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Received: June 10, 2010 , Accepted: July 29, 2010 , Published: August 8, 2010 // The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways. To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas. We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project. From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3. Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas. Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23. In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration. Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas. Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.

Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1 p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

p73 gene, a new p53 homologue, has been identified: it supposedly acts as tumour suppressor gene in neuroblastoma. To clarify whether p73 might be involved in lung carcinogenesis, we examined p73 expression in resected lung cancer and paired normal lung in 60 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We also examined p73 gene status in three representative cases using Southern blot, and p53 gene alteration in 49 cases using PCR-single-strand conformation polymorphism (PCR-SSCP) and direct sequence. In 87% of the cases (52/60) p73 expression in tumour was more than twice as high as that in paired normal lung tissues, and the difference between p73 expression in tumour and normal lung tissue was significant (P < 0.0001). However, Southern blot analysis revealed that none of the cases showed p73 gene amplification. Compared with clinicopathological characteristics, p73 expression correlates significantly with histological differences and age of patient, independently (P < 0.05). Concerning p53 gene status, 43% (21/49) showed p53 gene alteration, but there was no correlation between p73 overexpression and p53 gene alteration. Our results suggest that need for further functional analysis of the role of p73 in lung carcinogenesis. © 1999 Cancer Research Campaign

Neuroblastoma (NB) is a tumor of the sympathetic nervous system and is the most common extra-cranial tumor of childhood, accounting for 7% of all pediatric malignancies. Despite recent advances in therapeutics, outcome is still fatal for patients with aggressive NB and side-effects of treatment are severe. These are important reasons to gain further knowledge of the biology behind NB. Aims: The objective of this thesis was to explore genes and gene products that might contribute to initiation and progression of NB and possibly also other malignancies. Main focus has been on the chromosomal region 1p36.2-3 and participants of the PI3K/Akt signaling pathway. Results: Real-time expression analysis of 30 genes at 1p36.2-3 showed that TNFRSF9 and PIK3CD were down regulated in 1p-deleted compared to non-deleted NB tumors. Studies of the same region showed four genes (ERRFI, CASZ1, RBP7 and PIK3CD) possibly regulated by epigenetically means. Bisulphite sequencing of these four genes in NB cell lines and primary tumors showed that methylation probably is not involved but that histone deacetylation could be implicated in their regulation. Some rare sequence variants were also identified in ERRFI and PIK3CD. PIK3CD encodes a catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) that is involved in activation of Akt. Analysis of mRNA levels in a set of 88 genes associated to PI3K/Akt signaling showed that PDGFRA, PIK3R1, PIK3CD, PRKCBI, PRKCZ and EIF4EBP1 were differentially expressed comparing stage 1-2 to stage 4 NB. At the protein level a stage-dependent expression of the different catalytic isoforms were detected, where levels of p110α were higher in stage 4 tumors compared to stage 1-2, while the opposite was seen for p110δ. Stage 4 NB also had higher levels of phosphorylated Akt (T308 and S473) compared to low stage NB. Furthermore, levels of phosphorylated Akt T308 showed inverse correlation to protein levels of the tumor suppressor Pten. We have also identified a novel splice variant p37δ, encoded by PIK3CD. Usage of an alternative donor site leads to truncation in the RAS-binding domain and loss of the catalytic domain. Despite the truncation, p37δ interact with RAS and there is a strong correlation between protein levels of p37δ and RAS in primary cells. Expression of p37δ is increased in human cancers of the ovaries and colon and ubiquitous expression of the human p37δ in Drosophila increased the body size of the fly. Furthermore, over-expression of p37δ in HEK-293 and mouse embryonic fibroblasts increased proliferation and invasive properties compared to controls, indicating a role in tumorgenicity. Conclusion: Analysis of expression levels of genes and proteins could be used for pinpointing important genes and pathways. This thesis has added more knowledge about the genes at 1p36.2-3, a region commonly deleted in NB, as well as the PI3K/Akt signaling in NB. We have also described a new splice variant of p110δ that is expressed in human cancer and increases proliferation in vitro and in vivo.

