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Glioblastoma (GBM) comprises distinct subtypes characterized by their molecular profile. Mesenchymal identity in GBM has been associated with a comparatively unfavorable prognosis, primarily due to inherent resistance of these tumors to current therapies. The identification of molecular determinants of mesenchymal transformation could potentially allow for the discovery of new therapeutic targets. Zinc Finger and BTB Domain Containing 18 (ZBTB18/ZNF238/RP58) is a zinc finger transcriptional repressor with a crucial role in brain development and neuronal differentiation. Here, ZBTB18 is primarily silenced in the mesenchymal subtype of GBM through aberrant promoter methylation. Loss of ZBTB18 contributes to the aggressive phenotype of glioblastoma through regulation of poor prognosis–associated signatures. Restitution of ZBTB18 expression reverses the phenotype and impairs tumor-forming ability. These results indicate that ZBTB18 functions as a tumor suppressor in GBM through the regulation of genes associated with phenotypically aggressive properties. Implications: This study characterizes the role of the putative tumor suppressor ZBTB18 and its regulation by promoter hypermethylation, which appears to be a common mechanism to silence ZBTB18 in the mesenchymal subtype of GBM and provides a new mechanistic opportunity to specifically target this tumor subclass. Mol Cancer Res; 15(8); 998–1011. ©2017 AACR.

// Dieter H. Heiland 1, 2 , Roberto Ferrarese 1, 2 , Rainer Claus 3 , Fangping Dai 1, 2 , Anie P. Masilamani 1, 2 , Eva Kling 1, 2 , Astrid Weyerbrock 1, 2 , Teresia Kling 4 , Sven Nelander 4 , Maria S. Carro 1, 2 1 Department of Neurosurgery, Medical Center – University of Freiburg, Freiburg, Germany 2 Faculty of Medicine, University of Freiburg, Freiburg, Germany 3 Department of Hematology, Oncology, and Stem Cell Transplantation, University of Freiburg Medical Center, Freiburg, Germany 4 Department of Immunology, Genetics and Pathology and Science for Life Laboratories, University of Uppsala, Uppsala, Sweden Correspondence to: Maria S. Carro, email: maria.carro@uniklinik-freiburg.de Keywords: Glioblastoma, G-CIMP, DNMT1, p-c-Jun, mesenchymal Received: June 11, 2016 Accepted: December 15, 2016 Published: December 28, 2016 ABSTRACT High-grade gliomas (HGG) are the most common brain tumors, with an average survival time of 14 months. A glioma-CpG island methylator phenotype (G-CIMP), associated with better clinical outcome, has been described in low and high-grade gliomas. Mutation of IDH1 is known to drive the G-CIMP status. In some cases, however, the hypermethylation phenotype is independent of IDH1 mutation, suggesting the involvement of other mechanisms. Here, we demonstrate that DNMT1 expression is higher in low-grade gliomas compared to glioblastomas and correlates with phosphorylated c-Jun. We show that phospho-c-Jun binds to the DNMT1 promoter and causes DNA hypermethylation. Phospho-c-Jun activation by Anisomycin treatment in primary glioblastoma-derived cells attenuates the aggressive features of mesenchymal glioblastomas and leads to promoter methylation and downregulation of key mesenchymal genes (CD44, MMP9 and CHI3L1). Our findings suggest that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in Glioblastoma.

Dysregulation of the NF-κB transcription factor occurs in many cancer types. Krüppel-like family of transcription factors (KLFs) regulate the expression of genes involved in cell proliferation, differentiation and survival. Here, we report a new mechanism of NF-κB activation in glioblastoma through depletion of the KLF6 tumor suppressor. We show that KLF6 transactivates multiple genes negatively controlling the NF-κB pathway and consequently reduces NF-κB nuclear localization and downregulates NF-κB targets. Reconstitution of KLF6 attenuates their malignant phenotype and induces neural-like differentiation and senescence, consistent with NF-κB pathway inhibition. KLF6 is heterozygously deleted in 74.5% of the analyzed glioblastomas and predicts unfavorable patient prognosis suggesting that haploinsufficiency is a clinically relevant means of evading KLF6-dependent regulation of NF-κB. Together, our study identifies a new mechanism by which KLF6 regulates NF-κB signaling, and how this mechanism is circumvented in glioblastoma through KLF6 loss.

