The occurrence of brain metastases among breast cancer patients is currently rising with approximately 20-25% incidence rates, underlining the importance of the identification of new therapeutic and ...prognostic markers. We have previously screened for new markers for brain metastasis by array CGH. We found that loss of 11p15 is common among these patients. In this study, we investigated the clinical significance of loss of 11p15 in primary breast cancer (BC) and breast cancer brain metastases (BCBM). 11p15 aberration patterns were assessed by allelic imbalance (AI) analysis in primary BC (n = 78), BCBM (n = 21) and metastases from other distant sites (n = 6) using six different markers. AI at 11p15 was significantly associated with BCBM (p = 0.002). Interestingly, a subgroup of primary BC with a later relapse to the brain had almost equally high AI rates as the BCBM cases. In primary BC, AI was statistically significantly associated with high grade, negative hormone receptor status, and triple-negative (TNBC) tumors. Gene expression profiling identified PRKCDBP in the 11p15 region to be significantly downregulated in both BCBM and primary BC with brain relapse compared to primary tumors without relapse or bone metastasis (fdr<0.05). qRT-PCR confirmed these results and methylation was shown to be a common way to silence this gene. In conclusion, we found loss at 11p15 to be a marker for TNBC primary tumors and BCBM and PRKCDBP to be a potential target gene in this locus.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In adults, glioblastomas are the most lethal and most frequent malignant brain tumors, and the poor prognosis despite aggressive treatment indicates the need to establish novel targets for molecular ...intervention. The secreted growth factor pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) shows mitogenic, chemotactic, and transforming activity. Whereas PTN expression is tightly regulated during embryogenesis and is very limited in normal adult tissues, a marked PTN up-regulation is seen in tumors including glioblastomas. Likewise, the PTN receptor anaplastic lymphoma kinase (ALK) has been shown previously to be upregulated and functionally relevant in glioblastoma. In this study, we explore the antitumorigenic effects of the simultaneous ribozyme-mediated knockdown of both receptor and ligand. Various glioblastoma cell lines are analyzed for PTN and ALK expression. Beyond the individual efficacies of several specific ribozymes against PTN or ALK, respectively, antiproliferative and proapoptotic effects of a single gene targeting approach are strongly enhanced on double knockdown of both genes in vitro. More importantly, this results in the abolishment of tumor growth in an in vivo subcutaneous tumor xenograft model. Finally, the analysis of various downstream signaling pathways by antibody arrays reveals a distinct pattern of changes in the activation of signal transduction molecules on PTN/ALK double knockdown. Beyond the already known ones, it identifies additional pathways relevant for PTN/ALK signaling. We conclude that double targeting of PTN and ALK leads to enhanced antitumorigenic effects over single knockdown approaches, which offers novel therapeutic options owing to increased efficacy also after prolonged knockdown.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose: Vascular endothelial growth factor (VEGF)-A isa key mediator of angiogenesis in malignant gliomas. Soluble VEGF receptor
1 (sVEGFR-1) can complex VEGF-A and reduce its bioavailability. In ...several animal models sVEGFR-1 inhibited angiogenesis and
tumor growth. We analyzed the levels of endogenous sVEGFR-1 in gliomas of different malignancy grades in relation to tumor
vascularity and VEGF-A.
Experimental Design: The concentration of sVEGFR-1 was determined by ELISA in 104 gliomas and normal brain. Levels of sVEGFR-1 were compared with
malignancy grade, microvessel density, and VEGF-A concentration. Effects of sVEGFR-1 on glioma extract-induced endothelial
cell chemotaxis were analyzed in vitro .
Results: The concentration of sVEGFR-1 correlated with the malignancy grade and was 12-fold higher in glioblastomas than in diffuse
astrocytomas ( P < 0.001), with intermediate levels for anaplastic astrocytomas. VEGF-A levels were 30-fold higher ( P < 0.001) in glioblastomas than in diffuse astrocytomas. The sVEGFR-1:VEGF-A ratio was 0.27 in glioblastomas and 0.70 in diffuse
astrocytomas. Both sVEGFR-1 and VEGF-A correlated with microvessel density ( P < 0.001) and with each other ( P < 0.001); sVEGFR-1 and VEGF-A also correlated with each other when only glioblastomas were analyzed ( P = 0.001). In vitro , recombinant sVEGFR-1 inhibited endothelial cell chemotaxis induced by tumor extracts.
Conclusions: Although absolute levels of sVEGFR-1 are increased in the more malignant gliomas, the sVEGFR-1:VEGF-A ratio is decreased
2.6-fold in glioblastomas compared with diffuse astrocytomas, suggesting that the ensuing increased bioavailability of VEGF-A
favors angiogenesis. The inhibition of tumor extract-induced endothelial chemotaxis by sVEGFR-1 suggests that sVEGFR-1 could
be useful as an angiogenesis inhibitor in the specific context of human gliomas.
Purpose: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular
endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal ...antibody DC101 inhibited intracerebral glioblastoma growth
but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit
glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context
of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular
endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth.
