Supervisor: Dr. Isabelle Pirson
PIRSON project 1: MLK3 modulators in melanoma
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 modulate the MAPK pathways in context-dependent manner promoting apoptosis or proliferation, tumor invasion or growth. 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 and its downstream targets in melanoma are not well understood while of main interest for therapeutic purposes.
The project will aim to identify MLK3 modulators in melanoma by (1) Identifying the proximal MLK3 interactors through BioID assay in drug treated and non-treated melanoma cell lines (2) Analyzing MLK3 posttranslational modifications in melanoma cells (3) Characterize the impact of homozygous deletion of the protein and its overexpression on melanoma development/progression in vivo using zebrafish as a model
Aim 1 – In human melanoma cell lines, the expression of MLK3 fused to TurboID will allow to label the proximal interactors by biotinylation. They will be pulled-down and identified by mass spectrometry (MS platform in the Institute). Techniques used in the lab that the student will have the opportunity to learn: molecular cloning – cell culture – viral infection – proteic 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 – In zebrafish, melanoma will be induced in MLK3KO fishes compared to wt to evaluate tumor growth and metastasis. Primary tumors of the different genotypes will be studied morphologically. The techniques used in the lab that the student will have the opportunity to learn: fish raising and crossing– tumor induction – histology – FACS sorting – RNA seq.
The student will benefit from close collaboration with another PhD student 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 highimpact research stories. You are welcome to join our group!
PIRSON project 2 : Role of a RhoGTPase regulator in the progression of melanoma
RhoGTPases regulate cytoskeleton dynamics and vesicular trafficking and play key roles in cancer development and metastasis through activation of specific effectors. One of these effectors is overexpressed in various human cancers and promotes their survival but less is known about its molecular mechanisms of action. Zebrafish has emerged as a major model for studying developmental processes and cancer development. The objective of the present project is to use zebrafish as a model, to evaluate the role of a RhoGTPase regulator in the progression of melanoma, one of the most metastatic and aggressive human cancer.
The project will aim to decipher the role of this effector in melanoma in two aims: (1) evaluate its role on cell migration, invasion, proliferation and apoptosis in human melanoma cell lines through Knock-down and overexpression systems (2) characterize the impact of homozygous deletion of the protein and its overexpression on melanoma development/progression in vivo using zebrafish as a model
Aim 1 – In human melanoma cell lines, the expression of the protein will be modulated by shRNA stable expression or CRISPR/cas9 technology to analyze the proliferative and migratory cell behaviour. The techniques used in the lab that the student will have the opportunity to learn: molecular cloning – cell culture – viral infection– proliferation assays – migration/invasion assays- western blot.
Aim 2 – In zebrafish, to induce melanoma in already available KO fish compared to wt to evaluate the role of the RhoGTPase regulator in tumor growth and metastasis. Primary tumors of the different genotypes will be studied morphologically. The tumors will be transplanted in wt fish to study the metastatic tendency. Our leading experiment suggested that the wt tumor grow faster than KO one. Now we will address the reason of this by bulk, scRNAseq and histology.
The techniques used in the lab that the student will have the opportunity to learn: fish raising and crossing – histology – RNA preparation and RNAseq analysis. The student will benefit from close collaboration with another PhD student in our team pursuing a near project using similar techniques. Some of the techniques will be handled in close collaboration with the plateforms in the Faculty (MS, FACS, Imaging, RNAseq..). 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!