Identification of ZBTB18 as a novel colorectal tumor suppressor gene through genome-wide promoter hypermethylation analysis

Background Cancer initiation and progression are driven by genetic and epigenetic changes. Although genome/exome sequencing has significantly contributed to the characterization of the genetic driver alterations, further investigation is required to systematically identify cancer driver genes regulated by promoter hypermethylation. Results Using genome-wide analysis of promoter methylation in 45 colorectal cancer cell lines, we found that higher overall methylation levels were associated with microsatellite instability (MSI), faster proliferation and absence of APC mutations. Because epigenetically silenced genes could represent important oncogenic drivers, we used mRNA expression profiling of colorectal cancer cell lines and primary tumors to identify a subset of 382 (3.9%) genes for which promoter methylation was negatively associated with gene expression. Remarkably, a significant enrichment in zinc finger proteins was observed, including the transcriptional repressor ZBTB18. Re-introduction of ZBTB18 in colon cancer cells significantly reduced proliferation in vitro and in a subcutaneous xenograft mouse model. Moreover, immunohistochemical analysis revealed that ZBTB18 is frequently lost or reduced in colorectal tumors, and reduced ZBTB18 expression was found to be associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. Conclusions We identified a set of 382 genes putatively silenced by promoter methylation in colorectal cancer that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZBTB18 has tumor suppressor activity and is a novel prognostic marker for patients with locally advanced colorectal cancer.Peer reviewe

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

Tyrosine kinase; EPHA3; Colorectal cancerTirosina-cinasa; EPHA3; Càncer colorectalTirosina quinasa; EPHA3; Cáncer colorrectalEPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Mitigation of coupled wind-wave-earthquake responses of a 10 MW fixed-bottom offshore wind turbine

© 2020 Elsevier Ltd In this paper we present a study on the mitigation of dynamic responses of a 10 MW monopile offshore wind turbine under coupled wind-wave-earthquake excitations. We have developed and validated the generic seismic coupled analysis and structural control architecture tool to overcome the limitation of numerical tools when examining the wind-wave-earthquake coupling effects. We investigated the dynamic responses of a 10 MW monopile offshore wind turbine under different loading combinations and found that the earthquake loading increases the tower-top displacement and pile-cap moment by 47.6% and 95.1%, respectively, compared to the wind-wave-only condition. It is found that the earthquake-induced vibration in the fore-aft direction is mitigated by the wind and wave loadings due to the energy dissipated by the aerodynamic and hydrodynamic damping. In addition, the tower responses are dominated by the earthquake excitation. In order to alleviate the tower vibration induced by the earthquake, we implemented the structural control capability within the tool using tuned mass dampers. The tuned mass dampers with appropriately selected design parameters achieve a larger mitigation on the tower-top displacement for the earthquake-only condition compared to the coupled-loading scenario. The reason is that the tuned mass damper is only effective in mitigating tower vibration, and it is not capable of reducing the tower elastic deformation which is the major contribution of the tower displacement for the coupled-loading condition. In addition, we have found that a heavier tuned mass damper requires a lower tuned frequency to achieve a larger mitigation. A configuration for the mitigation control of the 10 MW offshore wind turbine is suggested by using a 5% mass ratio of the tuned mass damper

Mechanisms of inactivation of the tumour suppressor gene RHOA in colorectal cancer

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF- β /SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms

Identification of novel therapeutic targets and tumor suppressor genes in colon cancer using genome-wide high‐throughput approaches

