Doctoral Theses from Sahlgrenska Academy

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Browsing Doctoral Theses from Sahlgrenska Academy by Subject "1p36"

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    From 1p3 to PI3K - Studies of neuroblastoma
    (2011-01-19) Fransson, Susanne
    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.
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    The Neuroblastoma Genome and Epigenome - Patient Stratification and Identification of Candidate Genes
    (2009-09-30T12:53:07Z) Carén, Helena
    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.