Miguel Torres-Martín

Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (NRG1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan Low Density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Global profiling in vestibular schwannomas shows critical deregulation of microRNAs and upregulation in those included in chromosomal region 14q32

Background Vestibular schwannomas are benign tumors that arise from Schwann cells in the VIII cranial pair and usually present NF2 gene mutations and/or loss of heterozygosity on chromosome 22q. Deregulation has also been found in several genes, such as ERBB2 and NRG1. MicroRNAs are non-coding RNAs approximately 21 to 23 nucleotides in length that regulate mRNAs, usually by degradation at the post-transcriptional level. Methods We used microarray technology to test the deregulation of miRNAs and other non-coding RNAs present in GeneChip miRNA 1.0 (Affymetrix) over 16 vestibular schwannomas and 3 control-nerves, validating 10 of them by qRT-PCR. Findings Our results showed the deregulation of 174 miRNAs, including miR-10b, miR-206, miR-183 and miR-204, and the upregulation of miR-431, miR-221, miR-21 and miR-720, among others. The results also showed an aberrant expression of other non-coding RNAs. We also found a general upregulation of the miRNA cluster located at chromosome 14q32. Conclusion Our results suggest that several miRNAs are involved in tumor formation and/or maintenance and that global upregulation of the 14q32 chromosomal site contains miRNAs that may represent a therapeutic target for this neoplasm.

Genomic deletions at 1p and 14q are associated with an abnormal cDNA microarray gene expression pattern in meningiomas but not in schwannomas

The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5-69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas.

Homozygous deletion of TNFRSF4, TP73, PPAP2B and DPYD at 1p and PDCD5 at 19q identified by multiplex ligation-dependent probe amplification (MLPA) analysis in pediatric anaplastic glioma with questionable oligodendroglial component

BackgroundPediatric oligodendrogliomas are rare and appear to show a different molecular profile from adult tumors. Some gliomas display allelic losses at 1p/19q in pediatric patients, although less frequently than in adult patients, but this is rare in tumors with an oligodendroglial component. The molecular basis of this genomic abnormality is unknown in pediatric gliomas, but it represents a relatively common finding in pediatric oligodendroglioma-like neoplasms with leptomeningeal dissemination.ResultsMultiplex ligation-dependent probe amplification (MLPA) analysis using SALSA P088-B1 for the analysis of the 1p/19q allelic constitution in a pediatric anaplastic (oligodendro)-glioma showed homozygous co-deletion for markers: TNFRSF4 (located at 1p36.33), TP73 (1p36.32), PPAP2B (1pter-p22.1), DPYD (1p21.3), and PDCD5 (19q13.12), and hemizygous deletion of BAX (19q13.3-q13.4). No sequence changes for R132 and R172 of the IDH1/2 genes were identified.ConclusionsThe molecular findings in this pediatric anaplastic glioma do not allow for a clearly definitive pathological diagnosis. However, the findings provide data on a number of 1p/19q genomic regions that, because of homozygotic deletion, might be the location of genes that are important for the development and clinical evolution of some malignant gliomas in children.

cDNA microarray expression profile in vestibular schwannoma: correlation with clinical and radiological features

Vestibular schwannomas are benign tumors of the vestibular nerves accounting for 10% of intracranial tumors. Hearing loss, tinnitus, and disequilibrium are the most frequently encountered symptoms, but large tumors may lead to brain compression and severe complications. It is known that inactivation of merlin, the product of the NF2 tumor suppressor gene, is responsible for development of vestibular schwannoma, both in sporadic and in bilateral, syndromic, and familial cases. In a recent meta-analysis of constitutional and somatic NF2 alterations, Ahronowitz et al. [1] identified 1,070 small genetic changes detected primarily by exon scanning, 42 intragenic changes of one whole exon or larger, and 29 gross chromosomal rearrangements and whole-gene deletions. Genomic regions other than chromosome 22 have been reported to be involved in certain schwannomas, including loss of chromosome arm 1p, gain of 9q34, and gain of 17q [2e4]. (See Sandberg and Stone [5] for a comprehensive review of the cytogenetic changes in schwannomas.) In addition, methylation of CpG islands has been described as a mechanism for development and progression of vestibular schwannoma. Epigenetic silencing of the NF2 gene [6,7], as well as of other tumor-related genes, has been described, sometimes with clinical and radiological implications [8,9]. In recent years, microarray analysis, a high-throughput system, is being used in basic and applied research. cDNA microarrays allow simultaneous measurement of expression levels for thousands of genes in a tissue, and it has been successfully used in the evaluation of several tumors [10,11]. In vestibular schwannoma, there is very limited information regarding microarray experiments. Welling et al. [12] analyzed the cDNA microarray expression profile of seven vestibular schwannomas, compared with the profile of one vestibular nerve from one of the seven patients. Among 25,920 genes or expressed sequence tags, 42 genes were upregulated (e.g., SPARC, ENG, RHOB) and 8 genes were significantly downregulated (e.g., EZR [previously VIL2], RBMM59). In a cDNA microarray study of the differential expression of genes in cultured human schwannomas and Schwann cell lines, Hanemann et al. [13] identified 41 genes as either upregulated or downregulated more than twofold; using real-time polymerase chain reaction (PCR), they confirmed differential regulation of 13 genes. In another study, tissue microarray for cell cycle regulators

Expression analysis of tumor-related genes involved in critical regulatory pathways in schwannomas.

