Publications by authors named "Karine Bille"

24 Publications

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Single-cell RNA sequencing reveals intratumoral heterogeneity in primary uveal melanomas and identifies HES6 as a driver of the metastatic disease.

Cell Death Differ 2021 Jan 18. Epub 2021 Jan 18.

Université Côte d'Azur, Nice, France.

Intratumor heterogeneity has been recognized in numerous cancers as a major source of metastatic dissemination. In uveal melanomas, the existence and identity of specific subpopulations, their biological function and their contribution to metastasis remain unknown. Here, in multiscale analyses using single-cell RNA sequencing of six different primary uveal melanomas, we uncover an intratumoral heterogeneity at the genomic and transcriptomic level. We identify distinct transcriptional cell states and diverse tumor-associated populations in a subset of the samples. We also decipher a gene regulatory network underlying an invasive and poor prognosis state driven in part by the transcription factor HES6. HES6 heterogenous expression has been validated by RNAscope assays within primary human uveal melanomas, which further unveils the existence of these cells conveying a dismal prognosis in tumors diagnosed with a favorable outcome using bulk analyses. Depletion of HES6 impairs proliferation, migration and metastatic dissemination in vitro and in vivo using the chick chorioallantoic membrane assay, demonstrating the essential role of HES6 in uveal melanomas. Thus, single-cell analysis offers an unprecedented view of primary uveal melanoma heterogeneity, identifies bona fide biomarkers for metastatic cells in the primary tumor, and reveals targetable modules driving growth and metastasis formation. Significantly, our findings demonstrate that HES6 is a valid target to stop uveal melanoma progression.
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http://dx.doi.org/10.1038/s41418-020-00730-7DOI Listing
January 2021

ITGBL1 is a new immunomodulator that favors development of melanoma tumors by inhibiting natural killer cells cytotoxicity.

Mol Cancer 2021 01 7;20(1):12. Epub 2021 Jan 7.

Université Nice Côte d'Azur, INSERM U1065, Team1 Biology and pathologies of melanocytes. Equipe labellisée ARC 2019, 06000, Nice, France.

Resistances to immunotherapies remains a major hurdle towards a cure for melanoma in numerous patients. An increase in the mesenchymal phenotype and a loss of differentiation have been clearly associated with resistance to targeted therapies. Similar phenotypes have been more recently also linked to resistance to immune checkpoint therapies. We demonstrated here that the loss of MIcrophthalmia associated Transcription Factor (MITF), a pivotal player in melanocyte differentiation, favors the escape of melanoma cells from the immune system. We identified Integrin beta-like protein 1 (ITGBL1), a secreted protein, upregulated in anti-PD1 resistant patients and in MITF melanoma cells, as the key immunomodulator. ITGBL1 inhibited immune cell cytotoxicity against melanoma cells by inhibiting NK cells cytotoxicity and counteracting beneficial effects of anti-PD1 treatment, both in vitro and in vivo. Mechanistically, MITF inhibited RUNX2, an activator of ITGBL1 transcription. Interestingly, VitaminD3, an inhibitor of RUNX2, improved melanoma cells to death by immune cells. In conclusion, our data suggest that inhibition of ITGBL1 might improve melanoma response to immunotherapies.
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http://dx.doi.org/10.1186/s12943-020-01306-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789764PMC
January 2021

Regulation of Melanogenesis by the Amino Acid Transporter SLC7A5.

J Invest Dermatol 2020 Nov 30;140(11):2253-2259.e4. Epub 2020 Mar 30.

Université Nice Côte d'Azur, C3M, INSERM, U1065, Biology and pathologies of melanocytes, Nice, France. Electronic address:

Integration of chromatin immunoprecipitation-sequencing and microarray data enabled us to identify previously unreported MITF-target genes, among which the amino acid transporter SLC7A5 is also included. We reported that small interfering RNA-mediated SLC7A5 knockdown decreased pigmentation in B16F10 cells but neither affected morphology nor dendricity. Treatment with the SLC7A5 inhibitors 2-amino-2-norbornanecarboxylic acid (BCH) or JPH203 also decreased melanin synthesis in B16F10 cells. Our findings indicated that BCH was as potent as reference depigmenting agent, kojic acid, but acted through a different pathway not affecting tyrosinase activity. BCH also decreased pigmentation in human MNT1 melanoma cells or normal human melanocytes. Finally, we tested BCH on a more physiological model, using reconstructed human epidermis and confirmed a strong inhibition of pigmentation, revealing the clinical potential of SLC7A5 inhibition and positioning BCH as a depigmenting agent suitable for cosmetic or dermatological intervention in hyperpigmentation diseases.
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http://dx.doi.org/10.1016/j.jid.2020.03.941DOI Listing
November 2020

Lysosomal acid ceramidase ASAH1 controls the transition between invasive and proliferative phenotype in melanoma cells.

