RESUMO
An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.
Assuntos
Adenosina , Vesículas Extracelulares , Humanos , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Imunidade , Vesículas Extracelulares/metabolismo , InflamaçãoRESUMO
Nutrition is emerging as a promising therapeutic tool to modulate the immune system in health and disease. We propose that the timing of dietary interventions is probably what determines their success. In this context, we explore recent research that identifies the early phases of dietary intervention as critical time windows for modulating immunity and optimizing cancer therapy. Furthermore, we highlight how the timing of intervention can yield different outcomes. The data suggest that nutrient availability and absorption over a short period can significantly impact mammalian immune and even non-immune landscapes. This, in turn, can lead to changes in mucosal and systemic immunity, potentially exacerbating or ameliorating inflammation, and perhaps influencing tumor cells and their response to cancer therapies.
Assuntos
Dieta , Neoplasias , Animais , Humanos , Sistema Imunitário , Neoplasias/terapia , MamíferosRESUMO
Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.
Assuntos
Mucosa , Salmonella typhimurium , Humanos , Camundongos , Animais , Linfócitos T , Imunidade nas MucosasRESUMO
At mucosal barriers, the T helper 17 (TH17) cell population plays a fundamental role in controlling tissue homeostasis. The adaptability of this population to a more pro-inflammatory or anti-inflammatory function - that is, their functional plasticity and consequently heterogeneity - primarily depends on the environment. We would like to term this process environmental immune adaptation. Interfering with TH17 cell adaptation leads to pathological consequences, including development of immune-mediated inflammatory diseases or even cancer. Several molecular mechanisms have been shown to participate in this process and recently, a better understanding of the transcriptional and metabolic profiling of TH17 cells has shed light on a new level of complexity. Here, we offer a summary on the role of TH17 cell plasticity in inflammatory diseases and cancer as well as the latest discoveries and controversies regarding the mechanisms that control the adaptability of the TH17 cell population.
Assuntos
Plasticidade Celular , Neoplasias , Humanos , Neoplasias/metabolismo , Adaptação Fisiológica , Células Th17RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second most deadly cancer by 2040, owing to the high incidence of metastatic disease and limited responses to treatment1,2. Less than half of all patients respond to the primary treatment for PDAC, chemotherapy3,4, and genetic alterations alone cannot explain this5. Diet is an environmental factor that can influence the response to therapies, but its role in PDAC is unclear. Here, using shotgun metagenomic sequencing and metabolomic screening, we show that the microbiota-derived tryptophan metabolite indole-3-acetic acid (3-IAA) is enriched in patients who respond to treatment. Faecal microbiota transplantation, short-term dietary manipulation of tryptophan and oral 3-IAA administration increase the efficacy of chemotherapy in humanized gnotobiotic mouse models of PDAC. Using a combination of loss- and gain-of-function experiments, we show that the efficacy of 3-IAA and chemotherapy is licensed by neutrophil-derived myeloperoxidase. Myeloperoxidase oxidizes 3-IAA, which in combination with chemotherapy induces a downregulation of the reactive oxygen species (ROS)-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7. All of this results in the accumulation of ROS and the downregulation of autophagy in cancer cells, which compromises their metabolic fitness and, ultimately, their proliferation. In humans, we observed a significant correlation between the levels of 3-IAA and the efficacy of therapy in two independent PDAC cohorts. In summary, we identify a microbiota-derived metabolite that has clinical implications in the treatment of PDAC, and provide a motivation for considering nutritional interventions during the treatment of patients with cancer.
Assuntos
Carcinoma Ductal Pancreático , Microbiota , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/dietoterapia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/microbiologia , Glutationa Peroxidase/metabolismo , Neoplasias Pancreáticas/dietoterapia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/microbiologia , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismo , Triptofano/farmacologia , Triptofano/uso terapêutico , Neutrófilos/enzimologia , Autofagia , Metagenoma , Metabolômica , Transplante de Microbiota Fecal , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/uso terapêutico , Modelos Animais de Doenças , Vida Livre de Germes , Neoplasias PancreáticasRESUMO
A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Linfócitos T CD4-Positivos , FibroseRESUMO
During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.
