Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.

BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention

Mitochondrial reactive oxygen species are scavenged by Cockayne syndrome B protein in human fibroblasts without nuclear DNA damage

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role

Prognostic Impact of PHIP Copy Number in Melanoma: Linkage to Ulceration

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced distant metastasis-free survival (DMFS; P=0.01) and disease-specific survival (DSS; P=0.009) by Kaplan–Meier analyses. PHIP FISH scores were independently predictive of DMFS (P=0.03) and DSS (P=0.03). Increased PHIP copy number was an independent predictor of ulceration status (P=0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P<0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of lactate dehydrogenase 5, hypoxia-inducible factor 1 alpha subunit, and vascular endothelial growth factor, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis

Premature chromosome condensation revisited: A novel chemical approach permits efficient cytogenetic analysis of cancers

Chemical induction of premature chromosome condensation (PCC) was investigated and optimized to be able to analyze the chromosomal constitution of cancer cells independent of mitosis and with minimal culture artifacts. A potent protein phosphatase inhibitor, calyculin A, was used to induce PCC in normal diploid cells, in several established human tumor cell lines, and in cells isolated from freshly dissected adenomatous polyps of a patient with hereditary colorectal cancer. In parallel, mitotic arrest was pursued by use of Colcemid. In cell lines, a difference of up to 10-fold was found between frequency of cells with PCC induced by calyculin A (PCC index) and the mitotic index after treatment with Colcemid. In the fresh tumor specimens, Colcemid failed to result in metaphase formation, whereas a regimen of 80 nM calyculin A for 75 min, after only 2 days of culturing, resulted in a PCC index of 2-5%. pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization) was used for a detailed analysis of four cell lines treated with calyculin A, which proved that PCC spreads are amenable to molecular karyotyping, and a comparison between PCC spreads and metaphases from mitotic arrest revealed no discrepancies in karyotypes. pq-COBRA-FISH on PCC spreads from fresh colon tumor samples revealed only numerical and no structural abnormalities. Calyculin A-induced PCC combined with multicolor FISH gives a new opportunity for analysis of the chromosomal constitution of G1 and G2 cancer cells and may find application in the study of the role of chromosome instability in cancer development

Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

<div><p>Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm characterized by tumor cell invasion, robust angiogenesis, and a mean survival of 15 months. Human cytomegalovirus (HCMV) infection is present in >90% of GBMs, although the role the virus plays in GBM pathogenesis is unclear. We report here that HCMV pp71, a viral protein previously shown to promote cell cycle progression, is present in a majority of human GBMs and is preferentially expressed in the CD133+, cancer stem-like cell population. Overexpression of pp71 in adult neural precursor cells resulted in potent induction of stem cell factor (SCF), an important pro-angiogenic factor in GBM. Using double immunofluorescence, we demonstrate in situ co-localization of pp71 and SCF in clinical GBM specimens. pp71 overexpression in both normal and transformed glial cells increased SCF secretion and this effect was specific, since siRNA mediated knockdown of pp71 or treatment with the antiviral drug cidofovir resulted in decreased expression and secretion of SCF by HCMV-infected cells. pp71- induced upregulation of SCF resulted in downstream activation of its putative endothelial cell receptor, c-kit, and angiogenesis as measured by increased capillary tube formation <i>in vitro</i>. We demonstrate that pp71 induces a pro-inflammatory response via activation of NFΚB signaling which drives SCF expression. Furthermore, we show that pp71 levels and NFKB activation are selectively augmented in the mesenchymal subtype of human GBMs, characterized by worst patient outcome, suggesting that HCMV pp71-induced paracrine signaling may contribute to the aggressive phenotype of this human malignancy.</p></div

pp71 stimulation of SCF secretion requires activation of the NFKB signaling pathway.

<p><b>A</b>: U87 cells stably transduced with a retrovirus encoding pp71 (pLXSN-pp71) or control (pLXSN) were used for transcriptome profiling using an Affymetrix Gene 1.0 ST array. Ingenuity pathway analysis (IPA) for differentially expressed genes identified several targets of the NFKB signaling pathway as being upregulated (fold-increase in expression is displayed next to the molecule icon). <b>B</b>: NPCs transduced with rAD-pp71, vector control, or HCMV- infected were co-immunostained for pp71 and markers of NFKB activation (p100/p52, Cox2, and RelB). Cells were counterstained with DAPI. <b>C</b>: Primary GSC neurospheres were treated as indicated and total RNA was analyzed by TaqMan for SCF, CXCL12, IL8, and Myb expression levels. Rab14 levels were used for normalization. The percent change in expression relative to untreated control is displayed. The experiment was repeated three times with similar results. <b>D</b>: U87 cells were transfected with an SCF-promoter driven luciferase construct and then treated as indicated. Luminescence readouts from a representative experiment are shown. Each condition was run in triplicate and the experiment was repeated three times. Positive (RPL13) and negative (scrambled) control promoter driven luciferase constructs were used. Differences were not statistically significant by student t-test.</p

pp71 preferentially activates non-canonical NFKB signaling in endogenously infected glioblastoma tissues.

<p><b>A</b>: Primary GBM tissue was processed using a subcellular protein fractionation kit. An equivalent amount of protein from each fraction was analyzed by western blot for pp71, p65/RelA, p100/p52, Sox2 and histone. <b>B</b>: Primary GBMs classified as proneural or mesenchymal using TaqMan analysis of differentially expressed transcripts (described in C) were analyzed by western blot for pp71, p100/p52, NIK, Actin, Olig2, and CD44. <b>C</b>: TaqMan was performed on GBM cDNA using probes to known mesenchymal (CEBP, CHI3L1, TWIST) and proneural (OLIG2, PDGFRα, Sox11) genes. The results show ΔΔCt for each marker relative to Rab14 in the tissues analyzed.</p