Adrian Merlo

5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers.

Loss of heterozygosity on chromosome 9p21 is one of the most frequent genetic alterations identified in human cancer. The rate of point mutations of p16, a candidate suppressor gene of this area, is low in most primary tumours with allelic loss of 9p21. Monosomic cell lines with structurally unaltered p16 show methylation of the 5′ CpG island of p16. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5′ CpG island of p16 was also found in approximately 20% of different primary neoplasms, but not in normal tissues, potentially representing a common pathway of tumour suppressor gene inactivation in human cancers.

Frequent Co-Alterations of TP53, p16/CDKN2A, p14ARF, PTEN Tumor Suppressor Genes in Human Glioma Cell Lines.

In this study we established the simultaneous status of TP53, p16, p14ARF and PTEN tumor suppressor genes in 34 randomly chosen human glioma cell lines. Nine cell lines (26.4%) harbored mutations or deletions in all four tumor suppressor genes and 22 cell lines (64%) had alterations in at least three. Mutations/deletions were found at the following frequencies: TP53 (76.5%), p14ARF (64.7%), p16 (64,7%), PTEN (73.5%). Thus, there was a high incidence of alterations in the cellular pathways involving the p53 transcription factor (94.1%), the retinoblastoma protein (64.7%) and the PTEN phosphatase (73.5%) and 91% of cell lines carried mutations in two or more pathways. This provides the first clear genetic evidence that these tumor suppressors participate in biological pathways which are functioning separately/independently in glioma cells. The status of the gene alterations did not correlate with tumorigenicity in immunocompromized mice or any clinical parameters. Although the mutation rate was higher in glioma cell lines than that reported for glioma tissues, the alterations were molecularly representative of those found in adult de novo glioblastoma. This study highlights the importance of developing therapeutic approaches applicable to tumors with a broad range of genetic alterations and also provides an invaluable panel of glioma cell lines to make this possible.

p53 gene mutation and ink4a-arf deletion appear to be two mutually exclusive events in human glioblastoma.

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.

Tenascin-C is a novel RBPJkappa-induced target gene for Notch signaling in gliomas.

Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.

GSK3β Regulates Differentiation and Growth Arrest in Glioblastoma

Cancers are driven by a population of cells with the stem cell properties of self-renewal and unlimited growth. As a subpopulation within the tumor mass, these cells are believed to constitute a tumor cell reservoir. Pathways controlling the renewal of normal stem cells are deregulated in cancer. The polycomb group gene Bmi1, which is required for neural stem cell self-renewal and also controls anti-oxidant defense in neurons, is upregulated in several cancers, including medulloblastoma. We have found that Bmi1 is consistently and highly expressed in GBM. Downregulation of Bmi1 by shRNAs induced a differentiation phenotype and reduced expression of the stem cell markers Sox2 and Nestin. Interestingly, expression of glycogen synthase kinase 3 beta (GSK3β), which was found to be consistently expressed in primary GBM, also declined. This suggests a functional link between Bmi1 and GSK3β. Interference with GSK3β activity by siRNA, the specific inhibitor SB216763, or lithium chloride (LiCl) induced tumor cell differentiation. In addition, tumor cell apoptosis was enhanced, the formation of neurospheres was impaired, and clonogenicity reduced in a dose-dependent manner. GBM cell lines consist mainly of CD133-negative (CD133-) cells. Interestingly, ex vivo cells from primary tumor biopsies allowed the identification of a CD133- subpopulation of cells that express stem cell markers and are depleted by inactivation of GSK3β. Drugs that inhibit GSK3, including the psychiatric drug LiCl, may deplete the GBM stem cell reservoir independently of CD133 status.

Pathway Analysis of Glioblastoma Tissue after Preoperative Treatment with the EGFR Tyrosine Kinase Inhibitor Gefitinib—A Phase II Trial

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation. Mol Cancer Ther; 10(6); 1102–12. ©2011 AACR.

Somatic deletion mapping on chromosome 10 and sequence analysis of PTEN/MMAC1 point to the 10q25-26 region as the primary target in low-grade and high-grade gliomas

The 10q25-26 region between the dinucleotide markers D10S587 and D10S216 is deleted in glioblastomas and, as we have recently shown, in low-grade oligodendrogliomas. We further refined somatic mapping on 10q23-tel and simultaneously assessed the role of the candidate tumor suppressor gene PTEN/MMAC1 in glial neoplasms by sequence analysis of eight low-grade and 24 high-grade gliomas. These tumors were selected for partial or complete loss of chromosome 10 based on deletion mapping with increased microsatellite marker density at 10q23-tel. Three out of eight (38%) low-grade and 3/24 (13%) high-grade gliomas exclusively target 10q25-26. We did not find a tumor only targeting 10q23.3, and most tumors (23/32, 72%) showed large deletions on 10q including both regions. The sequence analysis of PTEN/MMAC1 revealed nucleotide alterations in 1/8 (12.5%) low-grade gliomas in a tumor with LOH at 10q21-qtel and in 5/21 (24%) high-grade gliomas displaying LOH that always included 10q23-26. Our refined mapping data point to the 10q25-26 region as the primary target on 10q, an area that also harbors the DMBT1 candidate tumor suppressor gene. The fact that we find hemizygous deletions at 10q25-qtel in low-grade astrocytomas and oligodendrogliomas – two histologically distinct entities of gliomas – suggests the existence of a putative suppressor gene involved early in glial tumorigenesis.

Local Targeting of Malignant Gliomas by the Diffusible Peptidic Vector 1,4,7,10-Tetraazacyclododecane-1-Glutaric Acid-4,7,10-Triacetic Acid-Substance P

Purpose: Malignant glial brain tumors consistently overexpress neurokinin type 1 receptors. In classic seed-based brachytherapy, one to several rigid 125I seeds are inserted, mainly for the treatment of small low-grade gliomas. The complex geometry of rapidly proliferating high-grade gliomas requires a diffusible system targeting tumor-associated surface structures to saturate the tumor, including its margins. Experimental Design: We developed a new targeting vector by conjugating the chelator 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid to Arg1 of substance P, generating a radiopharmaceutical with a molecular weight of 1,806 Da and an IC50 of 0.88 ± 0.34 nmol/L. Cell biological studies were done with glioblastoma cell lines. neurokinin type-1 receptor (NK1R) autoradiography was done with 58 tumor biopsies. For labeling, 90Y was mostly used. To reduce the “cross-fire effect” in critically located tumors, 177Lut and 213Bi were used instead. In a pilot study, we assessed feasibility, biodistribution, and early and long-term toxicity following i.t. injection of radiolabeled 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid substance P in 14 glioblastoma and six glioma patients of WHO grades 2 to 3. Results: Autoradiography disclosed overexpression of NK1R in 55 of 58 gliomas of WHO grades 2 to 4. Internalization of the peptidic vector was found to be specific. Clinically, the radiopharmeutical was distributed according to tumor geometry. Only transient toxicity was seen as symptomatic radiogenic edema in one patient (observation period, 7-66 months). Disease stabilization and/or improved neurologic status was observed in 13 of 20 patients. Secondary resection disclosed widespread radiation necrosis with improved demarcation. Conclusions: Targeted radiotherapy using diffusible peptidic vectors represents an innovative strategy for local control of malignant gliomas, which will be further assessed as a neoadjuvant approach.

EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms.

Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20–60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50–80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

Quantitative real-time PCR does not show selective targeting of p14(ARF) but concomitant inactivation of both p16(INK4A) and p14(ARF) in 105 human primary gliomas.

In many human cancers, the INK4A locus is frequently mutated by homozygous deletions. By alternative splicing this locus encodes two non-related tumor suppressor genes, p16INK4A and p14ARF (p19ARF in mice), which regulate cell cycle and cell survival in the retinoblastoma protein (pRb) and p53 pathways, respectively. In mice, the role of p16INK4A as the critical tumor suppressor gene at the INK4A locus was challenged when it was found that p19ARF only knock-out mice developed tumors, including gliomas. We have analysed the genetic status of the INK4A locus in 105 primary gliomas using both microsatellite mapping (MSM) and quantitative real-time PCR (QRT–PCR). Comparison of the results of the two methods revealed agreement in 67% of the tumors examined. In discordant cases, fluorescence in situ hybridization (FISH) analysis was always found to support QRT–PCR classification. Direct assessment of p14ARF exon 1β, p16INK4A exon 1α and exon 2 by QRT–PCR revealed 43 (41%) homozygous and eight (7%) hemizygous deletions at the INK4A locus. In 49 (47%) gliomas, both alleles were retained. In addition, QRT–PCR, but not MSM, detected hyperploidy in five (5%) tumors. Deletion of p14ARF was always associated with co-deletion of p16INK4A and increased in frequency upon progression from low to high grade gliomas. Shorter survival was associated with homozygous deletions of INK4A in the subgroup of glioblastoma patients older than 50 years of age (P=0.025, Anova test single factor, α=0.05).