Amplification and activating mutations of the epidermal growth factor receptor (EGFR) oncogene are molecular hallmarks of glioblastomas. We hypothesized that deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR.We analyzed 790 human glioblastomas for deletions, mutations, or expression of NFKBIA and EGFR. We studied the tumor-suppressor activity of NFKBIA in tumor-cell culture. We compared the molecular results with the outcome of glioblastoma in 570 affected persons.NFKBIA is often deleted but not mutated in glioblastomas; most deletions occur in nonclassical subtypes of the disease. Deletion of NFKBIA and amplification of EGFR show a pattern of mutual exclusivity. Restoration of the expression of NFKBIA attenuated the malignant phenotype and increased the vulnerability to chemotherapy of cells cultured from tumors with NFKBIA deletion; it also reduced the viability of cells with EGFR amplification but not of cells with normal gene dosages of both NFKBIA and EGFR. Deletion and low expression of NFKBIA were associated with unfavorable outcomes. Patients who had tumors with NFKBIA deletion had outcomes that were similar to those in patients with tumors harboring EGFR amplification. These outcomes were poor as compared with the outcomes in patients with tumors that had normal gene dosages of NFKBIA and EGFR. A two-gene model that was based on expression of NFKBIA and O(6)-methylguanine DNA methyltransferase was strongly associated with the clinical course of the disease.Deletion of NFKBIA has an effect that is similar to the effect of EGFR amplification in the pathogenesis of glioblastoma and is associated with comparatively short survival.

Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect and exhibit great potential in clinical tumor therapy.

BACKGROUND: Tissue-specific alternative splicing is known to be critical to emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionary-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence activity in signaling pathways to profound biological effect. Given that tissue-specific splicing has a determinative role in brain development and the enrichment of genes containing tissue-specific exons for proteins with roles in signaling and development, it is thus plausible that changes in such exons could rewire normal neurogenesis towards malignant transformation. METHODS: We used integrated molecular genetic and cell biology analyses, computational biology, animal modeling, and clinical patient profiles to characterize the effect of aberrant splicing of a brain-enriched alternative exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7) on oncogene regulation and brain tumorigenesis. RESULTS: We show that aberrant splicing of a tissue-specific cassette exon in ANXA7 diminishes endosomal targeting and consequent termination of the signal of the EGFR oncoprotein during brain tumorigenesis. Splicing of this exon is mediated by the ribonucleoprotein Polypyrimidine Tract-Binding Protein 1 (PTBP1), which is normally repressed during brain development but, we find, is excessively expressed in glioblastomas through either gene amplification or loss of a neuron-specific microRNA, miR-124. Silencing of PTBP1 attenuates both malignancy and angiogenesis in a stem cell-derived glioblastoma animal model characterized by a high native propensity to generate tumor endothelium or vascular pericytes to support tumor growth. We show that EGFR amplification and PTBP1 overexpression portend a similarly poor clinical outcome, further highlighting the importance of PTBP1-mediated activation of EGFR. CONCLUSIONS: Our data illustrate how anomalous splicing of a tissue-regulated exon in a constituent of an oncogenic signaling pathway eliminates its tumor suppressor function and promotes tumorigenesis. This paradigm of malignant glial transformation as a consequence of tissue-specific alternative exon splicing in a tumor suppressor, may have widespread applicability in explaining how changes in critical tissue-specific regulatory mechanisms reprogram normal development to oncogenesis. SECONDARY CATEGORY: n/a.

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.

In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5′-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5′-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.

Acetylation is one of the key chromatin modifications that control gene transcription during embryonic development and tumorigenesis. The types of genes sensitive to such modifications in vivo are not known to date. We investigated the expression of a number of genes involved in embryonic development after treatment with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, in the limbs of chicken embryos. Our results show that TSA affects the expression profiles of some genes that play important roles during limb development. The expression of BMP4, SF/HGF and Twist1 increased, whereas the expression of BMP2, FGF8, Shh, Scleraxis, Myf5 and MyoD was decreased or even inhibited. In contrast, the expression of Pax3, Paraxis, Msx1, CREB, and PCNA was not affected. Our results indicate that the chicken embryo can serve as an effective in vivo model for studying the effect of HDAC inhibitors on gene expression and can be helpful for understanding the role of chromatin remodeling and epigenetic control of gene expression.