Oncogene 2019 02 25;38(8):1282-1295. Epub 2018 Sep 25.

Team 1, Biology and Pathologies of Melanocytes, Equipe labellisée ARC 2015, Université Côte d'Azur, Inserm U1065, C3M, Nice, France.

Phenotypic plasticity and subsequent generation of intratumoral heterogeneity underly key traits in malignant melanoma such as drug resistance and metastasis. Melanoma plasticity promotes a switch between proliferative and invasive phenotypes characterized by different transcriptional programs of which MITF is a critical regulator. Here, we show that the acid ceramidase ASAH1, which controls sphingolipid metabolism, acted as a rheostat of the phenotypic switch in melanoma cells. Low ASAH1 expression was associated with an invasive behavior mediated by activation of the integrin alphavbeta5-FAK signaling cascade. In line with that, human melanoma biopsies revealed heterogeneous staining of ASAH1 and low ASAH1 expression at the melanoma invasive front. We also identified ASAH1 as a new target of MITF, thereby involving MITF in the regulation of sphingolipid metabolism. Together, our findings provide new cues to the mechanisms underlying the phenotypic plasticity of melanoma cells and identify new anti-metastatic targets.
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http://dx.doi.org/10.1038/s41388-018-0500-0DOI Listing
February 2019

Pivotal role of NAMPT in the switch of melanoma cells toward an invasive and drug-resistant phenotype.

Genes Dev 2018 03 22;32(5-6):448-461. Epub 2018 Mar 22.

U1065, Institut National de la Santé et de la Recherche Médicale (INSERM), Biology and Pathologies of Melanocytes, Equipe Labellisée L'Association pour la Recherche sur le Cancer (ARC) 2015, Université Nice Côte d'Azur, INSERM, Centre Méditerranéen de Médecine Moléculaire (C3M), 06204 Nice, France.

In BRAF melanoma cells, a global metabolomic analysis discloses a decrease in nicotinamide adenine dinucleotide (NAD) levels upon PLX4032 treatment that is conveyed by a STAT5 inhibition and a transcriptional regulation of the nicotinamide phosphoribosyltransferase (NAMPT) gene. NAMPT inhibition decreases melanoma cell proliferation both in vitro and in vivo, while forced NAMPT expression renders melanoma cells resistant to PLX4032. NAMPT expression induces transcriptomic and epigenetic reshufflings that steer melanoma cells toward an invasive phenotype associated with resistance to targeted therapies and immunotherapies. Therefore, NAMPT, the key enzyme in the NAD salvage pathway, appears as a rational target in targeted therapy-resistant melanoma cells and a key player in phenotypic plasticity of melanoma cells.
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http://dx.doi.org/10.1101/gad.305854.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900716PMC
March 2018

Deciphering the Role of Oncogenic MITFE318K in Senescence Delay and Melanoma Progression.

J Natl Cancer Inst 2017 08;109(8)

INSERM, U1065 (équipe 1), Equipe labélisée ARC 2016, C3M, Nice, France.

Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation ( MITFE318K ) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF E318K remained uncharacterized.

Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n = 3) or E318K (n = 4) MITF donors. We also used a newly generated Mitf E318K knock-in (KI) mouse model to assess the role of Mitf E318K (n = 7 to 13 mice per group) in tumor development in vivo and performed transcriptomic analysis of the tumors to identify the molecular mechanisms. Finally, using immortalized or normal melanocytes (wild-type or E318K MITF, n = 2 per group), we assessed the role of MITF E318K on the induction of senescence mediated by BRAF V600E . All statistical tests were two-sided.

