RESUMO
In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of ß1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.
Assuntos
Neoplasias do Colo/genética , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Sindecana-1/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Benzotiazóis/farmacologia , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Células HT29 , Humanos , Indóis/farmacologia , Integrina beta1/genética , Integrina beta1/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Sulfonamidas/farmacologia , Sindecana-1/antagonistas & inibidores , Sindecana-1/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Quinase do Ponto de Checagem 2/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Variações do Número de Cópias de DNA/genética , Detecção Precoce de Câncer/métodos , Feminino , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
OBJECTIVE: To evaluate the feasibility of detecting actionable gene mutations in circulating tumor DNA (ctDNA) in patients with advanced non-small-cell lung cancer (NSCLC) using targeted next-generation sequencing (NGS). MATERIALS AND METHODS: In total 50 plasma samples from patients newly diagnosed with advanced NSCLC or resistant to first-line tyrosine kinase inhibitors (TKIs) were subjected to deep sequencing on a seven-gene panel (BRAF, EGFR, ERBB2, KRAS, NRAS, PIK3CA, PTEN) incorporated with molecular barcodes to improve accuracy in variant detection. When possible, results were compared with those from matched tissue samples. RESULTS: At least one alteration in the ctDNA was detected in 44 out of 50 patients (88%); EGFR was the most frequently mutated gene. Half the total number of patients (50%, 25 of 50) had at least one actionable genetic alteration with targeted therapies available for treatment. Our results showed a high concordance rate of 81% in detection of EGFR mutation between 26 matched tissue and plasma samples. For progressive patients, from whom tissue is mostly unavailable, the resistant EGFR T790 M mutation was validated using the droplet digital polymerase chain reaction (ddPCR), yielding a concordance of 92% between alternative platforms. CONCLUSION: Our study demonstrated that therapeutically actionable mutations can be detected with high accuracy in ctDNA using NGS. This promising approach offers alternative and non-invasive diagnostic methods for treatment guidance and clinical monitoring.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , DNA Tumoral Circulante/análise , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Estudos de Viabilidade , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação/genética , Patologia Molecular , Guias de Prática Clínica como Assunto , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-2/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Genetic determinants of drug response remain stable throughout life and offer great promise to patient-tailored drug therapy. The adoption of pharmacogenetic (PGx) testing in patient care requires accurate, cost effective and rapid genotyping with clear guidance on the use of the results. Hence, we evaluated a 32 SNPs panel for implementing PGx testing in clinical laboratories. METHODS: We designed a 32-SNP panel for PGx testing in clinical laboratories. The variants were selected using the clinical annotations of the Pharmacogenomics Knowledgebase (PharmGKB) and include polymorphisms of CYP2C9, CYP2C19, CYP2D6, CYP3A5 and VKORC1 genes. The CYP2D6 gene allele quantification was determined simultaneously with TaqMan copy number assays targeting intron 2 and exon 9 regions. The genotyping results showed high call rate accuracy according to concordance with genotypes identified by independent analyses on Sequenome massarray and droplet digital PCR. Furthermore, 506 genomic samples across three major ethnic groups of Singapore (Malay, Indian and Chinese) were analysed on our workflow. RESULTS: We found that 98% of our study subjects carry one or more CPIC actionable variants. The major alleles detected include CYP2C9*3, CYP2C19*2, CYP2D6*10, CYP2D6*36, CYP2D6*41, CYP3A5*3 and VKORC1*2. These translate into a high percentage of intermediate (IM) and poor metabolizer (PM) phenotypes for these genes in our population. CONCLUSION: Genotyping may be useful to identify patients who are prone to drug toxicity with standard doses of drug therapy in our population. The simplicity and robustness of this PGx panel is highly suitable for use in a clinical laboratory.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Variação Genética , Alelos , Povo Asiático/genética , China , Ensaios Enzimáticos , Etnicidade/genética , Frequência do Gene , Loci Gênicos , Haplótipos/genética , Humanos , Índia , Malásia , Fenótipo , Reprodutibilidade dos Testes , SingapuraRESUMO
INTRODUCTION: While overexpression of syndecan-1 has been associated with aggressive breast cancer in the Caucasian population, the expression pattern of syndecan-1 in Asian women remains unclear. Triple-positive breast carcinoma, in particular, is a unique subtype that has not been extensively studied. We aimed to evaluate the role of syndecan-1 as a potential biomarker and prognostic factor for triple-positive breast carcinoma in Asian women. METHODS: Using immunohistochemistry, staining scores of 61 triplepositive breast carcinoma specimens were correlated with patients' clinicopathological variables such as age, ethnicity, tumour size, histological grade, lymph node status, lymphovascular invasion, associated ductal carcinoma in situ grade, recurrence and overall survival. RESULTS: Syndecan-1 had intense staining scores in triplepositive invasive ductal breast carcinomas when compared to normal breast tissue. On multivariate analysis, syndecan-1 epithelial total percentage and immunoreactivity score showed statistical correlation with survival (p = 0.02). CONCLUSION: The intense staining scores of syndecan-1 and their correlation with overall survival in patients with triple-positive breast carcinoma suggest that syndecan-1 may have a role as a biological and prognostic marker in patients with this specific subtype of breast cancer.