Assuntos
Interleucinas , Neoplasias Hepáticas , Células T Matadoras Naturais , Animais , Camundongos , Células Endoteliais/metabolismo , Interleucinas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , Neoplasias Colorretais/metabolismo , Interleucina 22RESUMO
Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)-restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8+ T cells engineered with the same TCR. TCR-iNKT cells achieved robust cancer control by simultaneously modulating intratumoral suppressive myeloid populations and killing malignant cells. This dual antitumor function was further enhanced when the iNKT cell agonist α-galactosyl ceramide (α-GalCer) was administered as a therapeutic booster through a platform that ensured controlled delivery at the tumor site, named multistage vector (MSV). These preclinical results support the combination of tumor-redirected TCR-iNKT cells and local α-GalCer boosting as a potential therapy for patients with cancer.
Assuntos
Células T Matadoras Naturais , Neoplasias , Receptores de Antígenos de Linfócitos T , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos , Engenharia Celular , Células Mieloides , Células T Matadoras Naturais/fisiologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/uso terapêutico , Microambiente TumoralRESUMO
Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Ligadas por GPI/metabolismo , Terapia de Imunossupressão , 5'-Nucleotidase/genética , Trifosfato de Adenosina , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Vesículas Extracelulares/imunologia , Humanos , Inflamação , Ativação Linfocitária , Camundongos , Linfócitos T , Linfócitos T Reguladores/imunologiaRESUMO
Isolation of cells from organs and tissues represents a challenge for many researchers. Cell yield, purity, and cell death are common problems associated with it, which greatly affect experimental results in terms of reproducibility and biological observations. Here, we describe state-of-the-art protocols of how to isolate CD4+ T cells from both human and murine organs and tissues, reducing at minimum cell death, while increasing at the same time cell yield and purity.
Assuntos
Separação Celular , Citometria de Fluxo , Tecido Linfoide/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Biomarcadores/metabolismo , Sobrevivência Celular , Enzimas/metabolismo , Humanos , Separação Imunomagnética , Camundongos Endogâmicos C57BL , Fenótipo , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/metabolismo , Fluxo de TrabalhoRESUMO
Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8+ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.
Assuntos
COVID-19/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Memória Imunológica , Pulmão/imunologia , Células Th17/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/complicações , COVID-19/patologia , Células Clonais , Humanos , Inflamação/etiologia , Inflamação/imunologia , Pulmão/patologia , Células Mieloides , Pneumonia Bacteriana/imunologia , Células Th17/imunologiaRESUMO
The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.
Assuntos
Ouro/química , Imunoterapia/métodos , Interleucina-12/metabolismo , Nanopartículas Metálicas/química , Adenocarcinoma/terapia , Animais , Células Cultivadas , Feminino , Fibrossarcoma/terapia , Masculino , Neoplasias Mamárias Animais/terapia , CamundongosRESUMO
The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet-) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease.
Assuntos
Anergia Clonal , Regulação Leucêmica da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Memória Imunológica/genética , Leucemia Mieloide Aguda/imunologia , Linfócitos T/imunologia , Adulto , Antígenos CD/genética , Antígenos CD/imunologia , Antineoplásicos/uso terapêutico , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Feminino , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores KIR/genética , Receptores KIR/imunologia , Recidiva , Indução de Remissão , Estudos Retrospectivos , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Linfócitos T/patologia , Transplante Homólogo , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
CD1d-restricted Natural Killer T (NKT) cells are regarded as sentinels of tissue integrity by sensing local cell stress and damage. This occurs via recognition of CD1d-restricted lipid antigens, generated by stress-related metabolic changes, and stimulation by inflammatory cytokines, such as IL-12 and IL-18. Increasing evidence suggest that this occurs mainly upon NKT cell interaction with CD1d-expressing cells of the Mononuclear Phagocytic System, i.e., monocytes, macrophages and DCs, which patrol parenchymatous organs and mucosae to maintain tissue homeostasis and immune surveillance. In this review, we discuss critical examples of this crosstalk, presenting the known underlying mechanisms and their effects on both cell types and the environment, and suggest that the interaction with CD1d-expressing mononuclear phagocytes in tissues is the fundamental job of NKT cells.