Neuroblastoma (NB) is a tumor of the sympathetic nervous system, and the most common extracranial tumor of childhood. The prognosis for high-stage NBs is still poor, with survival rates of about 35%. Side-effects of treatment in these young children can also be severe. It is therefore important to develop better tools for improved patient stratification as well as to identify new targets for therapy. Aims: Using genetic and epigenetic approaches, this thesis aimed to analyze candidate genes with potential involvment in the initation/progression of NB and to identify genes that can be used for improved patient stratification. Results: The six candidate genes located in chromosome region 1p36.22 were down-regulated in tumors from patients with an unfavorable outcome compared with a favorable. DNA methylation was shown not to be involved in the down-regulation of gene transcripts. In a more comprehensive analysis of 1p36, four genes, ERRFI1, PIK3CD, RBP7 and CASZ1, were up-regulated by epigenetic treatment. Bisulfite sequencing revealed that DNA methylation most likely was not involved, suggesting for the potential involvement of other epigenetic mechanisms such as histone deacetylation. Missense mutations were identified in PIK3CD and ERRFI1 and the down-regulated mRNA expression of PIK3CD and CASZ1 was detected in high-stage NB. CASZ1 plays a role in neural development and is therefore an interesting candidate for further study. In a genome-wide analysis of DNA methylation, a group of methylated genes for which we showed gene expression was affected by epigenetic treatment was selected for further analysis. A selected group, e.g. SCNN1A, PRKCDBP and KRT19 could be used to distinguish between patients with an unfavorable outcome from those with a favorable one. Whole-genome copy number analysis of NB tumors identified homozygous deletions in the CDKN2A and RBMS3 genes. Moreover, copy neutral loss of heterozygosity was rare, but could be detected in three chromosomal regions. Tumors with MYCN amplification and those with 11q deletion displayed very different genomic profiles. The 11q-deletion group had significantly more chromosomal breaks than the other group, indicative of an 11q localized chromosomal instability gene (CIN). This group also had a significantly higher age at diagnosis. The groups defined by 11q deletion, MYCN amplification and 17q gain were the only groups associated with poor patient outcome. Conclusions: Whole-genome profiles add valuable information about genomic aberrations, which are important prognostic factors in NB. Aberrant DNA methylation can be a very early event in tumor development as well as in tumor progression. It is therefore of great importance to learn more about both the genetic and epigenetic profiles of NB. This thesis has added to the current knowledge in these regards and has also identified important genetic aberrations, as well as aberrantly methylated genes. In the future, these aberrations could possibly be used in patient stratification, as biomarkers or as targets for therapy.

Aim: Neuroblastoma (NB) is the most common tumor during infancy. It arises from undifferentiated cells in the sympathetic nervous system and is characterized by both clinical and genetic heterogeneity. One of the features of NBs with unfavorable outcome is loss on distal chromosome 1p. The aim of this thesis was to identify genetic factors with relevance for NB tumorigenesis, with special emphasis on the tumor suppressor candidate region on chromosome 1p36.2.Results: All known genes (UBE4B, KIF1B, PGD, CORT, DFFA and PEX14) in the hotspot NB tumor suppressor gene region on 1p36.22 were screened for mutations by capillary-based sequencing of DNA from a large series of NB tumors. The potential tumor suppressor properties of the genes ENO1, ICAT and CASP9, located close to this region, were also analyzed. A few mutations with predicted deleterious effects on protein function were found in the apoptotic regulator DFFA and in the UBE4B gene, which is involved in selective protein degradation. We were also able to show that expression of ENO1, DFFA, UBE4B and CASP9, critical for induction of the apoptotic cascade, is down-regulated in tumors with unfavorable outcome. We identified a novel gene, denoted APITD1, in 1p36.22; two alternative transcripts of this gene were abundantly expressed in normal adult and fetal tissues. Primary NB tumors showed either very weak or no measurable APITD1 expression and the lowest levels were detected in tumors with unfavorable outcome. The coding sequence of APITD1 was well-conserved in different species and no coding variations were found in primary NB tumors. Anti-proliferative and apoptotic effects were observed after over-expression of APITD1 and ENO1 in NB cell lines and in embryonal kidney- derived cells. A mutated ENO1 construct was used to show that the glycolytic enzyme á-enolase had as strong an effect on cell proliferation as the other translational product, MBP1, which is a negative regulator of the c-Myc oncogene. Biallelic inactivation of the transcription factor PHOX2B on chromosome 4p was found in a patient with multifocal NB, emphasizing the importance of early embryologic differentiation pathways in tumors of neural crest origin. Conclusions: A few rare mutations were found in genes with possible tumor suppressor functions. Several genes on 1p36.2 were also shown to be down-regulated in aggressive tumors. We therefore propose that the poor prognosis for NB patients with 1p deletions is a syngenic effect of mutations or transcriptional silencing of several genes in this region.