Results: We demonstrated a decrease in endogenous MITF SUMOylation in melanocytes from MITF E318K patients (mean of cells with hypoSUMOylated MITF, MITF E318K vs MITF WT , 94% vs 44%, difference = 50%, 95% CI = 21.8% to 67.2%, P  = .004). The Mitf E318K mice were slightly hypopigmented (mean melanin content Mitf WT vs Mitf E318K/+ , 0.54 arbitrary units [AU] vs 0.36 AU, difference = -0.18, 95% CI = -0.36 to -0.007, P  = .04). We provided genetic evidence that Mitf E318K enhances BRaf V600E -induced nevus formation in vivo (mean nevus number for Mitf E318K , BRaf V600E vs Mitf WT , BRaf V600E , 68 vs 44, difference = 24, 95% CI = 9.1 to 38.9, P  = .006). Importantly, although Mitf E318K was not sufficient to cooperate with BRaf V600E alone in promoting metastatic melanoma, it accelerated tumor formation on a BRaf V600E , Pten-deficient background (median survival, Mitf E318K/+  = 42 days, 95% CI = 31 to 46 vs Mitf WT  = 51 days, 95% CI = 50 to 55, P  < .001). Transcriptome analysis suggested a decrease in senescence in tumors from Mitf E318K mice. We confirmed this hypothesis by in vitro experiments, demonstrating that Mitf E318K impaired the ability of human melanocytes to undergo BRAF V600E -induced senescence.

Conclusions: We characterized the functions of melanoma-associated MITF E318K mutations. Our results demonstrate that MITF E318K reduces the program of senescence to potentially favor melanoma progression in vivo.
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http://dx.doi.org/10.1093/jnci/djw340DOI Listing
August 2017

SIRT1 promotes proliferation and inhibits the senescence-like phenotype in human melanoma cells.

Oncotarget 2014 Apr;5(8):2085-95

INSERM, U1065 (équipe 1), Equipe labélisée Ligue Contre le Cancer, C3M, Nice, France.

SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF-induced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAF(V600E)-mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAF(V600E)-mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039147PMC
http://dx.doi.org/10.18632/oncotarget.1791DOI Listing
April 2014

Secretome from senescent melanoma engages the STAT3 pathway to favor reprogramming of naive melanoma towards a tumor-initiating cell phenotype.

Oncotarget 2013 Dec;4(12):2212-24

Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Equipe 1, Biologie et pathologies des mélanocytes: de la pigmentation cutanée au mélanome. Equipe labellisée Ligue 2013, Nice, F-06204, France.

Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG. This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features. The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development. We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties. Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naïve melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926821PMC
http://dx.doi.org/10.18632/oncotarget.1143DOI Listing
December 2013

Aurora B is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway and is a valuable potential target in melanoma cells.

J Biol Chem 2012 Aug 5;287(35):29887-98. Epub 2012 Jul 5.

Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Equipe 1, Biologie et Pathologies des Mélanocytes de la Pigmentation Cutanée au Mélanome, Nice F-06204, France.

Metastatic melanoma is a deadly skin cancer and is resistant to almost all existing treatment. Vemurafenib, which targets the BRAFV600E mutation, is one of the drugs that improves patient outcome, but the patients next develop secondary resistance and a return to cancer. Thus, new therapeutic strategies are needed to treat melanomas and to increase the duration of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor response. The ERK pathway controls cell proliferation, and Aurora B plays a pivotal role in cell division. Here, we confirm that Aurora B is highly expressed in metastatic melanoma cells and that Aurora B inhibition triggers both senescence-like phenotypes and cell death in melanoma cells. Furthermore, we show that the BRAF/ERK axis controls Aurora B expression at the transcriptional level, likely through the transcription factor FOXM1. Our results provide insight into the mechanism of Aurora B regulation and the first molecular basis of Aurora B regulation in melanoma cells. The inhibition of Aurora B expression that we observed in vemurafenib-sensitive melanoma cells was rescued in cells resistant to this drug. Consistently, these latter cells remain sensitive to the effect of the Aurora B inhibitor. Noteworthy, wild-type BRAF melanoma cells are also sensitive to Aurora B inhibition. Collectively, our findings, showing that Aurora B is a potential target in melanoma cells, particularly in those vemurafenib-resistant, may open new avenues to improve the treatment of metastatic melanoma.
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http://dx.doi.org/10.1074/jbc.M112.371682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436153PMC
August 2012

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma.

