RESUMO
BACKGROUND: Treatment with antipsychotics is associated with an increased risk of type 2 diabetes mellitus (T2D), and increased levels of inflammatory biomarkers are present in patients with T2D. We previously demonstrated that the glucagon-like peptide-1 receptor agonist liraglutide significantly reduced glucometabolic disturbances and body weight in prediabetic, overweight/obese schizophrenia-spectrum disorder patients treated with clozapine or olanzapine. This study aims to assess the involvement of cytokines in the therapeutic effects of liraglutide. METHODS: Serum concentrations of 10 cytokines (interferon-γ [IFN-γ], tumor necrosis factor-α, interleukin 1ß [IL-1ß], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, and IL-13) from fasting prediabetic and normal glucose-tolerant (NGT) patients with schizophrenia-spectrum disorders were measured using multiplexed immunoassays. Prediabetic patients were randomized to 16 weeks of treatment with liraglutide or placebo, and cytokines were measured again at the end of the treatment. RESULTS: IFN-γ (1.98 vs 1.17 pg/ml, P = .001), IL-4 (0.02 vs 0.01 pg/ml, P < .001), and IL-6 (0.73 vs 0.46 pg/ml, P < .001) were significantly higher in prediabetic (n = 77) vs NGT patients (n = 31). No significant changes in cytokine levels following treatment with liraglutide (n = 37) vs placebo (n = 40) were found. CONCLUSION: Prediabetic vs NGT patients with schizophrenia treated with clozapine or olanzapine had increased serum levels of several proinflammatory cytokines, further substantiating the link between inflammation and T2D. Treatment with liraglutide did not affect the investigated cytokines. Further testing of these findings in larger numbers of individuals is needed.
Assuntos
Clozapina , Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Esquizofrenia , Biomarcadores , Clozapina/uso terapêutico , Humanos , Hipoglicemiantes/uso terapêutico , Interleucina-4/uso terapêutico , Interleucina-6/uso terapêutico , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Olanzapina/uso terapêutico , Estado Pré-Diabético/induzido quimicamente , Estado Pré-Diabético/tratamento farmacológico , Esquizofrenia/tratamento farmacológicoRESUMO
It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.
Assuntos
Antibacterianos/uso terapêutico , Linfoma Cutâneo de Células T/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
BACKGROUND: Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL), is a lymphoproliferative disorder characterized by proliferation of malignant T cells in a chronic inflammatory environment in the skin. The nature of MF is still not fully understood, but aberrant microRNA (miR) expression and function seem to play an important role in the pathogenesis and disease progression and have been proposed as a putative disease marker. Recent studies have reported aberrant expression of miR-93 in situin MF lesions and linked dysregulated miR-93 expression to advanced stages of MF. However, the pathophysiological role of miR-93 in MF is unknown. OBJECTIVE: Here, we provide the first evidence that miR-93 targets the cell cycle regulator cyclin-dependent kinase inhibitor p21 and promotes growth of malignant T cells in MF. METHODS/RESULTS: Thus, inhibition of miR-93 in MF patient-derived malignant T-cell lines increases expression of p21 and inhibition of malignant proliferation. Notably, treatment with the histone deacetylase inhibitor Vorinostat (SAHA) reduces miR-93 expression and enhances p21 expression in the malignant T cells. Importantly, transfection with an miR-93 mimic partly blocks SAHA-induced p21 expression. CONCLUSIONS: we provide evidence that enhanced expression of the putative oncogenic miR, miR-93, represses the cell cycle inhibitor p21 and promotes proliferation of malignant T cells. Moreover, we demonstrate that SAHA triggers p21 expression - at least partly - through an inhibition of miR-93.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , MicroRNAs/antagonistas & inibidores , Micose Fungoide/patologia , Neoplasias Cutâneas/patologia , Vorinostat/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients. OBJECTIVES: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL). METHODS: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 - an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry. RESULTS: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells. CONCLUSIONS: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linfoma Cutâneo de Células T/patologia , Neoplasias Cutâneas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Inativação Gênica , Humanos , Indóis/farmacologia , Regiões Promotoras Genéticas , Piridonas/farmacologiaRESUMO
Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region ß chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.
