CD44 expressed by myeloid cells promotes glioma invasion.

Glioblastoma multiforme (GBM) is one of the most common and malignant brain tumors in adulthood with a median survival of only 15 months. This poor prognosis is related to GBM's ability to extensively infiltrate the surrounding brain parenchyma resulting in diffuse spread of neoplastic cells in the brain, responsible for high rate of recurrence. CD44 (Cluster of Differentiation 44) is a transmembrane protein, overexpressed in multiple cancer types, including gliomas, and implicated in cell motility, proliferation and angiogenesis. Multiple studies have investigated the role of CD44 in GBM cells and have highlighted a link between tumor malignancy and CD44 expression. However up to date, little is known of the role of CD44 on cells from the tumor microenvironment (TME). Here, we have investigated a potential role of CD44 in the TME in regards to GBM invasiveness. Using an ex-vivo organotypic brain slice invasion assay, we show that absence of CD44 from the TME impairs the ability of glioma cells to invade the surrounding brain parenchyma. By deleting CD44 in the astrocytic, endothelial and myeloid compartments, we show that it is specifically CD44 expression in myeloid cells that is responsible for the observed phenotype. Combining in vivo studies in cell-specific knock-out mice and in vitro analyses on primary microglia we demonstrate that myeloid CD44 is implicated in Toll Like Receptor 2 signaling and is a major regulator of Matrix metalloproteinase 9 expression.

Nutritional Calcium Supply Dependent Calcium Balance, Bone Calcification and Calcium Isotope Ratios in Rats.

Serum calcium isotopes (δ44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ44/42CaSerum with bone histology are unknown. We analyzed Ca and δ44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ44/42CaBone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ44/42CaBone was similar in the femora and ribs. At the time of sacrifice, δ44/42CaSerum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ44/42CaBone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ44/42Ca in growing rats. δ44/42CaSerum inversely correlated with markers of bone mineralization.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Carbon ion radiotherapy eradicates medulloblastomas with chromothripsis in an orthotopic Li-Fraumeni patient-derived mouse model.

BACKGROUND: Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. METHODS: We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). RESULTS: In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. CONCLUSION: The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.

Longitudinal MRI study after carbon ion and photon irradiation: shorter latency time for myelopathy is not associated with differential morphological changes.

BACKGROUND: Radiation-induced myelopathy is a severe and irreversible complication that occurs after a long symptom-free latency time if the spinal cord was exposed to a significant irradiation dose during tumor treatment. As carbon ions are increasingly investigated for tumor treatment in clinical trials, their effect on normal tissue needs further investigation to assure safety of patient treatments. Magnetic resonance imaging (MRI)-visible morphological alterations could serve as predictive markers for medicinal interventions to avoid severe side effects. Thus, MRI-visible morphological alterations in the rat spinal cord after high dose photon and carbon ion irradiation and their latency times were investigated. METHODS: Rats whose spinal cords were irradiated with iso-effective high photon (n = 8) or carbon ion (n = 8) doses as well as sham-treated control animals (n = 6) underwent frequent MRI measurements until they developed radiation-induced myelopathy (paresis II). MR images were analyzed for morphological alterations and animals were regularly tested for neurological deficits. In addition, histological analysis was performed of animals suffering from paresis II compared to controls. RESULTS: For both beam modalities, first morphological alterations occurred outside the spinal cord (bone marrow conversion, contrast agent accumulation in the musculature ventral and dorsal to the spinal cord) followed by morphological alterations inside the spinal cord (edema, syrinx, contrast agent accumulation) and eventually neurological alterations (paresis I and II). Latency times were significantly shorter after carbon ions as compared to photon irradiation. CONCLUSIONS: Irradiation of the rat spinal cord with photon or carbon ion doses that lead to 100% myelopathy induced a comparable fixed sequence of MRI-visible morphological alterations and neurological distortions. However, at least in the animal model used in this study, the observed MRI-visible morphological alterations in the spinal cord are not suited as predictive markers to identify animals that will develop myelopathy as the time between MRI-visible alterations and the occurrence of myelopathy is too short to intervene with protective or mitigative drugs.

In Vitro Characterization of a Novel Type of Radiopaque Doxorubicin-Loaded Microsphere.

