RESUMO
Extracellular vesicles act as shuttle vectors or signal transducers that can deliver specific biological information and have progressively emerged as key regulators of organized communities of cells within multicellular organisms in health and disease. Here, we survey the evolutionary origin, general characteristics, and biological significance of extracellular vesicles as mediators of intercellular signaling, discuss the various subtypes of extracellular vesicles thus far described and the principal methodological approaches to their study, and review the role of extracellular vesicles in tumorigenesis, immunity, non-synaptic neural communication, vascular-neural communication through the blood-brain barrier, renal pathophysiology, and embryo-fetal/maternal communication through the placenta.
Assuntos
Biomarcadores/metabolismo , Doença/genética , Vesículas Extracelulares/metabolismo , Comunicação Celular , Vesículas Extracelulares/genética , Predisposição Genética para Doença , Humanos , Imunidade , Transdução de SinaisRESUMO
BACKGROUND: Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations. METHODS: Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts. RESULTS: We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa ßGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response. CONCLUSIONS: Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Morte Celular Autofágica/efeitos dos fármacos , Morte Celular Autofágica/imunologia , Calreticulina/imunologia , Calreticulina/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/imunologia , Feminino , Galectinas/farmacologia , Xenoenxertos , Humanos , Vigilância Imunológica , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
PURPOSE: The role of non-genetic factors as modifiers of TP53-related hereditary breast cancer (BC) risk is debated. In this regard, little is known about the impact of germline TP53 mutations on BC in sub-Saharan Africa, where the disease often presents in non-contraceptive multiparous premenopausal women with extended history of breastfeeding. Herein, we report the germline TP53 mutations found in a series of 92 Sudanese premenopausal BC patients characterized for reproductive history. METHODS: The entire TP53 coding sequence, including intron-exon boundaries and UTRs, was analyzed via DHPLC and direct sequencing, and the association of TP53 genotypes with BC risk and with individual lifetime exposures to reproductive factors was investigated with statistical tools. RESULTS: The germline TP53 mutation spectrum comprised 20 variants, 15 in the non-coding and 5 in the coding region. The latter included a deleterious missense mutation, c.817C>T (p.Arg273Cys), in a unique patient, and the common and functionally relevant coding polymorphism at amino acid 72 [Pro72Arg (rs1042522)]. The non-coding mutations included c.919+1G>A, a known deleterious splice site mutation, also in a unique patient. Notably, the 2 carriers of deleterious TP53 mutations clustered in the subset of cases with stronger reproductive history relative to childbearing age. When analyzed in comparison to population controls, the codon 72 polymorphism did not reveal associations with BC. CONCLUSIONS: Our study suggests that the codon 72 Arg>Pro polymorphism is not implicated in premenopausal BC susceptibility, whereas multiparity and breastfeeding might be BC risk factors for carriers of deleterious TP53 mutations.
Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Reprodução/genética , Proteína Supressora de Tumor p53/genética , Adulto , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/fisiopatologia , Feminino , Testes Genéticos , Genótipo , Mutação em Linhagem Germinativa/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Paridade/genética , Gravidez , Pré-Menopausa/genética , Pré-Menopausa/fisiologia , Reprodução/fisiologia , História Reprodutiva , Sudão/epidemiologiaRESUMO
The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.
