Glioblastoma epigenomics discloses a complex biology and potential therapeutic targets.

Background and purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

ß-Adrenoreceptors in Human Cancers.

Cancer is the leading cause of death and represents a significant economic burden worldwide. The numbers are constantly growing as a result of increasing life expectancy, toxic environmental factors, and adoption of Western lifestyle. Among lifestyle factors, stress and the related signaling pathways have recently been implicated in the development of tumors. Here we present some epidemiological and preclinical data concerning stress-related activation of the ß-adrenoreceptors (ß-ARs), which contributes to the formation, sequential transformation, and migration of different tumor cell types. We focused our survey on research results for breast and lung cancer, melanoma, and gliomas published in the past five years. Based on the converging evidence, we present a conceptual framework of how cancer cells hijack a physiological mechanism involving ß-ARs toward a positive modulation of their own survival. In addition, we also highlight the potential contribution of ß-AR activation to tumorigenesis and metastasis formation. Finally, we outline the antitumor effects of targeting the ß-adrenergic signaling pathways, methods for which primarily include repurposed ß-blocker drugs. However, we also call attention to the emerging (though as yet largely explorative) method of chemogenetics, which has a great potential in suppressing tumor growth either by selectively modulating neuronal cell groups involved in stress responses affecting cancer cells or by directly manipulating specific (e.g., the ß-AR) receptors on a tumor and its microenvironment.

Epigenetic Suppression of the IL-7 Pathway in Progressive Glioblastoma.

BACKGROUND: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. METHODS: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. RESULTS: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. CONCLUSIONS: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target.

Wnt pathway markers in low-grade and high-grade gliomas.

BACKGROUND AND PURPOSE: Aberrant activation of the Wnt pathway contributes to differentiation and maintenance of cancer stem cells underlying gliomagenesis. The aim of our research was to determine as to what degrees some Wnt markers are expressed in gliomas of different grades, lineages and molecular subtypes. METHODS: Nine grade II, 10 grade III and 72 grade IV surgically removed, formalin-fixed paraffin-embedded glioma specimens were included. Mutation status of IDH1 codon 132 was defined by immunohistochemistry and pyrosequencing in all tumors. Grade II and III astrocytic and oligodendroglial tumors were further tested for the expression of p53 and ATRX by immunohistochemistry, and codeletion of 1p19q by fluorescent in situ hybridization. Expression levels of the non-canonical Wnt5a and Fzd2, and the canonical Wnt3a and beta-catenin Wnt pathway markers were determined by immunohistochemistry, and compared between subgroups stratified according to grade, lineage and the presence or absence of IDH1 R132H/C mutations. RESULTS: In the normal brain - grade II-IV glioma comparisons, a gradual increase was observed for the expressions of Wnt5a, Wnt3a, Fzd2 and beta-catenin. In the astroglial and oligodendroglial lineages of grade II and III gliomas, only the Wnt5a expression was significantly higher in the astroglial subgroup. Stratification according to the IDH1 status resulted in a significant increase of the Wnt3 expression in the wild type grade II-IV gliomas. CONCLUSION: These data extend previous observations and show a correlation of Wnt pathway activity with glioma grade. Further investigations of the Wnt marker expression regulation according to glioma lineage or IDH gene mutational status are in progress by using more exact molecular approaches.

Involvement of the Catecholamine Pathway in Glioblastoma Development.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor-ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2-ADRBK1/GRK2; dopamine receptor D2-DRD2; and synaptic vesicle monoamine transporter-SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary-recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

Neuroscience highlights: The mirror inside our brain.

Over the second half of the 19th century, numerous theories arose concerning mechanisms involved in understanding of action, imitative learning, language development and theory of mind. These explorations gained new momentum with the discovery of the so called "mirror neurons". Rizzolatti's work inspired large groups of scientists seeking explanation in a new and hitherto unexplored area of how we perceive and understand the actions and intentions of others, how we learn through imitation to help our own survival, and what mechanisms have helped us to develop a unique human trait, language. Numerous studies have addressed these questions over the years, gathering information about mirror neurons themselves, their subtypes, the different brain areas involved in the mirror neuron system, their role in the above mentioned mechanisms, and the varying consequences of their dysfunction in human life. In this short review, we summarize the most important theories and discoveries that argue for the existence of the mirror neuron system, and its essential function in normal human life or some pathological conditions.

Neuroscience highlights: Main cell types underlying memory and spatial navigation.

Interest in the hippocampal formation and its role in navigation and memory arose in the second part of the 20th century, at least in part due to the curious case of Henry G. Molaison, who underwent brain surgery for intractable epilepsy. The temporal association observed between the removal of his entorhinal cortex along with a significant part of hippocampus and the developing severe memory deficit inspired scientists to focus on these regions. The subsequent discovery of the so-called place cells in the hippocampus launched the description of many other functional cell types and neuronal networks throughout the Papez-circuit that has a key role in memory processes and spatial information coding (speed, head direction, border, grid, object-vector etc). Each of these cell types has its own unique characteristics, and together they form the so-called "Brain GPS". The aim of this short survey is to highlight for practicing neurologists the types of cells and neuronal networks that represent the anatomical substrates and physiological correlates of pathological entities affecting the limbic system, especially in the temporal lobe. For that purpose, we survey early discoveries along with the most relevant neuroscience observations from the recent literature. By this brief survey, we highlight main cell types in the hippocampal formation, and describe their roles in spatial navigation and memory processes. In recent decades, an array of new and functionally unique neuron types has been recognized in the hippocampal formation, but likely more remain to be discovered. For a better understanding of the heterogeneous presentations of neurological disorders affecting this anatomical region, insights into the constantly evolving neuroscience behind may be helpful. The public health consequences of diseases that affect memory and spatial navigation are high, and grow as the population ages, prompting scientist to focus on further exploring this brain region.

DNA CpG methylation in sequential glioblastoma specimens.

PURPOSE: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. METHODS: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. RESULTS: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. CONCLUSION: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets.

Longitudinal Characteristics of Glioblastoma in Genome-Wide Studies.

Glioblastoma is one of the deadliest tumors with barely over one-year median survival despite intensive efforts in defining its molecular characteristics and searching for innovative treatment strategies. While major progress has been made in cataloging cross-sectional genomic, transcriptomic and epigenomic features of the tumor, and inferring its main molecular pathways and niches for potential targeted intervention, we still do not have sufficient knowledge concerning evolutionary patterns and dynamics of molecular changes or the treatment-induced effects affecting glioblastoma biology. In this review, we summarize the results of recent longitudinal genomic, transcriptomic and epigenomic studies that brought us closer to a better understanding of this lethal disease. Evidence suggests that neuronal / glioma stem cells with accumulating mutations initiate glioblastoma development and recurrence, but the hypothetical models describing the courses that lead to established tumors have not been fully proven. Moving from the histopathological phenotype to the results of high resolution OMICS studies, we try to synthesize the currently available information from sequential glioblastoma analyses in order to highlight its multifaceted features and heterogenetity, as well as the expected complexity of potential treatment strategies that might once succeed.