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1.
Blood ; 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33512408

RESUMO

CLL remains incurable despite BCR-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate, which is activated by unmethylated CpG-DNA. Here, we show that plasma from CLL patients contains significantly more unmethylated DNA than plasma from healthy controls (p<0.0001) and that cell-free DNA levels correlate with the prognostic markers CD38, b2-microglobulin and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (TTFT: p=0.003, HR=4.0). We went on to show that TLR9 expression was associated with in-vitro CLL cell migration (p<0.001) and intracellular endosomal TLR9 strongly correlated with aberrant surface expression ((sTLR9); r=0.9). In addition, lymph node-derived CLL cells showed increased sTLR9 (p=0.016) and RNA sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility and inflammation in sTLR9hi cells. Mechanistically, the TLR9 agonist, ODN2006, promoted CLL cell migration (p<0.001) that was mediated, by p65 NF-kB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NSG mouse xenograft model. Finally, we showed that dual targeting of TLR9 and BTK was strongly synergistic (median CI=0.2 at ED50), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

2.
Breast Cancer Res ; 22(1): 126, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198803

RESUMO

BACKGROUND: Activating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment. MATERIALS AND METHODS: We knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR. RESULTS: By silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells. CONCLUSION: These data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

3.
Biochim Biophys Acta Rev Cancer ; 1874(2): 188429, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32956766

RESUMO

Neurotensin receptor-3 or sortilin is a vacuolar protein sorting 10 protein domain (Vps10p) has been firstly discovered in the human brain, it acts as receptor or co-receptor of the cell and traffics different proteins within the cell. Sortilin deregulation contributes to the development of several diseases, including neurological diseases and cancer. On the other hand, neurotrophins which are a family of proteins essential for the nervous system development, function and plasticity. The first discovered member is the nerve growth factor; other members are brain-derived growth factor, neurotrophin-3 and neurotrophin-4. Nerve growth factor and brain-derived growth factor are the common neurotrophins that have a role in cancer. Neurotrophins initiate their signals through interaction with tyrosine receptor kinases TrkA, TrkB, and TrkC; each member has an affinity for a specific receptor to stimulate cell survival, while the interaction with p75NTR initiates cell apoptosis pathway by forming a complex with sortilin and neurotrophin precursors. A number of therapeutic approaches are emerging to target the neurotrophins pathway as well as sortilin.

4.
FASEB J ; 34(9): 11844-11859, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32652768

RESUMO

Lactation-induced bone loss occurs due to high calcium requirements for fetal growth but skeletal recovery is normally achieved promptly postweaning. Dietary protein is vital for fetus and mother but the effects of protein undernutrition on the maternal skeleton and skeletal muscles are largely unknown. We used mouse dams fed with normal (N, 20%) or low (L, 8%) protein diet during gestation and lactation and maintained on the same diets (NN, LL) or switched from low to normal (LN) during a 28 d skeletal restoration period post lactation. Skeletal muscle morphology and neuromuscular junction integrity was not different between any of the groups. However, dams fed the low protein diet showed extensive bone loss by the end of lactation, followed by full skeletal recovery in NN dams, partial recovery in LN and poor bone recovery in LL dams. Primary osteoblasts from low protein diet fed mice showed decreased in vitro bone formation and decreased osteogenic marker gene expression; promoter methylation analysis by pyrosequencing showed no differences in Bmpr1a, Ptch1, Sirt1, Osx, and Igf1r osteoregulators, while miR-26a, -34a, and -125b expression was found altered in low protein fed mice. Therefore, normal protein diet is indispensable for maternal musculoskeletal health during the reproductive period.

