Katarina Ejeskär

Screening for gene mutations in a 500 kb neuroblastoma tumor suppressor candidate region in chromosome 1p; mutation and stage-specific expression in UBE4B/UFD2.

Deletion of a part of the short arm of chromosome 1 is one of the most common chromosomal rearrangements observed in neuroblastoma (NBL) tumors and it is associated with a poor prognosis. No NBL tumor suppressor gene has yet been identified in the region. Our shortest region of overlap of deletions, ranging from marker D1S80 to D1S244, was shown to partly overlap a 500 kb region that was homozygously deleted in a NBL cell line. We have screened seven genes known to reside in or very close to this overlap consensus region, UBE4B/UFD2, KIF1B, DFFA, PGD, CORT, PEX14, and ICAT, for coding mutations in NBL tumor DNA. A few deviations from the reference sequences were identified; most interestingly being a splice site mutation that was detected in UBE4B/UFD2 in a stage 3 NBL with a fatal outcome. This mutation was neither present in the patients constitutional DNA nor in any of 192 control chromosomes analysed. Also, the expression of UBE4B/UFD2 was markedly diminished in the high-stage/poor-outcome tumors as compared to the low-stage/favorable-outcome tumors. Overall, the number of amino-acid changes in the genes of the region was low, which shows that mutations in these genes are rare events in NBL development. Given the data presented here, UBE4B/UFD2 stands out as the strongest candidate NBL tumor suppressor gene in the region at this stage.

Analyses of apoptotic regulators CASP9 and DFFA at 1P36.2, reveal rare allele variants in human neuroblastoma tumours

The genes encoding Caspase-9 and DFF45 have both recently been mapped to chromosome region 1p36.2, that is a region alleged to involve one or several tumour suppressor genes in neuroblastoma tumours. This study presents an update contig of the ‘Smallest Region of Overlap of deletions’ in Scandinavian neuroblastoma tumours and suggests that DFF45 is localized in the region. The genomic organization of the human DFF45 gene, deduced by in-silico comparisons of DNA sequences, is described for the first time in this paper. In the present study 44 primary tumours were screened for mutation by analysis of the genomic sequences of the genes. In two out of the 44 tumours this detected in the DFFA gene one rare allele variant that caused a non-polar to a polar amino acid exchange in a preserved hydrophobic patch of DFF45. One case was hemizygous due to deletion of the more common allele of this polymorphism. Out of 194 normal control alleles only one was found to carry this variant allele, so in respect of it, no healthy control individual out of 97 was homozygous. Moreover, our RT–PCR expression studies showed that DFF45 is preferably expressed in low-stage neuroblastoma tumours and to a lesser degree in high-stage neuroblastomas. We conclude that although coding mutations of Caspase-9 and DFF45 are infrequent in neuroblastoma tumours, our discovery of a rare allele in two neuroblastoma cases should be taken to warrant further studies of the role of DFF45 in neuroblastoma genetics.

A cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation

BackgroundA common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region.ResultsThe current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development.ConclusionThe genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation.

Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma.

The phosphoinositide-3 kinase (PI3K) pathway plays a critical role in cancer cell growth and survival and has also been implicated in the development of the childhood cancer neuroblastoma. In neuroblastoma high mRNA expression of the PI3K catalytic isoform PIK3CD is associated to favorable disease. Yet, activation of Akt is associated with poor prognosis. Since the contribution of the numerous members of this pathway to neuroblastoma pathogenesis is mainly unknown, genes of the PI3K/Akt pathway were analyzed at the mRNA level through microarrays and quantitative real-time RT-PCR (TaqMan) and at the protein level using western blot analysis. Five genes showed lower mRNA expression in aggressive compared to more favorable neuroblastomas (PRKCZ, PRKCB1, EIF4EBP1, PIK3RI and PIK3CD) while the opposite was seen for PDGFRA. Clustering analysis shows that the expression levels of these six genes can predict aggressive disease. At the protein level, p110δ (encoded by PIK3CD) and p85α isomers (encoded by PIK3R1) were more highly expressed in favorable compared to aggressive neuroblastoma. Evaluation of the expression of these PI3K genes can predict aggressive disease, and indicates stage-dependent involvement of PI3K-pathway members in neuroblastoma.

p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors

The phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, proliferation and survival, and are frequently affected in human cancer. PI3K is composed of a catalytic subunit, p110, and a regulatory subunit, p85. The PI3K catalytic subunit p110δ is encoded by PIK3CD and contains p85- and RAS-binding domains, and a kinase domain. Here we present an alternatively spliced PIK3CD transcript encoding a previously unknown protein, p37δ, and demonstrate that this protein is expressed in human ovarian and colorectal tumors. p37δ retains the p85-binding domain and a fraction of the RAS-binding domain, lacks the catalytic domain, and has a unique carboxyl-terminal region. In contrast to p110δ, which stabilizes p85, p37δ promoted p85 sequestering. Despite the truncated RAS-binding domain, p37δ bound to RAS and we found a strong positive correlation between the protein levels of p37δ and RAS. Overexpressing p37δ, but not p110δ, increased the proliferation and invasive properties of HEK-293 cells and mouse embryonic fibroblasts. Cells overexpressing p37δ showed a quicker phosphorylation response of AKT and ERK1/2 following serum stimulation. Ubiquitous expression of human p37δ in the fruit fly increased body size, DNA content and phosphorylated ERK1/2 levels. Thus, p37δ appears to be a new tumor-specific isoform of p110δ with growth-promoting properties.

A novel 1p36.2 located gene, APITD1, with tumour-suppressive properties and a putative p53-binding domain, shows low expression in neuroblastoma tumours

Neuroblastoma is characterised by a lack of TP53 mutations and no other tumour suppressor gene consistently inactivated has yet been identified in this childhood cancer form. Characterisation of a new gene, denoted APITD1, in the neuroblastoma tumour suppressor candidate region in chromosome 1p36.22 reveals that APITD1 contains a predicted TFIID-31 domain, representing the TATA box-binding protein-associated factor, TAFII31, which is required for p53-mediated transcription activation. Two different transcripts of this gene were shown to be ubiquitously expressed, one of them with an elevated expression in foetal tissues. Primary neuroblastoma tumours of all different stages showed either very weak or no measurable APITD1 expression, contrary to the level of expression observed in neuroblastoma cell lines. A reduced pattern of expression was also observed in a set of various tumour types. APITD1 was functionally tested by adding APITD1 mRNA to neuroblastoma cells, leading to the cell growth to be reduced up to 90% compared to control cells, suggesting APITD1 to have a role in a cell death pathway. Furthermore, we determined the genomic organisation of APITD1. Automated genomic DNA sequencing of the coding region of the gene as well as the promoter sequence in 44 neuroblastoma tumours did not reveal any loss-of-function mutations, indicating that mutations in APITD1 is not a common abnormality of neuroblastoma tumours. We suggest that low expression of this gene might interfere with the ability for apoptosis through the p53 pathway.

Verification of microRNA expression in human endometrial adenocarcinoma.

BackgroundMicroRNAs are small non-coding RNAs that have been implicated in tumor initiation and progression. In a previous study we identified 138 miRNAs as differentially expressed in endometrial adenocarcinoma compared to normal tissues. One of these miRNAs was miRNA-34a, which regulates several genes involved in the Notch pathway, which is frequently altered in endometrial cancer. The aims of this study were to verify the differential expression of a subset of miRNAs and to scrutinize the regulatory role of mir-34a on the target genes NOTCH1 and DLL1.MethodsTwenty-five miRNAs that were previously identified as differentially expressed were subjected to further analysis using qPCR. To investigate the regulation of NOTCH1 and DLL1 by mir-34a, we designed gain- and loss-of-function experiments in Ishikawa and HEK293 cell lines by transfection with a synthetic mir-34a mimic and a mir-34a inhibitor.ResultsOf the 25 validated miRNAs, seven were down-regulated and 18 were up-regulated compared to normal endometrium, which was fully consistent with our previous findings. In addition, the up-regulation of mir-34a led to a significant decrease in mRNA levels of NOTCH1 and DLL1, while down-regulation led to a significant increase in mRNA levels of these two genes.ConclusionsWe verified both up-regulated and down-regulated miRNAs in the tumor samples, indicating various roles of microRNAs during tumor development. Mir-34a functions as a regulator by decreasing the expression of NOTCH1 and DLL1. Our study is the first to identify a correlation between mir-34a and its target genes NOTCH1 and DLL1 in endometrial adenocarcinoma.

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas

BACKGROUND The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation. METHODS Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests. RESULTS We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma. CONCLUSIONS Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.

Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition.

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

ABSTRACT The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.