Nikola Holtkamp

PIK3CA mutations in glioblastoma multiforme

Glioblastoma multiforme WHO grade IV is the most common and malignant variant of astrocytic tumors. Loss of heterozygosity of chromosome 10 and mutations in the tumor suppressor gene PTEN on 10q are molecular hallmarks of glioblastomas. Recently, mutations were identified in PIK3CA, encoding a protein that antagonizes the function of PTEN protein in the PI3K/Akt pathway. To address the question whether an exclusive mutation pattern can be observed in PIK3CA and PTEN, we determined the frequency of mutations in both genes. All coding exons were examined by single strand confirmation polymorphism and direct sequencing. Additionally, we analyzed chromosome 10 for loss of heterozygosity and evaluated the mutational status of TP53. In 70 glioblastomas, 5 (7%) PIK3CA mutations and 10 (14%) PTEN mutations were found. All mutations in PIK3CA located to exons 1, 9 and 20, thereby supporting the concept of mutational hot spot regions. In all but one glioblastoma, mutations were seen either in PIK3CA or in PTEN. In conclusion, the frequency of PIK3CA mutations in glioblastomas appears to be much lower than initially reported.

Identification of cDNAs from Japanese pufferfish (Fugu rubripes) and Atlantic salmon (Salmo salar) coding for homologues to tetrapod prion proteins

We identified cDNAs coding for homologues to tetrapod prion proteins (PrPs) in Atlantic salmon (Salmo salar) and Japanese pufferfish (Fugu rubripes), which were termed ‘similar to PrPs’ (stPrPs). Besides significant sequence homologies the fish stPrPs display characteristic structural features in common with tetrapod PrPs. In addition, two stPrPs were shown to be highly expressed in brain tissue. None of the so far identified PrP‐homologues of fish resembles doppel. Hence, the duplication of the PrP gene, which generated doppel, may have occurred not in fish but later in the tetrapod lineage. The identification of fish PrPs provides a basis to address concerns about a possible susceptibility of fish to prion infections.

EGFR and erbB2 in malignant peripheral nerve sheath tumors and implications for targeted therapy

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas with poor prognosis and limited treatment options. Evidence for a role of epidermal growth factor receptor (EGFR) and receptor tyrosine kinase erbB2 in MPNSTs led us to systematically study these potential therapeutic targets in a larger tumor panel (n = 37). Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization analysis revealed increased EGFR dosage in 28% of MPNSTs. ERBB2 and three tumor suppressor genes (PTEN [phosphatase and tensin homolog deleted on chromosome 10], CDKN2A [cyclin-dependent kinase inhibitor 2A], and TP53 [tumor protein p53]) were frequently lost or reduced. Reduction of CDKN2A was linked to appearance of metastasis. Comparison of corresponding neurofibromas and MPNSTs revealed an increase in genetic lesions in MPNSTs. No somatic mutations were found within tyrosine-kinase-encoding exons of EGFR and ERBB2. However, at the protein level, expression of EGFR and erbB2 was frequently detected in MPNSTs. EGFR expression was significantly associated with increased EGFR gene dosage. The EGFR ligands transforming growth factor alpha and EGF were more strongly expressed in MPNSTs than in neurofibromas. The effects of the drugs erlotinib and trastuzumab, which target EGFR and erbB2, were determined on MPNST cell lines. In contrast to trastuzumab, erlotinib mediated dose-dependent inhibition of cell proliferation. EGF-induced EGFR phosphorylation was attenuated by erlotinib. Summarized, our data indicate that EGFR and erbB2 are potential targets in treatment of MPNST patients.