Authors:
Corine Bertolotto Fabienne Lesueur Sandy Giuliano Thomas Strub Mahaut de Lichy Karine Bille Philippe Dessen Benoit d'Hayer Hamida Mohamdi Audrey Remenieras Eve Maubec Arnaud de la Fouchardière Vincent Molinié Pierre Vabres Stéphane Dalle Nicolas Poulalhon Tanguy Martin-Denavit Luc Thomas Pascale Andry-Benzaquen Nicolas Dupin Françoise Boitier Annick Rossi Jean-Luc Perrot Bruno Labeille Caroline Robert Bernard Escudier Olivier Caron Laurence Brugières Simon Saule Betty Gardie Sophie Gad Stéphane Richard Jérôme Couturier Bin Tean Teh Paola Ghiorzo Lorenza Pastorino Susana Puig Celia Badenas Hakan Olsson Christian Ingvar Etienne Rouleau Rosette Lidereau Philippe Bahadoran Philippe Vielh Eve Corda Hélène Blanché Diana Zelenika Pilar Galan François Aubin Bertrand Bachollet Céline Becuwe Pascaline Berthet Yves Jean Bignon Valérie Bonadona Jean-Louis Bonafe Marie-Noëlle Bonnet-Dupeyron Fréderic Cambazard Jacqueline Chevrant-Breton Isabelle Coupier Sophie Dalac Liliane Demange Michel d'Incan Catherine Dugast Laurence Faivre Lynda Vincent-Fétita Marion Gauthier-Villars Brigitte Gilbert Florent Grange Jean-Jacques Grob Philippe Humbert Nicolas Janin Pascal Joly Delphine Kerob Christine Lasset Dominique Leroux Julien Levang Jean-Marc Limacher Cristina Livideanu Michel Longy Alain Lortholary Dominique Stoppa-Lyonnet Sandrine Mansard Ludovic Mansuy Karine Marrou Christine Matéus Christine Maugard Nicolas Meyer Catherine Nogues Pierre Souteyrand Laurence Venat-Bouvet Hélène Zattara Valérie Chaudru Gilbert M Lenoir Mark Lathrop Irwin Davidson Marie-Françoise Avril Florence Demenais Robert Ballotti Brigitte Bressac-de Paillerets

Nature 2011 Oct 19;480(7375):94-8. Epub 2011 Oct 19.

1] INSERM, U895 (équipe 1), Equipe labélisée Ligue Contre le Cancer, C3M, 06204 Nice, France [2] Université of Nice Sophia-Antipolis, UFR Médecine, 06204 Nice, France [3] Centre Hospitalier Universitaire de Nice, Service de Dermatologie, 06204 Nice, France [4].

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.
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http://dx.doi.org/10.1038/nature10539DOI Listing
October 2011

Senescent cells develop a PARP-1 and nuclear factor-{kappa}B-associated secretome (PNAS).

Genes Dev 2011 Jun 6;25(12):1245-61. Epub 2011 Jun 6.

Biologie et Pathologies des Mélanocytes de la Pigmentation Cutanée au Mélanome, Equipe labellisée Ligue Nationale contre le Cancer, Nice, France.

Melanoma cells can enter the process of senescence, but whether they express a secretory phenotype, as reported for other cells, is undetermined. This is of paramount importance, because this secretome can alter the tumor microenvironment and the response to chemotherapeutic drugs. More generally, the molecular events involved in formation of the senescent-associated secretome have yet to be determined. We reveal here that melanoma cells experiencing senescence in response to diverse stimuli, including anti-melanoma drugs, produce an inflammatory secretory profile, where the chemokine ligand-2 (CCL2) acts as a critical effector. Thus, we reveal how senescence induction might be involved in therapeutic failure in melanoma. We further provide a molecular relationship between senescence induction and secretome formation by revealing that the poly(ADP-ribose) polymerase-1 (PARP-1)/nuclear factor-κB (NF-κB) signaling cascade, activated during senescence, drives the formation of a secretome endowed with protumoral and prometastatic properties. Our findings also point to the existence of the PARP-1 and NF-κB-associated secretome, termed the PNAS, in nonmelanoma cells. Most importantly, inhibition of PARP-1 or NF-κB prevents the proinvasive properties of the secretome. Collectively, identification of the PARP-1/NF-κB axis in secretome formation opens new avenues for therapeutic intervention against cancers.
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http://dx.doi.org/10.1101/gad.625811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127427PMC
June 2011

Microphthalmia-associated transcription factor controls the DNA damage response and a lineage-specific senescence program in melanomas.

