Molecular Genetics of Intracranial Meningiomas with Emphasis on Canonical Wnt Signalling.

Research over the last decade recognized the importance of novel molecular pathways in pathogenesis of intracranial meningiomas. In this review, we focus on human brain tumours meningiomas and the involvement of Wnt signalling pathway genes and proteins in this common brain tumour, describing their known functional effects. Meningiomas originate from the meningeal layers of the brain and the spinal cord. Most meningiomas have benign clinical behaviour and are classified as grade I by World Health Organization (WHO). However, up to 20% histologically classified as atypical (grade II) or anaplastic (grade III) are associated with higher recurrent rate and have overall less favourable clinical outcome. Recently, there is emerging evidence that multiple signalling pathways including Wnt pathway contribute to the formation and growth of meningiomas. In the review we present the synopsis on meningioma histopathology and genetics and discuss our research regarding Wnt in meningioma. Epithelial-to-mesenchymal transition, a process in which Wnt signalling plays an important role, is shortly discussed

Promjene u strukturi gena i izraženosti proteina DVL-1, DVL-2, DVL-3 te transkripcijskih faktora TCF-1 i LEF-1 u astrocitnim tumorima mozga [Changes in gene structure and protein expression of DVL-1, DVL-2, DVL-3 and transcription factors TCF-1 and LEF-1 in astrocytic brain tumors]

Astrocytomas are the most common primary tumors of the central nervous system. According to the WHO classification, there are four grades of astrocytoma, considering their histology, molecular characteristics and prognosis, where the grade IV (glioblastoma) is the deadliest form and still without effective therapy. The aim of this dissertation was to investigate changes in gene structure and protein expression of Dishevelled family and transcription factors TCF1 and LEF1, the molecular components of the Wnt signaling pathway, in astrocytic brain tumors and compare results with clinical parameters. Four selected microsatellite markers for DVL1 (D1S468 and D1S243), DVL2 (D17S960) and DVL3 (D3S1262) genes have shown a significant number of samples demonstrating MSI and LOH, genetic changes that contribute to the genome instability in cancer. Diffuse astrocytomas (WHO grade II) analyzed with D1S468 (p=0.008) have shown the highest percentage of MSI (53.8%), while LOH was the most common in glioblastoma (31.2%) analysed with D3S1262 (p=0.007). The results obtained on the total sample indicate that the microsatellite instability is constantly present, with a more frequent appearance in lower grades and may be the cause of astrocytoma formation, whereas large deletions are significantly associated with the highest grade and have a role in progression. The levels of DVL1, DVL2 and DVL3 protein expression did not show statistically significant correlation with genetic changes. Nevertheless, statistically significant differences in the number of cells with certain level of expression of a single protein between the grades were observed. Bivariate correlation of all analyzed proteins showed a statistically significant positive correlation between DVL3 and TCF1 (p=0.020), DVL3 and LEF1 (p=0.006), TCF1 and LEF1 (p=0.021), while DVL1 and DVL3 were negatively correlated (p=0.002). DVL1 was also negatively correlated (p<0.001) with astrocytoma grade, while DVL3 (p<0.001), TCF1 (p=0.008) and LEF1 (p<0.001) showed positive correlation with malignancy grade suggesting involvement of these proteins in malignant progression. These results contribute to a better understanding of the molecular profile of astrocytic brain tumors and could serve as molecular markers of progression

Genetic changes of MLH1 and MSH2 genes could explain constant findings on microsatellite instability in intracranial meningioma

Postreplicative mismatch repair safeguards the stability of our genome. The defects in its functioning will give rise to microsatellite instability. In this study, 50 meningiomas were investigated for microsatellite instability. Two major mismatch repair genes, MLH1 and MSH2, were analyzed using microsatellite markers D1S1611 and BAT26 amplified by polymerase chain reaction and visualized by gel electrophoresis on high-resolution gels. Furthermore, genes DVL3 (D3S1262), AXIN1 (D16S3399), and CDH1 (D16S752) were also investigated for microsatellite instability. Our study revealed constant presence of microsatellite instability in meningioma patients when compared to their autologous blood DNA. Altogether 38% of meningiomas showed microsatellite instability at one microsatellite locus, 16% on two, and 13.3% on three loci. The percent of detected microsatellite instability for MSH2 gene was 14%, and for MLH1, it was 26%, for DVL3 22.9%, for AXIN1 17.8%, and for CDH1 8.3%. Since markers also allowed for the detection of loss of heterozygosity, gross deletions of MLH1 gene were found in 24% of meningiomas. Genetic changes between MLH1 and MSH2 were significantly positively correlated (p = 0.032). We also noted a positive correlation between genetic changes of MSH2 and DVL3 genes (p = 0.034). No significant associations were observed when MLH1 or MSH2 was tested against specific histopathological meningioma subtype or World Health Organization grade. However, genetic changes in DVL3 were strongly associated with anaplastic histology of meningioma (χ2 = 9.14; p = 0.01). Our study contributes to better understanding of the genetic profile of human intracranial meningiomas and suggests that meningiomas harbor defective cellular DNA mismatch repair mechanisms

Optimal amplification conditions for D16S3399 polymorphic STS axin-1 gene marker

We investigated D16S3399 marker and affirmed it as a highly polymorphic marker useful for the analysis of the human axin-1 gene. Axin-1 acts as a tumor suppressor gene and its protein is an inhibitor of the Wnt signaling pathway. We report on heterozygosity status, alleles frequency observed in a preliminary group of Croatian subjects and the optimal amplification conditions for D16S3399 marker. The amplified CA repeat was confirmed by direct sequencing

