Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study.

PURPOSE: Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay. METHODS: It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases. RESULTS: The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ([Formula: see text] + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated. CONCLUSION: We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity.

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.

Unconventional Secretion of Heat Shock Proteins in Cancer.

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies.

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrP(C) interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrP(C)-STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrP(C)-STI1 binding is corrupted in neuronal cell lines persistently infected with PrP(Sc), as well as in primary cultured hippocampal neurons acutely exposed to PrP(Sc). Consistent with this, high levels of eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation were found in PrP(Sc)-infected cells and in neurons acutely exposed to PrP(Sc). These data indicate that modulation of protein synthesis is critical for PrP(C)-STI1 neurotrophic functions, and point to the impairment of this process during PrP(Sc) infection as a possible contributor to neurodegeneration.

High frequency of WNT-activated medulloblastomas with CTNNB1 wild type suggests a higher proportion of hereditary cases in a Latin-Iberian population.

Purpose: Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%-90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics. Methods: A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively. Results: WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants. Conclusion: We observed a lower incidence of CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.

Genome-wide translation patterns in gliomas: An integrative view.

Gliomas are the most frequent tumors of the central nervous system (CNS) and include the highly malignant glioblastoma (GBM). Characteristically, gliomas have translational control deregulation related to overactivation of signaling pathways such as PI3K/AKT/mTORC1 and Ras/ERK1/2. Thus, mRNA translation appears to play a dominant role in glioma gene expression patterns. The, analysis of genome-wide translated transcripts, together known as the translatome, may reveal important information for understanding gene expression patterns in gliomas. This review provides a brief overview of translational control mechanisms altered in gliomas with a focus on the current knowledge related to the translatomes of glioma cells and murine glioma models. We present an integrative meta-analysis of selected glioma translatome data with the aim of identifying recurrent patterns of gene expression preferentially regulated at the level of translation and obtaining clues regarding the pathological significance of these alterations. Re-analysis of several translatome datasets was performed to compare the translatomes of glioma models with those of their non-tumor counterparts and to document glioma cell responses to radiotherapy and MNK modulation. The role of recurrently altered genes in the context of translational control and tumorigenesis are discussed.

Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer.

Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.

Loss of 5'-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness.

The 5'-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients' clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients' clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP's role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.

Overexpression of mTOR and p(240-244)S6 in IDH1 Wild-Type Human Glioblastomas Is Predictive of Low Survival.

PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.

Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells.

Translational control by eIF2α in neurons: Beyond the stress response.

The translation of mRNAs is a tightly controlled process that responds to multiple signaling pathways. In neurons, this control is also exerted locally due to the differential necessity of proteins in axons and dendrites. The phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) is one of the mechanisms of translational control. The phosphorylation of eIF2α has classically been viewed as a stress response, halting translation initiation. However, in the nervous system this type of regulation has been related to other mechanisms besides stress response, such as behavior, memory consolidation and nervous system development. Additionally, neurodegenerative diseases have a major stress component, thus eIF2α phosphorylation plays a preeminent role and its modulation is currently viewed as a new opportunity for therapeutic interventions. This review consolidates current information regarding eIF2α phosphorylation in neurons and its impact in neurodegenerative diseases. © 2016 Wiley Periodicals, Inc.

Stress-Inducible Protein 1 (STI1): Extracellular Vesicle Analysis and Quantification.

This chapter is derived from our experience in the study of stress-Inducible Protein 1 (STI1) in extracellular vesicles. We used different techniques to isolate, explore, and characterize the extracellular vesicles that contained this protein. Ultracentrifugation and gel chromatography were used to isolate extracellular vesicles of different sizes, nanotracking particle analysis (NTA) determined number and size of vesicles, while flow cytometry and ELISA were used to determine the specific protein content of vesicles.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.

A proteína prion celular e seus ligantes - vitronectina, STI1 e laminina - nos mecanismos de plasticidade neuronal / The cellular prion protein and its ligants - vitronectin, STI1 and laminin - in the mechanisms of neuronal plasticity

Prions são agentes etiológicos das encefalopatias espongiformes transmissíveis, doenças que acometem tanto homens quanto animais. A proteína infecciosa, PrPsc, é uma isoforma de uma proteína celular normal denominada PrPc. As funções de PrPc ainda causam controvérsia na literatura, mas já foi demonstrada a participação de PrPc em uma variedade de fenômenos biológicos, como homeostase de íons cobre, proteção contra estresse oxidativo, sinalização celular e neuritogênese entre outros. A interação de PrPc com laminina, uma proteína de matriz extracelular, leva a formação e manutenção de neuritos em neurônios hipocampais. Seguindo este caminho, demonstramos no presente trabalho a interação de PrPc com outra proteína de matriz extracelular, vitronectina (Vn)...