RESUMO
Colorectal cancer (CRC) is a disease with high incidence and mortality. Colonoscopy is a gold standard among tests used for CRC traceability. However, serious complications, such as colon perforation, may occur. Non-invasive diagnostic procedures are an unmet need. We aimed to identify a plasma microRNA (miRNA) signature for CRC detection. Plasma samples were obtained from subjects (n = 109) at different stages of colorectal carcinogenesis. The patients were stratified into a non-cancer (27 healthy volunteers, 17 patients with hyperplastic polyps, 24 with adenomas), and a cancer group (20 CRC and 21 metastatic CRC). miRNAs (381) were screened by TaqMan Low-Density Array. A classifier based on four differentially expressed miRNAs (miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p) was able to discriminate cancer versus non-cancer cases. The overexpression of these miRNAs was confirmed by RT-qPCR, and a cross-study validation step was implemented using eight data series retrieved from Gene Expression Omnibus (GEO). In addition, another external data validation using CRC surgical specimens from The Cancer Genome Atlas (TCGA) was carried out. The predictive model's performance in the validation set was 76.5% accuracy, 59.4% sensitivity, and 86.8% specificity (area under the curve, AUC = 0.716). The employment of our model in the independent publicly available datasets confirmed a good discrimination performance in five of eight datasets (median AUC = 0.823). Applying this algorithm to the TCGA cohort, we found 99.5% accuracy, 99.7% sensitivity, and 90.9% specificity (AUC = 0.998) when the model was applied to solid colorectal tissues. Overall, we suggest a novel signature of four circulating miRNAs, i.e., miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p, as a predictive tool for the detection of CRC.
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Prostate cancer (PCa) is the second most common cancer in men. The indolent course of the disease makes the treatment choice a challenge for physicians and patients. In this study, a minimally invasive method was used to evaluate the potential of molecular markers in identifying patients with aggressive disease. Cell-free plasma samples from 60 PCa patients collected before radical prostatectomy were used to evaluate the levels of expression of eight genes (AMACR, BCL2, NKX3-1, GOLM1, OR51E2, PCA3, SIM2 and TRPM8) by quantitative real-time PCR. Overexpression of AMACR, GOLM1, TRPM8 and NKX3-1 genes was significantly associated with aggressive disease characteristics, including extracapsular extension, tumor stage and vesicular seminal invasion. A trio of genes (GOLM1, NKX3-1 and TRPM8) was able to identify high-risk PCa cases (85% of sensitivity and 58% of specificity), yielding a better overall performance compared with the biopsy Gleason score and prostate-specific antigen, routinely used in the clinical practice. Although more studies are required, these circulating markers have the potential to be used as an additional test to improve the diagnosis and treatment decision of high-risk PCa patients.
Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Neoplasias da Próstata/diagnóstico , RNA Mensageiro/sangue , Idoso , Biomarcadores Tumorais/genética , Brasil , Ácidos Nucleicos Livres/genética , Tomada de Decisão Clínica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Calicreínas/sangue , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica/genética , Seleção de Pacientes , Próstata/patologia , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , RNA Mensageiro/genética , Medição de Risco/métodosRESUMO
E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.
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Background: The differential diagnosis of thyroid nodules using fine-needle aspiration biopsy (FNAB) is challenging due to the inherent limitation of the cytology tests. The use of molecular markers has potential to complement the FNAB-based diagnosis and avoid unnecessary surgeries. In this study, we aimed to identify DNA methylation biomarkers and to develop a diagnostic tool useful for thyroid lesions. Methods: Genome-wide DNA methylation profiles (Illumina 450K) of papillary thyroid carcinoma (PTC = 60) and follicular thyroid carcinoma (FTC = 10) were compared with non-neoplastic thyroid tissue samples (NT = 50) and benign thyroid lesions (BTL = 17). The results were confirmed in publicly available databases from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) using the same DNA methylation platform. Two classifiers were trained to discriminate FTC and PTC from BTL. To increase the applicability of the method, six differentially methylated CpGs were selected and evaluated in 161 thyroid tumors and 69 BTL postsurgical specimens and 55 prospectively collected FNAB using bisulfite-pyrosequencing. Results: DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and FTC, respectively. The CpGs confirmed by GEO and TCGA databases showing high areas under the receiver operating characteristic curve in all sample sets were used to train our diagnostic classifier. The model based on six CpGs was able to differentiate benign from malignant thyroid lesions with 94.3% sensitivity and 82.4% specificity. A similar performance was found applying the algorithm to TCGA and GEO external data sets (91.3-97.4% sensitivity and 87.5% specificity). We successfully evaluated the classifiers using a bisulfite-pyrosequencing technique, achieving 90.7% sensitivity and 75.4% specificity in surgical specimens (five of six CpGs). The study comprising FNAB cytology materials corroborated the applicability and performance of the methodology, demonstrating 86.7% sensitivity and 89.5% specificity in confirmed malignant tumors, and 100% sensitivity and 89% specificity in cases with indeterminate cytology. Conclusions: A novel diagnostic tool with potential application in preoperative screening of thyroid nodules is reported here. The proposed protocol has the potential to avoid unnecessary thyroidectomies.