Cancer Res 2010 May 13;70(9):3813-22. Epub 2010 Apr 13.

Institut National de la Sante et de la Recherche Medicale U895, University of Nice Sophia-Antipolis, Nice, France.

Apoptosis and senescence are cellular failsafe programs that counteract excessive mitogenic signaling observed in cancer cells. Melanoma is known for its notorious resistance to apoptotic processes; therefore, senescence, which remains poorly understood in melanomas, can be viewed as a therapeutic alternative. Microphthalmia-associated transcription factor (MITF), in which its M transcript is specifically expressed in melanocyte cells, plays a critical role in melanoma proliferation, and its specific inhibition is associated with G(0)-G(1) growth arrest. Interestingly, decreased MITF expression has been described in senescent melanocytes, and we have observed an inhibition of MITF expression in melanoma cells exposed to chemotherapeutic drugs that induce their senescence. All these observations thereby question the role of MITF in controlling senescence in melanoma cells. Here, we report that long-term depletion of MITF in melanoma cells triggers a senescence program characterized by typical morphologic and biochemical changes associated with a sustained growth arrest. Further, we show that MITF-silenced cells engage a DNA damage response (DDR) signaling pathway, leading to p53 upregulation, which is critically required for senescence entry. This study uncovers the existence of a lineage-restricted DDR/p53 signaling pathway that is inhibited by MITF to prevent senescence and favor melanoma cell proliferation.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2913DOI Listing
May 2010

{alpha}MSH and Cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism.

J Biol Chem 2009 Jul 22;284(28):18699-706. Epub 2009 Apr 22.

From INSERM, U895, Centre Méditerranéen de Médecine Moléculaire (C3M), Equipe 1, Biology and Pathologies of Melanocytes, F-06204 Nice Cedex 3, France.

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.
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http://dx.doi.org/10.1074/jbc.M109.005819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707200PMC
July 2009

Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport.

J Biol Chem 2008 May 15;283(18):12635-42. Epub 2008 Feb 15.

INSERM U895, Biologie et Pathologies des Cellules Mélanocytaires de la Pigmentation Cutanée au Mélanome 28, avenue de Valombrose, 06107 Nice Cedex 2, France.

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field.
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http://dx.doi.org/10.1074/jbc.M800130200DOI Listing
May 2008

IGF1 promotes resistance to apoptosis in melanoma cells through an increased expression of BCL2, BCL-X(L), and survivin.

J Invest Dermatol 2008 Jun 13;128(6):1499-505. Epub 2007 Dec 13.

Institut National de la Santé et de la Recherche Médicale U597, Biologie et Pathologie des cellules mélanocytaires: de la pigmentation cutanée au mélanome, Nice, France.

IGF1 plays a key role in the development and growth of multiple tumors and in the prevention of apoptosis. In melanoma cells, IGF1 has been shown to mediate resistance to anoikis-induced apoptosis. However, the effect of IGF1 on other proapoptotic stimuli has never been reported. Further, the molecular mechanisms by which IGF1 mediates its prosurvival properties in melanoma cells remain unknown. Here, we demonstrate that IGF1 impairs the onset of tumor necrosis factor-related apoptosis-inducing ligand and staurosporine-induced apoptosis in melanoma cells expressing either wild-type or oncogenic B-Raf. Further, we show that IGF1 inhibits mitochondrial damage that occurs during apoptosis, thereby indicating that IGF1 acts at the level of mitochondria to mediate its antiapoptotic stimuli. Accordingly, IGF1 increases the mRNA levels and protein expression of antiapoptotic members of the BCL2 family--BCL2 and BCL-X(L)--and that of the inhibitor of apoptosis protein, survivin. Further, their specific silencing by small interfering RNA prevents the protective effect of IGF1. These findings therefore delineate the molecular mechanisms by which IGF1 mediates its prosurvival properties and provide a basis for clinical strategies designed to neutralize IGF1 or its target genes.
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http://dx.doi.org/10.1038/sj.jid.5701185DOI Listing
June 2008

Up-regulation of MET expression by alpha-melanocyte-stimulating hormone and MITF allows hepatocyte growth factor to protect melanocytes and melanoma cells from apoptosis.

