RESUMO
Anaplastic thyroid carcinoma (ATC) is an extremely difficult disease to tackle, with an overall patient survival of only a few months. The currently used therapeutic drugs, such as kinase inhibitors or immune checkpoint inhibitors, can prolong patient survival but fail to eradicate the tumor. In addition, the onset of drug resistance and adverse side-effects over time drastically reduce the chances of treatment. We recently showed that Twist1, a transcription factor involved in the epithelial mesenchymal transition (EMT), was strongly upregulated in ATC, and we wondered whether it might represent a therapeutic target in ATC patients. To investigate this hypothesis, the effects of harmine, a ß-carboline alkaloid shown to induce degradation of the Twist1 protein and to possess antitumoral activity in different cancer types, were evaluated on two ATC-derived cell lines, BHT-101 and CAL-62. The results obtained demonstrated that, in both cell lines, harmine reduced the level of Twist1 protein and reverted the EMT, as suggested by the augmentation of E-cadherin and decrease in fibronectin expression. The drug also inhibited cell proliferation and migration in a dose-dependent manner and significantly reduced the anchorage-independent growth of both ATC cell lines. Harmine was also capable of inducing apoptosis in BHT-101 cells, but not in CAL-62 ones. Finally, the activation of PI3K/Akt signaling, but not that of the MAPK, was drastically reduced in treated cells. Overall, these in vitro data suggest that harmine could represent a new therapeutic option for ATC treatment.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Harmina/farmacologia , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Proteína 1 Relacionada a Twist/genética , Fosfatidilinositol 3-Quinases , Neoplasias da Glândula Tireoide/tratamento farmacológicoRESUMO
Mutations in mitochondrial transfer RNA (mt-tRNA) genes are responsible for a wide range of syndromes, for which no effective treatment is available. We previously reported that transfection of the nucleotide sequence encoding for the 16-residue ß32_33 peptide from mitochondrial leucyl-tRNA synthetase ameliorates the cell phenotype caused by the mitochondrial tRNA mutations. In this work, we demonstrated that both the ß32_33 peptide linked with the known (L)-Phe-(D)-Arg-(L)-Phe-(L)-Lys (FrFK) mitochondrial penetrating sequence and, strikingly, the ß32_33 peptide per se, are able to penetrate both the plasma and mitochondrial membranes and exert the rescuing activity when exogenously administered to cells bearing the mutations m.3243A > G and m.8344A > G. These mutations are responsible for the most common and severe mt-tRNA-related diseases. In addition, we dissected the molecular determinants of constructs activity by showing that both the order of amino acids along the sequence and presence of positive charges are essential determinants of the peptide activity in cells and mt-tRNA structures stabilization in vitro. In view of future in vivo studies, this information may be required to design of ß32_33 peptide-mimetic derivatives. The ß32_33 and FrFK-ß32_33 peptides are, therefore, promising molecules for the development of therapeutic agents against diseases caused by the mt-tRNA point mutations.
Assuntos
Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Peptídeos/metabolismo , RNA de Transferência/metabolismo , Aminoácidos/metabolismo , Linhagem Celular , Humanos , Mutação Puntual/fisiologiaRESUMO
The transcription factor Nanog plays a critical role in the self-renewal of embryonic stem cells as well as in neural stem cells (NSCs). microRNAs (miRNAs) are also involved in stemness regulation. However, the miRNA network downstream of Nanog is still poorly understood. High-throughput screening of miRNA expression profiles in response to modulated levels of Nanog in postnatal NSCs identifies miR-17-92 cluster as a direct target of Nanog. Nanog controls miR-17-92 cluster by binding to the upstream regulatory region and maintaining high levels of transcription in NSCs, whereas Nanog/promoter association and cluster miRNAs expression are lost alongside differentiation. The two miR-17 family members of miR-17-92 cluster, namely miR-17 and miR-20a, target Trp53inp1, a downstream component of p53 pathway. To support a functional role, the presence of miR-17/20a or the loss of Trp53inp1 is required for the Nanog-induced enhancement of self-renewal of NSCs. We unveil an arm of the Nanog/p53 pathway, which regulates stemness in postnatal NSCs, wherein Nanog counteracts p53 signals through miR-17/20a-mediated repression of Trp53inp1.