Assuntos
Comunicação Celular , Suscetibilidade a Doenças , Sistema Fagocitário Mononuclear/imunologia , Sistema Fagocitário Mononuclear/metabolismo , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Especificidade de Órgãos/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Microambiente TumoralRESUMO
Heterotypic cellular and molecular interactions in the tumor microenvironment (TME) control cancer progression. Here, we show that CD1d-restricted invariant natural killer (iNKT) cells control prostate cancer (PCa) progression by sculpting the TME. In a mouse PCa model, iNKT cells restrained the pro-angiogenic and immunosuppressive capabilities of tumor-infiltrating immune cells by reducing pro-angiogenic TIE2+, M2-like macrophages (TEMs), and sustaining pro-inflammatory M1-like macrophages. iNKT cells directly contacted macrophages in the PCa stroma, and iNKT cell transfer into tumor-bearing mice abated TEMs, delaying tumor progression. iNKT cells modulated macrophages through the cooperative engagement of CD1d, Fas, and CD40, which promoted selective killing of M2-like and survival of M1-like macrophages. Human PCa aggressiveness associate with reduced intra-tumoral iNKT cells, increased TEMs, and expression of pro-angiogenic genes, underscoring the clinical significance of this crosstalk. Therefore, iNKT cells may control PCa through mechanisms involving differential macrophage modulation, which may be harnessed for therapeutically reprogramming the TME.
Assuntos
Antígenos CD40/metabolismo , Macrófagos/metabolismo , Células T Matadoras Naturais/imunologia , Neoplasias da Próstata/genética , Animais , Progressão da Doença , Humanos , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human ß-catenin cDNA encoding a nondegradable form of ß-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of ß-catenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UW-B cells was statistically significantly lower than that of all the other control cells (p < 0.01), (C) the mortality of irradiated UW-B cells was statistically significantly higher than that of the controls and their nonirradiated counterparts (p < 0.05), and (D) morphological features of neuronal differentiation were observed in the Wnt-activated cells. In tissue samples, the Ki-67 labeling index (LI) was lower in ß-catenin-positive samples compared to non-ß-catenin positive ones. The Ki-67 LI median (LI = 40) of the nuclear ß-catenin-positive tumor samples was lower than that of non-nuclear ß-catenin-positive samples (LI = 50), but the difference was not statistically significant. Overall, our data suggest that activation of the Wnt pathway reduces the proliferation and invasion of MBs and increases the tumor's radiosensitivity.
Assuntos
Proliferação de Células/fisiologia , Meduloblastoma/fisiopatologia , Meduloblastoma/radioterapia , Tolerância a Radiação/fisiologia , Proteínas Wnt/metabolismo , Adolescente , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Criança , Pré-Escolar , Imunofluorescência , Humanos , Lactente , Recém-Nascido , Antígeno Ki-67/metabolismo , Meduloblastoma/patologia , Microscopia Eletrônica de Transmissão , Invasividade Neoplásica/fisiopatologia , Neurogênese/fisiologia , Transfecção , beta Catenina/genética , beta Catenina/metabolismoRESUMO
EGFR mutations are strongly predictive of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor activity in non-small cell lung cancer (NSCLC), but resistance mechanisms are not completely understood. The interindividual variability in toxicity also points out to the need of novel pharmacogenetic markers to select patients before therapy. Therefore, we evaluated the associations between EGFR and AKT1 polymorphisms and outcome/toxicity in gefitinib-treated NSCLC patients. Polymorphic loci in EGFR, and AKT1, and EGFR and K-Ras mutations were assessed in DNA isolated from blood samples and/or paraffin-embedded tumor from 96 gefitinib-treated NSCLC patients. Univariate and multivariate analyses compared genetic variants with clinical efficacy and toxicity using Fisher's, log-rank test, and Cox's proportional hazards model. AKT1-SNP4 association with survival was also evaluated in 127 chemotherapy-treated/gefitinib-naive patients, whereas its relationship with AKT1 expression and gefitinib cytotoxicity was studied in 15 NSCLC cell lines. AKT1-SNP4 A/A genotype was associated with shorter time-to-progression (P = 0.04) and overall survival (P = 0.007). Multivariate analyses and comparison with the gefitinib-nontreated population underlined its predictive significance, whereas the in vitro studies showed the association of lower AKT1 mRNA levels with gefitinib resistance. In contrast, EGFR-activating mutations were significantly correlated with response, longer time-to-progression, and overall survival, whereas EGFR -191C/A (P < 0.001), -216 G/T (P < 0.01), and R497K (P = 0.02) polymorphisms were strongly associated with grade >1 diarrhea. AKT1-SNP4 A/A genotype seems to be a candidate biomarker of primary resistance, whereas EGFR -191C/A, -216G/T, and R497K polymorphisms are associated with diarrhea when using gefitinib in NSCLC patients, thus offering potential new tools for treatment optimization.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/genética , Quinazolinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/análise , Biomarcadores Farmacológicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Resistencia a Medicamentos Antineoplásicos/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/fisiologia , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/efeitos adversos , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3sigma, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza-irinotecan interaction.