PURPOSE: To evaluate and compare the material characteristics of a novel type of radiopaque doxorubicin-loaded microsphere (V-100) with radiopaque and non-radiopaque doxorubicin-loaded microspheres. MATERIALS AND METHODS: The prototype V-100 featuring inherent radiopacity and three available commercial controls (DC-Bead-LUMI™-70-150, Embozene-Tandem™-100 and DC-Bead™-M1) were analyzed before and after doxorubicin loading (37.5 mg doxorubicin/1 ml microspheres) in suspension with aqua and/or aqua/iodixanol-320. Study goals included inherent radiopacity [e.g., using conventional computed tomography (CT)], doxorubicin loading efficacy, morphology using light and fluorescence microscopy, size distribution using laser diffraction/light scattering, time-in-suspension, rheological properties using rheometer analysis, and microsphere stability observed over a period of 5 days after doxorubicin loading. RESULTS: V-100 showed good inherent radiopacity without adverse imaging artifacts. Under conventional CT, the quantitative radiopacity was as follows: 480.4 ± 2.9HU for V-100, 2432.7 ± 3.2HU for DC-Bead-LUMI™-70-150, 118.1 ± 3.0HU for Embozene-Tandem™-100, and 19.8 ± 1.5HU for DC-Bead™-M1. All of the types of microspheres showed a similar loading efficiency (> 98%) after 24 h; however, there were slower doxorubicin loading velocities for the radiopaque microspheres. The doxorubicin-loaded V-100 and Embozene-Tandem™-100 showed typical narrow-sized distributions. In aqua/iodixanol-320 suspension, doxorubicin-loaded V-100 showed the best suspension features and ideal deformability and elasticity characteristics. Similar to other microspheres, doxorubicin-loaded V-100 was very stable and storable for at least 5 days. CONCLUSION: V-100 is a promising novel type of radiopaque doxorubicin-loaded microsphere. Compared with the controls, V-100 shows good inherent radiopacity without adverse imaging artifacts and with comparable doxorubicin loading efficacy. Further advantages of V-100 include narrow-sized distribution and excellent suspension, rheology, and stability features.

The Novel X-Ray Visible Zein-Based Non-adhesive Precipitating Liquid Embolic HEIE1_2017: An Exploratory Study.

PURPOSE: To investigate the novel zein-based non-adhesive precipitating liquid embolic HEIE1_2017. MATERIALS AND METHODS: Zein-based liquid embolics are an own class of embolization material. In this study, HEIE1_2017, a novel zein-based liquid embolic, was investigated. Visibility was assessed in vitro in CT and MRI phantoms, embolization characteristics were assessed in vivo in the kidneys of 12 pigs. Components of HEIE1_2017 were zein as occlusion material, ethanol as solvent, and iodized oil as radiopaque material. HEIE1_2017 was used in pure (HEI-PURE) and manually modified (HEI-MOD) form and compared with 6% ethylene vinyl alcohol copolymer (EVOH). Different radiological methods (CT, MRI, DSA, cone-beam CT, and micro-CT) and histopathologic analyses were applied to compare visibility and vascular occlusion patterns. RESULTS: In CT phantoms, all embolics were definitely visible as hyperdense materials. In MRI phantoms, signal-to-noise ratio was highest for HEI-PURE, followed by HEI-MOD and EVOH. In all kidneys, embolization procedures were technically successful and without complications. In DSA, all embolics were definitely visible during and after embolization. Only EVOH caused substantial artifacts in cone-beam CT and CT. In micro-CT and histopathology, HEI-PURE showed a homogeneous occlusion from segmental arteries to glomerular capillaries. HEI-MOD demonstrated the deepest vascular penetration (up to the level of peritubular capillaries), but with an inhomogeneous distribution. For EVOH, there was inhomogeneous vascular occlusion from segmental arteries to glomerular capillaries. CONCLUSION: HEIE1_2017 is a promising novel zein-based liquid embolic. Further preclinical and clinical studies with higher case numbers and long-term follow-up are needed to further assess the value of this embolic material.

Inhibition of CD95/CD95L (FAS/FASLG) Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma.

