SFRP4 protein expression is reduced in high grade astrocytomas which is not caused by the methylation of its promoter.

Introduction: Epigenetics play a vital role in stratifying CNS tumors and gliomas. The importance of studying Secreted frizzled-related protein 4 (SFRP4) in gliomas is to improve diffuse glioma methylation profiling. Here we examined the methylation status of SFRP4 promoter and the level of its protein expression in diffuse gliomas WHO grades 2-4. Methods: SFRP4 expression was detected by immunohistochemistry and evaluated semi-quantitatively. In the tumor hot-spot area, the intensity of protein expression in 200 cells was determined using ImageJ (National Institutes of Health, United States). The assessment of immunopositivity was based on the IRS score (Immunoreactivity Score). Promoter methylation was examined by methylation specific-PCR (MSP) in fifty-one diffuse glioma samples and appropriate controls. Isolated DNA was treated with bisulfite conversion and afterwards used for MSP. Public databases (cBioPortal, COSMIC and LOVD) were searched to corroborate the results. Results and discussion: SFRP4 protein expression in glioblastomas was very weak or non-existent in 86.7% of samples, moderate in 13.3%, while strong expression was not observed. The increase in astrocytoma grade resulted in SFRP4 protein decrease (p = 0.008), indicating the loss of its antagonistic role in Wnt signaling. Promoter methylation of SFRP4 gene was found in 16.3% of cases. Astrocytomas grade 2 had significantly more methylated cases compared to grade 3 astrocytomas (p = 0.004) and glioblastomas (p < 0.001), which may indicate temporal niche of methylation in grade 2. Furthermore, the expression levels of SFRP4 were high in samples with methylated SFRP4 promoter and low or missing in unmethylated cases (Pearson's R = -0.413; p = 0.003). We also investigated the association of SFRP4 changes to key Wnt regulators GSK3ß and DKK3 and established a positive correlation between methylations of SFRP4 and GSK3ß (Pearson's R = 0.323; p = 0.03). Furthermore, SFRP4 expression was correlated to unmethylated DKK3 (Chi square = 7.254; p = 0.027) indication that Wnt signaling antagonist is associated to negative regulator's demethylation. Conclusion: The study contributes to the recognition of the significance of epigenetic changes in diffuse glioma indicating that restoring SFRP4 protein holds potential as therapeutic avenue. Reduced expression of SFRP4 in glioblastomas, not following promoter methylation pattern, suggests another mechanism, possible global methylation, that turns off SFRP4 expression in higher grades.

Genetics in ophthalmology: molecular blueprints of retinoblastoma.

This review presents current knowledge on the molecular biology of retinoblastoma (RB). Retinoblastoma is an intraocular tumor with hereditary and sporadic forms. 8,000 new cases of this ocular malignancy of the developing retina are diagnosed each year worldwide. The major gene responsible for retinoblastoma is RB1, and it harbors a large spectrum of pathogenic variants. Tumorigenesis begins with mutations that cause RB1 biallelic inactivation preventing the production of functional pRB proteins. Depending on the type of mutation the penetrance of RB is different. However, in small percent of tumors additional genes may be required, such as MYCN, BCOR and CREBBP. Additionally, epigenetic changes contribute to the progression of retinoblastoma as well. Besides its role in the cell cycle, pRB plays many additional roles, it regulates the nucleosome structure, participates in apoptosis, DNA replication, cellular senescence, differentiation, DNA repair and angiogenesis. Notably, pRB has an important role as a modulator of chromatin remodeling. In recent years high-throughput techniques are becoming essential for credible biomarker identification and patient management improvement. In spite of remarkable advances in retinoblastoma therapy, primarily in high-income countries, our understanding of retinoblastoma and its specific genetics still needs further clarification in order to predict the course of this disease and improve therapy. One such approach is the tumor free DNA that can be obtained from the anterior segment of the eye and be useful in diagnostics and prognostics.

Wnt Signaling Inhibitors and Their Promising Role in Tumor Treatment.