5.
Cancer Res ; 80(13): 2861-2873, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32393661

RESUMO

Although fibrotic stroma forms an integral component of pancreatic diseases, whether fibroblasts programmed by different types of pancreatic diseases are phenotypically distinct remains unknown. Here, we show that fibroblasts isolated from patients with pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), periampullary tumors, and adjacent normal (NA) tissue (N = 34) have distinct mRNA and miRNA profiles. Compared with NA fibroblasts, PDAC-associated fibroblasts were generally less sensitive to an antifibrotic stimulus (NPPB) and more responsive to positive regulators of activation such as TGFß1 and WNT. Of the disease-associated fibroblasts examined, PDAC- and CP-derived fibroblasts shared greatest similarity, yet PDAC-associated fibroblasts expressed higher levels of tenascin C (TNC), a finding attributable to miR-137, a novel regulator of TNC. TNC protein and transcript levels were higher in PDAC tissue versus CP tissue and were associated with greater levels of stromal activation, and conditioned media from TNC-depleted PDAC-associated fibroblasts modestly increased both PDAC cell proliferation and PDAC cell migration, indicating that stromal TNC may have inhibitory effects on PDAC cells. Finally, circulating TNC levels were higher in patients with PDAC compared with CP. Our characterization of pancreatic fibroblast programming as disease-specific has consequences for therapeutic targeting and for the manner in which fibroblasts are used in research. SIGNIFICANCE: Primary fibroblasts derived from various types of pancreatic diseases possess and retain distinct molecular and functional characteristics in culture, providing a series of cellular models for treatment development and disease-specific research.


Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Tenascina/genética , Tenascina/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais Cultivadas
6.
Oncol Lett ; 19(3): 2502-2507, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32194751

RESUMO

HURP gene encodes the hepatoma upregulated protein (HURP), a microtubule associated protein regulating mitotic spindle dynamics, which promotes chromosomal congression and alignment during mitosis, with a potential role in tumorigenesis. In the present study, HURP mRNA expression was investigated by reverse transcription-quantitative PCR in oropharyngeal squamous cell carcinoma (OPSCC). Primary OPSCC tumors from 107 patients and 48 adjacent normal tissues, as well as 12 respiratory tract cancer cell lines (9 head and neck squamous cell carcinoma, 2 lung cancer and 1 normal bronchial) were utilised in the present study. mRNA expression levels of HURP were higher in malignant OPSCC tissues compared with in normal mucosa (P<1×10-5) and significantly associated with sex and smoking status (P<0.0001). Vinorelbine in vitro toxicity at half-maximal inhibitory concentration (IC50) was measured in the 11 cancer cell lines using an MTT assay. Sensitivity to vinorelbine was significantly correlated with HURP expression (r=0.636; P=0.035). The data indicated that HURP overexpression is frequent in OPSCC tissues and associated with smoking. The correlation between HURP mRNA expression and vinorelbine in vitro response suggests that HURP is a potential modulator of vinorelbine response; therefore, it should be explored for its possible predictive value for the efficiency of vinorelbine treatment in this type of cancer.

7.
Br J Cancer ; 122(7): 1050-1058, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32020063

RESUMO

BACKGROUND: Long non-coding RNAs compose an important level of epigenetic regulation in normal physiology and disease. Despite the plethora of publications of lncRNAs in human cancer, the landscape is still unclear. METHODS: Microarray analysis in 44 NSCLC paired specimens was followed by qPCR-based validation in 29 (technical) and 38 (independent) tissue pairs. Cross-validation of the selected targets was achieved in 850 NSCLC tumours from TCGA datasets. RESULTS: Twelve targets were successfully validated by qPCR (upregulated: FEZF1-AS1, LINC01214, LINC00673, PCAT6, NUTM2A-AS1, LINC01929; downregulated: PCAT19, FENDRR, SVIL-AS1, LANCL1-AS1, ADAMTS9-AS2 and LINC00968). All of them were successfully cross validated in the TCGA datasets. Abnormal DNA methylation was observed in the promoters of FENDRR, FEZF1-AS1 and SVIL-AS1. FEZF1-AS1 and LINC01929 were associated with survival in the TCGA set. CONCLUSIONS: Our study provides through multiple levels of internal and external validation, a comprehensive list of dysregulated lncRNAs in NSCLC. We therefore envisage this dataset to serve as an important source for the lung cancer research community assisting future investigations on the involvement of lncRNAs in the pathogenesis of the disease and providing novel biomarkers for diagnosis, prognosis and therapeutic stratification.

