28 research outputs found
Growth factor receptor-Src-mediated suppression of GRK6 dysregulates CXCR4 signaling and promotes medulloblastoma migration
BACKGROUND: Metastasis in medulloblastoma (MB) is associated with poor survival. Recent genetic studies revealed MB to comprise distinct molecular subgroups, including the sonic hedgehog (SHH) subgroup that exhibits a relatively high rate of progression. To identify targeted therapeutics against metastasis, a better understanding of the regulation of MB cell migration is needed. G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through their regulation of G-protein coupled receptors (GPCRs) involved in growth factor (GF)-mediated cell migration. However, the specific roles and regulation of GRKs in MB have not been investigated. METHODS: Microarray mRNA analysis was performed for GRKs, GPCRs, and GFs in 29 human MB, and real time RT-PCR was used to detect GRK6 expression in MB cells. Lenti- or retro-virus infection, and siRNA or shRNA transfection, of MB cells was used to overexpress and knockdown target genes, respectively. Western blot was used to confirm altered expression of proteins. The effect of altered target protein on cell migration was determined by Boyden chamber assay and xCELLigence migration assays. RESULTS: We observed co-overexpression of PDGFRA, CXCR4, and CXCL12 in the SHH MB subtype compared to non-SHH MB (5, 7, and 5-fold higher, respectively). GRK6, which typically acts as a negative regulator of CXCR4 signaling, is downregulated in MB, relative to other GRKs, while the percentage of GRK6 expression is lower in MB tumors with metastasis (22%), compared to those without metastasis (43%). In SHH-responsive MB cells, functional blockade of PDGFR abolished CXCR4-mediated signaling. shPDGFR transfected MB cells demonstrated increased GRK6 expression, while PDGF or 10% FBS treatment of native MB cells reduced the stability of GRK6 by inducing its proteosomal degradation. Overexpression or downregulation of Src, a key mediator of GF receptor/PDGFR signaling, similarly inhibited or induced GRK6 expression, respectively. siRNA downregulation of GRK6 enhanced CXCR4 signaling and promoted MB migration, while lentiviral-GRK6 overexpression suppressed CXCR4 signaling, potentiated the effect of AMD3100, a CXCR4 antagonist, and impaired migration. CONCLUSIONS: Our findings demonstrate a novel mechanism of GF receptor/PDGFR-Src-mediated dysregulation of CXCR4 signaling that promotes MB cell migration, which could potentially be exploited for therapeutic targeting in SHH MB
Tumor-Infiltrating Lymphocytes in Glioblastoma Are Associated with Specific Genomic Alterations and Related to Transcriptional Class
Tumor-infiltrating lymphocytes (TILs) have prognostic significance in many cancers, yet their roles in glioblastoma (GBM) have not been fully defined. We hypothesized TILs in GBM are associated with molecular alterations, histologies and survival
Heterozygosity for Pten Promotes Tumorigenesis in a Mouse Model of Medulloblastoma
BACKGROUND: Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma. METHODOLOGY/PRINCIPAL FINDINGS: We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/-; Pten +/- medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/-; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression. CONCLUSIONS/SIGNIFICANCE: This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma
EphrinB1 expression is dysregulated and promotes oncogenic signaling in medulloblastoma
Eph receptors and ephrin ligands are master regulators of oncogenic signaling required for proliferation, migration, and metastasis. Yet, Eph/ephrin expression and activity in medulloblastoma (MB), the most common malignant brain tumor of childhood, remains poorly defined. We hypothesized that Eph/ephrins are differentially expressed by sonic hedgehog (SHH) and non-SHH MB and that specific members contribute to the aggressive phenotype. Affymetrix gene expression profiling of 29 childhood MB, separated into SHH (N=11) and non-SHH (N=18), was performed followed by protein validation of selected Eph/ephrins in another 60 MB and two MB cell lines (DAOY, D556). Functional assays were performed using MB cells overexpressing or deleted for selected ephrins. We found EPHB4 and EFNA4 almost exclusively expressed by SHH MB, whereas EPHA2, EPHA8, EFNA1 and EFNA3 are predominantly expressed by non-SHH MB. The remaining family members, except EFNB1, are ubiquitously expressed by over 70–90% MB, irrespective of subgroup. EFNB1 is the only member differentially expressed by 28% of SHH and non-SHH MB. Corresponding protein expression for EphB/ephrinB1 and B2 was validated in MB. Only ephrinB2 was also detected in fetal cerebellum, indicating that EphB/ephrinB1 expression is MB-specific. EphrinB1 immunopositivity localizes to tumor cells within MB with the highest proliferative index. EphrinB1 overexpression promotes EphB activation, alters F-actin distribution and morphology, decreases adhesion, and significantly promotes proliferation. Either silencing or overexpression of ephrinB1 impairs migration. These results indicate that EphrinB1 is uniquely dysregulated in MB and promotes oncogenic responses in MB cells, implicating ephrinB1 as a potential target
Antibodies to Senescent Antigen and C3 Are Not Required for Normal Red Blood Cell Lifespan in a Murine Model
High rate of concurrent BRAF-KIAA1549 gene fusion and 1p deletion in disseminated oligodendroglioma-like leptomeningeal neoplasms (DOLN)
Antibodies to Senescent Antigen and C3 Are Not Required for Normal Red Blood Cell Lifespan in a Murine Model
Red blood cells (RBCs) have a well-defined lifespan, indicating a mechanism by which senescent cells of a certain age are removed from circulation. However, the specifics by which senescent cells are recognized and removed are poorly understood. There are multiple competing hypotheses for this process, perhaps the most commonly cited is that senescent RBCs expose neoantigens [or senescent antigen(s)] that are then recognized by naturally occurring antibodies, which opsonize the senescent cells and result in clearance from circulation. While there are a large volume of published data to indicate that older RBCs accumulate increased levels of antibody on their surface, to the best of our knowledge, the causal role of such antibodies in clearance has not been rigorously assessed. In the current report, we demonstrate that RBC lifespan and clearance patterns are not altered in mice deficient in antibodies, in C3 protein, or missing both. These data demonstrate that neither antibody nor C3 is required for clearance of senescent RBCs, and questions if they are even involved, in a murine model of RBC lifespan