In a continuous search for the improvement of antitumor therapies, the inhibition of the Wnt signaling pathway has been recognized as a promising target. The altered functioning of the Wnt signaling in human tumors points to the strategy of the inhibition of its activity that would impact the clinical outcomes and survival of patients. Because the Wnt pathway is often mutated or epigenetically altered in tumors, which promotes its activation, inhibitors of Wnt signaling are being intensively investigated. It has been shown that knocking down specific components of the Wnt pathway has inhibitory effects on tumor growth in vivo and in vitro. Thus, similar effects are expected from the application of Wnt inhibitors. In the last decades, molecules acting as inhibitors on the pathway's specific molecular levels have been identified and characterized. This review will discuss the inhibitors of the canonical Wnt pathway, summarize knowledge on their effectiveness as therapeutics, and debate their side effects. The role of the components frequently mutated in various tumors that are principal targets for Wnt inhibitors is also going to be brought to the reader's attention. Some of the molecules identified as Wnt pathway inhibitors have reached early stages of clinical trials, and some have only just been discovered. All things considered, inhibition of the Wnt signaling pathway shows potential for the development of future therapies.

Bilateral Meningioma: A Case Report and Review of the Literature.

Here, we present a rarely seen example of bilateral meningiomas exhibiting different malignancy grades, I (meningothelial) and II (atypical), recorded in a 72-year-old patient. The presence of two separated lesions of different grades in a single patient can elucidate meningioma progression. To this end, the involvement of specific protein markers of epithelial to mesenchymal transition (EMT), the process responsible for progression, was tested in both tumors. Protein expression status of specific epithelial (E-cadherin) and mesenchymal markers (N-cadherin, SNAIL&SLUG and TWIST1) was investigated. Furthermore, markers that are connected to Wnt signaling pathway-beta-catenin, GSK3beta and DVL1-were also analyzed. For signs of neurofibromatosis and schwanomatosis genetic testing was performed. Immunohistochemistry evaluated by immunoreactivity score (IRS) was used to determine the signal strengths and proteins' location. Our results indicated that, in comparison to the grade I tumor, mesenchymal markers SNAIL and SLUG were upregulated in the atypical meningioma. TWIST1, beta-catenin and GSK3beta were upregulated in both grades, while E-cadherin was partially lost. A pronounced cadherin switch could not be established; however, N-cadherin showed widespread tissue presence. Genetic testing did not detect changes of NF2 or SMARCB1 genes denying germline origin of the lesions. The rare presence of two different grades in one patient elucidate previously unknown molecules involved in meningioma progression.

Decoding the Role of DVL1 in Intracranial Meningioma.

In the search for molecular candidates for targeted meningioma therapies, increasing attention has been paid to the role of signaling pathways in the development and progression of intracranial meningiomas. Although it is well known that the Wnt signaling pathway is involved in meningioma progression, the role of its central mediator, DVL1, is still unclear. In order to investigate the influence of DVL1 gene alterations on the progression of human intracranial meningioma, we focused on its central PDZ domain, which is responsible for DVL interaction with the Fzd receptor and the phosphorylation of DVL mediated through the casein kinases CK1 and CK2. A genetic analysis of genomic instability revealed the existence of microsatellite instability in 9.09% and the loss of heterozygosity in 6.06% of the samples. The sequencing of the PDZ gene region showed repetitive deletions of two bases located in intron 7 and exon 8, and a duplication in intron 8 in most samples, with different outcomes on the biological function of the DVL1 protein. Immunohistochemistry revealed that the nuclear expression of DVL1 was significantly correlated with a higher expression of active ß-catenin (p = 0.029) and a higher meningioma grade (p = 0.030), which leads to the conclusion that it could be used as biomarker for meningioma progression and the activation of the Wnt signaling pathway.

Integrative cBioPortal Analysis Revealed Molecular Mechanisms That Regulate EGFR-PI3K-AKT-mTOR Pathway in Diffuse Gliomas of the Brain.

