Publications by authors named "Javier S Castresana"

62 Publications

In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines.

Pharmaceuticals (Basel) 2021 Nov 19;14(11). Epub 2021 Nov 19.

Department of Health Sciences, Public University of Navarra (UPNA), IdiSNA (Navarra Institute for Health Research), 31008 Pamplona, Spain.

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9- and all-). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9- RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9--RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.
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http://dx.doi.org/10.3390/ph14111184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8624165PMC
November 2021

Molecular and Cellular Mechanisms of Glioblastoma.

Cells 2021 06 10;10(6). Epub 2021 Jun 10.

Molecular Pathology Research Unit, Virgen de la Salud Hospital, 45005 Toledo, Spain.

Glioblastoma is the most malignant primary brain tumor [...].
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http://dx.doi.org/10.3390/cells10061456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230415PMC
June 2021

Silencing of Histone Deacetylase 6 Decreases Cellular Malignancy and Contributes to Primary Cilium Restoration, Epithelial-to-Mesenchymal Transition Reversion, and Autophagy Inhibition in Glioblastoma Cell Lines.

Biology (Basel) 2021 May 26;10(6). Epub 2021 May 26.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Glioblastoma multiforme, the most common type of malignant brain tumor as well as the most aggressive one, lacks an effective therapy. Glioblastoma presents overexpression of mesenchymal markers Snail, Slug, and N-Cadherin and of the autophagic marker p62. Glioblastoma cell lines also present increased autophagy, overexpression of mesenchymal markers, Shh pathway activation, and lack of primary cilia. In this study, we aimed to evaluate the role of HDAC6 in the pathogenesis of glioblastoma, as HDAC6 is the most overexpressed of all HDACs isoforms in this tumor. We treated glioblastoma cell lines with siHDAC6. HDAC6 silencing inhibited proliferation, migration, and clonogenicity of glioblastoma cell lines. They also reversed the mesenchymal phenotype, decreased autophagy, inhibited Shh pathway, and recovered the expression of primary cilia in glioblastoma cell lines. These results demonstrate that HDAC6 might be a good target for glioblastoma treatment.
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http://dx.doi.org/10.3390/biology10060467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228543PMC
May 2021

Glioblastoma and MiRNAs.

Cancers (Basel) 2021 Mar 30;13(7). Epub 2021 Mar 30.

Interdisciplinary Brain Research Centre, Faculty of Medicine, Aligarh Muslim University, Aligarh 202002, India.

Glioblastoma (GB) is one of the most common types of lethal brain tumors. Although several treatment options are available including surgery, along with adjuvant chemo and radiotherapy, the disease has a poor prognosis and patients generally die within 14 months of diagnosis. GB is chemo and radio resistant. Thus, there is a critical need for new insights into GB treatment to increase the chance of therapeutic success. This is why microRNA (miRNA) is being potentially considered in the diagnosis and treatment of glioblastoma. The objective of our review is to provide a holistic picture of GB up-regulated and down-regulated miRNA, in relationship with the expression of other genes, cell signaling pathways, and their role in GB diagnosis and treatment. MiRNA treatment is being considered to be used against GB together with radiotherapy and chemotherapy. Moreover, the use of miRNA as a diagnostic tool has also begun. Knowing that miRNAs are isolated in almost all human body fluids and that there are more than 3000 miRNAs in the human genome, plus the fact that each miRNA controls hundreds of different mRNAs, there is still much study needed to explore how miRNAs relate to GB for its proliferation, progression, and inhibition.
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http://dx.doi.org/10.3390/cancers13071581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037240PMC
March 2021

APR-246 combined with 3-deazaneplanocin A, panobinostat or temozolomide reduces clonogenicity and induces apoptosis in glioblastoma cells.

Int J Oncol 2021 03 26;58(3):312-330. Epub 2021 Jan 26.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Glioblastoma is the most malignant brain tumor and presents high resistance to chemotherapy and radiotherapy. Surgery, radiotherapy and chemotherapy with temozolomide are the only treatments against this tumor. New targeted therapies, including epigenetic modulators such as 3‑deazaneplanocin A (DZ‑Nep; an EZH2 inhibitor) and panobinostat (a histone deacetylase inhibitor) are being tested in vitro, together with temozolomide. The present study combined APR‑246 with DZ‑Nep, panobinostat and teomozolomide in order to explore the possibility of restoring p53 function in mutated cases of glioblastoma. Following the Chou‑Talalay method it was demonstrated that APR‑246 acts in an additive manner together with the other compounds, reducing clonogenicity and inducing apoptosis in glioblastoma cells independently of p53 status.
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http://dx.doi.org/10.3892/ijo.2021.5177DOI Listing
March 2021

TP53, ATRX alterations, and low tumor mutation load feature IDH-wildtype giant cell glioblastoma despite exceptional ultra-mutated tumors.

Neurooncol Adv 2020 Jan-Dec;2(1):vdz059. Epub 2020 Jan 24.

Department of Pathology (Neuropathology) and Instituto de Investigación i+12, Hospital Universitario 12 de Octubre, Madrid, Spain.

Background: Giant cell glioblastoma (gcGBM) is a rare morphological variant of IDH-wildtype (IDHwt) GBM that occurs in young adults and have a slightly better prognosis than "classic" IDHwt GBM.

Methods: We studied 36 GBMs, 14 with a histopathological diagnosis of gcGBM and 22 with a giant cell component. We analyzed the genetic profile of the most frequently mutated genes in gliomas and assessed the tumor mutation load (TML) by gene-targeted next-generation sequencing. We validated our findings using The Cancer Genome Atlas (TCGA) data.

Results: p53 was altered by gene mutation or protein overexpression in all cases, while driver , , , or mutations were infrequent or absent. Compared to IDHwt GBMs, gcGBMs had a significant higher frequency of , , , and mutations, while lower frequency of amplification, deletion, and promoter mutation. Almost all tumors had low TML values. The high TML observed in only 2 tumors was consistent with and mutations. In the histopathological review of TCGA IDHwt, -mutant tumors identified giant cells in 37% of the cases. Considering our series and that of the TCGA, patients with -mutant gcGBMs had better overall survival than those with GBMs (log-rank test,  < .002).

Conclusions: gcGBMs have molecular features that contrast to "classic" IDHwt GBMs: unusually frequent mutations and few amplifications and deletions, especially in tumors with a high number of giant cells. TML is frequently low, although exceptional high TML suggests a potential for immune checkpoint therapy in some cases, which may be relevant for personalized medicine.
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http://dx.doi.org/10.1093/noajnl/vdz059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212869PMC
January 2020

Overexpression of alpha-synuclein promotes both cell proliferation and cell toxicity in human SH-SY5Y neuroblastoma cells.

J Adv Res 2020 May 22;23:37-45. Epub 2020 Jan 22.

School of Biological Sciences & Engineering, Yachay Tech University, Urcuquí, Ecuador.

Alpha-Synuclein (aSyn) is a chameleon-like protein. Its overexpression and intracellular deposition defines neurodegenerative α-synucleinopathies including Parkinson's disease. Whether aSyn up-regulation is the cause or the protective reaction to α-synucleinopathies remains unresolved. Remarkably, the accumulation of aSyn is involved in cancer. Here, the neuroblastoma SH-SY5Y cell line was genetically engineered to overexpress aSyn at low and at high levels. aSyn cytotoxicity was assessed by the MTT and vital-dye exclusion methods, observed at the beginning of the sub-culture of low-aSyn overexpressing neurons when cells can barely proliferate exponentially. Conversely, high-aSyn overexpressing cultures grew at high rates while showing enhanced colony formation compared to low-aSyn neurons. Cytotoxicity of aSyn overexpression was indirectly revealed by the addition of pro-oxidant rotenone. Pretreatment with partially reduced graphene oxide, an apoptotic agent, increased toxicity of rotenone in low-aSyn neurons, but, it did not in high-aSyn neurons. Consistent with their enhanced proliferation, high-aSyn neurons showed elevated levels of SMP30, a senescence-marker protein, and the mitosis Ki-67 marker. High-aSyn overexpression conferred to the carcinogenic neurons heightened tumorigenicity and resistance to senescence compared to low-aSyn cells, thus pointing to an inadequate level of aSyn stimulation, rather than the aSyn overload itself, as one of the factors contributing to α-synucleinopathy.
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http://dx.doi.org/10.1016/j.jare.2020.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016025PMC
May 2020

The synergistic effect of DZ‑NEP, panobinostat and temozolomide reduces clonogenicity and induces apoptosis in glioblastoma cells.

Int J Oncol 2020 Jan 30;56(1):283-300. Epub 2019 Oct 30.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Current treatment against glioblastoma consists of surgical resection followed by temozolomide, with or without combined radiotherapy. Glioblastoma frequently acquires resistance to chemotherapy and/or radiotherapy. Novel therapeutic approaches are thus required. The inhibition of enhancer of zeste homolog 2 (EZH2; a histone methylase) and histone deacetylases (HDACs) are possible epigenetic treatments. Temozolomide, 3‑deazaneplanocin A (DZ‑Nep; an EZH2 inhibitor) and panobinostat (an HDAC inhibitor) were tested in regular and temozolomide‑resistant glioblastoma cells to confirm whether the compounds could behave in a synergistic, additive or antagonistic manner. A total of six commercial cell lines, two temozolomide‑induced resistant cell lines and two primary cultures derived from glioblastoma samples were used. Cell lines were exposed to single treatments of the drugs in addition to all possible two‑ and three‑drug combinations. Colony formation assays, synergistic assays and reverse transcription‑quantitative PCR analysis of apoptosis‑associated genes were performed. The highest synergistic combination was DZ‑Nep + panobinostat. Triple treatment was also synergistic. Reduced clonogenicity and increased apoptosis were both induced. It was concluded that the therapeutic potential of the combination of these three drugs in glioblastoma was evident and should be further explored.
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http://dx.doi.org/10.3892/ijo.2019.4905DOI Listing
January 2020

Tubastatin A, an inhibitor of HDAC6, enhances temozolomide‑induced apoptosis and reverses the malignant phenotype of glioblastoma cells.

Int J Oncol 2019 May 1;54(5):1797-1808. Epub 2019 Mar 1.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Glioblastoma or grade IV astrocytoma is the most common and lethal form of glioma. Current glioblastoma treatment strategies use surgery followed by chemotherapy with temozolomide. Despite this, numerous glioblastoma cases develop resistance to temozolomide treatments, resulting in a poor prognosis for the patients. Novel approaches are being investigated, including the inhibition of histone deacetylase 6 (HDAC6), an enzyme that deacetylates α‑tubulin, and whose overexpression in glioblastoma is associated with the loss of primary cilia. The aim of the present study was to treat glioblastoma cells with a selective HDAC6 inhibitor, tubastatin A, to determine if the malignant phenotype may be reverted. The results demonstrated a notable increase in acetylated α‑tubulin levels in treated cells, which associated with downregulation of the sonic hedgehog pathway, and may hypothetically promote ciliogenesis in those cells. Treatment with tubastatin A also reduced glioblastoma clonogenicity and migration capacities, and accelerated temozolomide‑induced apoptosis. Finally, HDAC6 inhibition decreased the expression of mesenchymal markers, contributing to reverse epithelial‑mesenchymal transition in glioblastoma cells.
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http://dx.doi.org/10.3892/ijo.2019.4739DOI Listing
May 2019

Molecular Study of Long-Term Survivors of Glioblastoma by Gene-Targeted Next-Generation Sequencing.

J Neuropathol Exp Neurol 2018 08;77(8):710-716

Department of Pathology, Virgen de la Salud Hospital, Toledo, Spain.

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. Despite its high lethality, a small proportion of patients have a relatively long overall survival (OS). Here we report a study of a series of 74 GBM samples from 29 long-term survivors ([LTS] OS ≥36 months) and 45 non-LTS. Using next-generation sequencing, we analyzed genetic alterations in the genes most frequently altered in gliomas. Approximately 20% of LTS had a mutation in the IDH1 or IDH2 (IDH) genes, denoting the relevance of this molecular prognostic factor. A new molecular group of GBMs harbored alterations in ATRX or DAXX genes in the absence of driver IDH or H3F3A mutations. These patients tended to have a slightly better prognosis, to be younger at diagnosis, and to present frontal or temporal tumors, and, morphologically, to present giant tumor cells. A significant fraction of LTS GBM patients had tumors with 1 or more alterations in the relevant GBM signaling pathways (RTK/PI3K, TP53 and RB1). In these patients, the PDGFRA alteration is suggested to be a favorable molecular factor. Our findings here are relevant for developing future targeted therapies and for identifying molecular prognostic factors in GBM patients.
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http://dx.doi.org/10.1093/jnen/nly048DOI Listing
August 2018

Sonic hedgehog, Wnt, and brain-derived neurotrophic factor cell signaling pathway crosstalk: potential therapy for depression.

J Neurosci Res 2018 01 20;96(1):53-62. Epub 2017 Jun 20.

Interdisciplinary Brain Research Centre, Faculty of Medicine, Aligarh Muslim University, Aligarh, India.

There are various theories to explain the pathophysiology of depression and support its diagnosis and treatment. The roles of monoamines, brain-derived neurotrophic factor (BDNF), and Wnt signaling are well researched, but sonic hedgehog (Shh) signaling and its downstream transcription factor Gli1 are not well studied in depression. Shh signaling plays a fundamental role in embryonic development and adult hippocampal neurogenesis and also involved in the growth of cancer. In this article, we summarize the evidence for the Shh signaling pathway in depression and the potential crosstalk of Shh with Wnt and BDNF. Antidepressants are known to upregulate the adult hippocampal neurogenesis to treat depression. Shh plays an important role in adult hippocampal neurogenesis, and its downstream signaling components regulate the synthesis of Wnt proteins. Moreover, the expression of Gli1 and Smo is downregulated in depression. BDNF and Wnt signaling are also regulated by various available antidepressants, so there is the possibility that Shh may be involved in the pathophysiology of depression. Therefore, the crosstalk between the Shh, Wnt, and BDNF signaling pathways is being discussed to identify the potential targets. Specifically, the potential role of the Shh signaling pathway in depression is explored as a new target for better therapies for depression.
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http://dx.doi.org/10.1002/jnr.24104DOI Listing
January 2018

Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

Tumour Biol 2016 Sep 15;37(9):12359-12370. Epub 2016 Jun 15.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, Irunlarrea 1, Pamplona, 31008, Spain.

Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.
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http://dx.doi.org/10.1007/s13277-016-5119-6DOI Listing
September 2016

P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines.

Cancer Lett 2016 10 26;381(1):67-75. Epub 2016 Jul 26.

Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain. Electronic address:

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-β pathway, suggesting a therapeutic potential of P144 for GBM treatment.
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http://dx.doi.org/10.1016/j.canlet.2016.07.029DOI Listing
October 2016

Whole exome sequencing in a case of sporadic multiple meningioma reveals shared NF2, FAM109B, and TPRXL mutations, together with unique SMARCB1 alterations in a subset of tumor nodules.

Cancer Genet 2015 Jun 11;208(6):327-32. Epub 2015 Apr 11.

Research Unit, La Paz University Hospital, IdiPAZ, Madrid, Spain.

Meningiomas are common intracranial tumors derived from arachnoid cells. Multiple meningiomas are occasionally present even in patients with no history of neurofibromatosis type 2, a condition that can cause the formation of this neoplasm. Previous studies have shown that most multiple meningiomas are monoclonal in origin. In this study, exome sequencing was performed on four meningiomas and the corresponding peripheral blood DNA from a 61-year-old woman with sporadic multiple meningioma. At least three common mutational events (at the NF2, FAM109B, and TPRXL genes) were detected in the tumors' DNA when they were compared with the lymphocyte DNA from the patient as control. Additionally, an array of unique mutations was detected in each tumor, including in SMARCB1 in two of the samples, a gene whose alteration leads to the development of meningioma. Mutations in other genes, such as IRS4, GULP1, NHSL1, and C10orf53, accounted for one alteration in each meningioma nodule. Our data suggest a monoclonal origin of the meningiomas in this patient, although the numerous alterations contained in each sample indicated multiple secondary variable changes in each tumor nodule. Whether the alterations described in this work are drivers of tumorigenesis or are simply passengers requires further study.
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http://dx.doi.org/10.1016/j.cancergen.2015.03.012DOI Listing
June 2015

Molecular classification defines 4 prognostically distinct glioma groups irrespective of diagnosis and grade.

J Neuropathol Exp Neurol 2015 Mar;74(3):241-9

From the Molecular Pathology Research Unit (PM, MM, THI, BM) and Departments of Pathology (MM) and Neurosurgery (ARDL), Virgen de la Salud Hospital, Toledo; Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona (JSC); Department of Pathology, MD Anderson International, Madrid (JFG); Department of Pathology, University Hospital Complex of Vigo, Vigo (CF); Department of Pathology, University Clinic Hospital, Barcelona (TR); and IdiPaz Research Unit, La Paz University Hospital, Madrid (JAR), Spain.

According to World Health Organization criteria, diffuse gliomas are divided into several histological subtypes, including astrocytomas, oligodendrogliomas, and oligoastrocytomas, and 4 malignancy grades (I-IV). Molecular alterations, such as the isocitrate dehydrogenase gene (IDH) mutation or 1p/19q loss, are found in these tumors but are not included in the current classification system. Recently, mutation of α thalassemia/mental retardation syndrome X-linked (ATRX) gene and its loss of expression have been reported in infiltrating gliomas. We evaluated ATRX protein expression in 272 gliomas and its association with molecular and clinical features. Loss of ATRX expression was more common in tumors with an astrocytic component (astrocytomas II/III, 46.4%; oligoastrocytomas, 47.5%) but was uncommon in oligodendrogliomas (7.3%) and glioblastomas (0.9%). In astrocytic tumors, loss of ATRX expression was significantly associated with longer overall survival. Remarkably, on the basis of IDH mutation, 1p/19q codeletion, and ATRX expression, our study defined 4 molecularly and prognostically different groups of gliomas, showing the relevance of ATRX expression as a new marker for refining the molecular classification of gliomas and for distinguishing clinically distinct prognostic subgroups of patients.
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http://dx.doi.org/10.1097/NEN.0000000000000167DOI Listing
March 2015

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

Genes Chromosomes Cancer 2015 Apr 23;54(4):197-209. Epub 2014 Dec 23.

Molecular Neuro-oncogenetics Laboratory, Research Unit, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain.

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.
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http://dx.doi.org/10.1002/gcc.22232DOI Listing
April 2015

Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples.

Tumour Biol 2015 Apr 22;36(4):2383-91. Epub 2014 Nov 22.

Brain Tumor Biology Unit, University of Navarra School of Sciences, Pamplona, Spain.

Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease.
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http://dx.doi.org/10.1007/s13277-014-2846-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012430PMC
April 2015

Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

PLoS One 2014 13;9(11):e113105. Epub 2014 Nov 13.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231109PMC
December 2015

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

Oncol Rep 2014 Dec 3;32(6):2327-34. Epub 2014 Oct 3.