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells.Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767-76. ©2018 AACR.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy.

Influencing cancer metabolism by lifestyle changes is an attractive strategy as - if effective - exercise-induced problems may be less severe than those induced by classical anti-cancer therapies. Pursuing this idea, clinical trials evaluated the benefit of e.g. different diets such as the ketogenic diet, intermittent caloric restriction and physical exercise (PE) in the primary and secondary prevention of different cancer types. PE proved to be beneficial in the context of breast and colon cancer.Glioblastoma has a dismal prognosis, with an average overall survival of about one year despite maximal safe resection, concomitant radiochemotherapy with temozolomide followed by adjuvant temozolomide therapy. Here, we focused on the influence of PE as an isolated and adjuvant treatment in murine GB therapy.PE did not reduce toxic side effects of chemotherapy in mice administered in a dose escalating scheme as shown before for starvation. Although regular treadmill training on its own had no obvious beneficial effects, its combination with temozolomide was beneficial in the treatment of glioblastoma-bearing mice. As PE might partly act through the induction of reactive oxygen species, dihydroartemisinin - an approved anti-malarial drug which induces oxidative stress in glioma cells - was further evaluated in vitro and in vivo. Dihydroartemisinin showed anti-glioma activity by promoting autophagy, reduced the clonogenic survival and proliferation capacity of glioma cells, and prolonged the survival of tumor bearing mice. Using the reactive oxygen species scavenger n-acetyl-cysteine these effects were in part reversible, suggesting that dihydroartemisinin partly acts through the generation of reactive oxygen species.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

BACKGROUND: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. METHODS: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. RESULTS: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. CONCLUSION: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Cell-based therapy by implanted human bone marrow-derived mononuclear cells improved bone healing of large bone defects in rats.

UNLABELLED: QUESTION/AIM: Cell-based therapy by cultivated stem cells (mesenchymal stem cells [MSC] and endothelial progenitor cells [EPC]) in a large-sized bone defect has already shown improved vascularization and new bone formation. However, these methods are clinically afflicted with disadvantages. Another heterogeneous bone marrow cell population, the so-called human bone marrow-derived mononuclear cells (BMC), has nevertheless been used clinically and showed improved vascularization in ischemic limbs or in the myocardium. For clinical use, a certified process has been established; thus, BMC were isolated from bone marrow aspirate by density gradient centrifugation, washed, cleaned, and given back to patients within several hours. This investigation tested the ability of human BMC seeded on beta-tricalcium phosphate (ß-TCP) and placed into a large bone defect in rats to improve the bone healing process in vivo. METHODS: Human EPC were isolated from buffy coat, and MSC or BMC, respectively, were isolated from bone marrow aspirate by density gradient centrifugation. 1.0×10(6) cells were loaded onto 750 µL ß-TCP (0.7-1.4 mm). Large femoral defects (6 mm) in athymic rats were created surgically and stabilized with an internal fixateur. The remaining defects were filled with ß-TCP granules alone (group 1), ß-TCP+EPC/MSC (group 2), or ß-TCP+BMC (group 3). After 8 weeks, histomorphometric analysis (new bone formation), radiological microcomputer tomography analysis (bony bridging), and biomechanical testing (three-point bending) were achieved. Moreover, a tumorigenicity study was performed to evaluate the safety of BMC implantation after 26 weeks. For statistical analysis, the Kruskal-Wallis test was used. RESULTS: Eight weeks after implantation of EPC/MSC or BMC, respectively, we detected a more significant new bone formation compared to control. In group 2 and 3, bony bridging of the defect was seen. In the control group, more chondrocytes and osteoid were detected. In the BMC and EPC/MSC group, respectively, less chondrocytes and a significantly more advanced bone formation were observed. The biomechanical stability of the bone regenerate was significantly enhanced if BMC and EPC/MSC, respectively, were implanted compared to control. Moreover, no tumor formation was detected either macroscopically or histologically after 26 weeks of BMC implantation. DISCUSSION: Implanted BMC suggest that a heterogeneous cell population may provide a powerful cellular therapeutic strategy for bone healing in a large bone defect in humans.