Experimental Designs: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101),
EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations.
Results: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and
DC101. C225 inhibited glioblastoma cell migration in vitro , but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo , either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor
angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion.
Conclusions: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in
an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited
by simultaneous blockade of EGFR.
High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson's disease. STN-HFS exerts a powerful ...modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.
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Glioblastomas (GBM) are the most frequent and malignant human brain tumor type. Typically striking in adulthood, tumor progression is rapid, relentless, and ultimately leads to the patient's death ...within a year of diagnosis. The identification of transcriptionally regulated genes can lead to the discovery of targets for antibody or small-molecule-mediated therapy, as well as diagnostic markers. We prepared cDNA arrays that are specifically enriched for genes expressed in human brain tumors and profiled gene expression patterns in 14 individual tumor samples. Out of 25,000 clones arrayed, greater than 200 genes were found transcriptionally induced in glioblastomas compared to normal human brain tissue including the receptor tyrosine phosphatasezeta (RPTPzeta) and one of its ligands, pleiotrophin (Ptn). We confirmed by Northern blot analysis and immunohistochemistry that RPTPzeta is enriched in tumor samples. Knockdown of RPTPzeta by RNA interference studies established a functional role of RPTPzeta in cell migration. Our results suggest a novel function for RPTPzeta in regulating glioblastoma cell motility and point to the therapeutic utility of RPTPzeta as a target for antibody-mediated therapy of brain tumors.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon ...expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.
The dichotomy between glioblastoma cell migration and proliferation is regulated by various parameters including oxygen tension. In glioblastoma stem-like cells, hypoxia induces downregulation of ...pentose phosphate pathway (PPP) enzymes and a flux shift towards glycolysis. We investigated whether the 2 parallel glucose metabolic pathways are intrinsically linked with cell function and whether these pathways are mechanistically involved in regulating functional programs.
Enzyme expression, migration, and proliferation under hypoxia were studied in multiple cell types. Rapidly and slowly dividing or migrating glioblastoma cells were separated, and enzyme profiles were compared. Glucose-6-phosphate dehydrogenase (G6PD) and Aldolase C (ALDOC), the most strongly inversely regulated PPP and glycolysis enzymes, were knocked down by short hairpin RNA.
Hypoxia caused downregulation of PPP enzymes and upregulation of glycolysis enzymes in a broad spectrum of cancer and nonneoplastic cells and consistently stimulated migration while reducing proliferation. PPP enzyme expression was increased in rapidly dividing glioblastoma cells, whereas glycolysis enzymes were decreased. Conversely, glycolysis enzymes were elevated in migrating cells, whereas PPP enzymes were diminished. Knockdown of G6PD reduced glioblastoma cell proliferation, whereas ALDOC knockdown decreased migration. Enzyme inhibitors had similar effects. G6PD knockdown in a highly proliferative but noninvasive glioblastoma cell line resulted in prolonged survival of mice with intracerebral xenografts, whereas ALDOC knockdown shortened survival. In a highly invasive glioblastoma xenograft model, tumor burden was unchanged by either knockdown.
Cell function and metabolic state are coupled independently of hypoxia, and glucose metabolic pathways are causatively involved in regulating "go or grow" cellular programs.
Purpose: Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor MET are strongly up-regulated in malignant
gliomas. The SF/HGF-MET system contributes to glioma invasion and ...angiogenesis via autocrine and paracrine mechanisms. We
analyzed whether local treatment with NK4, an antagonistic fragment of SF/HGF, could inhibit glioma growth in vivo .
Experimental Design: A guide-screw system was used to implant tumor cells intracerebrally and to perform therapeutic injections. Mice received
daily intratumoral injections of NK4 or buffer as of day 1 or 7 after tumor cell injection until day 20. Functional effects
of NK4 on glioma and endothelial cells were analyzed in vitro .
Results: Tumor volume was reduced by 61.1% in mice treated with NK4 compared with controls when treatment was initiated on day 1 ( P < 0.05) and by 61.4% when treatment was initiated on day 7 ( P < 0.001). Intratumoral microvessel density was reduced by 64.9% when treatment started on day 1 and by 36.7% when it started
on day 7. The proliferative activity of the tumor cells was reduced by >30% regardless of when NK4-treatment was initiated.
The apoptotic fraction of tumor cells was increased 2-fold and 1.5-fold when animals were treated with NK4 as of day 1 or
day 7, respectively. In vitro , NK4 inhibited SF/HGF-induced glioblastoma, and endothelial cell migration and proliferation in a dose-dependent fashion.
Conclusion: NK4 inhibits glioblastoma growth in vivo , most likely via antimitogenic, antimotogenic, proapoptotic, and antiangiogenic mechanisms. Given the strong up-regulation
of SF/HGF and MET in human malignant gliomas, NK4 holds promise as a direct interstitial therapeutic agent for these fatal
tumors.
Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within ...the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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