Diffuse gliomas are a heterogeneous group of tumors with aggressive biological behavior and a lack of effective treatment methods. Despite new molecular findings, the differences between pathohistological types still require better understanding. In this in silico analysis, we investigated AKT1, AKT2, AKT3, CHUK, GSK3ß, EGFR, PTEN, and PIK3AP1 as participants of EGFR-PI3K-AKT-mTOR signaling using data from the publicly available cBioPortal platform. Integrative large-scale analyses investigated changes in copy number aberrations (CNA), methylation, mRNA transcription and protein expression within 751 samples of diffuse astrocytomas, anaplastic astrocytomas and glioblastomas. The study showed a significant percentage of CNA in PTEN (76%), PIK3AP1 and CHUK (75% each), EGFR (74%), AKT2 (39%), AKT1 (32%), AKT3 (19%) and GSK3ß (18%) in the total sample. Comprehensive statistical analyses show how genomics and epigenomics affect the expression of examined genes differently across various pathohistological types and grades, suggesting that genes AKT3, CHUK and PTEN behave like tumor suppressors, while AKT1, AKT2, EGFR, and PIK3AP1 show oncogenic behavior and are involved in enhanced activity of the EGFR-PI3K-AKT-mTOR signaling pathway. Our findings contribute to the knowledge of the molecular differences between pathohistological types and ultimately offer the possibility of new treatment targets and personalized therapies in patients with diffuse gliomas.

Methylation Patterns of DKK1, DKK3 and GSK3ß Are Accompanied with Different Expression Levels in Human Astrocytoma.

In the present study, we investigated genetic and epigenetic changes and protein expression levels of negative regulators of Wnt signaling, DKK1, DKK3, and APC as well as glycogen synthase kinase 3 (GSK3ß) and ß-catenin in 64 human astrocytomas of grades II-IV. Methylation-specific PCR revealed promoter methylation of DKK1, DKK3, and GSK3ß in 38%, 43%, and 18% of samples, respectively. Grade IV comprised the lowest number of methylated GSK3ß cases and highest of DKK3. Evaluation of the immunostaining using H-score was performed for ß-catenin, both total and unphosphorylated (active) forms. Additionally, active (pY216) and inactive (pS9) forms of GSK3ß protein were also analyzed. Spearman's correlation confirmed the prevalence of ß-catenin's active form (rs = 0.634, p < 0.001) in astrocytoma tumor cells. The Wilcoxon test revealed that astrocytoma with higher levels of the active pGSK3ß-Y216 form had lower expression levels of its inactive form (p < 0.0001, Z = -5.332). Changes in APC's exon 11 were observed in 44.44% of samples by PCR/RFLP. Astrocytomas with changes of APC had higher H-score values of total ß-catenin compared to the group without genetic changes (t = -2.264, p = 0.038). Furthermore, a positive correlation between samples with methylated DKK3 promoter and the expression of active pGSK3ß-Y216 (rs = 0.356, p = 0.011) was established. Our results emphasize the importance of methylation for the regulation of Wnt signaling. Large deletions of the APC gene associated with increased ß-catenin levels, together with oncogenic effects of both ß-catenin and GSK3ß, are clearly involved in astrocytoma evolution. Our findings contribute to a better understanding of the etiology of gliomas. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations.

Are We Benign? What Can Wnt Signaling Pathway and Epithelial to Mesenchymal Transition Tell Us about Intracranial Meningioma Progression.

Epithelial to mesenchymal transition (EMT), which is characterized by the reduced expression of E-cadherin and increased expression of N-cadherin, plays an important role in the tumor invasion and metastasis. Classical Wnt signaling pathway has a tight link with EMT and it has been shown that nuclear translocation of ß-catenin can induce EMT. This research has showed that genes that are involved in cadherin switch, CDH1 and CDH2, play a role in meningioma progression. Increased N-cadherin expression in relation to E-cadherin was recorded. In meningioma, transcription factors SNAIL, SLUG, and TWIST1 demonstrated strong expression in relation to E- and N-cadherin. The expression of SNAIL and SLUG was significantly associated with higher grades (p = 0.001), indicating their role in meningioma progression. Higher grades also recorded an increased expression of total ß-catenin followed by an increased expression of its active form (p = 0.000). This research brings the results of genetic and protein analyzes of important molecules that are involved in Wnt and EMT signaling pathways and reveals their role in intracranial meningioma. The results of this study offer guidelines and new markers of progression for future research and reveal new molecular targets of therapeutic interventions.

