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Medulloblastoma is the most common malignant tumor in the pediatric population. Its classification has incorporated key molecular variations alongside histological characterization. CD39 (also known as ENTPD1) and CD73 (also known as NT5E), enzymes of the purinergic signaling pathway, act in synergy to generate extracellular adenosine, creating an immunosuppressive tumor microenvironment. Our study examined the expression of mRNA of these genes in previously described transcriptome data sets of medulloblastoma patient samples from the Cavalli Cohort (n = 763). Survival distribution was estimated according to the Kaplan-Meier method using a median cut-off and log-rank statistics (p ≤ 0.05). In non-WNT and non-SHH medulloblastoma Group 4 (n = 264), the high expression of ENTPD1 and NT5E was significantly related to a lower overall survival (p = 2.7e-04; p = 2.6e-03). In the SHH-activated group (n = 172), the high expression of ENTPD1 was significantly related to lower overall survival (p = 7.8e-03), while the high expression of NT5E was significantly related to greater overall survival (p = 0.017). In the WNT group (n = 63), the expressions of ENTPD1 and NT5E were not significantly correlated with overall survival (p = 0.212; p = 0.101). In non-WNT and non-SHH medulloblastoma Group 3 (n = 113), the high expression of ENTPD1 was significantly related to greater survival (p = 0.034), while expression of NT5E was not significantly related to survival of patients (p = 0.124). This in silico analysis indicates that ENTPD1 (CD39) and NT5E (CD73) can be seen as potential prognostic markers and therapeutic targets for primary medulloblastomas in non-WNT and non-SHH Group 4.
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Glioblastoma (GB) is the most common primary brain tumor in adults and carries a dismal prognosis, despite the best available treatment. The 2021 WHO Classification of CNS tumors incorporated molecular profiling to better define the characteristics and prognosis of tumor types and subtypes. These recent advances in diagnosis have not yet resulted in breakthrough therapies capable of shifting the treatment paradigm. NT5E/CD73 is a cell surface enzyme that participates in a complex purinergic pathway in synergy with ENTPD1/CD39 producing extracellular adenosine (ADO) from ATP. ADO promotes tumor progression by inducing immunosuppression, stimulating adhesion, invasion, and angiogenesis. In this study, we performed an in silico analysis of 156 human glioblastoma samples in an unexplored public database to investigate the transcriptional levels of NT5E and ENTPD1. The analysis revealed a significant increase in transcription levels of the genes under study in GB samples versus non-tumor brain tissue samples, in concordance with previous studies. High transcriptional levels of NT5E or ENTPD1 were independently related to a decrease in overall survival (p = 5.4e-04; 1.1e-05), irrespective of the IDH mutation status. NT5E transcriptional levels were significantly higher in GB IDH wild-type patients compared to GB IDH-mutant; however, ENTPD1 levels showed no significant difference, p ≤ 0.001. This in silico study indicates the need for a deeper understanding of the purinergic pathway relation to GB development, also inspiring future population studies that could explore ENTPD1 and NT5E not only as prognostic markers but also as potential therapeutic targets.
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The ectoenzyme CD73, encoded by the NT5E gene, has emerged as a potential prognostic and therapeutic marker for papillary thyroid carcinoma (PTC), which has increased in incidence in recent decades. Here, from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA) database, we extracted and combined clinical features, levels of NT5E mRNA, and DNA methylation of PTC samples and performed multivariate and random forest analyses to evaluate the prognostic relevance and the potential of discriminating between adjacent non-malignant and thyroid tumor samples. As a result, we revealed that lower levels of methylation at the cg23172664 site were independently associated with BRAF-like phenotype (p = 0.002), age over 55 years (p = 0.012), presence of capsule invasion (p = 0.007) and presence of positive lymph node metastasis (LNM) (p = 0.04). The methylation levels of cg27297263 and cg23172664 sites showed significant and inversely correlations with levels of NT5E mRNA expression (r = -0.528 and r = -0.660, respectively), and their combination was able to discriminate between adjacent non-malignant and tumor samples with a precision of 96%-97% and 84%-85%, respectively. These data suggest that combining cg23172664 and cg27297263 sites may bring new insights to reveal new subsets of patients with papillary thyroid carcinoma.
Assuntos
Carcinoma Papilar , Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/genética , Metilação de DNA/genética , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Medicina de Precisão , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , 5'-Nucleotidase/genética , Proteínas Ligadas por GPI/genéticaRESUMO
The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan-Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , 5'-Nucleotidase/genética , Biomarcadores , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , PrognósticoRESUMO
Ecto-5'-nucleotidase (CD73) is an enzyme present on the surface of tumor cells whose primary described function is the production of extracellular adenosine. Due to the immunosuppressive properties of adenosine, CD73 is being investigated as a target for new antitumor therapies. We and others have described that CD73 is present at the surface of different CD8+ T cell subsets. Nonetheless, there is limited information as to whether CD73 affects CD8+ T cell proliferation and survival. In this study, we assessed the impact of CD73 deficiency on CD8+ T cells by analyzing their proliferation and survival in antigenic and homeostatic conditions. Results obtained from adoptive transfer experiments demonstrate a paradoxical role of CD73. On one side, it favors the expression of interleukin-7 receptor α chain on CD8+ T cells and their homeostatic survival; on the other side, it reduces the survival of activated CD8+ T cells under antigenic stimulation. Also, upon in vitro antigenic stimulation, CD73 decreases the expression of interleukin-2 receptor α chain and the anti-apoptotic molecule Bcl-2, findings that may explain the reduced CD8+ T cell survival observed in this condition. These results indicate that CD73 has a dual effect on CD8+ T cells depending on whether they are subject to an antigenic or homeostatic stimulus, and thus, special attention should be given to these aspects when considering CD73 blockade in the design of novel antitumor therapies.
