Elise Bonvin

Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)beta2 was up-regulated in the poorly pigmented cells. Furthermore, TGFbeta signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis.

Transcriptome-wide distribution and function of RNA hydroxymethylcytosine

Chemical modification of RNA for function Chemical modifications play an important role in modifying and regulating the function of DNA and RNA. Delatte et al. show that, in the fruit fly, many messenger RNAs (mRNAs) contain the modified base 5-hydroxymethylcytosine (5hmC). The chemical mark is added by the same enzyme that adds 5hmC to DNA. Because many mRNAs involved in neuronal development contain 5hmC, blocking the enzyme causes brain defects and is lethal. In vivo, RNA hydroxymethylation promotes mRNA translation. Science, this issue p. 282 Posttranscriptional modification of messenger RNAs (mRNAs) is prevalent in Drosophila and promotes mRNA translation. Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout (Cftr−/−) and F508del‐CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr−/− mice (23/25) while only a few Cftr+/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr−/− mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del‐CFTR and in newborn Cftr−/− and F508del‐CFTR mice. Tracheal and ventilatory function analyses showed in Cftr−/− mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del‐CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.

Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling.

Sodium 4-phenylbutyrate (4-PBA) has attracted a great deal of attention in cystic fibrosis (CF) pathology due to its capacity to traffic ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) to the cell membrane and restore CFTR chloride function at the plasma membrane of CF lung cells in vitro and in vivo. Using two different ΔF508-CFTR lung epithelial cell lines (CFBE41o- and IB3-1 cells, characterized with ΔF508-homozygous and heterozygous genotype, respectively) in vitro, 4-PBA induced an increase of proinflammatory cytokine interleukin (IL)-8 production in a concentration-dependent manner. This 4-PBA-induced IL-8 production was associated with a strong reduction of proteasome and nuclear factor-κB transcriptional activities in the two ΔF508-CFTR lung cells either in a resting state or after tumor necrosis factor-α stimulation. In contrast, a strong increase of activator protein-1 transcriptional activity was observed. The inhibition of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and c-Jun-NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), respectively, was associated with a reduction (2–3.5-fold) of IL-8 production in both ΔF508-CFTR lung cell lines treated with 4-PBA. No significant change of IL-8 production was observed after an inhibition of p38 MAPK with 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190). Therefore, we suggest that inhibition of both ERK1/2 and JNK signaling may be a means to strongly reduce 4-PBA-induced IL-8 production in combination with 4-PBA treatment to restore CFTR Cl- channel function in lung epithelial cells of patients with CF.

A Phosphatidylinositol 3-Kinase–Pax3 Axis Regulates Brn-2 Expression in Melanoma

ABSTRACT Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/β-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.

Impact of nutrition on phenotype in CFTR-deficient mice.

To elucidate the impact of nutrition in cystic fibrosis (CF), we compared the phenotypic traits of Cftr–/– mice fed either a lipid-enriched liquid diet (Peptamen) or a standard chow combined with polyethylenglycol osmotic laxative (PEG), two strategies commonly used to prevent intestinal obstruction in CF mice. Survival, growth, liver, and ventilatory status were determined in Cftr–/– and Cftr+/+ mice, followed-up until 120 d. Ventilation was recorded in conscious animals using whole-body plethysmography. We found that the survival rate was similar in Peptamen and PEG Cftr–/– mice. Cftr–/– mice had lower minute ventilation than Cftr+/+ mice, whatever the diet. Both Cftr–/– and Cftr+/+ mice fed Peptamen displayed preadult growth delay compared with PEG-treated animals. Despite subsequent growth catch-up, Cftr–/– mice remained smaller than Cftr+/+ mice, whatever the diet. All Peptamen fed Cftr–/– mice showed hepatomegaly and liver steatosis, which also occurred but to a lesser extent in Peptamen fed Cftr+/+ animals. Therefore, while both treatment strategies are similarly efficient to avoid high mortality at weaning, Peptamen induces preadult growth delay and liver steatosis. These effects of diet are important to consider in future animal studies and also prompt to evaluate high-energy diets in CF patients.

Phosphorylation of BRN2 Modulates Its Interaction with the Pax3 Promoter To Control Melanocyte Migration and Proliferation

ABSTRACT MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS→BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.

Abnormal TRAF4 and TMEM16a expression during tracheal development in CFTR-deficient mice

Apart of CF, CFTR gene mutations have also been associated with CFTR-related disorders, such as congenital absence of the vas deferens (CAVD). The most common CAVD mutation is the IVS8(T)5 variant in CFTR gene intron 8 that alters the efficiency with which the splice acceptor is used. The aim of the present study was to analyze and compare the levels of CFTR mRNA transcripts lacking exon 9 obtained from testicular and nasal biopsies of CAVD patients. A total of 23 testicular biopsies were analyzed: 12 from CAVD and 10 from patients with conserved spermatogenesis (controls). In 4 CAVD patients, nasal biopsies were also analyzed. The relative amounts of accurately spliced transcripts (+9) and transcripts lacking exon 9 (−9) were evaluated by real-time PCR, using a multiplex based assay (TaqMan). We detected in all samples the presence of the 2 CFTR transcripts, +9 and −9. However, patients carrying IVS8(T)5 variant produced higher proportions of transcripts −9 than patients carrying the IVS8(T)7 or IVS8(T)9. Additionally, it was observed that the proportion of transcripts −9 was higher in testicular biopsies than in the nasal epithelium of the same patient. As previously reported, we confirmed that the degree of CFTR exon 9 skipping was inversely correlated with the IVS8(T)n polymorphic tract. Moreover, the level of CFTR mRNA transcripts lacking exon 9 was increased in testicular biopsies when compared with nasal biopsies from the same individual. Thus, differential efficiency between different tissues expressing CFTR may explain the reproductive tract abnormalities, such as CAVD observed in our patients, and absence of pathologic changes in other CF-associated organs.

Glucocorticoids resistance in Cystic Fibrosis

Introduction Cystic Fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasian population. The gene mutated in CF encodes a chloride channel named CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Lung dysfunction is the main cause of mortality in CF patients. Infection combined with inflammation lead to progressive destruction of the respiratory epithelium. Glucocorticoids (GC) are anti-inflammatory molecules commonly used to treat inflammation but with controversial efficiency among patients. The lack of efficiency could be due to resistance to GC already observed in others respiratory diseases. To test this hypothesis, we study the key steps in GC activation pathway in the lung to identify the molecular basis of such dysregulation. Our present studies focus on the regulation of the expression of the GR isoforms. Methods CF bronchial epithelial cell lines (IB3-1) and CF corrected (S9) were incubated with IL-1β (10ng/ml) with or without dexamethasone (dex, 1μM). Rates of secreted IL-8 were dosed by ELISA NF-κB activation was measured using NF-κB promotor coupled to luciferase. Expression of GR isoforms was measured by quantitative RT-PCR. Results In presence of IL-1β, dex can restore basal secretion of IL-8 at 16h in S9, whereas IB3-1 barely respond. Moreover, no effect of dex on NF-κB activation is observed in CF cells. There is a differential regulation of the GR isoforms expression at 16h in the two cell lines. The study of the GR activation window of activation in inflammatory conditions is currently in progress. Conclusions In this study we have shown a different regulation of inflammation by GC Our preliminary study on GR expression also suggests differences between CF and non-CF cell lines. The present results require to be assessed in other cellular models and from CF lung biopsies. We will subsequently investigate the possible dysregulation of other key steps in GC activation pathway such as GR phosphorylation and nuclear translocation.