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Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Ciclo Celular , Proliferação de Células , Células Cultivadas , Cerebelo/citologia , Proteínas de Choque Térmico/genética , Proteínas de Homeodomínio/genética , Camundongos , MicroRNAs/genética , Proteína Homeobox Nanog , Células-Tronco Neurais/citologiaRESUMO
The association of Epstein-Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR-21 is induced after in vitro EBV infection. The PU.1 binding site in miR-21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR-21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR-21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR-21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/genética , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , MicroRNAs/genética , Animais , Sítios de Ligação/genética , Ciclina D3/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo/genética , Herpesvirus Humano 4 , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/virologia , Regiões Promotoras GenéticasRESUMO
FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Células de Sertoli/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Western Blotting , Antígenos CD4/biossíntese , Antígenos CD4/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Imunoprecipitação da Cromatina , Citometria de Fluxo , Fatores de Transcrição Forkhead/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Masculino , Proteínas de Membrana/genética , Camundongos , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/genética , Proteínas Serrate-Jagged , Supressão Genética , Transfecção , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3É, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3É+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.
Assuntos
Progressão da Doença , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptor Notch3 , Receptores CXCR4 , Timócitos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Receptor Notch3/genética , Receptor Notch3/metabolismo , Timócitos/metabolismo , Timócitos/citologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Camundongos , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Humanos , Camundongos Transgênicos , Transdução de Sinais , Diferenciação Celular/genéticaRESUMO
INTRODUCTION: Vascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc. METHODS: To demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-ß and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression. RESULTS: We showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-ß and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-ß was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression. CONCLUSIONS: BM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.
Assuntos
Endotélio Vascular/citologia , Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica/fisiologia , Fenótipo , Medicina Regenerativa/métodos , Escleroderma Sistêmico/fisiopatologia , Actinas/metabolismo , Becaplermina , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Primers do DNA/genética , Relação Dose-Resposta a Droga , Citometria de Fluxo , Imunofluorescência , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Pericitos/fisiologia , Proteínas Proto-Oncogênicas c-sis/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Escleroderma Sistêmico/terapia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Notch receptors deeply influence T-cell development and differentiation, and their dysregulation represents a frequent causative event in "T-cell acute lymphoblastic leukemia" (T-ALL). "Myeloid-derived suppressor cells" (MDSCs) inhibit host immune responses in the tumor environment, favoring cancer progression, as reported in solid and hematologic tumors, with the notable exception of T-ALL. Here, we prove that Notch-signaling deregulation in immature T cells promotes CD11b+Gr-1+ MDSCs in the Notch3-transgenic murine model of T-ALL. Indeed, aberrant T cells from these mice can induce MDSCs in vitro, as well as in immunodeficient hosts. Conversely, anti-Gr1-mediated depletion of MDSCs in T-ALL-bearing mice reduces proliferation and expansion of malignant T cells. Interestingly, the coculture with Notch-dependent T-ALL cell lines, sustains the induction of human CD14+HLA-DRlow/neg MDSCs from healthy-donor PBMCs that are impaired upon exposure to gamma-secretase inhibitors. Notch-independent T-ALL cells do not induce MDSCs, suggesting that Notch-signaling activation is crucial for this process. Finally, in both murine and human models, IL-6 mediates MDSC induction, which is significantly reversed by treatment with neutralizing antibodies. Overall, our results unveil a novel role of Notch-deregulated T cells in modifying the T-ALL environment and represent a strong premise for the clinical assessment of MDSCs in T-ALL patients.
Assuntos
Células Supressoras Mieloides , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Antígenos HLA-DR/metabolismo , Humanos , Camundongos , Transdução de Sinais , Linfócitos TRESUMO
OBJECTIVE: Bone metastasis is a clinically important outcome of prostate carcinoma (PC). We focused on the phenotypic and functional characterization of a particularly aggressive phenotype within the androgen-independent bone metastasis-derived PC3 cell line. These cells, originated from the spontaneous conversion of a CD44-negative subpopulation, stably express the CD44v8-10 isoform (CD44v8-10pos) and display stem cell-like features and a marked invasive phenotype in vitro that is lost upon CD44v8-10 silencing. METHODS: Flow cytometry, enzyme-linked immunoassay, immunofluorescence, and Western blot were used for phenotypic and immunologic characterization. Real-time quantitative polymerase chain reaction and functional assays were used to assess osteomimicry. RESULTS: Analysis of epithelial-mesenchymal transition markers showed that CD44v8-10pos PC3 cells surprisingly display epithelial phenotype and can undergo osteomimicry, acquiring bone cell phenotypic and behavioral traits. Use of specific siRNA evidenced the ability of CD44v8-10 variant to confer osteomimetic features, hence the potential to form bone-specific metastasis. Moreover, the ability of tumors to activate immunosuppressive mechanisms which counteract effective immune responses is a sign of the aggressiveness of a tumor. Here we report that CD44v8-10pos cells express programmed death ligand 1, a negative regulator of anticancer immunity, and secrete exceptionally high amounts of interleukin-6, favoring osteoclastogenesis and immunosuppression in bone microenvironment. Notably, we identified a novel pathway activated by CD44v8-10, involving tafazzin (TAZ) and likely the Wnt/TAZ axis, known to play a role in upregulating osteomimetic genes. CONCLUSIONS: CD44v8-10 could represent a marker of a more aggressive bone metastatic PC population exerting a driver role in osteomimicry in bone. A novel link between TAZ and CD44v8-10 is also shown.
RESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric malignancy that arises from the transformation of immature T-cell progenitors and has no definitive cure. Notch signaling governs many steps of T cell development and its dysregulation represents the most common causative event in the pathogenesis of T-ALL. The activation of canonical NF-κB pathway has been described as a critical downstream mediator of Notch oncogenic functions, through the sustaining of tumor cell survival and growth. The potential role of Notch/NF-κB partnership is also emerging in the generation and function of regulatory T cells (Tregs) in the context of cancer. However, little is known about the effects of combined mutations of Notch and NF-κB in regulating immune-environment and progression of T-ALL. To shed light on the topics above we generated double-mutant mice, harboring conventional knock-out mutation of NF-κB1/p50 on the genetic background of a transgenic model of Notch-dependent T-ALL. The immunophenotyping of double-mutant mice demonstrates that NF-κB1 deletion inhibits the progression of T-ALL and strongly modifies immune-environment of the disease. Double-mutant mice display indeed a dramatic reduction of pre-leukemic CD4+CD8+ (DP) T cells and regulatory T cells (Tregs) and, concurrently, the rising of an aggressive myeloproliferative trait with a massive expansion of CD11b+Gr-1+ cells in the periphery, and an accumulation of the granulocyte/monocyte progenitors in the bone-marrow. Interestingly, double-mutant T cells are able to improve the growth of CD11b+Gr-1+ cells in vitro, and, more importantly, the in vivo depletion of T cells in double-mutant mice significantly reduces the expansion of myeloid compartment. Our results strongly suggest that the myeloproliferative trait observed in double-mutant mice may depend on non-cell-autonomous mechanism/s driven by T cells. Moreover, we demonstrate that the reduction of CD4+CD8+ (DP) T cells and Tregs in double-mutant mice relies on a significant enhancement of their apoptotic rate. In conclusion, double-mutant mice may represent a useful model to deepen the knowledge of the consequences on T-ALL immune-environment of modulating Notch/NF-κB relationships in tumor cells. More importantly, information derived from these studies may help in the refinement of multitarget therapies for the disease.
Assuntos
NF-kappa B/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Receptores Notch/imunologia , Microambiente Tumoral/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , NF-kappa B/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores Notch/genética , Transdução de Sinais/fisiologiaRESUMO
The Notch signaling pathway plays multiple roles in driving T-cell fate decisions, proliferation, and aberrant growth. NF-κB is a cell-context key player interconnected with Notch signaling either in physiological or in pathological conditions. This review focuses on how the multilayered crosstalk between different Notches and NF-κB subunits may converge on Foxp3 gene regulation and orchestrate CD4+ regulatory T (Treg) cell function, particularly in a tumor microenvironment. Notably, Treg cells may play a pivotal role in the inhibition of antitumor immune responses, possibly promoting tumor growth. A future challenge is represented by further dissection of both Notch and NF-κB pathways and consequences of their intersection in tumor-associated Treg biology. This may shed light on the molecular mechanisms regulating Treg cell expansion and migration to peripheral lymphoid organs thought to facilitate tumor development and still to be explored. In so doing, new opportunities for combined and/or more selective therapeutic approaches to improve anticancer immunity may be found.
Assuntos
NF-kappa B/imunologia , Neoplasias/imunologia , Receptores Notch/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Movimento Celular/imunologia , Proliferação de Células , Humanos , Neoplasias/patologia , Linfócitos T Reguladores/patologiaRESUMO
Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through ß-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4+CD8+ cells, by a "thymus-autonomous" mechanism.
Assuntos
Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch3/metabolismo , Receptores CXCR4/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Movimento Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Camundongos , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismoRESUMO
In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.
RESUMO
To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1-mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.