[cat] El càncer colorectal és una malaltia causada per canvis genètics i epigenètics. La inactivació de gens supressors de tumors i l'activació d'oncogens són fites clau en la progressió tumoral. Els pacients amb càncer en estadi III o IV són sotmesos a cirurgia seguida de quimioteràpia. No obstant això, només el 30% dels pacients mostren una resposta objectiva fins i tot als millors agents quimioterapèutics disponibles. En aquest estudi es van utilitzar assajos d'alt rendiment de tot el genoma per caracteritzar millor els aspectes importants de la progressió oncogènica, com la desregulació de la proliferació i l'expressió aberrant causada per mecanismes epigenètics. La ràpida proliferació tumoral s'associa al pitjor pronòstic del pacient, aquí hem caracteritzat la signatura transcripcional de les cèl.lules de càncer colorectal amb ràpida proliferació de cèl.lules en un intent d'identificar els gens importants per sostenir el creixement tumoral i que podrien ser utilitzats com a dianes terapèutiques. Per comprendre millor els mecanismes subjacents a la profunda reprogramació transcripcional observada en les cèl.lules canceroses, es va investigar l'associació entre els nivells de metilació de l'ADN en els promotors i els nivells d'expressió d'aquests gens en línies cel.lulars de càncer colorectal. Els resultats d'aquest estudi poden contribuir a la identificació de noves dianes quimioterapèutics per a pacients amb càncer colorectal, i la caracterització de nous gens / vies amb activitat supressora de tumors, que són silenciats epigeneticament.[eng] Colorectal cancer is a disease caused by genetic and epigenetic changes. Inactivation of tumor suppressor genes and activation of oncogenes are key landmarks in tumor progression. However, the list of tumor suppressor genes and oncogenes is far from complete, even in the case of the tumor types that are best characterized, such as colorectal cancer. Colorectal cancer is the second most frequent cause of cancer-related death in the Western world and is a serious health issue for the European Union. Patients having stage III or IV cancer undergo surgery followed by chemotherapy. However, the clinical management of these patients is far from optimal, and only about 30 % of the patients show an objective response to even the best chemotherapeutic agents available. In this study genome-wide high throughput assays were used to better characterize important aspects of the oncogenic progression such as deregulation of proliferation and aberrant expression caused by epigenetic mechanisms. Because rapid tumor proliferation is associated with poor patient prognosis, here we characterized the transcriptional signature of rapidly proliferating colorectal cancer cells in an attempt to identify genes important to sustain tumor growth and that could be used as novel therapeutic targets. The proliferation rate of 52 colorectal cancer cell lines was determined and genome-wide expression profiling of a subset of these lines was assessed by microarray analysis. The expression of 1,290 genes was significantly correlated with the growth rates of colorectal cancer cells. These included genes involved in cell cycle, RNA processing/splicing and protein transport. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and protoporphyrinogen oxidase (PPDX) were shown to have higher expression in faster growing cancer cells. Importantly, pharmacological and genetic inhibition of GAPDH or PPDX reduced the growth of colon cancer cells in vitro and in vivo. To better understand the mechanisms underlying the profound transcriptional reprogramming observed in cancer cells, we investigated the association between the levels of DNA methylation in the promoters of >14,000 genes and the levels of expression of these genes in a panel of 45 colorectal cancer cell lines. A group of cell lines with significantly higher methylation levels was observed, supporting the notion that there is a group of colorectal tumors with a CpG methylator phenotype (CIMP+). A significant negative regulation between methylation and expression levels was observed for 1,409 genes, suggesting that these genes are silenced during the tumorigenic process through this epigenetic mechanism. A significant number of these genes were zinc finger proteins, suggesting an important role of these DNA-binding proteins on the tumorigenic process. Strikingly, approximately one fourth of these genes are not associated with CpG islands, indicating that DNA methylation outside these CpG rich regions is an important mechanism regulating gene expression and significantly contribute to tumor progression. In addition, we postulate that at least some of those genes have tumor suppressor activity. As a proof-of-concept, we show that restoration of the expression of ZNF238, a gene showing a significant methylation/expression correlation, resulted in reduced growth of colon cancer cells in vitro and in vivo. In conclusion, in this study we shed new light on the mechanisms underlying the uncontrolled proliferation of colon cancer cells and the expression reprograming imposed in these cells through CpG methylation. The results of this study may contribute to the identification of novel chemotherapeutic targets for patients with colorectal cancer, and the characterization of novel genes/pathways with tumor suppressor activity, that are epigenetically silenced

Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix to short-chain fatty acids by human faecal microbiota in vitro

International audienc

Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix to short-chain fatty acids by human faecal microbiota in vitro

International audienc