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Relevant functions of DeUBiquitinases (DUBs) in phenotypic plasticity and migration of therapy-resistant melanoma

ABG-106433 Stage master 2 / Ingénieur 4 mois 500
22/06/2022
The Mediterranean Center for Molecular Medicine (C3M), Nice, France
Nice Provence-Alpes-Côte d'Azur France
  • Biologie
  • Biochimie
skin cancer, melanoma, migration, invasion, plasticity, therapie
24/12/2022

Établissement recruteur

The Mediterranean Center for Molecular Medicine (C3M), created in January 2008, is a joint research unit under the governance of Inserm (National Institute of Health and Medical Research) and Université Côte d'Azur (UCA). This research centre is dedicated to translational research into cancer, cardiometabolic diseases and infectious diseases. The association of researchers and clinicians working in C3M creates a synergy based on a common project at the interface between fundamental resarch and clinical research. The C3M is made up of thirteen research teams that maintain fruitful collaborations with doctors and clinicians from a dozen clinical departments of the Nice University hospitals.

Description

This internship will be supervised by Inserm researcher Mickael Ohanna (33 publications, H-Index 20, including Nature cell biology, Nature communication, Cancer Research, Genes and Development). 

Equipe d’accueil : Equipe 11 ‘’Microenvironnement, Signalisation et Cancer’’ (UCA, Inserm U1065 / C3M)

Tuteur: Dr Ohanna Mickael (CRCN, Inserm) /  Tel : 04 89 15 38 53

 

Cell migration is a fundamental process during embryonic development, immune surveillance, and wound healing, while alterations in migratory programs have been linked to cancer metastasis. In the context of cutaneous melanoma, which is the most aggressive form of skin cancer, its metastatic spread to other secondary sites and its growth into vital organs contribute to the mortality of melanoma patients. Although immunotherapy and targeted therapies (MAPK pathway inhibitors) have radically changed the management of metastatic melanoma, offering new hope, however, after a regression phase, progressive signs of tumor growth can be detected several months after the first administration of treatment due to the development of drug resistance. Thus, the treatment of metastasis remains a major challenge in the management of melanoma.

To metastasize, melanoma cells residing in the skin must travel great distances to reach vital organs. This is often accompanied by phenotypic changes with increased cell motility, migration and invasiveness. Melanoma cells have been shown to be particularly plastic in their ability to differentiate into several distinct, reversible and stable phenotypic cell states using a variety of molecular adaptation mechanisms, such as aberrant activation or/and acquisition of proteins involved in the mesenchymal-like migratory phenotype (Snail/Slug, PRRX1, ZEB1, Twist1, MITF, VIM, FN) from initiation and progression to metastasis and treatment resistance (Pedri D et al FEBS 2021). Our work shows that this mesenchymal phenotypic adaptation of melanoma cells adjusts both as a function of metabolism, physical and structural cues from the tumor environment and in response to drug treatments (Ohanna M et al Gene Dev 2018, Berestjuk I et al EMBO Mol Med 2022, Girard A et al Cancer Res 2020). Thus understanding how this dynamic change in mesenchymal migratory characteristics of tumor cells, not only explains the benefits and mechanisms by which tumor cell disseminate, escape therapy, but offers a therapeutic window to provide durable responses in melanoma treatment. 

Among the leads, the markers responsible for this phenotypic reprogramming such as the major transcriptional factors Snail/Slug, ZEB1/ZEB2, MITF and Twist1 are known to be extremely labile proteins and their protein levels are tightly controlled by the enzymes involved in ubiquitination (E3 ligases) and deubiquitination (DUBs). To date, no DUBs involved and essential for the maintenance of the mesenchymal phenotype related to therapeutic resistance have been identified in melanoma. Because these DUBs are putative drug targets, their identification and characterization in this process of mesenchymal phenotype adaptation will allow to propose new and durable therapeutic targets in combination with current therapies. Thus, using a library of siRNAs (Short Interfering RNA) targeting DUBs, we have identified those involved in the disruption of migration of melanoma cells resistant to inhibitors of the MAPK pathway. 

Thus, the objective of this thesis is to characterize and integrate deubiquitinating enzymes (DUBs) and their substrates in the processes related to mesenchymal phenotypic plasticity of melanoma cells resistant to targeted therapies. This approach will identify novel post-translational molecular mechanisms responsible for both metastatic dissemination and development of resistance.

Objectives: 

W1 - Investigate and validate the biological role of identified DUBs on migration/invasion of resistant melanoma cells: this study uses genetic (loss and overexpression) and functional experiments on various highly or poorly metastatic and treatment-resistant melanoma cell lines based on cell migration/invasion promoting approaches (Scratch assay and migration and invasion chamber experiments). The clinical relevance (survival in patients, in tumor progression in patients) and disruption of functional enrichment (GSEA) of the identified DUB will be confronted to public databases by bioinformatics analysis (cbioportal,GEO)

W2- Determine the molecular mechanisms related to the modulation of mesenchymal migration characteristics of resistant melanoma cells: Evaluate the modulation of selected DUB (loss and overexpression) and the consequences on cellular parameters (cell morphology, directionality ratio, Speeds, displacement) and mesenchymal migration characteristics such as expression of β-catenin markers, vimentin (VIM), fibronectin (FN) as well as associated signaling pathways (pAKT, TGFB-b, NF-KB, Smad), transcription factors (ZEB1, Snail, MITF, Slug and Twist) and matrix metalloproteinases (MMP) such as MMP2, MMP7 and MMP9

W3- Identification, validation and functional integration of the substrate: the identification of the substrate of the selected DUB will be done by immunoprecipitation and co-IP in association with the establishment of a molecular and functional enrichment network (mass spectrometry, cytoscape), linked to the characteristics of the mesenchymal migration

W4- Involvement of the selected DUB in the process of resistance to treatment linked to mesenchymal migratory adaptation in vitro and in vivo: this part will be done using cellular and pharmacological approaches (cell death & viability/BRAF signaling inhibitor) and experimental in mice (study of the tumor capacity of lines lacking the targeted DUB (sh-DUB) in the presence or absence of the treatment) 

The internship will be done in the team "Microenvironment, Signaling and Cancer" under the direction of Dr Marcel Deckert (DR Inserm). The team is composed of 4 Inserm researchers, 1 CNRS researcher, 1 post-doctoral fellow, 4 PhD students and 2 engineers, within the C3M (Centre Méditerranéen de Médecine Moléculaire).

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Profil

This offer is designed for University students interested in a Master 1 or Master 2 degree, essentially students in the Health biology curriculum. 

Profile sought:
- Motivated, autonomous, dynamic, with a positive and sharing attitude as a team member 
- Theoretical knowledge of the techniques covered: classical techniques of cell biology (cell culture, transfection, siRNA) and molecular biology (RT-PCR), biochemistry (Western Blotting) Skills for the job:
- Organize work to meet schedules and deadlines
- Ability to use standard calculation and statistical software.
- Participation in meetings and scientific monitoring
- Mastery of writing and presentation techniques (Word, PowerPoint)

Prise de fonction

27/01/2023
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