Brain metastases from lung cancer show increased expression of DVL1, DVL3 and beta-catenin and down-regulation of E-cadherin

The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p=0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p=0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain

The cellular story of dishevelleds

Dishevelled (DVL) proteins, three of which have been identified in humans, are highly conserved components of canonical and noncanonical Wnt signaling pathways. These multifunctional proteins, originally discovered in the fruit fly, through their different domains mediate complex signal transduction: DIX (dishevelled, axin) and PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains serve for canonical beta-catenin signaling, while PDZ and DEP (dishevelled, Egl-10, pleckstrin) domains serve for non-canonical signaling. In canonical or beta-catenin signaling, DVL forms large molecular supercomplexes at the plasma membrane consisting of Wnt-Fz-LRP5/6-DVLAXIN. This promotes the disassembly of the beta-catenin destruction machinery, beta-catenin accumulation, and consequent activation of Wnt signaling. Therefore, DVLs are considered to be key regulators that rescue cytoplasmic beta-catenin from degradation. The potential medical importance of DVLs is in both human degenerative disease and cancer. The overexpression of DVL has been shown to potentiate the activation of Wnt signaling and it is now apparent that up-regulation of DVLs is involved in several types of cancer

Roll-to-Roll pilot line for large-scale manufacturing of microfluidic devices

Roll-to-roll (R2R) technologies with roller-based nanoimprinting methods enable manufacturing of highly cost-effective and large-scale sheets of flexible polymer film with precise structures on a micro- and nanoscale 1. Areas that can benefit strongly from such large scale technologies are microfluidics, biosensors, and lab-on-chip products for point of care diagnostics, drug discovery and food control. Here, R2R fabrication could greatly reduce production costs and increase manufacturing capacity with respect to currently used products. A pilot line with this technology is investigated in the European Horizon 2020 project R2R Biofluidics and its capabilities are tested on two Demonstrators: - Demonstrator 1: In-vitro diagnostic chip with imprinted microfluidic channels based on optical chemiluminescence measurement by photodetectors. - Demonstrator 2: Neuronal cell culture plate with imprinted cavities and channels for controlled culturing and fluorescence imaging of neurons, for high throughput drug screening. Please click Additional Files below to see the full abstract

High-throughput roll-to-roll production of polymer biochips for multiplexed DNA detection in point-of-care diagnostics

Roll-to-roll UV nanoimprint lithography has superior advantages for high-throughput manufacturing of micro- or nano-structures on flexible polymer foils with various geometries and configurations. Our pilot line provides large-scale structure imprinting for cost-effective polymer biochips (4500 biochips/hour), enabling rapid and multiplexed detections. A complete high-volume process chain of the technology for producing structures like μ-sized, triangular optical out-couplers or capillary channels (width: from 1 μm to 2 mm, height: from 200 nm up to 100 μm) to obtain biochips (width: 25 mm, length: 75 mm, height: 100 μm to 1.5 mm) was described. The imprinting process was performed with custom-developed resins on polymer foils with resin thicknesses ranging between 125–190 μm. The produced chips were tested in a commercial point-of-care diagnostic system for multiplexed DNA analysis of methicillin resistant Staphylococcus aureus (e.g., mecA, mecC gene detections). Specific target DNA capturing was based on hybridisation between surface bound DNA probes and biotinylated targets from the sample. The immobilised biotinylated targets subsequently bind streptavidin–horseradish peroxidase conjugates, which in turn generate light upon incubation with a chemiluminescent substrate. To enhance the light out-coupling thus to improve the system performance, optical structures were integrated into the design. The limits-of-detection of mecA (25 bp) for chips with and without structures were calculated as 0.06 and 0.07 μM, respectively. Further, foil-based chips with fluidic channels were DNA functionalised in our roll-to-roll micro-array spotter following the imprinting. This straightforward approach of sequential imprinting and multiplexed DNA functionalisation on a single foil was also realised for the first time. The corresponding foil-based chips were able to detect mecA gene DNA sequences down to a 0.25 μM concentration.This research was supported by R2R BIOFLUIDICS project (http://www.r2r-biofluidics.eu/) under Horizon 2020 European Union (EU) Research and Innovation Programme with grant agreement no 646260. The research was also partially supported by NextGenMicrofluidics project (https:// www. nextgenmicrofluidics.eu/) under HORIZON2020 with grant agreement no 862092. The authors cordially thank Gerburg Schider & Gerhard Mohr, Markus Postl, Paul Patter and Alexander Wheeldon (JOANNEUM RESEARCH – Materials, Weiz, Austria) for revising the manuscript, preparing all the chip and R2R pilot line illustrations, taking the photographs and providing technical support, respectively. The authors are also grateful to Christian Wolf and Johannes Götz (JOANNEUM RESEARCH – Materials, Weiz, Austria) for their supports in the fluidic design and R2R UV-NIL structuring, respectively. We further kindly thank Alba Simon Munoz and Robert Fay (SCIENION AG, Berlin, Germany) for providing the illustration of the R2R micro-spotting line. PT specially thanks Ege Ozgun (NANOTAM, Bilkent University, Ankara, Turkey) for critically reading the manuscript

Brain Metastases from Lung Cancer Show Increased Expression of DVL1, DVL3 and Beta-Catenin and Down-Regulation of E-Cadherin

The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p = 0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p = 0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain