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Detailed Characterization of Alterations of Chromosomes 7, 9, and 10 in Glioblastomas as Assessed by Single-Nucleotide Polymorphism Arrays

Glioblastomas are cytogenetically heterogeneous tumors that frequently display alterations of chromosomes 7, 9p, and 10q. We used high-density (500K) single-nucleotide polymorphism arrays to investigate genome-wide copy number alterations and loss of heterozygosity in 35 primary glioblastomas. We focused on the identification and detailed characterization of alterations involving the most frequently altered chromosomes (chromosomes 7, 9, and 10), the identification of distinct prognostic subgroups of glioblastomas based on the cytogenetic patterns of alteration for these chromosomes, and validation of their prognostic impact in a larger series of tumors from public databases. Gains of chromosome 7 (97%), with or without epidermal growth factor receptor (EGFR) amplification, and losses of chromosomes 9p (83%) and 10 (91%) were the most frequent alterations. Such alterations defined five different cytogenetic groups with a significant effect on patient survival; notably, EGFR amplification (29%) was associated with a better survival among older patients, as confirmed by multivariate analysis of a larger series of glioblastomas from the literature. In addition, our results provide further evidence about the relevance of other genes (eg, EGFR, CDKN2A/B, MTAP) in the pathogenesis of glioblastomas. Altogether, our results confirm the cytogenetic heterogeneity of glioblastomas and suggest that their stratification based on combined assessment of cytogenetic alterations involving chromosomes 7, 9, and 10 may contribute to the prognostic evaluation of glioblastomas.

Gene expression profiles of human glioblastomas are associated with both tumor cytogenetics and histopathology

Despite the increasing knowledge about the genetic alterations and molecular pathways involved in gliomas, few studies have investigated the association between the gene expression profiles (GEP) and both cytogenetics and histopathology of gliomas. Here, we analyzed the GEP (U133Plus2.0 chip) of 40 gliomas (35 astrocytic tumors, 3 oligodendrogliomas, and 2 mixed tumors) and their association with tumor cytogenetics and histopathology. Unsupervised and supervised analyses showed significantly different GEP in low- vs high-grade gliomas, the most discriminating genes including genes involved in the regulation of cell proliferation, apoptosis, DNA repair, and signal transduction. In turn, among glioblastoma multiforme (GBM), 3 subgroups of tumors were identified according to their GEP, which were closely associated with the cytogenetic profile of their ancestral tumor cell clones: (i) EGFR amplification, (ii) isolated trisomy 7, and (iii) more complex karyotypes. In summary, our results show a clear association between the GEP of gliomas and tumor histopathology; additionally, among grade IV astrocytoma, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone. Further studies in larger series of patients are necessary to confirm our observations.

Amplified and Homozygously Deleted Genes in Glioblastoma: Impact on Gene Expression Levels

Background Glioblastoma multiforme (GBM) displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown. Methodology Single-nucleotide polymorphism (SNP)-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46), and to evaluate the impact of copy number alterations (CNA) on mRNA levels of the genes involved. Principal Findings Recurrent amplicons were detected for chromosomes 7 (50%), 12 (22%), 1 (11%), 4 (9%), 11 (4%), and 17 (4%), whereas homozygous deletions involved chromosomes 9p21 (52%) and 10q (22%). Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2), while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1–q15 and 4q12, respectively). Despite homozygous del(9p21) and del(10q23.31) included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene. Conclusions Overall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.

Intratumoral patterns of clonal evolution in gliomas

Few studies have explored the patterns of clonal evolution in gliomas. Here, we investigate the cytogenetic patterns of intratumoral clonal evolution of gliomas and their impact on tumor histopathology and patient survival. Cytogenetic analysis of 90 gliomas was performed in individual tumor cells (>200 cells/tumor) using multicolor (N = 16 probes) interphase—FISH. Overall, chromosome gains were more frequent than chromosome losses. Gains of chromosome 7 and/or EGFR amplification were detected in 91% of the cases, whereas del(9p21) (77%) and del(10q23) (78%) were the most frequent chromosome losses. Virtually, all cases (99%) showed ≥2 tumor cell clones, with higher numbers among high- versus low-grade gliomas (p = 0.001). Nine different cytogenetic patterns were found in the ancestral tumor clones. In most gliomas, ancestral clones showed abnormalities of chromosome 7, 9p, and/or 10q and cytogenetic evolution consisted of acquisition of additional abnormalities followed by tetraploidization. Conversely, early tetraploidization was associated with low-grade astrocytomas—2/3 pilocytic and 3/6 grade II diffuse astrocytomas—and combined loss of 1p36/19q13 with oligodendrogliomas, respectively; both aberrations were associated with a better patient outcome (p = 0.03). Overall, our results support the existence of different pathways of intratumoral evolution in gliomas

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

The long non-coding RNA HOTAIR is transcriptionally activated by HOXA9 and is an independent prognostic marker in patients with malignant glioma

The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2′-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity

ABSTRACT One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem‐like cells (GSCs), known to be chemo‐ and radio‐resistant. GSCs and non‐stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub‐populations. Nucleolin overexpression has been recently associated with breast cancer sub‐populations with different stem‐like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells. Different levels of nucleolin expression resulted in a 3.4‐fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin‐binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4‐, NANOG‐positive GSC, and in the corresponding non‐stem GBM cells, as well as in SOX2‐positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5‐ or 4.6‐fold higher compared to the non‐targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient‐derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM. HIGHLIGHTSNucleolin is overexpressed in GSC sub‐populations and non‐stem glioblastoma cells;Nucleolin enables intracellular targeted drug delivery in glioblastoma cells;Nucleolin is overexpressed in glioblastoma patient‐derived samples.

Identification of copy number variations of chromosomes 7, 9 and 10 in human glioblastomas by SNP-arrays.

1 pagina.-- Poster presentado al 16o International Charles Heidelberger Symposium on Cancer Research celebrado en Coimbre (Portugal) del 26 al 28 de Septiembre de 2010.-- et al.