RESUMO
Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/fisiopatologia , Mesilato de Imatinib/uso terapêutico , Paraganglioma/tratamento farmacológico , Paraganglioma/fisiopatologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mesilato de Imatinib/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Organogênese/efeitos dos fármacos , Organogênese/fisiologia , Paraganglioma/genética , Paraganglioma/patologia , Cultura Primária de Células , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND & AIMS: Studies of the transcriptional networks that regulate nuclear receptor-mediated proliferation of quiescent hepatocytes could lead to new information about liver growth and hepatoprotective strategies. METHODS: We used quantitative real-time PCR to analyze expression of neuron-derived orphan receptor 1 (Nor-1) and its target genes during liver regeneration after hepatectomy in mice, and in hepatocellular carcinoma (HCC) samples from patients. We used adenoviral vectors to express Nor-1 in normal liver (Ad/CMV/V5-Nor-1), or reduce its level with small hairpin RNAs (Ad/BLOCK-iT/Nor-1(small hairpin RNA)) after partial hepatectomy. RESULTS: Levels of Nor-1 messenger RNA and protein, and transcription of Nor-1 target genes (Ccnd1 and Vcam-1), increased during the late priming and proliferative phases of liver regeneration after partial hepatectomy. Levels of NOR-1 messenger RNA and transcription of its target gene CCND1 and of the NOR-1 subfamily member NUR-77 also increased in human HCC samples compared with paired HCC-free tissue. Ad-Nor-1(small hairpin RNA) reduced the hepatocyte proliferation after hepatectomy. Overexpression of Nor-1 in normal livers of mice induced proliferation of quiescent hepatocytes independently of interleukin-6 and tumor necrosis factor-α signaling. In gene expression profile analysis, Nor-1 altered expression of genes involved in the cell cycle, proliferation, and tumorigenesis. CONCLUSIONS: In mice, the orphan nuclear receptor Nor-1 activates proliferation of quiescent hepatocytes and is required for hepatocyte proliferation after partial hepatectomy. Nor-1 and its gene targets are also up-regulated in human HCC samples. Nor-1 activates a transcriptional program that induces hepatocyte proliferation independently of inflammatory signaling pathways.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Hepatócitos/citologia , Neoplasias Hepáticas/metabolismo , Regeneração Hepática/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Receptores de Esteroides/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Animais , Carcinoma Hepatocelular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/genética , Hepatectomia , Humanos , Neoplasias Hepáticas/genética , Regeneração Hepática/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , RNA Mensageiro/análise , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The present perspective article proposes an etiopathological model for multiple sclerosis pathogenesis and progression associated with the activation of human endogenous retroviruses. We reviewed preclinical, clinical, epidemiological, and evolutionary evidence indicating how the complex, multi-level interplay of genetic traits and environmental factors contributes to multiple sclerosis. We propose that endogenous retroviruses transactivation acts as a critical node in disease development. We also discuss the rationale for combined anti-retroviral therapy in multiple sclerosis as a disease-modifying therapeutic strategy. Finally, we propose that the immuno-pathogenic process triggered by endogenous retrovirus activation can be extended to aging and aging-related neurodegeneration. In this regard, endogenous retroviruses can be envisioned to act as epigenetic noise, favoring the proliferation of disorganized cellular subpopulations and accelerating system-specific "aging". Since inflammation and aging are two sides of the same coin (plastic dis-adaptation to external stimuli with system-specific degree of freedom), the two conditions may be epiphenomenal products of increased epigenomic entropy. Inflammation accelerates organ-specific aging, disrupting communication throughout critical systems of the body and producing symptoms. Overlapping neurological symptoms and syndromes may emerge from the activity of shared molecular networks that respond to endogenous retroviruses' reactivation.
Assuntos
Retrovirus Endógenos , Esclerose Múltipla , Humanos , Retrovirus Endógenos/patogenicidade , Retrovirus Endógenos/genética , Esclerose Múltipla/virologia , Esclerose Múltipla/etiologia , Envelhecimento , Animais , Epigênese GenéticaRESUMO
BACKGROUND: An incremental, loosely planned development approach is often used in bioinformatic studies when dealing with custom data analysis in a rapidly changing environment. Unfortunately, the lack of a rigorous software structuring can undermine the maintainability, communicability and replicability of the process. To ameliorate this problem we propose the Leaf system, the aim of which is to seamlessly introduce the pipeline formality on top of a dynamical development process with minimum overhead for the programmer, thus providing a simple layer of software structuring. RESULTS: Leaf includes a formal language for the definition of pipelines with code that can be transparently inserted into the user's Python code. Its syntax is designed to visually highlight dependencies in the pipeline structure it defines. While encouraging the developer to think in terms of bioinformatic pipelines, Leaf supports a number of automated features including data and session persistence, consistency checks between steps of the analysis, processing optimization and publication of the analytic protocol in the form of a hypertext. CONCLUSIONS: Leaf offers a powerful balance between plan-driven and change-driven development environments in the design, management and communication of bioinformatic pipelines. Its unique features make it a valuable alternative to other related tools.