Mismatch Repair Pathway, Genome Stability and Cancer.

The acquisition of genomic instability is one of the key characteristics of the cancer cell, and microsatellite instability (MSI) is an important segment of this phenomenon. This review aims to describe the mismatch DNA repair (MMR) system whose deficiency is responsible for MSI and discuss the cellular roles of MMR genes. Malfunctioning of the MMR repair pathway increases the mutational burden of specific cancers and is often involved in its etiology, sometimes as an influential bystander and sometimes as the main driving force. Detecting the presence of MSI has for a long time been an important part of clinical diagnostics, but has still not achieved its full potential. The MSI blueprints of specific tumors are useful for precize grading, evaluation of cancer chance and prognosis and to help us understand how and why therapy-resistant cancers arise. Furthermore, evidence indicates that MSI is an important predictive biomarker for the application of immunotherapy.

Nucleotide variations of TP53 exon 4 found in intracranial meningioma and in silico prediction of their significance.

The aim of the present study was to identify TP53 exon 4 mutations in patients with meningioma and to investigate their potential association with specific tumor pathology. Nucleotide alterations were investigated in 48 meningiomas via the direct sequencing of TP53 exon 4 in patient tumor and blood samples using the DNA Sanger method with the BigDyeTerminator v3.1 Cycle Sequencing kit and Applied Biosystems 3730XL apparatus. The results revealed that TP53 exon 4 was frequently altered in meningioma, occurring in 60.4% of the patients investigated. A total of 18 different alterations were detected in the meningioma samples assessed in the current study. The majority of these appeared more than once and some were repeatedly identified in several patients. Changes at codons 72 (c.215G>C) and 62 (c.186delA) were highly prevalent, occurring in 44.8% of patients. Other changes detected via frequency analysis included: Five substitutions on codon 105 (c.315C>T); four insertions on codon 70 (c.209_210insG); three insertions on codon 64 (c.190C>G), 82 (245C>T; 245delC; 243_244insA) and 104 (c.312G>A); and two insertions on codons 108 (c.322G>C), 71 (c.213C>A), 73 (c.217G>A), 91 (c.271T>C) and 100 (c.300G>T). Codons 68 (c.202_203insT), 77 (c.229C>T), 88 (c.263C>G) and 92 (c.276C>A) were altered once. Alterations on codons 82, 91, 108, 104, 105, 70 and 92 were characterized as possibly damaging by PolyPhen-2 and Mutation Taster2 tools. The current study also demonstrated that nucleotide alterations were significantly associated with the loss of p53 expression (P=0.04) and female patients (P=0.049), particularly codon 72. The results present novel data on the mutational spectrum of TP53 in meningeal brain tumors.

Comparable Genomic Copy Number Aberrations Differ across Astrocytoma Malignancy Grades.