Characterization of the amplicon on chromosomal segment 4q12 in glioblastoma multiforme

A subset of glioblastomas (GBMs) carry gene amplifications on chromosomal segment 4q12. To characterize this amplicon in detail, we analyzed a set of 100 samples consisting of 65 GBMs, 10 WHO grade III astrocytomas, 12 oligodendrogliomas, and 13 glioma cell cultures. We applied multiplex ligation-dependent probe amplification to determine the gene dosage of PDGFRA, KIT, and KDR and the flanking genes USP46, RASL11B, LNX1, CHIC2, SEC3L1, and IGFBP7. The amplicon was highly variable in size and copy number and extended over a region of up to 5 Mb. Amplifications on 4q12 were observed in 15% of GBMs and 23% of GBM cell cultures but not in 22 other gliomas. We analyzed transcription and translation of some genes within this amplicon. Gene amplification generally correlated with high transcript levels but did not necessarily result in increased protein levels. However, we detected frequent expression of proteins encoded by PDGFRA, KIT, and KDR in GBMs and GBM cell cultures independent of the amplification status. Future treatment of GBM patients may include drugs targeting multiple kinases that are encoded by genes on chromosomal segment 4q12.

Subclassification of nerve sheath tumors by gene expression profiling

Nerve sheath tumors are the most common tumors of Neurofibromatosis type 1 (NF1) patients. Dermal neurofibromas develop in nearly all NF1‐patients, whereas plexiform neurofibromas are only observed in one‐third of the patients. NF1‐patients have about a 10% lifetime risk for developing malignant pheripheral nerve sheath tumors (MPNST). The origin of these tumors is thought to be the Schwann cell lacking functional neurofibromin. However, additional genetic alterations are likely to modulate tumor biology and to contribute to individual nerve sheath tumor entities. To gain insight into the molecular events and to determine whether these tumors can be classified according to gene expression profiles, we performed expression analysis applying cDNA array technology. Nine dermal neurofibromas, 7 plexiform neurofibromas, ten MPNST and two MPNST cell cultures were examined. All tumors but 6 sporadic MPNST were obtained from NF1‐patients. We detected significant differences in gene expression patterns between neurofibromas and MPNST and between dermal neurofibromas and plexiform neurofibromas. Tumor class prediction agreed in all but one case with histological and clinical classification. NF1‐associated and sporadic MPNST could not be distinguished by their gene expression patterns. We present a panel of discriminating genes that may assist subclassification of nerve sheath tumors.

Differentially expressed genes in neurofibromatosis 1-associated neurofibromas and malignant peripheral nerve sheath tumors

Neurofibromas represent one of the hallmarks of neurofibromatosis 1 (NF1) patients. Tumor progression of neurofibromas to malignant peripheral nerve sheath tumors (MPNST) is a frequent and life threatening complication. To learn more about processes involved in malignant transformation, we evaluated differential gene expression in plexiform neurofibroma and MPNST from the same NF1 patient. Suppression subtractive hybridization (SSH) yielded 133 differentially expressed genes confirmed by reverse Northern blotting. Virtual Northern blots were employed to validate 23 genes. To independently verify differential expression, immunohistochemical analyses with antibodies to matrix metalloproteinase 13 (MMP13), platelet-derived growth factor receptor alpha (PDGFRA) and fibronectin (FN1) were performed on 9 dermal and 9 plexiform neurofibromas and 16 MPNST from 19 NF1 patients. All three proteins proved to be up-regulated in MPNST. MMP13 expression was observed in 44% of MPNST but was absent in neurofibromas. PDGFRA was expressed in all tumors, but the number of cells expressing it was below 30% in neurofibromas and over 50% in MPNST. Likewise, FN1 was expressed in all tumors, but less than 30% of the cells in neurofibromas and more than 70% of the cells in MPNST exhibited antibody binding. Our data point to several genes not previously recognized to be differentially expressed, and provide a framework for future studies on progression-associated gene expression in low- and high-grade nerve sheath tumors.

Fine mapping of chromosome 22q tumor suppressor gene candidate regions in astrocytomas

Astrocytomas and glioblastomas are the most frequent primary brain tumors in adults. Mutations and altered expression of multiple genes have been found to contribute to the genesis of these tumors. However, many factors in the genesis of astrocytic gliomas are not resolved yet. The frequent losses on several chromosomes indicate the role of still unidentified tumor suppressor genes. Loss of heterozygosity (LOH) on 22q has been described in up to 30% of astrocytic tumors and may be associated with progression to anaplasia. In a first step, information from the nearly finished physical sequence of chromosome 22 were used to map LOH data from 22q deletion studies on different tumor entities to identify potential tumor suppressor gene candidate regions. Next, a series of 153 astrocytic gliomas was examined with 11 polymorphic markers spanning these regions. Forty‐nine (32%) astrocytic gliomas exhibited LOH on 22q, 17 (35%) of which lost heterozygosity for all markers and 32 (65%) of which carried interstitial or partial deletions. Two regions were identified on the physical DNA sequence. The centromeric region spans 3 Mb and the telomeric region 2.7 Mb. The reduced size of these regions now allows direct analysis of all genes included. We already performed mutation analysis on 4 candidate genes from these regions (MYO18B, DJ1042K10.2, MKL1 and EP300), but did not find any mutations in astrocytic tumors.

