Pax and Mitf in Signalling, Eye Development and Melanoma

Simon Saule

Simon Saule Team leader Tel:

We study the genetic control of neuronal differentiation and of pigmentary differentiation. This work has two major biological applications, one relating to the development of ocular function and the other relating to the development of ocular tumours (uveal melanoma and retinoblastoma).

Mitf (for microphthalmia) is a Myc-type transcription factor (a bHLHZip factor) expressed in melanocytes and is essential for the proliferation/differentiation of these cells. Subcellular localisation of this transcription factor encoding distinct isoforms may also be important for its function. To study this point, we have prepared vectors encoding biologically active Mitf proteins fused to GFP, making it possible to study the kinetics of Mitf transport between the nucleus and the cytoplasm. GFP can be destroyed by photobleaching in a small cellular volume within the nucleus or cytoplasm, with no effect on cell viability (Fig. 1). It is then possible to measure the fluorescence corresponding to the repopulation of the bleached zone by new Mitf-GFP fusion proteins; consequently, the diffusion dynamics can be studied (fluorescence recovery after photobleaching).

Figure 1 : expérience de FRAP réalisée sur des cellules BHK21 transfectées avec un vecteur codant l’isoforme MitfA-GFP. P-GFPNF : quantité de protéine MitfA-GFP libre dans le noyau au temps t; P-GFPNB, quantité de protéine MitfA-GFP liée dans le noyau au temps t; P-GFPc: quantité de protéine MitfA-GFP le cytoplasme au temps t. Kimp, constant d’import nucléaire; Kexp, constant d’export nucléaire; Kon, constante de liaison à la chromatine; Koff, constant de dissociation de ces structures nucléaires.
Figure 1 : FRAP experiment with BHK21 cells transfected with MitfA-GFP NF(t): amount of free MITF-GFP in the nucleus at time t NB(t): amount of bound MITF-GFP in the nucleus at time t C(t): amount of MITF-GFP in the cytoplasm at time t kimp: nuclear import constant for MITF-GFP kexp: nuclear export constant for MITF-GFP kon: constant for the binding of MITF-GFP to nuclear structures koff: constant for the dissociation of MITF-GFP from nuclear structures

Uveal Melanoma (UM) is the most frequent and aggressive ocular primary tumor in adults with 6 new cases per million per year. The gene expression signature identifies two molecular classes of UM with low risk (Class 1) and high risk (Class 2) of metastasis essentially located in the liver. The use of cytogenetics, genomic and transcriptomic studies have conducted to the identification of genes more specifically associated with, and hopefully responsible for metastasis.

Our team is interested in understanding how cytosqueleton dynamics, proteases and DNA reparation processes are used to facilitate cell migration and invasion.

Our general objective has been to dissect the mechanisms leading to uveal melanoma metastasis. We have shown that phosphatase PTP4A3 known to promote cell migration and invasion in cancer is differentially expressed between the two class of UM and predictive of metastasis. It is now essential to identify the targets of this phosphatase to understand how the UM become metastatic.

Our plan is to analyze the effect of PTP4A3 on targets phosphorylation and their biochemical pathways to understand the control of cell migration and invasion.

Our specific approach to understanding metastasis development is based on tools that combine cell biology, molecular biology and developmental biology. We examine the effects of up and down regulation of genes of interest in cell migration in vitro by time lapse videomicroscopy and cell invasion using an in vivo model of chick embryo chorioallentoïd membrane to assess for uveal melanoma cells intravasation.

 

Key publications

Year of publication 2017

Malika Foy, Océane Anézo, Simon Saule, Nathalie Planque (2017 Mar 13)

PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells.

Experimental cell research : DOI : S0014-4827(17)30117-9

Year of publication 2015

Ewa Kotula, Nathalie Berthault, Celine Agrario, Marie-Christine Lienafa, Anthony Simon, Florent Dingli, Damarys Loew, Vonick Sibut, Simon Saule, Marie Dutreix (2015 May 29)

DNA-PKcs plays role in cancer metastasis through regulation of secreted proteins involved in migration and invasion.

Cell cycle (Georgetown, Tex.) : 1961-72 : DOI : 10.1080/15384101.2015.1026522

Year of publication 2013

Selma Maacha, Nathalie Planque, Cécile Laurent, Caterina Pegoraro, Océane Anezo, Frédérique Maczkowiak, Anne H Monsoro-Burq, Simon Saule (2013 Dec 23)

Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

PloS one : e84717 : DOI : 10.1371/journal.pone.0084717

Year of publication 2011

Jérôme Couturier, Simon Saule (2011 Oct 21)

Genetic determinants of uveal melanoma.

Developments in ophthalmology : 150-65 : DOI : 10.1159/000328270
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 (2011 Feb 1)

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

Nature : 94-8 : DOI : 10.1038/nature10539

Year of publication 2010

Cécile Laurent, Fabien Valet, Nathalie Planque, Licia Silveri, Selma Maacha, Océane Anezo, Philippe Hupe, Corine Plancher, Cécile Reyes, Benoit Albaud, Audrey Rapinat, David Gentien, Jérôme Couturier, Xavier Sastre-Garau, Laurence Desjardins, Jean-Paul Thiery, Sergio Roman-Roman, Bernard Asselain, Emmanuel Barillot, Sophie Piperno-Neumann, Simon Saule (2010 Dec 6)

High PTP4A3 phosphatase expression correlates with metastatic risk in uveal melanoma patients.

Cancer research : 666-74 : DOI : 10.1158/0008-5472.CAN-10-0605
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