A collection of intracranial astrocytomas of different malignancy grades was analyzed for copy number aberrations (CNA) in order to identify regions that are driving cancer pathogenesis. Astrocytomas were analyzed by Array Comparative Genomic Hybridization (aCGH) and bioinformatics utilizing a Bioconductor package, Genomic Identification of Significant Targets in Cancer (GISTIC) 2.0.23 and DAVID software. Altogether, 1438 CNA were found of which losses prevailed. On our total sample, significant deletions affected 14 chromosomal regions, out of which deletions at 17p13.2, 9p21.3, 13q12.11, 22q12.3 remained significant even at 0.05 q-value. When divided into malignancy groups, the regions identified as significantly deleted in high grades were: 9p21.3; 17p13.2; 10q24.2; 14q21.3; 1p36.11 and 13q12.11, while amplified were: 3q28; 12q13.3 and 21q22.3. Low grades comprised significant deletions at 3p14.3; 11p15.4; 15q15.1; 16q22.1; 20q11.22 and 22q12.3 indicating their involvement in early stages of tumorigenesis. Significantly enriched pathways were: PI3K-Akt, Cytokine-cytokine receptor, the nucleotide-binding oligomerization domain (NOD)⁻like receptor, Jak-STAT, retinoic acid-inducible gene (RIG)-I-like receptor and Toll-like receptor pathways. HPV and herpex simplex infection and inflammation pathways were also represented. The present study brings new data to astrocytoma research amplifying the wide spectrum of changes that could help us identify the regions critical for tumorigenesis.

Different behaviour of DVL1, DVL2, DVL3 in astrocytoma malignancy grades and their association to TCF1 and LEF1 upregulation.

Key regulators of the Wnt signalling, DVL1, DVL2 and DVL3, in astrocytomas of different malignancy grades were investigated. Markers for DVL1, DVL2 and DVL3 were used to detect microsatellite instability (MSI) and gross deletions (LOH), while immunohistochemistry and immunoreactivity score were used to determine the signal strengths of the three DVL proteins and transcription factors of the pathway, TCF1 and LEF1. Our findings demonstrated that MSI at all three DVL loci was constantly found across tumour grades with the highest number in grade II (P = 0.008). Collectively, LOHs were more frequent in high-grade tumours than in low grade ones. LOHs of DVL3 gene were significantly associated with grade IV tumours (P = 0.007). The results on protein expressions indicated that high-grade tumours expressed less DVL1 protein as compared with low grade ones. A significant negative correlation was established between DVL1 expression and malignancy grades (P < 0.001). The expression of DVL2 protein was found similar across grades, while DVL3 expression significantly increased with malignancy grades (P < 0.001). The signal strengths of expressed DVL1 and DVL3 were negatively correlated (P = 0.002). However, TCF1 and LEF1 were both significantly upregulated and increasing with astrocytoma grades (P = 0.001). A positive correlation was established between DVL3 and both TCF1 (P = 0.020) and LEF1 (P = 0.006) suggesting their joint involvement in malignant progression. Our findings suggest that DVL1 and DVL2 may be involved during early stages of the disease, while DVL3 may have a role in later phases and together with TCF1 and LEF1 promotes the activation of Wnt signalling.

Hypermethylation of Secreted Frizzled Related Protein 1 gene promoter in different astrocytoma grades.

AIM: To identify the involvement of Secreted Frizzled Related Protein 1 (SFRP1) promoter hypermethylation in different malignancy grades of astrocytoma and assess its association with beta-catenin, lymphoid-enhancer factor 1, and T-cell factor 1. METHODS: Twenty-six astrocytoma samples were collected from 2008-2015. Promoter hypermethylation was evaluated by methylation-specific polymerase-chain-reaction and protein expression by immunohistochemistry and stereological analysis. The staining intensity was scored by comparing immunoreactivity with normal tissue and by using 10% and 50% cut-offs. RESULTS: SFRP1 promoter methylation was found in 32% of astrocytomas. The number of hypermethylated samples increased in higher astrocytoma grades and was the highest in glioblastoma (P=0.042 compared to other astrocytoma grades). There was 45.8% of samples with the lack of or weak expression of SFRP1 protein and 29.2% with strong expression. Samples with methylated promoter expressed significantly less SFRP1 than samples with unmethylated promoter (P=0.031). Beta-catenin expression levels were elevated. Yet, glioblastomas with unmethylated SFRP1 promoter had significantly less beta-catenin (P=0.033). Strong expression of lymphoid-enhancer factor 1 was associated to higher astrocytoma grades (P=0.006). CONCLUSION: SFRP1 gene was epigenetically silenced in glioblastomas when compared to low astrocytoma grades, which may suggest that the lack of its protein is involved in astrocytoma 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.