8.
Sci Rep ; 9(1): 11992, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427592

RESUMO

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.


Assuntos
Transformação Celular Neoplásica , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/patologia , Biomarcadores , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Senescência Celular/genética , Mapeamento Cromossômico , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Progressão da Doença , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Instabilidade Genômica , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Mutação , Estadiamento de Neoplasias , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
9.
J Thorac Oncol ; 14(8): 1360-1369, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31009812

RESUMO

INTRODUCTION: Inherited susceptibility to lung cancer risk in never-smokers is poorly understood. The major reason for this gap in knowledge is that this disease is relatively uncommon (except in Asians), making it difficult to assemble an adequate study sample. In this study we conducted a genome-wide association study on the largest, to date, set of European-descent never-smokers with lung cancer. METHODS: We conducted a two-phase (discovery and replication) genome-wide association study in never-smokers of European descent. We further augmented the sample by performing a meta-analysis with never-smokers from the recent OncoArray study, which resulted in a total of 3636 cases and 6295 controls. We also compare our findings with those in smokers with lung cancer. RESULTS: We detected three genome-wide statistically significant single nucleotide polymorphisms rs31490 (odds ratio [OR]: 0.769, 95% confidence interval [CI]: 0.722-0.820; p value 5.31 × 10-16), rs380286 (OR: 0.770, 95% CI: 0.723-0.820; p value 4.32 × 10-16), and rs4975616 (OR: 0.778, 95% CI: 0.730-0.829; p value 1.04 × 10-14). All three mapped to Chromosome 5 CLPTM1L-TERT region, previously shown to be associated with lung cancer risk in smokers and in never-smoker Asian women, and risk of other cancers including breast, ovarian, colorectal, and prostate. CONCLUSIONS: We found that genetic susceptibility to lung cancer in never-smokers is associated to genetic variants with pan-cancer risk effects. The comparison with smokers shows that top variants previously shown to be associated with lung cancer risk only confer risk in the presence of tobacco exposure, underscoring the importance of gene-environment interactions in the etiology of this disease.


Assuntos
Cromossomos Humanos Par 5 , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Telomerase/genética , Estudos de Casos e Controles , Europa (Continente)/epidemiologia , Feminino , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Técnicas de Genotipagem/métodos , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco
10.
Carcinogenesis ; 40(4): 500-512, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-30624614

RESUMO

There is increasing evidence that stromal myofibroblasts play a key role in the tumour development however, the mechanisms by which they become reprogrammed to assist in cancer progression remain unclear. As cultured cancer-associated myofibroblasts (CAMs) retain an ability to enhance the proliferation and migration of cancer cells in vitro, it is possible that epigenetic reprogramming of CAMs within the tumour microenvironment may confer long-term pro-tumourigenic changes in gene expression. This study reports the first comparative multi-omics analysis of cancer-related changes in gene expression and DNA methylation in primary myofibroblasts derived from gastric and oesophageal tumours. In addition, we identify novel CAM-specific DNA methylation signatures, which are not observed in patient-matched adjacent tissue-derived myofibroblasts, or corresponding normal tissue-derived myofibroblasts. Analysis of correlated changes in DNA methylation and gene expression shows that different patterns of gene-specific DNA methylation have the potential to confer pro-tumourigenic changes in metabolism, cell signalling and differential responses to hypoxia. These molecular signatures provide new insights into potential mechanisms of stromal reprogramming in gastric and oesophageal cancer, while also providing a new resource to facilitate biomarker identification and future hypothesis-driven studies into mechanisms of stromal reprogramming and tumour progression in solid tumours.


Assuntos
Biomarcadores Tumorais/genética , Epigênese Genética , Neoplasias Esofágicas/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Miofibroblastos/patologia , Neoplasias Gástricas/patologia , Movimento Celular , Proliferação de Células , Metilação de DNA , Epigenômica , Neoplasias Esofágicas/genética , Humanos , Miofibroblastos/metabolismo , Neoplasias Gástricas/genética , Células Tumorais Cultivadas , Microambiente Tumoral
11.
Clin Epigenetics ; 11(1): 6, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642396

RESUMO

BACKGROUND: Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. METHODS: Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. RESULTS: Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10-9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10-7), and Tenascin X (TNXB) (p = 6.30 × 10-6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. CONCLUSIONS: We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Degeneração Macular/genética , Proteínas Proto-Oncogênicas/genética , Tenascina/genética , Fatores Genéricos de Transcrição/genética , Fatores de Transcrição TFII/genética , Sequenciamento Completo do Genoma/métodos , Idoso , Autopsia , Estudos de Casos e Controles , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Epitélio Pigmentado da Retina/química
12.
Mol Oncol ; 13(4): 701-724, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30444046

RESUMO

The KRAB-ZNF (Krüppel-associated box domain zinc finger) gene family is composed of a large number of highly homologous genes, gene isoforms, and pseudogenes. The proteins encoded by these genes, whose expression is often tissue-specific, act as epigenetic suppressors contributing to the addition of repressive chromatin marks and DNA methylation. Due to its high complexity, the KRAB-ZNF family has not been studied in sufficient detail, and the involvement of its members in carcinogenesis remains mostly unexplored. In this study, we aimed to provide a comprehensive description of cancer-associated KRAB-ZNFs using publicly available The Cancer Genome Atlas pan-cancer datasets. We analyzed 6727 tumor and normal tissue samples from 16 cancer types. Here, we showed that a small but distinctive cluster of 16 KRAB-ZNFs is commonly upregulated across multiple cancer cohorts in comparison to normal samples. We confirmed these observations in the independent panels of lung and breast cancer cell lines and tissues. This upregulation was also observed for most of the KRAB-ZNF splicing variants, whose expression is simultaneously upregulated in tumors compared to normal tissues. Finally, by analyzing the clinicopathological data for breast and lung cancers, we demonstrated that the expression of cancer-associated KRAB-ZNFs correlates with patient survival, tumor histology, and molecular subtyping. Altogether, our study allowed the identification and characterization of KRAB-ZNF factors that may have an essential function in cancer biology and thus potential to become novel oncologic biomarkers and treatment targets.


Assuntos
Perfilação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias/genética , Transcriptoma/genética , Dedos de Zinco , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Metilação de DNA/genética , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sobrevida
13.
J Thorac Oncol ; 13(10): 1483-1495, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29981437

RESUMO

BACKGROUND: Genome-wide association studies are widely used to map genomic regions contributing to lung cancer (LC) susceptibility, but they typically do not identify the precise disease-causing genes/variants. To unveil the inherited genetic variants that cause LC, we performed focused exome-sequencing analyses on genes located in 121 genome-wide association study-identified loci previously implicated in the risk of LC, chronic obstructive pulmonary disease, pulmonary function level, and smoking behavior. METHODS: Germline DNA from 260 case patients with LC and 318 controls were sequenced by utilizing VCRome 2.1 exome capture. Filtering was based on enrichment of rare and potential deleterious variants in cases (risk alleles) or controls (protective alleles). Allelic association analyses of single-variant and gene-based burden tests of multiple variants were performed. Promising candidates were tested in two independent validation studies with a total of 1773 case patients and 1123 controls. RESULTS: We identified 48 rare variants with deleterious effects in the discovery analysis and validated 12 of the 43 candidates that were covered in the validation platforms. The top validated candidates included one well-established truncating variant, namely, BRCA2, DNA repair associated gene (BRCA2) K3326X (OR = 2.36, 95% confidence interval [CI]: 1.38-3.99), and three newly identified variations, namely, lymphotoxin beta gene (LTB) p.Leu87Phe (OR = 7.52, 95% CI: 1.01-16.56), prolyl 3-hydroxylase 2 gene (P3H2) p.Gln185His (OR = 5.39, 95% CI: 0.75-15.43), and dishevelled associated activator of morphogenesis 2 gene (DAAM2) p.Asp762Gly (OR = 0.25, 95% CI: 0.10-0.79). Burden tests revealed strong associations between zinc finger protein 93 gene (ZNF93), DAAM2, bromodomain containing 9 gene (BRD9), and the gene LTB and LC susceptibility. CONCLUSION: Our results extend the catalogue of regions associated with LC and highlight the importance of germline rare coding variants in LC susceptibility.


Assuntos
Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco
14.
Sci Rep ; 7(1): 5821, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28725028

RESUMO

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.


Assuntos
Culex/genética , Variações do Número de Cópias de DNA/genética , Genes de Insetos , Canais de Sódio Disparados por Voltagem/genética , Animais , Dosagem de Genes , Haplótipos/genética , Reprodutibilidade dos Testes
16.
Oncol Lett ; 13(6): 4463-4468, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28588715

RESUMO

Deregulation of mitotic spindle genes has been reported to contribute to the development and progression of malignant tumours. The aim of the present study was to explore the association between the expression profiles of Aurora kinases (AURKA, AURKB and AURKC), cytoskeleton-associated protein 5 (CKAP5), discs large-associated protein 5 (DLGAP5), kinesin-like protein 11 (KIF11), microtubule nucleation factor (TPX2), monopolar spindle 1 kinase (TTK), and ß-tubulins (TUBB) and (TUBB3) genes and clinicopathological characteristics in human non-small cell lung carcinoma (NSCLC). Reverse transcription-quantitative polymerase chain reaction-based RNA gene expression profiles of 132 NSCLC and 44 adjacent wild-type tissues were generated, and Cox's proportional hazard regression was used to examine associations. With the exception of AURKC, all genes exhibited increased expression in NSCLC tissues. Of the 10 genes examined, only AURKA was significantly associated with prognosis in NSCLC. Multivariate Cox's regression analysis demonstrated that AURKA mRNA expression [hazard ratio (HR), 1.81; 95% confidence interval (CI), 1.16-2.84; P=0.009], age (HR, 1.03; 95% CI, 1.00-1.06; P=0.020), pathological tumour stage 2 (HR, 2.43; 95% CI, 1.16-5.10; P=0.019) and involvement of distal nodes (pathological node stage 2) (HR, 3.14; 95% CI, 1.24-7.99; P=0.016) were independent predictors of poor prognosis in patients with NSCLC. Poor prognosis of patients with increased AURKA expression suggests that those patients may benefit from surrogate therapy with AURKA inhibitors.

17.
Stem Cells Transl Med ; 6(5): 1321-1331, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28456008

RESUMO

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331.


Assuntos
Fibroblastos/citologia , Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Epigênese Genética/genética , Humanos
18.
J Cancer Res Clin Oncol ; 143(7): 1133-1141, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28258342

RESUMO

PURPOSE: Collagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies. METHODS: Hereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues. RESULTS: We demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (p < 1 × 10-4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions. CONCLUSION: In conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/biossíntese , Colágeno Tipo I/biossíntese , Neoplasias Pulmonares/patologia , Proteína Desacopladora 2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Transporte/genética , Hipóxia Celular/fisiologia , Colágeno Tipo I/genética , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Transcriptoma , Proteína Desacopladora 2/genética , Regulação para Cima
19.
Br J Cancer ; 116(5): 592-599, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28095398

RESUMO

BACKGROUND: Taxanes are mitotic poisons widely used in the treatment of non-small cell lung cancer (NSCLC), however, little is known about potential molecular modulators of response to these compounds. Aurora B (AURKB) is a critical regulator of the mitotic spindle assembly, previously shown overexpressed in NSCLC. Here we investigated the hypothesis that AURKB expression modulates the efficacy of taxanes in NSCLC cells. METHODS: AURKB mRNA expression was determined by qPCR in 132 frozen NSCLC tissues and nine NSCLC cell lines. Aurora B expression was knocked down in cell lines using multiple shRNA constructs. Barasertib was used to specifically inhibit AURKB activity, determined by the level of H3S10 phosphorylation. RESULTS: Frequent AURKB mRNA upregulation was observed in NSCLC tissues (P<0.0001), being more prominent in squamous carcinomas (P<0.0001). Aurora B expression in cell lines strongly correlated with sensitivity to both docetaxel (P=0.004) and paclitaxel (P=0.007). Aurora B knockdown derivatives consistently showed a dose-dependent association between low-AURKB expression and resistance to paclitaxel. Specific chemical inhibition of Aurora B activity also demonstrated a strong dose-dependent efficiency in triggering paclitaxel resistance. CONCLUSIONS: Aurora B activity is an important modulator of taxane response in NSCLC cells. This may lead to further insights into taxane sensitivity of NSCLC tumours.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Aurora Quinase B/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Organofosfatos/farmacologia , Quinazolinas/farmacologia , Regulação para Cima/efeitos dos fármacos
20.
PLoS One ; 11(11): e0165567, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27802312

RESUMO

BACKGROUND: Carboxyl-ester lipase (CEL) contributes to fatty acid ethyl ester metabolism, which is implicated in alcoholic pancreatitis. The CEL gene harbours a variable number of tandem repeats (VNTR) region in exon 11. Variation in this VNTR has been linked to monogenic pancreatic disease, while conflicting results were reported for chronic pancreatitis (CP). Here, we aimed to investigate a potential association of CEL VNTR lengths with alcoholic CP. METHODS: Overall, 395 alcoholic CP patients, 218 patients with alcoholic liver cirrhosis (ALC) serving as controls with a comparable amount of alcohol consumed, and 327 healthy controls from Germany and the United Kingdom (UK) were analysed by determination of fragment lengths by capillary electrophoresis. Allele frequencies and genotypes of different VNTR categories were compared between the groups. RESULTS: Twelve repeats were overrepresented in UK ACP patients (P = 0.04) compared to controls, whereas twelve repeats were enriched in German ALC compared to alcoholic CP patients (P = 0.03). Frequencies of CEL VNTR lengths of 14 and 15 repeats differed between German ALC patients and healthy controls (P = 0.03 and 0.008, respectively). However, in the genotype and pooled analysis of VNTR lengths no statistical significant association was depicted. Additionally, the 16-16 genotype as well as 16 repeats were more frequent in UK ALC than in alcoholic CP patients (P = 0.034 and 0.02, respectively). In all other calculations, including pooled German and UK data, allele frequencies and genotype distributions did not differ significantly between patients and controls or between alcoholic CP and ALC. CONCLUSIONS: We did not obtain evidence that CEL VNTR lengths are associated with alcoholic CP. However, our results suggest that CEL VNTR lengths might associate with ALC, a finding that needs to be clarified in larger cohorts.


Assuntos
Lipase/genética , Cirrose Hepática Alcoólica/genética , Repetições Minissatélites , Pancreatite Alcoólica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Éxons , Feminino , Predisposição Genética para Doença , Genótipo , Alemanha/epidemiologia , Humanos , Cirrose Hepática Alcoólica/epidemiologia , Masculino , Pessoa de Meia-Idade , Pancreatite Alcoólica/epidemiologia , Reino Unido/epidemiologia
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