Assuntos
Biologia Computacional/métodos , Software , Variações do Número de Cópias de DNARESUMO
BACKGROUND & AIMS: Germline variations in allele-specific expression (ASE) are associated with highly penetrant familial cancers, but their role in common sporadic cancers is unclear. ASE of adenomatous polyposis coli (APC) is associated with pathogenesis of familial adenomatous polyposis. We investigated whether moderate variations in ASE of APC contribute to common forms of colorectal cancer (CRC). METHODS: Denaturing high-performance liquid chromatography was used to analyze germline ASE of APC in blood samples from patients with CRC (cases, n = 53) and controls (n = 68). Means, medians, and variances of ASE were compared. Variants in the APC gene region also were analyzed. RESULTS: The distribution of ASE differed significantly between groups; cases had significantly larger amounts of variance than controls (P = .0004). Risk for CRC increased proportionally with the degree of deviation from the mean. The odds ratio for individuals with levels of ASE that deviated more than 1 standard deviation from the mean was 3.97 (95% confidence interval, 1.71-9.24; P = .001); for those with levels greater than 1.645 standard deviations, the odds ratio was 13.46 (95% confidence interval, 1.76-609.40; P = .005). Sequence analysis revealed that a patient with a high level of ASE who did not have a family history of CRC carried a nonsense mutation in APC (p.Arg216X). Genotype analysis of APC associated multiple single-nucleotide polymorphisms with ASE values and/or variance among cases, but not controls. Cis variants, therefore, might account for some of the variance in ASE of APC. CONCLUSIONS: Patients with CRC have a larger variance in germline levels of ASE in APC than controls; large distances from the mean ASE were associated with risk for common forms of CRC.
Assuntos
Neoplasias Colorretais/genética , Genes APC , Variação Genética , Adulto , Idoso , Análise de Variância , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Códon sem Sentido , Análise Mutacional de DNA , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Itália , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Medição de Risco , Fatores de RiscoRESUMO
Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.
Assuntos
Epigênese Genética/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Paraganglioma/genética , Paraganglioma/patologia , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Western Blotting , Caspases/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Lentivirus/genética , Análise em Microsséries , Microscopia Imunoeletrônica , Nervos Periféricos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Succinato Desidrogenase/genética , TransfecçãoRESUMO
Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the proinflammatory enzymes 5lipoxygenase (5LO) and cyclooxygenase2 (COX2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffinembedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5LO and COX2. The CRC cell lines Caco2 and LS174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or antiinflammatory drug celecoxib (CCX) and analyzed by reverse transcriptionquantitative PCR and immunofluorescence for 5LO, COX2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX2, 5LO and KI67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5LO and COX2 transcript and proteins in both Caco2 and LS174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX2, 5LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.