Postsurgical adjuvant tumor therapy by combining anti-angiopoietin-2 and metronomic chemotherapy limits metastatic growth.

Antiangiogenic tumor therapy has failed in the adjuvant setting. Here we show that inhibition of the Tie2 ligand angiopoietin-2 (Ang2) effectively blocks metastatic growth in preclinical mouse models of postsurgical adjuvant therapy. Ang2 antibody treatment combines well with low-dose metronomic chemotherapy (LDMC) in settings in which maximum-dose chemotherapy does not prove effective. Mechanistically, Ang2 blockade could be linked to quenching the inflammatory and angiogenic response of endothelial cells (ECs) in the metastatic niche. Reduced EC adhesion molecule and chemokine expression inhibits the recruitment of tumor-promoting CCR2(+)Tie2(-) metastasis-associated macrophages. Moreover, LDMC contributes to therapeutic efficacy by inhibiting the recruitment of protumorigenic bone marrow-derived myeloid cells. Collectively, these data provide a rationale for mechanism-guided adjuvant tumor therapies.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy.

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1.

Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

The fibrillin-1 hypomorphic mgR/mgR murine model of Marfan syndrome shows severe elastolysis in all segments of the aorta.

OBJECTIVE: Fibrillin-1 hypomorphic mice (mgR/mgR) are accepted as a model of Marfan syndrome. Phenotypic investigations of this mouse have not previously included quantification of phenotypic features and detailed examinations of the histopathology other than in the ascending aorta. METHODS: We developed a quantitative polymerase chain reaction assay to genotype the mice. Necropsy was performed on 50 male mice after natural death. We then sacrificed 10 mgR/mgR and 10 wild-type mice at 14-19 weeks to perform in vivo computed tomographic scans (n = 3) and microscopic examinations (n = 7). Four aortic segments (ascending, descending, pararenal, and infrarenal aorta) were excised. Each segment was divided into four subsegments and analyzed with Van Gieson staining. The number of elastin breaks and internal aortic diameter were determined twice in randomized, blinded fashion. RESULTS: Computed tomographic scans of mgR/mgR mice revealed aneurysm formation in the ascending aorta and kyphoscoliosis. Elastolysis was present in all four aortic segments of mgR/mgR but was rarely observed in wild-type mice (P < .001). The diameter of the ascending aorta was larger in mgR/mgR than in wild-type mice (P = .01), but para- and infrarenal aortic diameter were even smaller in mgR/mgR mice (P < .001 and P = .01, respectively). Exploratory gene expression analysis showed a number of differentially expressed genes with overrepresentation of immune-related functions. Quantitative polymerase chain reaction analysis confirmed upregulation of selected genes in both the ascending aorta and the abdominal aorta. CONCLUSIONS: Our findings suggest that mgR/mgR mice could be a useful model to study aortic abnormalities in segments other than the ascending aorta in order to understand the molecular mechanisms of aortic disease in Marfan syndrome.

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

MicroRNA-182 promotes leptomeningeal spread of non-sonic hedgehog-medulloblastoma.

The contribution of microRNAs to the initiation, progression, and metastasis of medulloblastoma (MB) remains poorly understood. Metastatic dissemination at diagnosis is present in about 30% of MB patients, and is associated with a dismal prognosis. Using microRNA expression profiling, we demonstrate that the retinal miR-183-96-182 cluster on chromosome 7q32 is highly overexpressed in non-sonic hedgehog MBs (non-SHH-MBs). Expression of miR-182 and miR-183 is associated with cerebellar midline localization, and miR-182 is significantly overexpressed in metastatic MB as compared to non-metastatic tumors. Overexpression of miR-182 in non-SHH-MB increases and knockdown of miR-182 decreases cell migration in vitro. Xenografts overexpressing miR-182 invaded adjacent normal tissue and spread to the leptomeninges, phenotypically reminiscent of clinically highly aggressive large cell anaplastic MB. Hence, our study provides strong in vitro and in vivo evidence that miR-182 contributes to leptomeningeal metastatic dissemination in non-SHH-MB. We therefore reason that targeted inhibition of miR-182 may prevent leptomeningeal spread in patients with non-SHH-MB.

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor.

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.