Supervisor: Dr. Isabelle Pirson
Melanoma is one of the most metastatic and multidrug resistant cancer making it the first leading cause of death from skin tumors. In melanoma, the most frequent mutations in BRAF and NRAS genes constitutively activate the MAPK pathway and tumor initiation. A key regulator of this pathway, the kinase MLK3, can activate the MAPKs and promote proliferation and invasion of various cancer cell types. MLK3 has been proposed to be implicated in resistance to BRAF-inhibitors treatments in human melanoma and proposed as a modulator of melanoma cell invasion. Despite evidence of its involvement in cancer progression, the precise mechanisms of upstream regulation of MLK3 in melanoma are not well understood while of main interest for therapeutic purposes.
The project will aim to identify MLK3 modulators in melanoma upon drug treatment by
(1) Identifying the proximal MLK3 interactors through BioID assay in drug treated and resistant melanoma cell lines
(2) Analyzing molecular regulations of endogenous MLK3 upon drug treatment
(3) Confirming the impact of the MLK3 modulator(s) identified on drug sensitivity in human melanoma cell lines and zebrafish xenograft.
Aim 1 – In human melanoma cell lines, the expression of MLK3 fused to TurboID will allow to label the proximal interactor by biotinylation. They will be pulled-down and identify by mass spectrometry (done by the platform in the Institute). The techniques used in the lab that the student will have the opportunity to learn: molecular cloning – cell culture – viral infection – protein pull-down – MS results analysis.
Aim 2 – The regulation of MLK3 by phosphorylation and/or ubiquitination will be studied in human melanoma cells with or without treatment with the drugs. Point mutagenesis for the posttranslational modifications identified will be performed to confirm MLK3 modulation. The techniques: cell culture – proteins purification – western blot – immunoprecipitation – molecular biology.
Aim 3 – Using siRNA/shRNA the identified interactors of MLK3 will be knocked-down in melanoma cells to evaluate their role in melanoma drug sensitivity in vitro (cell culture) and in vivo (transplantation) in zebrafish that emerged as
a major model for studying cancer initiation/progression and drug discovery e.g. in melanoma studies. The techniques: cell culture – growth and migration invasion tests on cells – zebrafish handling – tumor cell xenotransplantation.
The student will benefit from close collaboration with another PhD student in our team pursuing a project using similar techniques focused on studying the role a RhoB GTPase effector in melanoma. We have in hands the tools to pursue actively this exciting project and all the equipment, skills and expertise to conduct the research with success are available in our Institute. Multiple meetings are organized : at the team or lab level to improve your presentation skills and discuss the technical aspects of your work and in the Faculty where international speakers are invited to present each week very high impact research stories. You are welcome to join our group!