Molecular Neuro-Oncogenetics Laboratory, Research Unit, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain.

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.
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http://dx.doi.org/10.3892/or.2014.3526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240498PMC
December 2014

On habit and the mind-body problem. The view of Felix Ravaisson.

Front Hum Neurosci 2014 9;8:684. Epub 2014 Sep 9.

Department of Biochemistry and Genetics, University of Navarra School of Sciences Pamplona, Spain.

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http://dx.doi.org/10.3389/fnhum.2014.00684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158773PMC
September 2014

Quantitative method for in vitro matrigel invasiveness measurement through image analysis software.

Mol Biol Rep 2014 Oct 3;41(10):6335-41. Epub 2014 Jul 3.

Brain Tumor Biology Unit-CIFA, University of Navarra School of Sciences, Pamplona, Spain.

The determination of cell invasion by matrigel assay is usually evaluated by counting cells able to pass through a porous membrane and attach themselves to the other side, or by an indirect quantification of eluted specific cell staining dye by means of optical density measurement. This paper describes a quantitative analytical imaging approach for determining the invasiveness of tumor cells using a simple method, based on images processing with the public domain software, ImageJ. Images obtained by direct capture are split into the red channel, and the generated image is used to measure the area that cells cover in the picture. To overcome the several disadvantages that classical cell invasion determinations present, we propose this method because it generates more accurate and sensitive determinations, and it could be a reasonable option for improving the quality of the results. The cost-effective alternative method proposed is based on this simple and robust software that is worldwide affordable.
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http://dx.doi.org/10.1007/s11033-014-3556-0DOI Listing
October 2014

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.

Mol Cytogenet 2014 Jan 6;7(1). Epub 2014 Jan 6.

Molecular Neuro-oncogenetics Laboratory, Research Unit-Unidad de Investigación, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain.

Background: Pediatric 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.

Results: Multiplex 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.

Conclusions: The 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.
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http://dx.doi.org/10.1186/1755-8166-7-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905963PMC
January 2014

Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide-lomeguatrib combination.

Mol Biol Rep 2014 Feb 25;41(2):697-703. Epub 2013 Dec 25.

Department of Neurosurgery, School of Medicine, Ankara University, Ankara, Turkey.

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ-lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ-lomeguatrib (50 μM) was used in AA cell lines, IC50 values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib-TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.
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http://dx.doi.org/10.1007/s11033-013-2908-5DOI Listing
February 2014

Gene expression analysis of aberrant signaling pathways in meningiomas.

Oncol Lett 2013 Jul 23;6(1):275-279. Epub 2013 May 23.

Neuro-Oncogenetics Laboratory, Research Unit, Madrid, Spain ;

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, , which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningioma.
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http://dx.doi.org/10.3892/ol.2013.1363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3742750PMC
July 2013

NF2 genetic alterations in sporadic vestibular schwannomas: clinical implications.

Otol Neurotol 2013 Sep;34(7):1355-61

Department of Otolaryngology, Molecular Neuro-Oncogenetics Laboratory, La Paz University Hospital, IdiPAZ, Madrid, Spain.

Hypothesis: NF2 gene alterations may have a clinical impact in non-NF2 vestibular schwannomas (VSs).

Background: It has been suggested that NF2 mutations might correlate with clinical expression of VS in NF2 patients. The aim of this study was to analyze the impact of genetic alterations in the NF2 gene on epidemiologic, clinical, and radiologic features of patients with sporadic VS. The association between cigarette consumption and the molecular genetic findings was also studied.

Methods: The study group consisted of 51 patients who underwent surgery for removal of vestibular schwannoma in our institution between January 2006 and December 2010. Five highly polymorphic microsatellite DNA markers were used to observe the frequency of loss of heterozygosity (LOH) in chromosome 22. The NF2 gene mutations were detected using polymerase chain reaction amplification and denaturing high-performance liquid chromatography analysis (PCR/dHPLC), and direct sequencing of NF2. Multiplex ligation-dependent probe amplification (MLPA) of the NF2 gene was also performed.

