Stéphanie Mottier

Mapping and identification of essential gene functions on the X chromosome of Drosophila

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large‐scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large‐scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X‐chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P‐element‐tagged enhancers is described and discussed.

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.

Angiotensin-2 receptors (AT1-R and AT2-R), new prognostic factors for renal clear-cell carcinoma?

Background:The growth factor Angiotensin-2 signals through Angiotensin receptor type 1 (AT1-R) in a broad range of cell types and tumours and through the type-2 receptor (AT2-R) in a more restricted group of cell types. Although numerous forms of cancer have been shown to overexpress AT1-R, expression of AT1-R and AT2-R by human renal clear-cell carcinoma (RCCC) is not well understood. In this study, the expression of both angiotensin receptors was quantified in a retrospective series of RCCC and correlated with prognostic factors.Methods:Angiotensin receptor type 1 and AT2-R expressions were quantified on tumour tissues by immunohistochemistry (IHC), western blot and quantitative reverse transcriptase PCR (qRT–PCR). IHC results were correlated to Fuhrmans grade and patient progression-free survival (PFS).Results:A total of 84 RCCC were analysed. By IHC, AT1-R and AT2-R were expressed to a greater level in high-grade tumours (AT1-R: P<0.001, AT2-R: P<0.001). Univariate analysis showed a correlation between PFS and AT1-R or AT2-R expression (P=0.001). By multivariate analysis, only AT2-R expression correlated with PFS (HR 1.021, P=0.006) and cancer stage (P<0.001). By western blot, AT1-R and AT1-R were also found to be overexpressed in higher Fuhrmans grade (P<0.01 and P=0.001 respectively). By qRT–PCR, AT1-R but not AT2-R mRNA were downregulated (P=0.001 and P=0.118, respectively).Conclusion:Our results show that AT1-R and AT2-R proteins are overexpressed in the most aggressive forms of RCCC and that AT2-R expression correlates with PFS. AT1-R or AT2-R blockage could, therefore, offer novel directions for anti-RCCC therapy.

KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines

The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with 131I and 125I monoclonal antibody A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response. Using DNA microarrays, we have identified the GPA33 gene as a target of PPARγ in HT29‐Cl.16E colon cancer cells. Treatment of HT29‐Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPARγ agonist GW7845 induced a 2‐ to 6‐fold increase in GPA33 mRNA as determined by real‐time PCR. This induction was also found in HT29‐Cl.16E cells treated with rosiglitazone and ciglitazone and was prevented by cotreatment with the PPARγ antagonist GW9662, indicating that this regulation was PPARγ dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDXl, CDX2 and KLF5 expression was not modified by PPARγ activation. By contrast, a significant increase in KLF4 was seen, both at mRNA and protein levels. Furthermore, chromatin immunoprecipitation studies demonstrated that an increased amount of KLF4 protein was bound to the GPA33 promoter in cells treated with rosiglitazone. Finally, downregulation of KLF4 expression by siRNA reduced rosiglitazone‐induced GPA33 expression. This indicates that PPARγ activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPARγ activation leads to increased (p21WAF1/Cip1 and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPARγ‐regulated genes.

Description of 2 angiogenic phenotypes in clear cell renal cell carcinoma

Angiogenesis in clear cell renal cell carcinoma has received recent focus with the development of antiangiogenic therapies. Although tumor progression is known to be correlated with intratumoral and plasma levels of vascular endothelial growth factor-A, the role of tumor induced-angiogenesis remains unclear in these tumors. We analyzed the vascular network in a cohort of 73 clear cell renal cell carcinoma cases using endothelial immunostaining. We studied protein expression of vascular endothelial growth factor, Von Hippel Lindau, and carbonic anhydrase IX by immunohistochemistry, Von Hippel Lindau gene alteration by sequencing, deletion- and methylation-specific Multiplex Ligation-dependent Probe Amplification, and gene expression by pangenomic microarray and quantitative polymerase chain reaction in a subcohort of 39 clear cell renal cell carcinoma cases. We described 2 distinct angiogenic phenotypes in comparison with the normal kidney vasculature: low and high angiogenic phenotypes. The low angiogenic phenotype was associated with more aggressive prognostic factors such as T3 to T4 (62% versus 31%, P=.002), N+ (29% versus 3% P=.004), M+ (53% versus 21%, P=.004) stages, Fuhrman grade (grade 3-4: 91% versus 36%, P<.001), and intratumoral vascular endothelial growth factor expression (74% versus 28%, P<.001); was less associated with Von Hippel Lindau inactivation (56% versus 80%, P=.03); and was a predictor of poor prognosis in terms of progression-free, cancer-specific, and overall survival (log-rank test, P=.002, P=.011, and P=.035, respectively). The low angiogenic phenotype was also associated with a relative down-regulation of gene expression (platelet-derived growth factor D, N-acetyl transferase 8, and N-acetyl transferase 8 B). In conclusion, the histologic and molecular distinction between these 2 angiogenic phenotypes could help to better understand the biologic behavior of clear cell renal cell carcinoma angiogenesis and could be analyzed in a prospective study of the effects of antiangiogenic drugs.