J Biol Chem 2007 May 19;282(19):14140-7. Epub 2007 Mar 19.

INSERM, U597, Biologie et Pathologies des Cellules Mélanocytaires: de la Pigmentation Cutanée au Mélanome, Equipe labellisée par la Ligue Nationale contre le Cancer, Nice Cedex 2, France.

The MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. In mouse melanoma cells, Met expression is increased by alphaMSH via the activation of the cAMP pathway. However, the mechanism by which cAMP regulates MET and the biological consequences of this increase were not known. In the present report, we show that alphaMSH regulates MET expression in both human melanocytes and mouse melanoma cells through a transcriptional mechanism that requires MITF. Furthermore, the adenovirus driven expression of MITF is sufficient to increase MET in melanoma cells. Functional analysis of the MET promoter allows us to identify an E-box motif conserved in both human and mouse promoter that mediates the effect of MITF. Interestingly, up-regulation of MET expression by cAMP leads to an exacerbated HGF signaling and allows HGF to protect melanocytes and melanoma cells from apoptosis. Thus, physiological stimuli or pathological events that would induce MITF expression may lead to increased MET expression thereby favoring melanoma survival. These observations strengthen the roles of MITF and MET in melanoma development.
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http://dx.doi.org/10.1074/jbc.M611563200DOI Listing
May 2007

The cleavage of microphthalmia-associated transcription factor, MITF, by caspases plays an essential role in melanocyte and melanoma cell apoptosis.

Genes Dev 2005 Sep;19(17):1980-5

INSERM U597, Biologie et Pathologie des cellules mélanocytaires: de la pigmentation cutanée aux mélanomes, Ligue Nationale contre le Cancer, Equipe labellisée 2001, 06107 NICE Cedex 2, France.

Microphthalmia-associated transcription factor (MITF) M-form is a melanocyte-specific transcription factor that plays a key role in melanocyte development, survival, and differentiation. Here, we identified MITF as a new substrate of caspases and we characterized the cleavage site after Asp 345 in the C-terminal domain. We show that expression of a noncleavable form of MITF renders melanoma cells resistant to apoptotic stimuli, and we found that the C-terminal fragment generated upon caspase cleavage is endowed with a proapoptotic activity that sensitizes melanoma cells to death signals. The proapoptotic function gained by MITF following its processing by caspases provides a tissue-restricted means to modulate death in melanocyte and melanoma cells.
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http://dx.doi.org/10.1101/gad.335905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1199569PMC
September 2005

Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells.

J Cell Biol 2005 Jul 27;170(1):49-59. Epub 2005 Jun 27.

INSERM U597, Biologie et physiopathologie des cellules mélanocytaires, Faculty of Medicine, 06107 Nice cedex 2, France.

In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.
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http://dx.doi.org/10.1083/jcb.200501067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171372PMC
July 2005

Cyclic AMP promotes a peripheral distribution of melanosomes and stimulates melanophilin/Slac2-a and actin association.

FASEB J 2004 Jun 1;18(9):989-91. Epub 2004 Apr 1.

INSERM U597, Biologie et Pathologie des Celllules Mélanocytaires, Faculté de Médecine, Nice, France.

Melanosomes are melanin-containing organelles that belong to a recently individualized group of lysosome-related organelles. Recently, numerous reports have dissected the molecular mechanisms that control melanosome transport, but nothing was known about the possible regulation of melanosome distribution by exogenous physiological stimulus. In the present report, we demonstrate that a physiological melanocyte-differentiating agent such as alpha-melanocyte-stimulating hormone, through the stimulation of the cAMP pathway, induces a rapid centrifugal transport of melanosomes, leading to their accumulation at the dendrite tips of melanocytes. Interestingly, the small GTP binding proteins of the p21Rho family and one of their effectors, p160 Rho-associated kinase, but not PKA, play a key role in redistribution of melanosomes at the extremities of the dendrites. Further, we have investigated, at the molecular level, the effect of cAMP on the different proteins involved in the control of melanosome transport. We demonstrate that cAMP stimulates the expression of Rab27a and rapidly increases the interaction of the melanophilin/Slac2-a with actin. Thus, we propose that the stimulation of the interaction between melanophilin/Slac2-a and actin would allow the rapid accumulation of melanosomes in the actin-rich region of the dendrite extremities.
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http://dx.doi.org/10.1096/fj.03-1240fjeDOI Listing
June 2004

Microphthalmia associated transcription factor is a target of the phosphatidylinositol-3-kinase pathway.

