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2-RTK signaling : structural and functional approaches (Jérôme Vicogne)

photoRTKv2

- Receptor Tyrosine Kinase (RTK) functional and structural analysis.

- Ligand-Receptor interactions.

- Role of Heparan Sulphates proteoglycans (HSPG) and Heparanase activity in HGF/MET signaling, and particularly, deciphering the role HS binding sites involved in the HGF/MET complex formation.

- Transmembrane protein synthesis and purification.

- Recombinant and total chemical protein synthesis.

- Design of micro and nano-systems for biodetection.

MET (or HGF-R), the tyrosine kinase receptor (RTK) for hepatocyte growth factor (HGF), controls cell growth, invasion and survival. Its deregulation is associated with the acquisition of tumorigenic properties but also invasive phenotype. The involvement of MET in numerous human tumors is now established and interfering with its activation is therefore a potential strategy for developing therapeutics against tumorigenesis or metastatic processes. To this end, it is essential to understand the mechanisms leading to MET activation, and in particular, the binding events taking place in the early steps of the interaction of MET with its ligand HGF.

MET is a disulfide-linked heterodimer composed of an extracellular 50 kDa alpha-chain and a transmembrane 145 kDa beta-chain. HGF is considered as a bivalent ligand containing a high affinity binding site for MET in the alpha-chain and a low affinity binding site in the beta-chain. Cooperation between the two binding sites is required for proper MET activation. The alpha-chain of HGF is constituted by a N-terminal domain followed by 4 kringle domains (K domains). The N and K1 domain (NK1) is a natural variant of HGF and is known to contain the high affinity HGF binding site for MET. Since NK1 is a potent agonist, this quaternary structure is supposed to be responsible for MET dimerization and activation induced by HGF.

Heparane sulfates from proteoglycans (HSPG) also play a critical role in HGF-MET signalling since they bind HGF but also MET. HSPG probably regulate HGF-MET signalling in promoting HGF cell surface distribution by non-specific electrostatic interactions but also in acting as co-receptors favouring MET clusters. 

The goal of our project is to study the individual role of N and K1 subdomains as well as HS in the activation of MET receptor using an innovative chemical biology approach. For this task, we are using engineered N and K1 HGF sub-domains produced by total chemical synthesis (see CBC team).

We are studying the binding properties of N and K1 constructs for various extracellular MET constructs using SPR (Surface Plasmon Resonance) and ALPHAScreenÒ technologies. The biological activities of N and K1 constructs are pursued using various in vitro cellular assays (MET activation and signalling, various phenotypic assays such as scattering, survival, morphogenesis…).

Therefore this project is intrinsically interdisciplinary using multiple technical approaches taking advantage of the multiple and complementary skills (Chemistry, Biophysics and Cell Biology) present in CBC team.

 

3-S.P.E.S. : Study of Prostate Epithelial Stem cells (Roland Bourette)

 Research in the SPES team (currently funded by the « Région Nord-Pas de Calais ») centres on the isolation and characterization of prostate epithelial stem cells. The SPES team is involved in ONCO LILLESite de Recherche Intégrée sur le Cancer, in the basic research program « Tumor stem cells as factors of persistence and relapse » of the main integrated research program « Tumor dormancy and persistence ».

 Adult somatic stem cells represent a rare population within tissues that self-renew and differentiate into the mature cells types of the organ or tissue in which they reside. It has been postulated that adult stem cells also may contribute to cancer development and metastasis as a so-called cancer stem cell, which retains some stem cell properties. Thus, stem cells are of interested because of their potential to repair damaged tissues, and because of their purported role in tumor initiation and maintenance.

 Although prostate cancer is the most common malignancy diagnosed in males in the Western world (approximately 1 in 6 men), the cell of origin of the disease remains unclear. Studying normal and cancer prostate stem cells and their properties will hopefully lead both to a better understanding of tumor formation and to the development of new targets for advanced disease.

 In collaboration with Dr. Larry Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, USA), we used the s-SHIP promoter to tag and isolate a population of mouse epithelial prostate cells exhibiting multiple stem/progenitor cell properties, including regenerative capability upon transplantation. This approach has been also used successfully for the identification of mammary stem cells. Our objective is now to characterize further this purified prostate stem cell population to elucidate the molecular circuitry that contributes to the maintenance of normal prostate stem cells. In parallel, we are using a similar experimental approach to tag, isolate and study potential human cancer stem cells derived from prostate tumors.

Prostate 1 prostate 2

Immunohistochemical analysis of prostate sections from 6-day old transgenic mice stained with phaloidin-Alexa594 (red) ; green cells expressed GFP under the control of s-SHIP promoter.

The other research focus is on colony-stimulating factor-1 receptor (CSF-1R) and its role in prostate cancer.

 CSF-1R is a member of the family of receptor tyrosine kinases (RTKs) that are key regulators of normal cellular processes since they lie at the head of a signal transduction cascade that modulate cell survival, proliferation, differentiation, adherence and migration. Consequently, uncontrolled RTK signaling plays a critical role in the development of many type of cancers, which made RTKs attractive targets of cancer therapy.

 CSF-1R (also known as macrophage-CSF or CD115) is the main regulator of the production, survival, and function of monocytes and macrophages. In addition to this role in hematopoiesis and bone resorption, CSF-1 and CSF-1R play a role in cells of nonhematopoietic origin. CSF-1R is expressed in normal placental trophoblast epithelium and its activation by the locally high levels of CSF-1 produced by the endometrial epithelium is essential for nomal embryonic implantation and placental development.

 More important, expression of CSF-1R and/or its ligand has been documented in several human tumors (breast, ovarian, and prostate cancers) and may facilitate tumor progression and metastasis. Our objective is to investigate the roles of CSF-1R in prostate cancer cells using both in vitro and in vivo studies. As a cellular model, we use the C2H mouse prostate cell line established from the spontaneous autochthonous TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model.

TRAMP C2H 

TRAMP C2H clonal cell line in 2D culture.