Assuntos
Enterotoxinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-17/biossíntese , Proteínas de Neoplasias/metabolismo , Fator de Transcrição STAT3/metabolismo , Síndrome de Sézary/metabolismo , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Síndrome de Sézary/patologia , Linfócitos T/patologiaRESUMO
B-lymphoid tyrosine kinase (BLK) is a non-receptor tyrosine kinase belonging to the SRC family kinases. BLK is known to be functionally involved in B-cell receptor signaling and B-cell development. New evidence suggests that B-lymphoid tyrosine kinase is ectopically expressed and is a putative oncogene in cutaneous T-cell lymphoma and other T-cell malignancies. However, little is known about the role of BLK in lymphomagenesis, and the oncogenic function seems to depend on the cellular context. Importantly, BLK is also ectopically expressed in other hematological and multiple non-hematological malignancies including breast, kidney, and lung cancers, suggesting that BLK could be a new potential target for therapy. Here, we studied the oncogenic potential of human BLK. We found that engrafted Ba/F3 cells stably expressing constitutive active human BLK formed tumors in mice, whereas neither Ba/F3 cells expressing wild type BLK nor non-transfected Ba/F3 cells did. Inhibition of BLK with the clinical grade and broadly reacting SRC family kinase inhibitor dasatinib inhibited growth of BLK-induced tumors. In conclusion, our study provides evidence that human BLK is a true proto-oncogene capable of inducing tumors, and we demonstrate a novel BLK activity-dependent tumor model suitable for studies of BLK-driven lymphomagenesis and screening of novel BLK inhibitors in vivo.
Assuntos
Carcinogênese/genética , Ativação Linfocitária/genética , Linfoma Cutâneo de Células T/genética , Quinases da Família src/genética , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Cutâneo de Células T/patologia , Camundongos , Proto-Oncogene Mas , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/biossínteseRESUMO
Staphylococcus aureus and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8+ T cells play a crucial role in anti-cancer responses and high CD8+ T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8+ T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8+ T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8+ T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL.
Assuntos
Toxinas Bacterianas , Linfócitos T CD8-Positivos , Proteínas Hemolisinas , Linfoma Cutâneo de Células T , Neoplasias Cutâneas , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Humanos , Leucócitos Mononucleares , Linfoma Cutâneo de Células T/imunologia , Neoplasias Cutâneas/imunologia , Staphylococcus aureusRESUMO
The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by a subset of chronically activated memory T cells and plays an important role in their activation and proliferation. Here, we show that primary malignant T cells isolated from patients with Sézary syndrome (SS) express Kv1.3 and are sensitive to potent Kv1.3 inhibitors ShK and Vm24, but not sensitive to a less potent inhibitor [N17A/F32T]-AnTx. Kv1.3 blockade inhibits CD3/CD28-induced proliferation and IL-9 expression by SS cells in a concentration-dependent manner. In parallel, CD3/CD28-mediated CD25 induction is inhibited, whereas Kv1.3 blockade has no effect on apoptosis or cell death as judged by Annexin V and PI staining. In conclusion, we provide the first evidence that malignant T cells in SS express functional Kv1.3 channels and that Kv1.3 blockade inhibits activation-induced proliferation as well as cytokine and cytokine receptor expression in malignant T cells, suggesting that Kv1.3 is a potential target for therapy in SS.
RESUMO
Staphylococcus aureus is implicated in disease progression in cutaneous T-cell lymphoma (CTCL). Here, we demonstrate that malignant T cell lines derived from CTCL patients as well as primary malignant CD4+ T cells from Sézary syndrome patients are considerably more resistant to alpha-toxin-induced cell death than their non-malignant counterparts. Thus, in a subset of Sézary syndrome patients the ratio between malignant and non-malignant CD4+ T cells increases significantly following exposure to alpha-toxin. Whereas toxin-induced cell death is ADAM10 dependent in healthy CD4+ T cells, resistance to alpha-toxin in malignant T cells involves both downregulation of ADAM10 as well as other resistance mechanisms. In conclusion, we provide first evidence that Staphylococcus aureus derived alpha-toxin can tilt the balance between malignant and non-malignant CD4+ T cells in CTCL patients. Consequently, alpha-toxin may promote disease progression through positive selection of malignant CD4+ T cells, identifying alpha-toxin as a putative drug target in CTCL.
RESUMO
Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2/metabolismo , Linfócitos T/fisiologia , Animais , Sinalização do Cálcio , Morte Celular , Células Cultivadas , Difosfonatos/farmacologia , Humanos , Camundongos , Naftalenossulfonatos/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptor Cross-Talk , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7/genética , Transgenes/genéticaRESUMO
Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with a median life expectancy of less than 4 years. Although initial treatment responses are often good, the vast majority of patients with SS fail to respond to ongoing therapy. We hypothesize that malignant T cells are highly heterogeneous and harbor subpopulations of SS cells that are both sensitive and resistant to treatment. Here, we investigate the presence of single-cell heterogeneity and resistance to histone deacetylase inhibitors (HDACi) within primary malignant T cells from patients with SS. Using single-cell RNA sequencing and flow cytometry, we find that malignant T cells from all investigated patients with SS display a high degree of single-cell heterogeneity at both the mRNA and protein levels. We show that this heterogeneity divides the malignant cells into distinct subpopulations that can be isolated by their expression of different surface antigens. Finally, we show that treatment with HDACi (suberanilohydroxamic acid and romidepsin) selectively eliminates some subpopulations while leaving other subpopulations largely unaffected. In conclusion, we show that patients with SS display a high degree of single-cell heterogeneity within the malignant T-cell population, and that distinct subpopulations of malignant T cells carry HDACi resistance. Our data point to the importance of understanding the heterogeneous nature of malignant SS cells in each individual patient to design combinational and new therapies to counter drug resistance and treatment failure.