Assuntos
Metilação de DNA , Neoplasias da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/diagnóstico , Adenocarcinoma Folicular/diagnóstico , Adulto , Idoso , Biópsia por Agulha Fina , Ilhas de CpG , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.
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Neoplasias do Sistema Nervoso Central/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Pessoa de Meia-Idade , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
BACKGROUND: DNA methylation in miRNA genes has been reported as a mechanism that may cause dysregulation of mature miRNAs and consequently impact the gene expression. This mechanism is largely unstudied in papillary thyroid carcinomas (PTC). METHODS: To identify differentially methylated miRNA-encoding genes, we performed global methylation analysis (Illumina 450 K), integrative analysis (TCGA database), data confirmation (pyrosequencing and RT-qPCR), and functional assays. RESULTS: Methylation analysis revealed 27 differentially methylated miRNA genes. The integrative analyses pointed out miR-21 and miR-146b as potentially regulated by methylation (hypomethylation and increased expression). DNA methylation and expression patterns of miR-21 and miR-146b were confirmed as altered, as well as seven of 452 mRNAs targets were down-expressed. The combined methylation and expression levels of miR-21 and miR-146b showed potential to discriminate malignant from benign lesions (91-96% sensitivity and 96-97% specificity). An increased expression of miR-146b due to methylation loss was detected in the TPC1 cell line. The miRNA mimic transfection highlighted putative target mRNAs. CONCLUSIONS: The increased expression of miR-21 and miR-146b due to loss of DNA methylation in PTC resulted in the disruption of the transcription machinery and biological pathways. These miRNAs are potential diagnostic biomarkers, and these findings provide support for future development of targeted therapies.
Assuntos
Metilação de DNA , MicroRNAs/genética , Câncer Papilífero da Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/diagnóstico , Regulação para Cima , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Análise de Sequência de DNA , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
Androgen receptor (AR) is a member of the steroid and nuclear family receptor that acts as transcription factor. AR signaling plays pivotal role in the development and progression of prostate cancer. However, the role of AR in penile cancer (PeCa) is poorly explored. Our previous molecular studies unveiled frequent AR mRNA loss in PeCa, which was further predicted as a major driver alteration in this neoplasm. Herein, we assessed the AR protein expression in 59 usual PeCa tissues and 42 surrounding normal tissues (SNT) by immunohistochemistry using a tissue microarray. In a paired analysis, we found a total absence of nuclear AR expression in PeCa while 95.2% of SNT samples presented strong nuclear AR expression (P < 0.001). Interestingly, 17 of 42 PeCa presented weak or moderate cytoplasmic AR staining, contrasting with 5 of 42 SNT (P = 0.008). Increased levels of AR cytoplasmic expression were related with poor prognosis features including advanced clinical staging (P = 0.044), compromised surgical margins (P = 0.005), and pathological inguinal node status (P = 0.047). Furthermore, AR cytoplasmic expression was also related with shorter overall survival (P = 0.032). In conclusion, the frequent loss of nuclear AR protein levels suggests a potential function in PeCa development. Based on this result, the androgen deprivation therapy is not indicated for PeCa patients. In addition, the AR cytoplasmic expression found in a significant number of cases (40.5%) showed prognostic value and pathways activated by the non-genomic AR signaling may represent a promising therapeutic strategy.