Assuntos
Neoplasias Colorretais , Infecções por Citomegalovirus , Humanos , Araquidonato 5-Lipoxigenase/genética , Células CACO-2 , Ciclo-Oxigenase 2/genética , Antígeno Ki-67 , Proteínas Proto-Oncogênicas p21(ras)/genética , Celecoxib/farmacologia , Citomegalovirus/genética , Ganciclovir , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/genética , Neoplasias Colorretais/genética , Receptores ErbBRESUMO
Head and neck paragangliomas (HNPGLs), rare chemoresistant tumors curable only with surgery, are strongly influenced by genetic predisposition, hence patients and relatives require lifetime follow-up with MRI and/or PET-CT because of de novo disease risk. This entails exposure to electromagnetic/ionizing radiation, costs, and organizational challenges, because patients and relatives are scattered far from reference centers. Simplified first-line screening strategies are needed. We employed flow injection analysis tandem mass spectrometry, as used in newborn metabolic screening, to compare the plasma metabolic profile of HNPGL patients (59 samples, 56 cases) and healthy controls (24 samples, 24 cases). Principal Component Analysis (PCA) and Partial Least Discriminant Analysis (PLS-DA) highlighted a distinctive HNPGL signature, likely reflecting the anaplerotic conversion of the TCA cycle to glutaminolysis and catabolism of branched amino acids, DNA damage and deoxyadenosine (dAdo) accumulation, impairment of fatty acid oxidation, switch towards the Warburg effect and proinflammatory lysophosphatidylcholines (LPCs) signaling. Statistical analysis of the metabolites that most impacted on PLS-DA was extended to 10 acoustic neuroma and 2 cholesteatoma patients, confirming significant differences relative to the HNPGL plasma metabolomic profile. The best confusion matrix from the ROC curve built on 2 metabolites, dAdo and C26:0-LPC, provided specificity of 94.29% and sensitivity of 89.29%, with positive and negative predictive values of 96.2% and 84.6%, respectively. Analysis of dAdo and C26:0-LPC levels in dried venous and capillary blood confirmed that dAdo, likely deriving from 2'-deoxy-ATP accumulated in HNPGL cells following endogenous genotoxic damage, efficiently discriminated HNPGL patients from healthy controls and acoustic neuroma/cholesteatoma patients on easily manageable dried blood spots.
RESUMO
BACKGROUND: SEL1L gene product is implicated in the endoplasmic reticulum (ER)-associated protein degradation and Unfolded Protein Response pathways. This gene and associated miRNAs have been indicated as predictive and prognostic markers of pancreatic cancer. AIM: Explore the role of SEL1L in colorectal cancer (CRC) progression. METHODS: SEL1L expression was analysed immunohistochemically in 153 adenomas and 71 CRCs from African American and North Italian patients. The distribution of stained cells was determined by computing median and inter quartile range. The receiver operating characteristics plot was used as discriminate power of SEL1L expression, CRC diagnosis and the effects on patient survival. RESULTS: SEL1L was low in normal mucosa and confined to few scattered cells at the base crypt of the villi and in the foveolar glandular compartment. The highest levels were in Paneth cells within the lysosomes. The enterocytic progenitor cells and mature enterocytes showed less cytoplasmic staining. In CRCs, SEL1L expression significantly correlated with the progression from adenoma to carcinoma (P = 0.0001) being stronger in well-to-moderately differentiated cancers. No correlation was found with other clinicopathological characteristics or ethnicity. CONCLUSIONS: SEL1L expression is a potential CRC tissue biomarker since its expression is significantly higher in adenoma cells with respect to normal mucosa. The levels of expression decrease sensibly in undifferentiated CRC cancers. Interestingly, Paneth cells contain high levels of SEL1L protein that could indicate pre-neoplastic mucosa undergoing neoplastic transformation. Since SEL1L's major function lies within ER stress and active ERAD response, it may identify CRCs with differentiated secretory phenotype and acute cellular stress.