Assuntos
Depsipeptídeos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Inibidores de Histona Desacetilases/farmacologia , Síndrome de Sézary , Linfócitos T , Vorinostat/farmacologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Sézary/tratamento farmacológico , Síndrome de Sézary/metabolismo , Síndrome de Sézary/patologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Deficient expression of SATB1 hampers thymocyte development and results in inept T-cell lineages. Recent data implicate dysregulated SATB1 expression in the pathogenesis of mycosis fungoides, the most frequent variant of cutaneous T-cell lymphoma. Here, we report on a disease stage-associated decrease of SATB1 expression and an inverse expression of STAT5 and SATB1 in situ. STAT5 inhibited SATB1 expression through induction of microRNA-155. Decreased SATB1 expression triggered enhanced expression of IL-5 and IL-9 (but not IL-6 and IL-32), whereas increased SATB1 expression had the opposite effect, indicating that the microRNA-155 target SATB1 is a repressor of IL-5 and IL-9 in malignant T cells. In accordance, inhibition of STAT5 and its upstream activator JAK3 triggered increased SATB1 expression and a concomitant suppression of IL-5 and IL-9 expression in malignant T cells. In conclusion, we provide a mechanistic link between the proto-oncogenic JAK3/STAT5/microRNA-155 pathway, SATB1, and cytokines linked to CTCL severity and progression, indicating that SATB1 dysregulation is involved in cutaneous T-cell lymphoma pathogenesis.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/genética , MicroRNAs/metabolismo , Micose Fungoide/genética , Neoplasias Cutâneas/genética , Linfócitos T/imunologia , Linhagem Celular Tumoral , Estudos de Coortes , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Interleucina-5/imunologia , Interleucina-5/metabolismo , Interleucina-9/imunologia , Interleucina-9/metabolismo , Janus Quinase 3/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/imunologia , Micose Fungoide/imunologia , Micose Fungoide/patologia , Estadiamento de Neoplasias , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/metabolismoRESUMO
Short chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are products of microbial macronutrients fermentation that distribute systemically and are believed to modulate host immune responses. Recent data have indicated that certain SCFAs, such as butyrate and propionate, directly modulate human dendritic cell (DC) function. Given the role of DCs in initiating and shaping the adaptive immune response, we now explore how SCFAs affect the activation of antigen-specific CD8+ T cells stimulated with autologous, MART1 peptide-pulsed DC. We show that butyrate reduces the frequency of peptide-specific CD8+ T cells and, together with propionate, inhibit the activity of those cells. On the contrary, acetate does not affect them. Importantly, butyrate and propionate inhibit the production of IL-12 and IL-23 in the DCs and exogenous IL-12 fully restores the activation of the MART-1-specific CD8+ T cells, whereas IL-23 has no effect. In conclusion, these results point to a pivotal role of butyrate and propionate in modulating CD8+ T cell activation via the inhibition of IL-12 secretion from DCs. These findings reveal a novel mechanism whereby bacterial fermentation products may modulate CD8+ T cell function with possible implications in anti-cancer immunotherapy.