The p73 gene has been mapped to 1p36.33, a region which is frequently deleted in a wide variety of neoplasms including tumours of neuroectodermal origin. The p73 protein shows structural and functional homology to p53. For these reasons, p73 was considered as a positional and functional candidate tumour suppressor gene. Thus far, mutation analysis has provided no evidence for involvement of p73 in oligodendrogliomas, lung carcinoma, oesophageal carcinoma, prostatic carcinoma and hepatocellular carcinoma. In neuroblastoma, two mutations have been observed in a series of 140 tumours. In view of the occurrence of 1p deletions in Merkel cell carcinoma (MCC) and the location of p73 we decided to search for mutations in the p73 gene in five MCC cell lines and ten MCC tumours to test potential tumour suppressor function for this gene in MCC. In view of the possible complementary functions of p73 and TP53 we also examined the status of the TP53 gene. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in four MCCs. In one MCC tumour, a mis-sense mutation located in the NH2-terminal transactivation region of the p73 gene was found. These results show that p73, analogous to neuroblastoma, is infrequently mutated in MCC. This is also the first report in which the role of TP53 in MCC has been investigated by sequencing the entire coding region of TP53. TP53 mis-sense mutations and one non-sense mutation were detected in three of 15 examined MCCs, suggesting that TP53 mutations may play a role in the pathogenesis or progression of a subset of MCCs. Moreover, typical UVB induced C to T mutations were found in one MCC cell line thus providing further evidence for sun-exposure in the aetiology of this rare skin cancer. © 2000 Cancer Research Campaign

Accumulating evidence has demonstrated that aberration of the p53 tumour-suppressor gene is one of the pivotal genetic events in hepatocellular carcinogenesis. Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined. © 1999 Cancer Research Campaign

Allelic loss of the short arm of chromosome 1 has been observed frequently in a wide spectrum of cancers, most frequently in oligodendroglioma. In our previous studies, we evaluated 177 oligodendroglial tumor samples and identified the AJAP1 gene (formerly Shrew1) in the consensus region of deletion. AJAP1 is a transmembrane protein found in adheren junctions and functions to inhibit glioma cell adhesion and migration. Whereas a putative tumor suppressor gene, we did not detect AJAP1 gene mutations. In subsequent studies, we found that AJAP1 was underexpressed in oligodendrogliomas relative to normal brain tissues. Bioinformatic analysis revealed the presence of CpG islands in the promoter of AJAP1. Methylation analysis of the AJAP1 promoter identified hypermethylation in 21 % of oligodendrogliomas (n = 27), and the degree of methylation correlated with low levels of AJAP1 expression (P = 0.045). The AJAP1 promoter was also highly methylated in a wide spectrum of cell lines (n = 22), including cell lines of glioblastoma. Analysis of the National Cancer Institute's REMBRANDT dataset, which contains 343 glioma samples, indicated that low AJAP1 gene expression was associated with decreased survival. Thus, both genetic (gene deletion) and epigenetic alterations (promoter methylation) are likely mechanisms that inactivate the putative tumor suppressor AJAP1 in gliomas, which contributes to poor prognosis.