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 MAP-kinase pathway1, 2. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown3. We report frequent somatic mutations in the heterotrimeric G protein alpha subunit, GNAQ, in blue nevi (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.

Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRAS(G12V)) but not its downstream target BRAF (BRAF(V600E)), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16(INK4a) nor classical senescence markers--such as foci of heterochromatin or DNA damage--were able to account for the specific response of melanocytes to HRAS(G12V). Instead, HRAS(G12V)-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.

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UV irradiation induces histone variant H2AX phosphorylated on serine 139 (γH2AX) foci and high levels of pan-nuclear γH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of γH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of γH2AX response: a minority of γH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of γH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear γH2AX without foci of either γH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear γH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear γH2AX were further increased by loss of the bypass polymerase Pol η and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damagebinding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced γH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear γH2AX, a preapoptotic signal associated with ATM-and JNK-dependent apoptosis during replication. The formation of γH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.T o maintain the integrity of genomic information under threat of DNA damage, cells employ a range of responses including DNA repair, replication bypass, recombination, cell-cycle checkpoints, and senescence or apoptosis (1, 2). These responses are described by the all-embracing concept of the DNA damage response (DDR) (1). DNA double-strand breaks (DSBs) are important but not the only starting lesion for the DDR.In eukaryotic cells, the DDR is rapidly initiated by the PI3K kinases ataxia-telangiectasia and Rad3-related (ATR) and ataxiatelangiectasia mutated kinase (ATM) (3, 4). ATR is recruited to single-stranded DNA regions at stalled replication forks in response to replication stress (5-8); ATM is activated primarily in response to DSBs (3, 9-11). ATR and ATM phosphorylate serine threonine protein kinase and CHK2, respectively, which activate cell-cycle checkpoints and p53, leading to a p53-dependent apoptotic pathway (12). This kinase cascade phosphorylates serine 139 and tyrosine 142 of the histone variant H2AX. After ionizing radiation accumulates in foci, phosphorylated H2AX (γH2AX) serine threonine protein kinase colocalizes with other markers of DSBs (53BP1, pNBS1, MDC1, and Brca1) in immunofluorescent microscopy (IF) (13-16). γH2AX foci are frequently adopted as quantitative markers for DSBs in IF (17, 18). However, the exact correspondence between γH2AX and D...

Human cytomegalovirus (HCMV) infections are seen often in glioblastoma multiforme (GBM) tumors, but whether the virus contributes to GBM pathogenesis is unclear. In this study, we explored an oncogenic role for the G protein-coupled receptor-like protein US28 encoded by HCMV that we found to be expressed widely in human GBMs. Immunohistochemical and RT-PCR approaches established that US28 was expressed in ~60% of human GBM tissues and primary cultures examined. In either uninfected GBM cells or neural progenitor cells, thought to be the GBM precursor cells, HCMV infection or US28 overexpression was sufficient to promote secretion of biologically active VEGF and to activate multiple cellular kinases which promote glioma growth and invasion, including phosphorylated STAT3 and e-NOS. Consistent with these findings, US28 overexpression increased primary GBM cell invasion in Matrigel. Notably, this invasive phenotype was further enhanced by exposure to RANTES/CCL5, a US28 ligand, associated with poor patient outcome in GBM. Conversely, RNAi-mediated knockdown of US28 in human glioma cells persistently infected with HCMV led to an inhibition in VEGF expression and glioma cell invasion in response to CCL5 stimulation. Analysis of clinical GBM specimens further revealed that US28 co-localized in situ with several markers of angiogenesis and inflammation, including VEGF, p-STAT3, COX2 and e-NOS. Taken together, our results indicate that US28 expression from HCMV contributes to GBM pathogenesis by inducing an invasive, angiogenic phenotype. Additionally, these findings argue that US28-CCL5 paracrine signaling may contribute to glioma progression and they suggest that targeting US28 may provide therapeutic benefits in GBM treatment.

The hSNF5 subunit of human SWI/SNF ATP-dependent chromatin remodeling complexes is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). Here, we report that loss of hSNF5 function in MRT-derived cells leads to polyploidization and chromosomal instability. Re-expression of hSNF5 restored the coupling between cell cycle progression and ploidy checkpoints. In contrast, cancer-associated hSNF5 mutants harboring specific single amino acid substitutions exacerbated poly-and aneuploidization, due to abrogated chromosome segregation. We found that hSNF5 activates the mitotic checkpoint through the p16 INK4a-cyclinD/CDK4-pRb-E2F pathway. These results establish that poly-and aneuploidy of tumor cells can result from mutations in a chromatin remodeler. Received January 12, 2005; revised version accepted January 27, 2005. ATP-dependent chromatin remodeling factors are critical components of the elaborate machinery that controls gene expression in eukaryotic cells (Becker and Horz 2002). The multisubunit SWI/SNF complex is the prototypical chromatin remodeling factor, present in all eukaryotes (Mohrmann and Verrijzer 2005). Human SNF5 (hSNF5, also known as Ini1, Baf47, or SmarcB1) encodes for a universal SWI/SNF subunit and tumor suppressor that is mutated in malignant rhabdoid tumors (MRTs) (Versteege et al. 1998;Klochendler-Yeivin et al. 2002;Roberts and Orkin 2004). MRTs are rare but highly aggressive pediatric cancers with a high mortality rate.Carriers of germline mutations are predisposed to various cancers and, consistent with a classic tumor suppressor phenotype, the wild-type allele is either lost or deleted in a large proportion of tumors (Biegel et al. 1999; Sevenet et al. 1999a,b;Taylor et al. 2000). hSNF5 mutations are also associated with a number of neoplasms other than MRTs (Grand et al. 1999; Sevenet et al. 1999a,b;Roberts and Orkin 2004). SNF5 inactivation studies in mice established its requirement during early embryogenesis and its role as a tumor suppressor (Klochendler-Yeivin et al. 2000;Roberts et al. 2000;Guidi et al. 2001;Roberts et al. 2002).Several studies found that re-expression of hSNF5 in MRT-derived cell lines caused an accumulation in G0/ G1, cellular senescence, and apoptosis (Ae et al. 2002;Betz et al. 2002;Versteege et al. 2002;Zhang et al. 2002;Oruetxebarria et al. 2004). These effects are largely the result of direct transcriptional activation of the tumor suppressor p16INK4a by hSNF5, which appears to be both necessary and sufficient for reduced cell proliferation and induction of cellular senescence and apoptosis (Oruetxebarria et al. 2004). p16INK4a controls the activity of pRb via inhibition of the cyclin D1-CDK4 kinase, which phosphorylates pRb (Lowe and Sherr 2003). Tumor suppressor pRb is a corepressor that is tethered to a broad range of genes by the E2F transcription factors. Hyperphosphorylation of pRb causes its dissociation from E2F, and relieves its antiproliferative activities. In addition to genes required for cell cycle progression from G1 to...

BackgroundAlthough p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene. Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway.MethodsExpression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes. Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b. The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group). Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing.ResultsExpression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment. MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition.ConclusionsOur results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.

Background:Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we explore the oncogenic effects of BPTF in melanoma.Methods:The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines. Immunoblotting was used to assess the expression of various proteins regulated by BPTF. The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group). The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses. All statistical tests were two-sided.Results:shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents.Conclusions:These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.