-IV aGVHD. Among these, rs17473423 (P = 1.20 × 10(-11)) at 12p12.1 within the KRAS locus showed the most significant association in the subgroup, sharing HLA-DQB1*06:01. Our result suggested that a GWAS can be successfully applied to identify allele mismatch associated with aGVHD development, contributing to the understanding of the genetic basis of aGVHD.
Assuntos
Doença Enxerto-Hospedeiro/genética , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Menor/genética , Adolescente , Adulto , Idoso , Alelos , Criança , Pré-Escolar , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Cadeias beta de HLA-DQ/genética , Teste de Histocompatibilidade , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Detachment of nonmalignant intestinal epithelial cells from the extracellular matrix (ECM) triggers their growth arrest and, ultimately, apoptosis. In contrast, colorectal cancer cells can grow without attachment to the ECM. This ability is critical for their malignant potential. We found previously that detachment-induced growth arrest of nonmalignant intestinal epithelial cells is driven by their detachment-triggered autophagy, and that RAS, a major oncogene, promotes growth of detached cells by blocking such autophagy. In an effort to identify the mechanisms of detachment-induced autophagy and growth arrest of nonmalignant cells we found here that detachment of these cells causes upregulation of ATG3 and that ATG3 upregulation contributes to autophagy and growth arrest of detached cells. We also observed that when ATG3 expression is artificially increased in the attached cells, ATG3 promotes neither autophagy nor growth arrest but triggers their apoptosis. ATG3 upregulation likely promotes autophagy of the detached but not that of the attached cells because detachment-dependent autophagy requires other detachment-induced events, such as the upregulation of ATG7. We further observed that those few adherent cells that do not die by apoptosis induced by ATG3 become resistant to apoptosis caused by cell detachment, a property that is critical for the ability of normal epithelial cells to become malignant. We conclude that cell-ECM adhesion can switch ATG3 functions: when upregulated in detached cells in the context of other autophagy-promoting events, ATG3 contributes to autophagy. However, when overexpressed in the adherent cells, in the circumstances not favoring autophagy, ATG3 triggers apoptosis.
Assuntos
Apoptose , Autofagia , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Intestinos/citologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Proteína 7 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Adesão Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Intestino Delgado/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Sintases/metabolismo , Interferência de RNA , Ratos , Enzimas Ativadoras de Ubiquitina/metabolismo , Regulação para CimaRESUMO
ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers. Since ZFAT has three to five consecutive tandem zinc-fingers, which may cooperatively function as a unit, we also determined two tandemly arrayed zinc-finger structures, between ZF2 to ZF4 and ZF3 to ZF5. Our NMR spectroscopic analysis detected the interaction between ZF4 and ZF5, which are connected by an uncommon linker sequence, KKIK. The ZF4-ZF5 linker restrained the relative structural space between the two zinc-fingers in solution, unlike the other linker regions with determined structures, suggesting the involvement of the ZF4-ZF5 interfinger linker in the regulation of ZFAT function.
Assuntos
Proteínas de Ligação a DNA/química , Conformação Proteica , Fatores de Transcrição/química , Dedos de Zinco/genética , Sequência de Aminoácidos/genética , Animais , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tireoidite Autoimune/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. ß2-glycoprotein I (ß2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact ß2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize ß2GPI/HLA class II complexes in the absence of phospholipids. In situ association between ß2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against ß2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that ß2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis.
Assuntos
Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica , Antígenos de Histocompatibilidade Classe II/imunologia , Complexos Multiproteicos/imunologia , beta 2-Glicoproteína I/imunologia , Adulto , Idoso , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Células HEK293 , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/imunologia , beta 2-Glicoproteína I/metabolismoRESUMO
We hypothesized that the compatibility of each HLA loci between donor and patient induced divergent transplant-related immunologic responses, which attributed to the individualized manifestation of clinical outcomes. Here, we analyzed 7898 Japanese pairs transplanted with T-cell-replete marrow from an unrelated donor with complete HLA allele typing data. Multivariable competing risk regression analyses were conducted to evaluate the relative risk (RR) of clinical outcomes after transplantation. A significant RR of HLA allele mismatch compared with match was seen with HLA-A, -B, -C, and -DPB1 for grade III-IV acute graft-versus-host disease (GVHD), and HLA-C for chronic GVHD. Of note, only HLA-C and HLA-DPB1 mismatch reduced leukemia relapse, and this graft-versus-leukemia effect of HLA-DPB1 was independent of chronic GVHD. HLA-DRB1 and HLA-DQB1 double (DRB1_DQB1) mismatch was revealed to be a significant RR for acute GVHD and mortality, whereas single mismatch was not. Thus, the number of HLA-A, -B, -C, -DPB1, and DRB1_DQB1 mismatches showed a clear-cut risk difference for acute GVHD, whereas the number of mismatches for HLA-A, -B, -C, and DRB1_DQB1 showed the same for mortality. In conclusion, we determined the biological response to HLA locus mismatch in transplant-related immunologic events, and provide a rationale for use of a personalized algorithm for unrelated donor selection.
Assuntos
Transplante de Medula Óssea , Teste de Histocompatibilidade , Histocompatibilidade/fisiologia , Leucemia/terapia , Doadores não Relacionados , Adolescente , Adulto , Idoso , Alelos , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/estatística & dados numéricos , Criança , Pré-Escolar , Feminino , Loci Gênicos/imunologia , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Cadeias beta de HLA-DP/genética , Cadeias beta de HLA-DP/imunologia , Cadeias beta de HLA-DQ/genética , Cadeias beta de HLA-DQ/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Humanos , Lactente , Recém-Nascido , Leucemia/epidemiologia , Leucemia/genética , Leucemia/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Hashimoto thyroiditis (HT) and Graves' disease (GD) share some immunological features. Determining the genetic basis that distinguishes HT from GD is key for a better understanding of the differences between these two related diseases. AIM: The aim of this study was to identify a non-HLA susceptibility locus that is specific to either HT or GD. DESIGN: We performed a two-stage genome-wide comparison between HT and GD in Japan. During the discovery stage, we performed a logistic regression analysis adjusting for sex using 727 413 single nucleotide polymorphisms (SNPs) for 265 HT and 261 GD patients. During the replication stage, 35 SNPs were analyzed for 181 HT and 286 GD cases. A combined meta-analysis was performed using the results from these two stages. An SNP showing a genome-wide significant level was further analyzed using 1363 healthy controls to determine the specificity of susceptibility. RESULTS: A genome-wide direct comparison between HT and GD revealed an SNP at the VAV3 locus with genome-wide significant association signals (rs7537605: P(combined) = 3.90 × 10(-8); odds ratio(combined) = 1.77; 95% confidence interval = 1.44-2.17). An association analysis using healthy controls showed that rs7537605 is significantly associated with HT (P = 1.24 × 10(-5); odds ratio = 1.60; 95% confidence interval = 1.30-1.97) but not with GD (P = .50), suggesting that the variant specifically affects susceptibility to HT. CONCLUSION: A genome-wide direct comparison between HT and GD revealed an HT-specific variant within VAV3 in the Japanese. Considering physiological roles of VAV3, such as a guanine nucleotide exchange factor, our finding provides new insight into the molecular mechanism of HT.