Assuntos
Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica , Neoplasias Colorretais/diagnóstico , Proteínas/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/diagnóstico , Adenoma/metabolismo , Adenoma/patologia , Western Blotting , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Taxa de Sobrevida , Análise Serial de Tecidos , Resposta a Proteínas não DobradasRESUMO
BACKGROUND & AIMS: The WNT-adenomatous polyposis coli system controls cell fate in the intestinal epithelium, where compartment-specific genes tightly regulate proliferation, migration, and differentiation. Nuclear receptors are transcription factors functioning as sensors of hormones and nutrients that are known to contribute to colon cancer progression. Here we mapped the messenger RNA (mRNA) abundance and the epithelial localization of the entire nuclear receptor family in mouse and human intestine. METHODS: We used complementary high-resolution in situ hybridization and systematic real-time quantitative polymerase chain reaction in samples of normal distal ileum and proximal colon mucosa and tumors obtained from mouse and human adenomatous polyposis coli-initiated tumor models (ie, Apc(Min/+) mice and familial adenomatous polyposis patients) and in cellular models of human colon cancer. RESULTS: We first defined for each receptor an expression pattern based on its transcript localization in the distal ileum and the proximal colon. Then, we compared the mRNA levels between normal intestinal epithelium and neoplastic intestinal tissue. After analyzing the correspondence between mouse and human tumor samples plus genetically modified human colon cancer cells, we used complementary graphic and statistical approaches to present a comprehensive overview with several classification trees for the nuclear hormone receptor intestinal transcriptome. CONCLUSIONS: We defined the intestinal nuclear hormone receptor map, which indicates that the localization pattern of a receptor in normal intestine predicts the modulation of its expression in tumors. Our results are useful to select those nuclear receptors that could be used eventually as early diagnostic markers or targeted for clinical intervention in intestinal polyposis and cancer.
Assuntos
Adenoma/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Células Epiteliais/metabolismo , Íleo/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adenoma/patologia , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Adolescente , Adulto , Animais , Colo/patologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Íleo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Adulto JovemRESUMO
The crypt-villus axis represents the essential unit of the small intestine, which integrity and functions are fundamental to assure tissue and whole-body homeostasis. Disruption of pathways regulating the fine balance between proliferation and differentiation results in diseases development. Nowadays, it is well established that microRNAs (miRNAs) play a crucial role in the homeostasis maintenance and perturbation of their levels may promote tumor development. Here, by using microarray technology, we analysed the miRNAs differentially expressed between the crypt and the villus in mice ileum. The emerged miRNAs were further validated by Real Time qPCR in mouse model (ApcMin/+), human cell lines and human tissue samples (FAP) of colorectal cancer (CRC). Our results indicated that miRNAs more expressed in the villi compartment are negatively regulated in tumor specimens, thus suggesting a close association between these microRNAs and the differentiation process. Particularly, from our analysis let-7e appeared to be a promising target for possible future therapies and a valuable marker for tumor staging, being upregulated in differentiated cells and downregulated in early-stage colonic adenoma samples.
Assuntos
Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais/patologia , MicroRNAs/metabolismo , Adenoma/genética , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40-CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.
RESUMO
Juvenile breast cancer is rare and poorly known. We studied a series of five breast cancer patients diagnosed within 25 years of age that included two adolescents, 12- and 15-years-old, and 3 young women, 21-, 21-, and 25-years-old, respectively. All cases were scanned for germline mutations along the entire BRCA1/2 coding sequences and TP53 exons 4-10, using protein truncation test, denaturing high performance liquid chromatography and direct sequencing. Paraffin-embedded primary tumors (available for 4/5 cases), and a distant metastasis (from the 15-years-old) were characterized for histological and molecular tumor subtype, human papilloma virus (HPV) types 16/18 E6 sequences and tumor-associated mutations in TP53 exons 5-8. A BRCA2 germline mutation (p.Ile2490Thr), previously reported in breast cancer and, as compound heterozygote, in Fanconi anemia, was identified in the 21-year-old patient diagnosed after pregnancy, negative for cancer family history. The tumor was not available for study. Only germline