Marie-Dominique Galibert

The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression

The stress‐activated signalling cascade leading to phosphorylation of the p38 family of kinases plays a crucial role during development and in the cellular response to a wide variety of stress‐inducing agents. Although alterations in gene expression characteristic of the stress response require the regulation of key transcription factors by the p38 family, few downstream targets for this signalling pathway have been identified. By examining the ability of pigment cells to respond to UV irradiation as part of the UV‐induced tanning response, we show that while the microphthalmia‐associated transcription factor Mitf regulates basal Tyrosinase expression, it is the ubiquitous basic helix–loop–helix‐leucine zipper transcription factor Usf‐1 that is required for the UV activation of the Tyrosinase promoter. Consistent with this we demonstrate that Usf‐1 is phosphorylated and activated by the stress‐responsive p38 kinase. The results suggest that activation of Usf‐1 by p38 at a wide variety of viral and cellular promoters will provide a link between stimuli as diverse as UV irradiation, glucose, viral infection and pro‐inflammatory cytokines, and the changes in gene expression associated with the stress response.

UV-induced Expression of Key Component of the Tanning Process, the POMC and MC1R Genes, Is Dependent on the p-38-activated Upstream Stimulating Factor-1 (USF-1)

Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the α-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.

The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion.

During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E. Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.

Aryl Hydrocarbon Receptor- and Calcium-dependent Induction of the Chemokine CCL1 by the Environmental Contaminant Benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1β and tumor necrosis factor-α. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.

α-Melanocyte-Stimulating Hormone Counteracts the Suppressive Effect of UVB on Nrf2 and Nrf-Dependent Gene Expression in Human Skin

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether alpha-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm(2)) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. alpha-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, gamma-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of alpha-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by alpha-MSH. Importantly, alpha-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by alpha-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of alpha-MSH and perhaps of related melanocortin peptides.

Integrative genome‐wide analysis reveals a robust genomic glioblastoma signature associated with copy number driving changes in gene expression

Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 “cis‐acting DNA targeted genes” corresponding to genomic aberrations with direct copy‐number‐driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non‐neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis‐acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

A 4-Gene Signature Associated with Clinical Outcome in High-Grade Gliomas

Purpose: Gene expression studies provide molecular insights improving the classification of patients with high-grade gliomas. We have developed a risk estimation strategy based on a combined analysis of gene expression data to search for robust biomarkers associated with outcome in these tumors. Experimental Design: We performed a meta-analysis using 3 publicly available malignant gliomas microarray data sets (267 patients) to define the genes related to both glioma malignancy and patient outcome. These biomarkers were used to construct a risk-score equation based on a Cox proportional hazards model on a subset of 144 patients. External validations were performed on microarray data (59 patients) and on RT-qPCR data (194 patients). The risk-score model performances (discrimination and calibration) were evaluated and compared with that of clinical risk factors, MGMT promoter methylation status, and IDH1 mutational status. Results: This interstudy cross-validation approach allowed the identification of a 4-gene signature highly correlated to survival (CHAF1B, PDLIM4, EDNRB, and HJURP), from which an optimal survival model was built (P < 0.001 in training and validation sets). Multivariate analysis showed that the 4-gene risk score was strongly and independently associated with survival (hazard ratio = 0.46; 95% CI, 0.26–0.81; P = 0.007). Performance estimations indicated that this score added beyond standard clinical parameters and beyond both the MGMT methylation status and the IDH1 mutational status in terms of discrimination (C statistics, 0.827 versus 0.835; P < 0.001). Conclusion: The 4-gene signature provides an independent risk score strongly associated with outcome of patients with high-grade gliomas. Clin Cancer Res; 17(2); 317–27. ©2011 AACR.

Merkel Cell Polyomavirus Small T Antigen mRNA Level Is Increased following In Vivo UV-Radiation

Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer involving Merkel cells. Recently, a new human polyomavirus was implicated in MCC, being present in 80% of the samples analyzed. In virus-positive MCC, the Merkel cell polyomavirus (MCPyV) is clonally integrated into the patients DNA, and carries mutations in its large T antigen, leading to a truncated protein. In non-symptomatic tissue MCPyV can reside at very low levels. MCC is also associated with older age, immunosuppression and sun exposure. However, the link with solar exposure remains unknown, as the precise mechanism and steps involved between time of infection by MCPyV and the development of MCC. We thus investigated the potential impact of solar simulated radiation (SSR) on MCPyV transcriptional activity. We screened skin samples of 20 healthy patients enrolled in a photodermatological protocol based on in vivo-administered 2 and 4 J/cm2 SSR. Two patients were infected with two new variants of MCPyV, present in their episomal form and RT-QPCR analyses on SSR-irradiated skin samples showed a specific and unique dose-dependent increase of MCPyV small t antigen transcript. A luciferase based in vitro assay confirmed that small t promoter is indeed UV-inducible. These findings demonstrate that solar radiation has an impact on MCPyV mRNA levels that may explain the association between MCC and solar exposure.

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1 -positive leukemia

BackgroundThe t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.ResultsWe compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI – highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings.ConclusionGene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.

Naturally occurring melanomas in dogs as models for non-UV pathways of human melanomas.

Spontaneously occurring melanomas are frequent in dogs. They appear at the same localizations as in humans, i.e. skin, mucosal sites, nail matrix and eyes. They display variable behaviors: tumors at oral localizations are more frequent and aggressive than at other anatomical sites. Interestingly, dog melanomas are associated with strong breed predispositions and overrepresentation of black‐coated dogs. Epidemiological analysis of 2350 affected dogs showed that poodles are at high risk of developing oral melanoma, while schnauzers or Beauce shepherds mostly developped cutaneous melanoma. Clinical and histopathological analyses were performed on a cohort of 153 cases with a 4‐yr follow‐up. Histopathological characterization showed that most canine tumors are intradermal and homologous to human rare morphological melanomas types – ‘nevocytoid type’ and ‘animal type’‐. Tumor cDNA sequencing data, obtained from 95 dogs for six genes, relevant to human melanoma classification, detected somatic mutations in oral melanoma, in NRAS and PTEN genes, at human hotspot sites, but not in BRAF. Altogether, these findings support the relevance of the dog model for comparative oncology of melanomas, especially for the elucidation of non‐UV induced pathways.