Systemically administered human bone marrow-derived mesenchymal stem home into peripheral organs but do not induce neuroprotective effects in the MCAo-mouse model for cerebral ischemia

Mesenchymal stem cells (MSC) from bone marrow induce neuroprotective effects and improve clinical symptoms in animal models for acute cerebral ischemia. So far only few data are available from the murine system. Moreover, no data exist regarding neuroprotective effects depending on the application route. Because most preclinical trials regarding restorative therapy in stroke are performed in mice, we aimed to investigate the neuroprotective capacities of human MSC (hMSC) in the middle cerebral artery occlusion (MCAo)-mouse model of cerebral ischemia. As systemic transplantation of MSC could provide a gentle therapeutic procedure for the (mostly elderly) stroke patients, we analyzed effects of this application at a clinically relevant time point. Bone marrow-derived hMSCs were administered intravenously 24 h after MCAo. Mortality and clinical outcome of the transplanted mice did not differ from PBS-treated controls. After 3 and 7 days hMSC were robustly detected in lung, spleen, kidney and intestine, but not in the brain. MRI measurements revealed no differences in infarct size in hMSC injected animals compared to controls. In the neurogenic subventricular zone and the dentate gyrus no significant increase of endogenous cell proliferation was detected following systemic hMSC transplantation. This data further prove the week neurogenic and neuroprotective effect and the limitations of systemically administered hMSCs in cerebral ischemia.

Involvement of Hif-1 in desferrioxamine-induced invasion of glioblastoma cells

Glioblastoma multiforme are highly invasive brain tumors. Experimental approaches focus on unravelling the mechanisms of invasion, this being a major reason for the poor prognosis of these tumors. Our previous results hinted towards involvement of the iron metabolism in invasion. In this study, we examined the effect of iron depletion on the invasive phenotype of glioblastoma cells. Transwell Matrigel invasion assays were used to monitor iron-dependent invasion of human glioblastoma cell lines U373MG and DBTRG05MG. Intracellular iron concentrations were modulated by applying desferrioxamine (DFO) and ferric ammonium citrate (FAC). We detected enhanced invasion of glioblastoma cells upon DFO-induced iron depletion. Treatment of cells with FAC strongly inhibited invasion. DFO treatment resulted in hypoxia-inducible factor 1 (Hif-1)-mediated induction of urokinase plasminogen activator receptor and matrix metalloproteinase 2. Further, RNA interference-mediated repression of urokinase plasminogen activator receptor inhibited DFO-induced invasion. Our data demonstrate a direct effect of DFO on Hif-1 expression resulting in activation of factors associated with ECM degradation and invasion of glioma cells. These findings caution on utilization of DFO and other iron chelators in the treatment of tumors with invasive potential.

Evaluation of drugs for treatment of prion infections of the central nervous system.

Prion diseases are fatal and at present there are neither cures nor therapies available to delay disease onset or progression in humans. Inspired in part by therapeutic approaches in the fields of Alzheimers disease and amyotrophic lateral sclerosis, we tested five different drugs, which are known to efficiently pass through the blood-brain barrier, in a murine prion model. Groups of intracerebrally prion-challenged mice were treated with the drugs curcumin, dapsone, ibuprofen, memantine and minocycline. Treatment with antibiotics dapsone and minocycline had no therapeutic benefit. Ibuprofen-treated mice showed severe adverse effects, which prevented assessment of therapeutic efficacy. Mice treated with low- but not high-dose curcumin and mice treated with memantine survived infections significantly longer than untreated controls (P<0.01). These results encourage further research efforts to improve the therapeutic effect of these drugs.