polymorphisms in BRCA1/2 and/or TP53 were detected in the other cases. The tumors of the 15- and 12-years-old were, respectively, classified as glycogen-rich carcinoma with triple negative subtype and as secretory carcinoma with basal subtype. The tumors of the 25-year-old and of the other 21-year-old were, respectively, diagnosed as infiltrating ductal carcinoma with luminal A subtype and as lobular carcinoma with luminal B subtype. No somatic TP53 mutations were found, but tumor-associated HPV 16 E6 sequences were retrieved from the 12- and 25-year-old, while both HPV 16 and HPV 18 E6 sequences were found in the tumor of the 15-year-old and in its associated metastasis. Blood from the 15- and 25-year-old, diagnosed with high-stage disease, resulted positive for HPV 16 E6. All the HPV-positive cases were homozygous for arginine at TP53 codon 72, a genotype associated with HPV-related cancer risk, and the tumors showed p16(INK4A) immunostaining, a marker of HPV-associated cancers. Notably menarche at 11 years was reported for the two adolescents, while the 25-year-old was diagnosed after pregnancy and breast-feeding. Our data suggest that high-risk HPV infection is involved in a subset of histopathologically heterogeneous juvenile breast carcinomas associated with menarche or pregnancy and breast-feeding. Furthermore we implicate BRCA2 in a juvenile breast carcinoma diagnosed at 21 years of age, 4 years after an early full-term pregnancy, in absence of cancer family history.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/virologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Carcinoma Lobular/virologia , Criança , Primers do DNA/química , Primers do DNA/genética , DNA Viral/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Humanos , Técnicas Imunoenzimáticas , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/secundário , Neoplasias Ovarianas/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Reação em Cadeia da Polimerase , Gravidez , Fatores de Risco , Adulto JovemRESUMO
Helicobacter pylori (Hp) is the major recognized risk factor for non-cardia gastric cancer (GC), but only a fraction of infected subjects develop GC, thus GC risk might reflect other genetic/environmental cofactors and/or differences in virulence among infectious Hp strains. Focusing on a high GC risk area of Northern Italy (Cremona, Lombardy) and using archived paraffin-embedded biopsies, we investigated the associations between the Hp vacA and cagA genotype variants and gastric intraepithelial neoplasia (GIN, 33 cases) versus non-neoplastic gastroduodenal lesions (NNGDLs, 37 cases). The glmM gene and the cagA and vacA (s and m) genotypes were determined by polymerase chain reaction (PCR) and sequencing. Hp was confirmed in 37/37 (100%) NNGDLs and detected in 9/33 GINs (27%), consistently with the well-known Hp loss in GC. CagA was detected in 4/9 Hp-positive GINs and in 29/37 NNGDLs. The vacA s1a and m1 subtypes were more common in GINs than in NNGDLs (6/7 vs. 12/34, p=0.014, for s1a; 7/7 vs. 18/34, p=0.020 for m1), with significant vacA s genotype-specific variance. The GIN-associated vacA s1a sequences clustered together, suggesting that aggressive Hp strains from a unique founder contribute to GC in the high-risk area studied.
RESUMO
BACKGROUND: Altered germline expression of genes may represent a powerful marker of genetic or epigenetic predisposition to cancer or other diseases. METHODS: We developed and validated a method of nonfluorescent primer extension that uses a single dideoxynucleotide and denaturing HPLC (DHPLC) to analyze the relative allele expression. We devised 5 independent assays for measuring allele-specific expression (ASE) to exploit different markers of mismatch repair genes MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]. We initially confirmed method reproducibility with genomic DNA (gDNA) from individuals heterozygous for a frequent single-nucleotide polymorphism in the MLH1 gene. After this preliminary validation with gDNA, we confirmed assay reproducibility with cDNA templates from control individuals. Relative allele expression was estimated by comparing the heights of the peaks corresponding to the 2 alleles. Results obtained with gDNA templates were used to normalize cDNA results. RESULTS: With these DHPLC-based primer-extension assays, we detected and confirmed a 5-fold imbalance in MLH1 allele expression in a mutation-negative patient with hereditary nonpolyposis colorectal cancer and in another patient with a modest degree of imbalance in MLH1 expression. Among control individuals, the relative expression of MLH1 alleles displayed a narrow range of variation. CONCLUSIONS: Independent DHPLC-based primer-extension assays for measuring and confirming ASE can be developed for different sequence variants of interest. This DHPLC application provides a cost-effective method for detecting ASE in cases for which conventional screening fails to detect pathogenic mutations in candidate genes and may be applicable for confirming ASE revealed by other methods, such as those used for transcriptome-wide analyses. .