PurposeGene expression array analysis is providing key data on the potential candidate genes and biological pathways involved in schwannoma origin and development. In this way we performed expression array studies on tumor-related genes in schwannomas.MethodsThe GE Array Q Series HS-006 (SuperArray, Bethesda, MD, USA) was used to determine the expression levels of 96 genes corresponding to 6 primary biological regulatory pathways in a series of 23 schwannomas.ResultsWe identified 15 genes down-regulated, primarily corresponding to signal transduction functions, and 26 genes up-regulated, most frequently involving cell adhesion functions.ConclusionsIn addition to the NF2 inactivation (considered as an early step), variations of other biological regulatory pathways might play a key role in schwannoma.

The Molecular Biology of Vestibular Schwannomas and Its Association with Hearing Loss: A Review

Hearing loss is the most common symptom in patients with vestibular schwannoma (VS). In the past, compressive mechanisms caused by the tumoral mass and its growth have been regarded as the most likely causes of the hearing loss associated with VS. Interestingly, new evidence proposes molecular mechanisms as an explanation for such hearing loss. Among the molecular mechanisms proposed are methylation of TP73, negative expression of cyclin D1, expression of B7-H1, increased expression of the platelet-derived growth factor A, underexpression of PEX5L, RAD54B, and PSMAL, and overexpression of CEA. Many molecular mechanisms are involved in vestibular schwannoma development; we review some of these mechanisms with special emphasis on hearing loss associated with vestibular schwannoma.

Global expression profile in low grade meningiomas and schwannomas shows upregulation of PDGFD, CDH1 and SLIT2 compared to their healthy tissue

Schwannomas and grade I meningiomas are non-metastatic neoplasms that share the common mutation of gene NF2. They usually appear in neurofibromatosis type 2 patients. Currently, there is no drug treatment available for both tumors, thus the use of wide expression technologies is crucial to identify therapeutic targets. Affymetrix Human Gene 1.0 ST was used to test global gene expression in 22 meningiomas, 31 schwannomas and, as non-tumoral controls, 3 healthy meningeal tissues, 8 non-tumoral nerves and 1 primary Schwann cell culture. A non-stringent P-value cut-off and fold change were used to establish deregulated genes. We identified a subset of genes that were upregulated in meningiomas and schwannomas when compared to their respectively healthy tissues, including PDGFD, CDH1 and SLIT2. Thus, these genes should be thoroughly studied as targets in a possible combined treatment.

Genome-wide methylation analysis in vestibular schwannomas shows putative mechanisms of gene expression modulation and global hypomethylation at the HOX gene cluster

Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein‐coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA‐21, Met Proto‐Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.

Mutational analysis of the CITED4 gene in glioblastomas

Allelic losses at 1p appear as a characteristic feature of glial tumors (glioblastoma, astrocytoma, oligodendroglioma, and mixed forms) and, together with 19q losses, represent a prognostic parameter predictive for chemosensitivity and survival primarily in anaplastic oligodendrogliomas [1e4]. Although several genes located at 1p ( p73, CAMTA1, p18, hRAD54, Patched2, Riz1, KIF1b) have been analyzed previously for inactivating mutations related to glioma development, no specific candidate genes have been identified [5e10]. CITED4 (CREB-binding protein/ p300-interacting transactivator with E/D-rich tail 4) is located at 1p34wp35 and encodes a 184eamino acid protein [11]. It is a member of the CITED family, which includes four identified genes, although only three of them are present in mammals (including humans): CITED1, 2, and 4 [12]. All family members share the presence of the CITED domain, which is able to interact with CBP (CREB-binding protein) and p300 [13]. These proteins are transcriptional co-activators that act in two ways to increase gene transcription: (1) by binding transcription factors with RNA polymerase II and (2) by acting like acetyltransferases [14]. Proteins CBP and p300 are able to act on the nucleosome [15]. CITED4 generally has a nuclear location, but cytoplasmatic translocation or loss of nuclear expression has been observed in breast cancer development, in which case it might represent a prognostic marker [13]. Therefore, CITED4 has been proposed as a candidate gene involved in neoplasms characterized by 1p loss. Tews and co-workers [16] recently reported on mutational (in 45 samples) and methylation (in 62 samples) studies of this gene. This glioma series primarily included tumors with major oligodendroglial components, and 15 different CITED4 polymorphisms, mostly single nucleotide exchanges, but no mutations were identified. In parallel, aberrant methylation of the CITED4-associated CpG island was primarily found in oligodendrogliomas with 1p/19q losses that generally showed at least 50% CITED4 reduced expression [16]. Although less frequent than in oligodendroglial tumors, allelic losses at 1p have also been described in astrocytomas (lowgrade and anaplastic) and glioblastomas [1,17], the most malignant form of glial neoplasms. Thus, we performed a mutational study of CITED4 in a series of 24 glial tumors (22 primary glioblastomas, 1 low-grade astrocytoma, and its recurrent secondary glioblastoma) using polymerase chain reaction/single-strand conformation polymorphism