Results: An NF2 mutation was identified in 49%, 22q LOH in 57%, and MLPA alterations in 13.7% of the cases. One mutational hit was present in 27%, and 2 hits were present in 45% of the tumors. No association was found between the type of NF2 mutation and relevant clinical parameters. The presence of NF2 mutations detected by PCR/dHPLC was associated with no complaint of hearing loss at the time of diagnosis (p = 0.023), with subjective aural fullness (p = 0.022) and with an absence of tumor involvement of the internal auditory canal (p = 0.029). Patients with NF2 mutations had lower mean corrected PTA thresholds compared with those with no NF2 mutation (p = 0.037). Inactivation of the NF2 gene by mutation, MLPA, or LOH was more frequent in smokers when compared with never smokers (p = 0.048).

Conclusion: NF2 mutations may play a role in the pathophysiology of hearing loss as well as in the pattern of growth of VS. Cigarette smoking in patients with VS seems to play a role in both the risk of developing the tumor and also in its genetic profile. More studies are needed to corroborate these results and, more broadly, to establish links between molecular and clinical data.
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http://dx.doi.org/10.1097/MAO.0b013e318298ac79DOI Listing
September 2013

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

PLoS One 2013 11;8(6):e65868. Epub 2013 Jun 11.

Neuro-Oncology Laboratory, Research Unit, La Paz University Hospital, IdiPAZ, Madrid, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065868PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679163PMC
January 2014

Codeletion of 1p and 19q determines distinct gene methylation and expression profiles in IDH-mutated oligodendroglial tumors.

Acta Neuropathol 2013 Aug 21;126(2):277-89. Epub 2013 May 21.

Molecular Pathology Research Unit, Department of Pathology, Virgen de la Salud Hospital, Avda. Barber 30, 45004, Toledo, Spain.

Oligodendroglial tumors (OTs) are primary brain tumors that show variable clinical and biological behavior. The 1p/19q codeletion is frequent in these tumors, indicating a better prognosis and/or treatment response. Recently, the prognostically favorable CpG island methylator phenotype (CIMP) in gliomas (G-CIMP+) was associated with mutations in the isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 (IDH) genes, as opposed to G-CIMP- tumors, highlighting the relevance of epigenetic mechanisms. We performed a whole-genome methylation study in 46 OTs, and a gene expression study of 25 tumors, correlating the methylation and transcriptomic profiles with molecular and clinical variables. Here, we identified two different epigenetic patterns within the previously described main G-CIMP+ profile. Both IDH mutation-associated methylation profiles featured one group of OTs with 1p/19q loss (CD-CIMP+), most of which were pure oligodendrogliomas, and a second group with intact 1p/19q and frequent TP53 mutation (CIMP+), most of which exhibited a mixed histopathology. A third group of OTs lacking the CIMP profile (CIMP-), and with a wild-type IDH and an intact 1p/19q, similar to the G-CIMP- subgroup, was also observed. The three CIMP groups presented a significantly better (CD-CIMP+), intermediate (CIMP+) or worse (CIMP-) prognosis. Furthermore, transcriptomic analyses revealed CIMP-specific gene expression signatures, indicating the impact of genetic status (IDH mutation, 1p/19q codeletion, TP53 mutation) on gene expression, and pointing to candidate biomarkers. Therefore, the CIMP profiles contributed to the identification of subgroups of OTs characterized by different prognoses, histopathologies, molecular features and gene expression signatures, which may help in the classification of OTs.
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http://dx.doi.org/10.1007/s00401-013-1130-9DOI Listing
August 2013

Changes in gene expression profiling of apoptotic genes in neuroblastoma cell lines upon retinoic acid treatment.

PLoS One 2013 1;8(5):e62771. Epub 2013 May 1.

Department of Health Sciences, Public University of Navarra, Pamplona, Spain.

To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641123PMC
November 2013
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