Roberto Ferrarese

NFKBIA Deletion in Glioblastomas

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

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.

Integrative Modeling Reveals Annexin A2-mediated Epigenetic Control of Mesenchymal Glioblastoma

Glioblastomas are characterized by transcriptionally distinct subtypes, but despite possible clinical relevance, their regulation remains poorly understood. The commonly used molecular classification systems for GBM all identify a subtype with high expression of mesenchymal marker transcripts, strongly associated with invasive growth. We used a comprehensive data-driven network modeling technique (augmented sparse inverse covariance selection, aSICS) to define separate genomic, epigenetic, and transcriptional regulators of glioblastoma subtypes. Our model identified Annexin A2 (ANXA2) as a novel methylation-controlled positive regulator of the mesenchymal subtype. Subsequent evaluation in two independent cohorts established ANXA2 expression as a prognostic factor that is dependent on ANXA2 promoter methylation. ANXA2 knockdown in primary glioblastoma stem cell-like cultures suppressed known mesenchymal master regulators, and abrogated cell proliferation and invasion. Our results place ANXA2 at the apex of a regulatory cascade that determines glioblastoma mesenchymal transformation and validate aSICS as a general methodology to uncover regulators of cancer subtypes.

c-Jun-N-terminal phosphorylation regulates DNMT1 expression and genome wide methylation in gliomas.

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.

Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression

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.

The aberrant splicing of BAF45d links splicing regulation and transcription in glioblastoma

Background Glioblastoma, the most aggressive primary brain tumor, is genetically heterogeneous. Alternative splicing (AS) plays a key role in numerous pathologies, including cancer. The objectives of our study were to determine whether aberrant AS could play a role in the malignant phenotype of glioma and to understand the mechanism underlying its aberrant regulation. Methods We obtained surgical samples from patients with glioblastoma who underwent 5-aminolevulinic fluorescence-guided surgery. Biopsies were taken from the tumor center as well as from adjacent normal-appearing tissue. We used a global splicing array to identify candidate genes aberrantly spliced in these glioblastoma samples. Mechanistic and functional studies were performed to elucidate the role of our top candidate splice variant, BAF45d, in glioblastoma. Results BAF45d is part of the switch/sucrose nonfermentable complex and plays a key role in the development of the CNS. The BAF45d/6A isoform is present in 85% of over 200 glioma samples that have been analyzed and contributes to the malignant glioma phenotype through the maintenance of an undifferentiated cellular state. We demonstrate that BAF45d splicing is mediated by polypyrimidine tract-binding protein 1 (PTBP1) and that BAF45d regulates PTBP1, uncovering a reciprocal interplay between RNA splicing regulation and transcription. Conclusions Our data indicate that AS is a mechanism that contributes to the malignant phenotype of glioblastoma. Understanding the consequences of this biological process will uncover new therapeutic targets for this devastating disease.

Abstract 3202: PTBP1-mediated aberrant splicing of a brain-specific exon in ANXA7 promotes brain tumorigenesis.

Glioblastoma multiforme (GBM) is the most common and malignant type of brain tumor in adults and is nearly uniformly fatal, with a median survival of one year. Diffuse infiltration, extensive neo-angiogenesis, uncontrolled cellular proliferation, presence of necrotic regions and resistance to apoptosis are all hallmark features of these tumors. The complex nature of GBMs is worsened by an incomplete understanding of its cellular origin and of the molecular determinants of its aggressiveness. This has contributed to make this cancer one of the most difficult to treat. Alternative splicing of pre-mRNA is an important mechanism to increase the diversity of protein functions; its dysregulation leads to the aberrant expression of specific protein isoforms, often associated with cancer. A relatively small number of splicing factors regulates the production of different splice variants of many proteins. In cancer, alterations in the expression of few splicing factors may lead to the aberrant splicing of several targets including oncogenes and tumor suppressors, which may contribute to the transformed cellular phenotype. Here, we investigate the tumorigenic role of the heterogeneous nuclear ribonucleoprotein PTBP1, a splicing factor commonly overexpressed in GBM. In the present study we used RNA immunoprecipitation and Exon Trap assay to describe how PTBP1 drives the aberrant splicing of a brain-specific exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7). By differential overexpression of ANXA7 splice variants in tumor cell lines, we showed that expression of ANXA7 variant B, normally expressed in brain, controls EGFR pathway; conversely, the tumor variant ANXA7-vA does not stop EGFR activation cascade. Immunostaining experiments demonstrated that, compared to ANXA7-vB, ANXA7-vA reduces the endosomal targeting of EGFR and prolonging the activation of its signaling pathway. Knocking down PTBP1 in tumor cell lines restored the expression of ANXA7-vB and mimicked the effect of ANXA7-vB overexpression on EGFR trafficking and pathway activation. Furthermore, we performed in vivo experiments to show that PTBP1 increased expression has a general effect on tumor aggressiveness as it drives angiogenesis and promotes invasiveness. Finally, we assessed the association of PTBP1 expression and clinical outcome and we found that patients with high PTBP1 expression had a significantly worse survival rate. Our study highlights the important role the splicing factors play in cancer. In fact, the mechanism we describe here could represent a common paradigm of malignant transformation which could possibly lead to the development of new therapeutic strategies for GBM treatment. Citation Format: Roberto Ferrarese, Eva Bug, Daniel Maticzka, Wilfried Reichardt, Maria Stella Carro, Markus Bredel. PTBP1-mediated aberrant splicing of a brain-specific exon in ANXA7 promotes brain tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3202. doi:10.1158/1538-7445.AM2013-3202