Assuntos
Butiratos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Interleucina-12/metabolismo , Propionatos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Humanos , Interleucina-23/metabolismo , Antígeno MART-1/metabolismoRESUMO
PURPOSE: Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9-producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown. EXPERIMENTAL DESIGN: We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice. RESULTS: Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes. CONCLUSIONS: Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328-39. ©2016 AACR.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Interleucina-9/biossíntese , Micose Fungoide/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fatores Reguladores de Interferon/genética , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Linfócitos T Reguladores/imunologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Linfoma Cutâneo de Células T/metabolismo , MicroRNAs/biossíntese , Fator de Transcrição STAT5/metabolismo , Neoplasias Cutâneas/metabolismo , Feminino , Humanos , Linfoma Cutâneo de Células T/genética , Masculino , MicroRNAs/genética , Fator de Transcrição STAT5/genética , Neoplasias Cutâneas/genéticaRESUMO
Aberrant activation of Janus kinase-3 (Jak3) and its key down-stream effectors, Signal Transducer and Activator of Transcription-3 (STAT3) and STAT5, is a key feature of malignant transformation in cutaneous T-cell lymphoma (CTCL). However, it remains only partially understood how Jak3/STAT activation promotes lymphomagenesis. Recently, non-coding microRNAs (miRNAs) have been implicated in the pathogenesis of this malignancy. Here, we show that (i) malignant T cells display a decreased expression of a tumor suppressor miRNA, miR-22, when compared to non-malignant T cells, (ii) STAT5 binds the promoter of the miR-22 host gene, and (iii) inhibition of Jak3, STAT3, and STAT5 triggers increased expression of pri-miR-22 and miR-22. Curcumin, a nutrient with anti-Jak3 activity and histone deacetylase inhibitors (HDACi) also trigger increased expression of pri-miR-22 and miR-22. Transfection of malignant T cells with recombinant miR-22 inhibits the expression of validated miR-22 targets including NCoA1, a transcriptional co-activator in others cancers, as well as HDAC6, MAX, MYCBP, PTEN, and CDK2, which have all been implicated in CTCL pathogenesis. In conclusion, we provide the first evidence that de-regulated Jak3/STAT3/STAT5 signalling in CTCL cells represses the expression of the gene encoding miR-22, a novel tumor suppressor miRNA.
Assuntos
Janus Quinase 3/metabolismo , Linfoma Cutâneo de Células T/genética , MicroRNAs/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Genes Supressores de Tumor , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , MicroRNAs/administração & dosagem , MicroRNAs/biossíntese , MicroRNAs/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , TransfecçãoRESUMO
Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.
Assuntos
Linfoma Cutâneo de Células T/metabolismo , Fator de Transcrição STAT4/metabolismo , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Inflamação/patologia , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Pele/patologia , Dermatopatias/patologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Regulação para Cima/efeitos dos fármacos , VorinostatRESUMO
Eosinophil granulocytes have been implicated in anticancer immunity but recent data indicate that eosinophils can also promote cancer. Herein, we studied eosinophils in skin lesions from 43 patients with mycosis fungoides (MF). The presence of eosinophils correlated with disease stage: 78% of patients with advanced disease displayed eosinophil infiltration, whereas this was only seen in 11% of patients with patches (p<0.01), and in 48% of those with plaque disease. Importantly, 72% of patients with positive staining for phospho-signal-transducer-and-activator-of-transcription (pY-STAT3) in malignant T-cells also stained positively for eosinophils, whereas this was only observed in 28% of pY-STAT3-negative patients (p<0.01). Notably, malignant T-cells expressed eosinophilic activation and trafficking factors: High-mobility group BOX-1 protein (HMGB1) and interleukin 5 (IL5). STAT3 siRNA profoundly inhibited IL5 but not HMGB1 expression. In conclusion, these data suggest that malignant T-cells orchestrate accumulation and activation of eosinophils supporting the notion of STAT3 being a putative target for therapy.
Assuntos
Eosinófilos/patologia , Micose Fungoide/metabolismo , Micose Fungoide/patologia , Fator de Transcrição STAT3/metabolismo , Biópsia , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-5/genética , Interleucina-5/metabolismo , Micose Fungoide/genética , Micose Fungoide/imunologia , Interferência de RNA , Fator de Transcrição STAT3/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Skin lesions from mycosis fungoides (MF) patients display an increased expression of interleukin-15 (IL-15), IL-17F, and other cytokines implicated in inflammation and malignant cell proliferation in cutaneous T-cell lymphoma (CTCL). In the leukemic variant of CTCL, Sézary syndrome (SS), IL-2 and IL-15 trigger activation of the Jak-3/STAT3 pathway and transcription of IL17A gene, whereas it is unknown what causes IL-15 expression, Jak3/STAT3 activation, and production of IL-17F in MF. Here, we studied the expression and regulation of IL-15 and its relation to IL-17F in MF cell lines and skin lesions from 60 MF patients. We show that: (1) the spontaneous IL-15 mRNA expression is resistant to Jak3 and STAT3 inhibitors at concentrations that profoundly inhibit STAT3 activation and IL-17F mRNA expression; (2) anti-IL-15 antibody blocks STAT3 activation induced by exogenous IL-15 in non-malignant MF T cells, whereas the spontaneous STAT3 activation and IL-17F expression in malignant T cells is not inhibited; (3) patients display heterogeneous IL-15/IL-17F mRNA expression patterns in skin lesions; and (4) IL-15 expression (in contrast to IL-17F) is not associated with progressive disease. Taken together, these findings indicate that IL-15 and IL-17F are differentially regulated and expressed in MF. We propose that IL-15 and IL-17F are markers for different inflammatory environments and play distinct roles in the development and progression of MF.