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CD39 and CD73 are ectoenzymes that dephosphorylate ATP into its metabolites; ADP, AMP, and adenosine, and thus are considered instrumental in the development of immunosuppressive microenvironments. We have previously shown that within the CD8+ T cell population, naïve and memory cells express the CD73 ectonucleotidase, while terminally differentiated effector cells are devoid of this enzyme. This evidence suggests that adenosine might exert an autocrine effect on CD8+ T cells during T cell differentiation. To study the possible role of CD73 and adenosine during this process, we compared the expression of the adenosinergic signaling components, the phenotype, and the functional properties between CD73-deficient and WT CD8+ T cells. Upon activation, we observed an upregulation of CD73 expression in CD8+ T cells along with an upregulation of the adenosine A2A receptor. Interestingly, when we differentiated CD8+ T cells to Tc1 cells in vitro, we observed that these cells produce adenosine and that CD73-deficient cells present a higher cytotoxic potential evidenced by an increase in IFN-γ, TNF-α, and granzyme B production. Moreover, CD73-deficient cells presented a increased glucose uptake and higher mitochondrial respiration, indicating that this ectonucleotidase restrict the mitochondrial capacity in CD8+ T cells. In agreement, when adoptively transferred, antigen-specific CD73-deficient CD8+ T cells were more effective in reducing the tumor burden in B16.OVA melanoma-bearing mice and presented lower levels of exhaustion markers than wild type cells. All these data suggest an autocrine effect of CD73-mediated adenosine production, limiting differentiation and cytotoxic T cells' metabolic fitness.
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Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.
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Adenosina/metabolismo , Terapia de Imunossupressão , Células-Tronco Mesenquimais/citologia , Telomerase/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD , Apirase , Diferenciação Celular , Linhagem Celular Transformada , Espaço Extracelular/química , Regulação da Expressão Gênica , Humanos , Células Jurkat , Ratos Wistar , Telomerase/genéticaRESUMO
Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-ß and adenosinergic signaling contributes to the phenotypic transition of cells and to renal fibrosis in DN models. We evaluated the role of TGF-ß on NT5E gene expression coding for the ecto-5`-nucleotidase CD73, the limiting enzyme in extracellular adenosine production. We showed that high d-glucose may predispose HK-2 cells towards active transcription of the proximal promoter region of the NT5E gene while additional TGF-ß results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-ß with occupancy by the p300 co-activator and the phosphorylated forms of the Smad2/3 complex and RNA Pol II. Transcriptional induction at NT5E in response to TGF-ß was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-ß in HK-2 cells. Interestingly, we found increased CD73 content in urinary extracellular vesicles only in diabetic patients with renal repercussions. Further, CD73-mediated AMPase activity was increased in the urinary sediment of DN patients. We conclude that the NT5E gene is a target of the profibrotic TGF-ß cascade and is a traceable marker of progressive DN.
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5'-Nucleotidase/genética , Nefropatias Diabéticas/genética , Fibrose/genética , Fator de Crescimento Transformador beta/genética , Adenosina/biossíntese , Biomarcadores/metabolismo , Linhagem Celular , Nefropatias Diabéticas/patologia , Proteína p300 Associada a E1A/genética , Epigênese Genética/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose/patologia , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Nucleotidases/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genéticaRESUMO
The incidence of papillary thyroid carcinoma (PTC) has been increasing, which raised the interest in its molecular pathways. Although the high expression of ecto-5'-nucleotidase (NT5E) gene expression and NT5E enzymatic activity in several types of cancer is associated with tumor progression, its role in PTC remains unknown. Here, we investigated the AMP hydrolysis in human normal thyroid cells and PTC cells, in primary culture, and the association of NT5E expression with clinical aspects of PTC patients. AMPase activity was higher in thyroid cells isolated from PTC, as compared to normal thyroid (Pâ¯=â¯0.0063). Significant correlation was observed between AMPase activity and NT5E levels in primary thyroid cell cultures (râ¯=â¯0.655, Pâ¯=â¯0.029). NT5E expression was higher in PTC than in the adjacent non-malignant thyroid tissue (Pâ¯=â¯0.0065) and were positively associated with metastatic lymph nodes (Pâ¯=â¯0.0007), risk of recurrence (Pâ¯=â¯0.0033), tumor size (Pâ¯=â¯0.049), and nodular hyperplasia in the adjacent thyroid parenchyma, when compared to normal thyroid or lymphocytic thyroiditis (Pâ¯=â¯0.0146). After adjusting for potential confounders, the malignant/non-malignant paired expression ratio of NT5E mRNA was independently associated with metastatic lymph nodes (Pâ¯=â¯0.0005), and tumor size (P=0.0005). In addition, the analysis of PTC described in the TCGA database also showed an association between higher expression of NT5E and metastatic lymph nodes, and tumor microinvasion. These results support the hypothesis that NT5E have a role in PTC microenvironment and might be a potential target for PTC therapy.
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5'-Nucleotidase/metabolismo , Linfonodos/enzimologia , Linfonodos/patologia , Metástase Linfática/patologia , Câncer Papilífero da Tireoide/enzimologia , Câncer Papilífero da Tireoide/patologia , 5'-Nucleotidase/genética , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Nucleotidases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Câncer Papilífero da Tireoide/genética , Glândula Tireoide/patologiaRESUMO
The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.