J Invest Dermatol 2003 Oct;121(4):831-6

INSERM U385, Biologie et Physiopathologie de la Peau, Nice, France.

In B16 melanoma cells, cyclic adenosine monophosphate inhibits the phosphatidylinositol-3-kinase and the phosphatidylinositol-3-kinase inhibitor, LY294002, stimulates melanogenesis. However, the molecular mechanisms, by which phosphatidylinositol-3-kinase inhibition increases melanogenesis remained to be identified. In this study, we show that LY294002 up-regulates the expression of the melanogenic enzymes, tyrosinase and Tyrp1, through a transcriptional mechanism that involves microphthalmia associated transcription factor, a basic helix-loop-helix transcription factor, which plays a key role in melanocyte survival and differentiation. Further, we observe that LY294002 increases the intracellular content of microphthalmia associated transcription factor, thereby demonstrating that microphthalmia associated transcription factor is also a convergence point of the phosphatidylinositol-3-kinase signaling pathway. Finally, our results indicate that LY294002 controls microphthalmia associated transcription factor at the transcriptional level through distal regulatory element that remain to be identified. Interestingly, we have recently reported that cAMP-elevating agents, through a phosphatidylinositol-3-kinase/AKT inhibition and a glycogen synthase kinase 3beta activation, may stimulate microphthalmia associated transcription factor binding to its target sequence, suggesting that inhibition of the phosphatidylinositol-3-kinase is implicated in the stimulation of melanogenesis at different levels. Thus, the results presented in this report strengthen the importance of the phosphatidylinositol-3-kinase pathway in the regulation of melanogenesis and emphasize the complexity of the cyclic adenosine monophosphate signaling that controls melanocyte differentiation and melanogenesis.
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http://dx.doi.org/10.1046/j.1523-1747.2003.12420.xDOI Listing
October 2003

Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli syndrome.

J Biol Chem 2003 Mar 16;278(13):11386-92. Epub 2003 Jan 16.

INSERM U385, Biologie et Physiopathologie de la Peau, Faculté de Médecine, Avenue de Valombrose, 06107, Nice cedex 2, France.

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.
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http://dx.doi.org/10.1074/jbc.M211996200DOI Listing
March 2003

Glycogen synthase kinase 3beta is activated by cAMP and plays an active role in the regulation of melanogenesis.

J Biol Chem 2002 Sep 1;277(37):33690-7. Epub 2002 Jul 1.

INSERM U385, Biologie et Physiopathologie de la peau, IFR 50, 28 avenue de Valombrose, 06107 NICE Cedex 2, France.

In human and mouse, cAMP plays a key role in the control of pigmentation. cAMP, through the activation of protein kinase A, increases the expression of microphthalmia-associated transcription factor (MITF), which in turn stimulates tyrosinase gene expression, to allow melanin synthesis. Beyond this simplified scheme, cAMP inhibits phosphatidylinositol 3-kinase (PI3K), and inhibition of PI3K, by a specific inhibitor, stimulates melanogenesis. However, the link between the PI3K pathway and melanogenesis remained to be elucidated. In this report, we showed that cAMP, through a protein kinase A-independent mechanism, led to inhibition of AKT phosphorylation and activity. Consistent with the role of AKT in the regulation of glycogen synthase kinase 3beta (GSK3beta), cAMP decreased the phosphorylation of GSK3beta and stimulated its activity. Further, experiments were performed to investigate the role of GSK3beta in the regulation of MITF expression and function. We observed that GSK3beta regulated neither MITF promoter activity nor the intrinsic transcriptional activity of MITF but synergized with MITF to activate the tyrosinase promoter. Additionally, lithium, a GSK3beta inhibitor, impaired the response of the tyrosinase promoter to cAMP, and cAMP increased the binding of MITF to the M-box. Taking into account that GSK3beta phosphorylates MITF and increases the ability of MITF to bind its target sequence, our results indicate that activation of GSK3beta by cAMP facilitates MITF binding to the tyrosinase promoter, thereby leading to stimulation of melanogenesis.
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http://dx.doi.org/10.1074/jbc.M202939200DOI Listing
September 2002