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The alternative TrkAIII splice variant is expressed by murine and human thymus. Alternative TrkAIII splicing predominates in postembryonic day E13 (E17 and E18), postnatal murine (3 week and 3 month) and human thymuses, with TrkAIII mRNA expressed by selected thymocyte subsets and thymic epithelial cells (TECs) and a 100 kDa immunoprecipitable TrkAIII-like protein detected in purified thymocyte and whole thymus extracts. FACS and immunohistochemical analysis indicate a non-cell surface localisation for the TrkAIII-like protein in cortical CD4+/CD8+ double positive and, to a lesser extent, single positive thymocyte subsets at the cortex/medulla boundary and in Hassle's corpuscles, reticular epithelial and dendritic cells of the thymic medulla. TrkA(I/II) expression, on the other hand, predominates in sub-capsular regions of the thymus. TrkAIII-like immunoreactivity at the cortex/medulla boundary associates with regions of thymocyte proliferation and not apoptosis. A potential role for thymic hypoxia in thymocyte alternative TrkAIII splicing is supported by reversal to TrkAI splicing by normoxic but not hypoxic culture and induction of Jurkat T cell alternative TrkAIII splicing by the hypoxia mimic CoCl2. In contrast, TEC expression of TrkAIII predominates in both normoxic and hypoxic culture conditions. The data support a potential role for TrkAIII in thymic development and function, of particular relevance to intermediate stage CD4+/CD8+ thymocyte subsets and TECs, which potentially reflects a reversible thymocyte and more permanent TEC adaptation to thymic environment. Since intracellular TrkAIII neither binds nor responds to NGF and can impede regular NGF/TrkA signalling (Tacconelli et al., Cancer Cell, 2004), its expression would be expected to provide an alternative and/or impediment to regular NGF/TrkA signalling within the developing and developed thymus of potential functional importance.
Assuntos
Receptor trkA/genética , Receptor trkA/metabolismo , Timo/citologia , Timo/metabolismo , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Células Cultivadas , Embrião de Mamíferos , Células Epiteliais/fisiologia , Citometria de Fluxo/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Células Jurkat , Camundongos , Neuroblastoma , Extratos do Timo/metabolismoRESUMO
The Notch pathway represents a conserved signal transduction machinery that is straightforward and based on a few elements (ligands, receptors, transducers). However, the existence of multiple control levels of the Notch signaling final outcome makes it strictly context dependent and dose dependent. The function of Notch as a regulator of cell development and differentiation, as well as the aberrant consequences of its modulation, either positive or negative, is well established. In this review, we will discuss our current knowledge about Notch-dependent regulation of generation and function of 2 subsets of the immunoregulatory system, namely regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Then, we will focus on an unforeseen mechanism that may unveil an additional way of Notch to govern the surrounding environment in cancer.
Assuntos
Células Supressoras Mieloides/imunologia , Receptores Notch/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diferenciação Celular/imunologia , Humanos , Células Supressoras Mieloides/citologia , Linfócitos T Reguladores/citologiaRESUMO
INTRODUCTION: To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc). METHODS: We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression. RESULTS: ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-ß) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-ß, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs. CONCLUSION: We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Esclerodermia Difusa/metabolismo , Actinas/genética , Actinas/metabolismo , Adulto , Becaplermina , Western Blotting , Células Cultivadas , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclerodermia Difusa/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
Chemoresistance is a major obstacle to effective therapy against colorectal cancer (CRC) and may lead to deadly consequences. The metabolism of CRC cells depends highly on the p38 MAPK pathway, whose involvement in maintaining a chemoresistant behavior is currently being investigated. Our previous studies revealed that p38α is the main p38 isoform in CRC cells. Here we show that p38α pharmacological inhibition combined with cisplatin administration decreases colony formation and viability of cancer cells and strongly increases Bax-dependent apoptotic cell death by activating the tumor suppressor protein FoxO3A. Our results indicate that FoxO3A activation up-regulates transcription of its target genes (p21, PTEN, Bim and GADD45), which forces both chemosensitive and chemoresistant CRC cells to undergo apoptosis. Additionally, we found that FoxO3A is required for apoptotic cell death induction, as confirmed by RNA interference experiments. In animal models xenografted with chemoresistant HT29 cells, we further confirmed that the p38-targeted dual therapy strategy produced an increase in apoptosis in cancer tissue leading to tumor regression. Our study uncovers a major role for the p38-FoxO3A axis in chemoresistance, thereby suggesting a new therapeutic approach for CRC treatment; moreover, our results indicate that Bax status may be used as a predictive biomarker.
Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Cisplatino/farmacologia , Feminino , Citometria de Fluxo , Imunofluorescência , Proteína Forkhead Box O3 , Células HT29 , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile.