Identification of different mechanisms leading to PAX6 down-regulation as potential events contributing to the onset of Hirschsprung disease

Hirschsprung disease (HSCR) is attributed to a failure of neural crest derived cells to migrate, proliferate, differentiate or survive in the bowel wall during embryonic Enteric Nervous System (ENS) development. This process requires a wide and complex variety of molecules and signaling pathways which are activated by transcription factors. In an effort to better understand the etiology of HSCR, we have designed a study to identify new transcription factors participating in different stages of the colonization process. A differential expression study has been performed on a set of transcription factors using Neurosphere-like bodies from both HSCR and control patients. Differential expression levels were found for CDYL, MEIS1, STAT3 and PAX6. A significantly lower expression level for PAX6 in HSCR patients, would suit with the finding of an over-representation of the larger tandem (AC)m(AG)n repeats within the PAX6 promoter in HSCR patients, with the subsequent loss of protein P300 binding. Alternatively, PAX6 is a target for DNMT3B-dependant methylation, a process already proposed as a mechanism with a role in HSCR. Such decrease in PAX6 expression may influence in the proper function of signaling pathways involved in ENS with the confluence of additional genetic factors to the manifestation of HSCR phenotype.Instituto de Salud Carlos III (ISCIII), Ministerio de Economía y Competitividad PI1301560Consejería de Innovación, Ciencia y Empresa CTS-744

Overexpression of canonical prefoldin associates with the risk of mortality and metastasis in non-small cell lung cancer

Canonical prefoldin is a protein cochaperone composed of six di erent subunits (PFDN1 to 6). PFDN1 overexpression promotes epithelial–mesenchymal transition (EMT) and increases the growth of xenograft lung cancer (LC) cell lines. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic. First, the mRNA expression of 518 non-small cell LC (NSCLC) cases from The Cancer Genome Atlas (TCGA) database was evaluated. Patients with PFDN1 overexpression had lower overall survival (OS; 45 vs. 86 months; p = 0.034). We then assessed the impact of PFDN expression on outcome in 58 NSCLC patients with available tumor tissue samples. PFDN1, 3, and 5 overexpression were found in 38% (n = 22), 53% (n = 31), and 41% (n = 24) of tumor samples. PFDN1, 3, and 5 overexpression were significantly associated with lower OS, lower disease-free survival (DFS), and lower distant metastasis-free survival (DMFS) for PFDN1 and 3 with a trend for PFDN5. In multivariate analysis, PFDN5 retained significance for OS (hazard ratio (HR) 2.56; p = 0.007) and PFDN1 for DFS (HR 2.53; p = 0.010) and marginally for DMFS (HR 2.32; p = 0.053). Our results indicate that protein response markers, such as PFDN1, 3, and 5, may complement mRNA signatures and be useful for determining the most appropriate therapy for NSCLC patients

Identificación y análisis de nuevos genes y mecanismos moleculares implicados en la Enfermedad de Hirschsprung

Falta palabras claveEn la presente tesis doctoral se propone la realización de estudios de expresión en cultivos de precursores entéricos para profundizar en el conocimiento del desarrollo del SNE, en el contexto de HSCR, y así llevar a cabo una mejor caracterización de los mismos y realizar la búsqueda de nuevos genes implicados en la patología, basándonos en diferencias de expresión de genes en estudios de pacientes y controles. A partir de dichos estudios de expresión se han obtenido diferencias en cuanto a niveles de expresión entre pacientes y controles en siete genes descritos en células madre pluripotentes humanas (DNMT3B, FN1, LAMC1, PAX6, NESTINA, ECAM, SEMA3A) y cuatro factores de transcripción descritos en células embrionarias humanas (CDYL, MEIS1, STAT3 y PAX6). De todos ellos se ha estudiado con más detalle el papel de la metiltransferasa DNMT3B encargada de establecer los patrones de metilación de novo durante el desarrollo embrionario, en relación con el desarrollo del SNE en el contexto de HSCR. Y por otro lado, se han estudiado las causas subyacentes a la disminución en los niveles de expresión de PAX6 obtenida en precursores entéricos procedentes de pacientes HSCR, cuya actividad principal es regular la proliferación y diferenciación celular. Así mismo, por primera vez se demuestra la implicación de un mecanismo epigenético en relación con el desarrollo de la enfermedad de Hirschsprung dada la hipometilación global obtenida en precursores entéricos de pacientes, evento que potencialmente puede inducir alteraciones en la activación de factores de transcripción, entre ellos PAX6, así como la sobreexpresión de genes críticos en la regulación del crecimiento celular

Identification of different mechanisms leading to PAX6 down-regulation as potential events contributing to the onset of Hirschsprung disease

Hirschsprung disease (HSCR) is attributed to a failure of neural crest derived cells to migrate, proliferate, differentiate or survive in the bowel wall during embryonic Enteric Nervous System (ENS) development. This process requires a wide and complex variety of molecules and signaling pathways which are activated by transcription factors. In an effort to better understand the etiology of HSCR, we have designed a study to identify new transcription factors participating in different stages of the colonization process. A differential expression study has been performed on a set of transcription factors using Neurosphere-like bodies from both HSCR and control patients. Differential expression levels were found for CDYL, MEIS1, STAT3 and PAX6. A significantly lower expression level for PAX6 in HSCR patients, would suit with the finding of an over-representation of the larger tandem (AC)m(AG)n repeats within the PAX6 promoter in HSCR patients, with the subsequent loss of protein P300 binding. Alternatively, PAX6 is a target for DNMT3B-dependant methylation, a process already proposed as a mechanism with a role in HSCR. Such decrease in PAX6 expression may influence in the proper function of signaling pathways involved in ENS with the confluence of additional genetic factors to the manifestation of HSCR phenotype.This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain (PI1301560) and Regional Ministry of Innovation, Science and Enterprise of the Autonomous Government of Andalucia (CTS-7447). MVE-R is supported by fellowship PI11/00533 from ISCIII.Peer reviewe

Four new loci associations discovered by pathway-based and network analyses of the genome-wide variability profile of Hirschsprung¿s disease

Abstract Finding gene associations in rare diseases is frequently hampered by the reduced numbers of patients accessible. Conventional gene-based association tests rely on the availability of large cohorts, which constitutes a serious limitation for its application in this scenario. To overcome this problem we have used here a combined strategy in which a pathway-based analysis (PBA) has been initially conducted to prioritize candidate genes in a Spanish cohort of 53 trios of short-segment Hirschsprung’s disease. Candidate genes have been further validated in an independent population of 106 trios. The study revealed a strong association of 11 gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other HSCR-related processes. Among the preselected candidates, a total of 4 loci, RASGEF1A, IQGAP2, DLC1 and CHRNA7, related to signal transduction and migration processes, were found to be significantly associated to HSCR. Network analysis also confirms their involvement in the network of already known disease genes. This approach, based on the study of functionally-related gene sets, requires of lower sample sizes and opens new opportunities for the study of rare diseases.This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain (PI1001290); Spanish Ministry of Economy and Competitiveness (BIO2008-04212), GVA-FEDER (PROMETEO/2010/001) and Consejeria de Innovación Ciencia y Empresa de la Junta de Andalucia (CTS-7447). The CIBER de Enfermedades Raras is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness. LG-A and MVE-R are supported by fellowships PFIS FI10/00020 and FI11/00533 from ISCIII respectively.Peer Reviewe

Association of single nucleotide polymorphisms at HSPB1 rs7459185 and TGFB1 rs11466353 with radiation esophagitis in lung cancer

[Background and purpose] Radiochemotherapy (RCT) success in lung cancer (LC) can be limited due to the onset of adverse effects in the adjacent normal tissue such as radiation-induced esophageal toxicity (RIET). Therefore, specific biomarkers to customize the RCT dose administration and esophageal toxicity prediction are necessary to improve treatment effectiveness.[Materials and methods] 47 LC patients prospectively recruited between 2012 and 2016 from 3 institutions were genotyped for 7 SNPs along TGFB1 and HSPB1 genes seeking an association with RIET risk development. Kaplan–Meier cumulative probability and Cox proportional hazards analyses were used to evaluate the effect of TGFB1 and HSPB1 genotypes on such risk.[Results] Multivariate analyses showed that patients carrying the HSPB1 rs7459185 CC genotype were associated with a significantly higher risk of acute grade 3 RIET than those carrying the GG/GC genotypes (HR = 17.73; 95% CI = 2.896–108.49; p = 0.002). LC patients who received higher (>median) volume of esophagus exposed to 30 Gy and harboring the rs7459185 GG/GC genotypes showed a significantly lower RIET incidence (p 60 Gy) radiation dose who presented the rs11466353 GG genotype had a significantly lower RIET incidence (p = 0.025).[Conclusion] The presence of different rs7459185/rs11466353 genotypes in LC patients associated with RIET risk and may be useful biomarkers along with other risk factors for guiding therapy intensity in an individualized therapy.This work was supported by grants from Instituto de Salud Carlos III (PI13/01155, PI16/02104) and Consejeria de Salud of the Junta de Andalucia (PI-0096-2012), Spain.Peer reviewe

Prognostic value of the TGFβ1 rs4803455 single nucleotide polymorphism in small cell lung cancer

[Background] Small cell lung cancer (SCLC) is one of the greatest therapeutic challenges of oncology. Potential associations between single nucleotide polymorphisms in heat shock protein β1 (HSPB1) and transforming growth factor β1 (TGFβ1) and survival have been investigated.[Methods] A prospective multicenter study of 94 patients with SCLC treated between 2013 and 2016 was conducted. Clinical, tumour-related, therapeutic, and genetic (9 SNPs of TGFβ1 gene and 5 of HSPB1 gene) variables were analyzed.[Results] The cohort included 77 men and 17 women with a median age of 61 years. Eighty percent presented with limited stage at diagnosis and received thoracic radiation with a median dose of 45 Gy (twice-daily radiation in 42%). Forty-seven percent received concurrent platinum-based chemotherapy and 57% received prophylactic cranial irradiation (PCI). Overall survival (OS) was 34% at 2 years and 16% at 3 years. In multivariate analysis, the rs4803455:CA genotype of the TGFβ1 gene showed a statistically significant association with lower disease-free survival (DFS; hazard ratio [HR] 3.13; confidence interval [CI] 1.19–8.17; p = 0.020) and higher local recurrence (HR 3.80; CI 1.37–10.5; p = 0.048), and a marginal association with lower OS (HR 1.94; CI 0.98–3.83; p = 0.057). A combined analysis showed that patients receiving PCI and carrying the rs4803455:CA genotype had statistically significant lower OS (p < 0.001) and DFS (p < 0.001) than patients receiving PCI and carrying the rs4803455:AA genotype.[Conclusions] Genetic analysis showed the CA genotype of TGFβ1 SNP rs4803455 was associated with worse prognosis in patients with SCLC and could be considered as a potential biomarker.Peer reviewe

Overexpression of canonical prefoldin associates with the risk of mortality and metastasis in non-small cell lung cancer

Canonical prefoldin is a protein cochaperone composed of six different subunits (PFDN1 to 6). PFDN1 overexpression promotes epithelial–mesenchymal transition (EMT) and increases the growth of xenograft lung cancer (LC) cell lines. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic. First, the mRNA expression of 518 non-small cell LC (NSCLC) cases from The Cancer Genome Atlas (TCGA) database was evaluated. Patients with PFDN1 overexpression had lower overall survival (OS; 45 vs. 86 months; p = 0.034). We then assessed the impact of PFDN expression on outcome in 58 NSCLC patients with available tumor tissue samples. PFDN1, 3, and 5 overexpression were found in 38% (n = 22), 53% (n = 31), and 41% (n = 24) of tumor samples. PFDN1, 3, and 5 overexpression were significantly associated with lower OS, lower disease-free survival (DFS), and lower distant metastasis-free survival (DMFS) for PFDN1 and 3 with a trend for PFDN5. In multivariate analysis, PFDN5 retained significance for OS (hazard ratio (HR) 2.56; p = 0.007) and PFDN1 for DFS (HR 2.53; p = 0.010) and marginally for DMFS (HR 2.32; p = 0.053). Our results indicate that protein response markers, such as PFDN1, 3, and 5, may complement mRNA signatures and be useful for determining the most appropriate therapy for NSCLC patients.This work was co-funded by the Spanish Ministry of Economy and Competitiveness (BFU2017-85420-R to JCR and BFU2016-7772-C3-1-P to SC) and by the Andalusian Government (BIO-271) and the University of Seville (US-1256285 to SC) in cooperation with the European Regional Development Fund.Peer reviewe