Paraffin-embedded tissue is less accurate than frozen section analysis for determining VHL mutational status in sporadic renal cell carcinoma

INTRODUCTION Literature controversies exist regarding the prognostic value of VHL mutations. The objective was to compare paraffin-embedded and frozen section specimens for VHL mutations detection and to evaluate the reliability of DNA analysis in formalin-fixed tissues. METHODS Seventy-six patients with clear cell renal cell carcinoma (RCC) previously assessed for VHL status from frozen samples were included. Seventy-three tumor samples were known to be mutated for VHL. DNA was extracted and an electrophoresis was performed to determine DNA quality. The whole coding sequence was synthesized by double PCR amplification followed by sequencing. Sequencing results were compared with those previously determined from frozen samples. RESULTS DNA could be extracted from the 76 paraffin samples. DNA quality was highly degraded and significantly less amplified by PCR in 34.2%, resulting in no sequence available for analysis in 57.7% and discordance with frozen samples in 42.3% of the cases respectively. VHL mutations were found in 52.1% of the whole paraffin samples whereas 98% were mutated; 72% could be sequenced, resulting in 69.1% of VHL mutations in this subset. Only half of observed mutations were fully consistent with frozen analysis in the 3 exons. Neomutations were found in 10.5% and 28.9% of known mutations in frozen samples were not detected in paraffin blocks. Only DNA quality significantly influenced PCR amplification and sequencing. CONCLUSION Tumoral DNA extraction and VHL mutation analysis can be performed from formalin-fixed paraffin-embedded (FFPE) tissue in RCC. But mutations identified tissues are not strictly concordant with those from frozen analysis and therefore results obtained from FFPE samples should be interpreted with care.

HtrA3 is regulated by 15-deoxy-Delta12,14-prostaglandin J2 independently of PPARgamma in clear cell renal cell carcinomas.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPARgamma ligand 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-beta family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPARgamma agonists, did not induce HtrA3 expression, and the PPARgamma antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPARgamma. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPARgamma. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene.

Differential expression of genes related to HFE and iron status in mouse duodenal epithelium.

Iron absorption, distribution, use, and storage are thought to be tightly regulated since altered iron stores may lead to cellular damage and disease. HFE, the hereditary hemochromatosis gene product, is expressed in the crypts of the duodenum, but the molecular mechanism by which it contributes to the inhibition of iron absorption is still unknown. In this study we aimed to identify transcriptional profiles in the duodenal epithelium of Hfe−/− mice. We used dedicated microarrays to compare gene expression among the duodenum of Hfe−/− mice, induced iron overload mice, and control mice. We found 151 differentially expressed genes and unknown sequences between Hfe−/− mice and normal littermates. Gene profiling revealed a gene subset more specific for Hfe inactivation. The functional annotation of upregulated genes highlighted that mucus production and cell maintenance may account for the influence of Hfe on epithelium integrity and luminal iron uptake.

Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways.

In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal-epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases.

PPARγ is functionally expressed in clear cell renal cell carcinoma.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been demonstrated to exert an inhibitory effect on cell growth in several tumor models, including clear cell renal cell carcinoma (CCRCC). PPARγ has therefore been proposed to be a potential therapeutic target. Thus, the PPARγ gene must be expressed and not altered in cancer cells. We have therefore analyzed tumor specimens collected from 63 patients with CCRCC who underwent partial or total nephrectomy. The multiplex ligation-dependent probe amplification (MLPA) assay was used to detect deletions in the PPARγ gene. The majority of the tumors (48/63; 76.2%) did not present alterations. Two samples (3.2%) presented a deletion of the non-coding exon A1. Nine samples (14.3%) showed large heterozygous deletions in chromosome 3p including PPARγ. Potential mutations were analyzed by DNA sequencing of the 6 coding exons of the PPARγ gene. No mutation was found in exons 1-5. In exon 6, a silent polymorphism was detected in 14 samples (22.2%). CCRCC were found to express the PPARγ1 isoform. The expression level of PPARγ was measured by real-time quantitative PCR. A significantly reduced transcript level was associated with an elevated Fuhrman grade. Finally, we analyzed the expression of angiopoietin-like 4, a known PPARγ target gene, in CCRCC cell lines cultured in the presence of rosiglitazone, a PPARγ agonist. A strong induction was found in the 3 cell lines tested, indicating that PPARγ is functional in all these cell lines. In conclusion, we show here that PPARγ is expressed and functional in CCRCC, prerequisites for being a potential target for CCRCC treatment.