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Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Neoplasias Penianas/diagnóstico , Neoplasias Penianas/metabolismo , Receptores Androgênicos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/mortalidade , Estudos de Casos e Controles , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Penianas/mortalidade , Prognóstico , Análise de Sobrevida , Análise Serial de TecidosRESUMO
Prostate cancer is a heterogeneous disease with high levels of clinical and gene heterogeneity, consequently offering several targets for therapy. Dogs with naturally occurring prostate cancer are useful models for molecular investigations and studying new treatment efficacy. Three genes and proteins associated with the WNT pathway (ß-catenin, APC and E-cadherin) and Caveolin-1 (CAV-1) were evaluated in canine pre-neoplastic proliferative inflammatory atrophy (PIA), prostate cancer and metastatic disease. The APC gene methylation status was also investigated. As in human prostate cancer, cytoplasmic and nuclear ß-catenin, which are fundamental for activating the canonical WNT pathway, were found in canine prostate cancer and metastasis. Membranous E-cadherin was also lost in these lesions, allowing cellular migration to the stroma and nuclear localization of ß-catenin. In contrast to human prostate tumours, no APC downregulation or hypermethylation was found in canine prostate cancer. The CAV-1 gene and protein overexpression were found in canine prostate cancer, and as in humans, the highest levels were found in Gleason scores ≥8. In conclusion, as with human prostate cancer, ß-catenin and E-cadherin in the WNT pathway, as well as Caveolin-1, are molecular drivers in canine prostate cancer. These findings provide additional evidence that dogs are useful models for studying new therapeutic targets in prostate cancer.
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Caveolina 1/metabolismo , Doenças do Cão/genética , Metástase Neoplásica/genética , Neoplasias da Próstata/veterinária , beta Catenina/metabolismo , Animais , Caderinas , Metilação de DNA , Doenças do Cão/metabolismo , Cães , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , beta Catenina/genéticaRESUMO
BACKGROUND: Among the sex hormones, oestrogen may play a role in colorectal cancer, particularly in conjunction with oestrogen receptor-ß (ERß). The expression of ERß isoform variants and their correlations with familial adenomatous polyposis (FAP) syndrome and sporadic colorectal carcinomas are poorly described. METHODS: This study aimed to investigate the expression levels of the ERß1, ERß2, ERß4 and ERß5 isoform variants using quantitative RT-PCR (921 analyses) in FAP, normal mucosa, adenomatous polyps and sporadic colorectal carcinomas. RESULTS: Decreased expression of ERß isoforms was identified in sporadic polyps and in sporadic colorectal cancer as well as in polyps from FAP syndrome patients compared with normal tissues (p < 0.001). In FAP patients, ERß1 and ERß5 isoforms showed significant down-expression in polyps (p < 0.001) compared with matched normal tissues. However, no differences were observed when sporadic colorectal carcinomas were compared to normal mucosa tissues. These findings suggest an association of the ERß isoform variants in individuals affected by germline mutations of the APC gene. Progressively decreased expression of ERß was found in polyps at early stages of low-grade dysplasia, followed by T1-T2 and T3-T4 tumours (p < 0.05). In sporadic colorectal cancer, the loss of expression was an independent predictor of recurrence, and ERß1 and ERß5 expression levels were associated with better disease-free survival (p = 0.002). CONCLUSION: These findings may provide a better understanding of oestrogens and their potential preventive and therapeutic effects on sporadic colorectal cancer and cancers associated with FAP syndrome.
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Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Polipose Adenomatosa do Colo/mortalidade , Polipose Adenomatosa do Colo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Bases de Dados Genéticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Isoformas de Proteínas , Isoformas de RNA , Análise de Sequência de DNARESUMO
Circulating nucleic acids are found in free form in body fluids and may serve as minimally invasive tools for cancer diagnosis and prognosis. Only a few studies have investigated the potential application of circulating mRNAs and microRNAs (miRNAs) in prostate cancer (PCa). The Cancer Genome Atlas (TCGA) database was used for an in silico analysis to identify circulating mRNA and miRNA as potential markers of PCa. A total of 2,267 genes and 49 miRNAs were differentially expressed between normal and tumor samples. The prediction analyses of target genes and integrative analysis of mRNA and miRNA expression revealed eleven genes and eight miRNAs which were validated by RT-qPCR in plasma samples from 102 untreated PCa patients and 50 cancer-free individuals. Two genes, OR51E2 and SIM2, and two miRNAs, miR-200c and miR-200b, showed significant association with PCa. Expression levels of these transcripts distinguished PCa patients from controls (67% sensitivity and 75% specificity). PCa patients and controls with prostate-specific antigen (PSA) ≤ 4.0 ng/mL were discriminated based on OR51E2 and SIM2 expression levels. The miR-200c expression showed association with Gleason score and miR-200b, with bone metastasis, bilateral tumor, and PSA > 10.0 ng/mL. The combination of circulating mRNA and miRNA was useful for the diagnosis and prognosis of PCa.