Purpose: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001 and investigated its effects on telomerase and telomeres. Experimental Design: We compared telomere length to KML001 cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species–related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. Results: We established a significant inverse correlation (r2 = 0.9) between telomere length and cytotoxicity. KML001 exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by γ-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. Conclusions: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.

Interference with telomerase and telomere maintenance is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single-stranded 3′ overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends. We report here the effects of a 3,6,9-trisubstituted acridine compound, BRACO-19, on telomerase function in vitro and in vivo. The biological activity of BRACO-19 was evaluated in the human uterus carcinoma cell line UXF1138L, which has very short telomeres (2.7 kb). In vitro, nuclear human telomerase reverse transcriptase (hTERT) expression was drastically decreased after 24 hours, induction of cellular senescence and complete cessation of growth was seen after 15 days, paralleled by telomere shortening of ca. 0.4 kb. In vivo, BRACO-19 was highly active as a single agent against early-stage (68 mm3) tumors in a s.c. growing xenograft model established from UXF1138L cells, if given chronically at 2 mg per kg per day i.p. BRACO-19 produced growth inhibition of 96% compared with controls accompanied by partial regressions (P < 0.018). Immunostaining of xenograft tissues showed that this response was paralleled by loss of nuclear hTERT protein expression and an increase in atypical mitoses indicative of telomere dysfunction. Cytoplasmic hTERT expression and its colocalization with ubiquitin was observed suggesting that hTERT is bound to ubiquitin and targeted for enhanced degradation upon BRACO-19 treatment. This is in accord with a model of induced displacement of telomerase from the telomere. The in vitro and in vivo data presented here is consistent with the G-quadruplex binding ligand BRACO-19 producing an anticancer effect by inhibiting the capping and catalytic functions of telomerase.

Although studies performed in the past decade have contributed to a better classification and characterization of GBM, the overall survival and outcome of GBM patients have not seen major improvement. The mesenchymal signature is described as the subtype of GBM with the worst prognosis, primarily due to inherent resistance of the tumors to contemporary therapies. The identification of molecular determinants of mesenchymal transformation could potentially allow for the discovery of new therapeutic targets. The Zing Finger And BTB Domain Containing 18 (ZBTB18; formerly ZNF238) is a transcriptional repressor with a crucial role in brain development and neuronal differentiation. Consistent with our previous study, which provided preliminary evidence of a role for ZBTB18 in a network of mesenchymal transformation in GBM, our new data have discovered epigenetic silencing, that is promoter methylation, as a mechanism to downregulate ZBTB18 in these tumors. We show that ZBTB18 functions as a master regulator of a mesenchymal gene signature in GBM and that loss of ZBTB18 contributes to the highly invasive mesenchymal phenotype. Conversely, re-expression of ZBTB18 reverses the phenotype and impairs tumor-forming ability. Overall, our results support a tumor suppressor role of ZBTB18 in the brain and identify promoter hypermethylation as a mechanism to silence ZBTB18 in the mesenchymal subtype of GBM, which provides a new mechanistic opportunity to specifically target this tumor subclass.

Tissue-specific alternative splicing is critical to the emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence signaling pathways to profound biological effect. In the brain, Annexin A7 isoform 1 (ANXA7-I1) is exclusively expressed in mature neurons, while isoform 2 (ANXA7-I2) in which exon 6 is skipped, is expressed in glial and progenitor cells. We show that lineage-specific splicing of the cassette exon 6 in the membrane-binding tumor suppressor ANXA7diminishes endosomal targeting and consequent signal termination of the EGFR oncoprotein during brain tumor progression. Splicing of this exon is mediated by Polypyrimidine Tract-Binding Protein 1 (PTBP1), a ribonucleoprotein normally repressed during neuronal development but which we found to be highly expressed also in glioblastomas through loss of a brain-enriched microRNA, miR-124, and gene amplification. Here, we show that the PTBP1-ANXA7 splicing-EGFR signal activation axis promotes in vitro cell migration and invasion, and tumor angiogenesis in vivo. In glioblastoma, ANXA7 splicing is likely inherited from a potential tumor-initiating ancestor but this trait is further exploited through accumulation of mutations that enhance EGFR signaling. Our data illustrate how lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates its tumor suppressor function and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can contribute to reprogramming normal development to oncogenesis.