> Cell polarity is defined by the ability of a cell to be morphologically, structurally and functionally organized along a so-called polarity axis. Polarity is a critical parameter in most cellular functions such as cell division and cell differentiation. It is also a key step leading to the initiation and the regulation of cell migration. The biological importance of cell polarity is reflected by its crucial role during development and also by the fact that loss of polarity is a hallmark of cancer cells. However, the link between loss of cell polarity and oncogenesis is not entirely clear. Perturbation of cell polarity is likely to have dramatic consequences in the regulation of cell division, differentiation and migration and may thus be a key event during cancer formation and evolution.
In order to better understand the relationship between cell polarity and oncogenesis, we study the molecular mechanisms that control cell polarity in astrocytes and astrocyte-derived tumors, gliomas.
> Astrocytes are major glial cells of the central nervous system. Under pathological situations involving inflammation of the cerebral tissue, astrocytes become reactive, polarize and migrate in the direction of the inflammatory site. Astrocytes or their progenitors can give rise to astrocytomas and glioblastomas, which are extremely invasive brain tumors and are associated with a very poor prognosis. To decipher signalling pathways that control polarity in normal and tumorous astrocytes, we analyze cell polarization during migration. Cell migration is a fundamental process that allows cell reorganization during development, delivery of immune cells to infection sites, wound healing or else dissemination of tumor cells during metastasis. Cell polarity is an essential feature of migrating cells in which intracellular organelles and cytoskeletal elements are organized along a rear-front polarity axis. Establishment and regulation of this polarity axis are key to the initiation and the regulation of cell migration.
Our first aim is to decipher the fundamental molecular mechanisms controlling cell polarity in normal cells
We use primary astrocytes in an in vitro migration assays to analyse polarity pathways.
Extracellular cues that control cell polarity: Cellular contacts with the extracellular matrix and cell-cell interactions.
> We have previously demonstrated that integrin-dependent signals were essential to induce polarization of wound-edge cells (S.Etienne-Manneville et al. Cell 2001). More recently we have also shown that anisotropic distribution of cadherin-mediated cell-cell contacts was sufficient to promote cell polarization (Dupin et al. J. Cell Biol 2009).
Adherens junctions control the localization of integrin-mediated focal adhesions to ensure the spatial restriction of integrin signals and correct cell orientation.
Image showing cadherin (green), centrosome (red) and nucleus (blue) staining in polarized astrocytes. Cells were plated on fibronectin coated circle micropattern. Upon junction formation, the nucleus move towards cell-cell contacts and the nucleus centrosome axis becomes oriented towards the free cell edge.
> The distribution of integrin- and cadherin-mediated contacts control cell polarity by influencing centrosome and also nucleus position. Nuclear movement depends on the microfilaments and also on intermediate filaments. We show that retrograde flow of actin fibers coming from the cell leading edge induces the accumulation of intermediate filaments in front of the nuclear envelope and thereby pushes the nucleus towards the cell rear (Dupin et al. J Cell Sci. 2011)
The regulation and function of key polarity proteins: Cdc42, the Par complex, the Scrib complex.
> Integrin mediated signals lead to the recruitment and activation of the small GTPase Cdc42 at the leading edge of migrating astrocytes and to the activation of the downstream effectors Par6 and aPKC to promote the reorientation of the centrosome in front of the nucleus in the direction of migration (Etienne-Manneville et al., Nature, 2003).
>The polarity protein and tumor suppressor Scrib is a key element in the regulation of Cdc42 by integrins (Osmani et al. Curr Biol, 2006) and thus plays a key role in the polarization of migrating cells. Scrib can interact and recruit at the leading edge the Cdc42-specific exchange factor bPIX.
> We have recently shown that bPIX and Cdc42 are present in intracellular vesicles that are directed towards the leading edge in a Arf6-dependent manner. Cdc42 is then activated following the interaction of PIX with plasma membrane-associated Scrib. These results illustrate the essential role of vesicular trafficking in cell polarization (Osmani et al. J. Cell Biol. 2010)
Polarized organization of the cytoskeleton and its function during cell migration.
Microtubules have been shown to play a key role during astrocyte directed migration. Microtubules elongate in the forming protrusion and the microtubule network, together with the centrosome, polarize towards the leading edge of the cells.
Image showing microtubule (green), centrosome (red) and nucleus (blue) staining in polarized migrating astrocytes.
> Cdc42-Par6-aPKC pathway controls the polarization of the microtubule network. aPKC activity is required for the clustering of the tumor suppressor APC (Adenomatous Polyposis Coli) at the plus ends of leading edge microtubules. Simultaneously, aPKC induces the recruitment of the polarity protein Dlg at the basal plasma membrane of the cell front. The APC-Dlg interaction that follows allows the capture of microtubules at the cell cortex (Etienne-Manneville, J. Cell Biol. 2005).
> Dlg also interacts with a protein called GKAP which bridges Dlg with the microtubule associated motor Dynein. The Dlg-GKAP-Dynein complex anchors microtubules and may provide the pulling forces necessary to bring the centrosome in front of the nucleus (Manneville, Jehanno et al. J. Cell Biol. 2009)
The exact function of such a polarized microtubule network remains unclear. We are investigating its function in polarized membrane traffic and in the polarized organization of the other cytoskeletal elements, actin and intermediate filaments.
Our second research goal is to determine whether alteration of polarity proteins can promote oncogenesis and how loss of polarity may participate in tumor development, in particular in tumor spreading.
In collaboration with the Group of J-Y Delattre (INSERM UMRS 975/CNRS UMR 7225/UPMC Groupe Hospitalier Pitié-Salpêtrière, Paris, France), we have analysed the expression levels of polarity proteins in astrocytomas and glioblastomas. We have observed systematic alterations of key polarity proteins in high grade tumors. We are investigating whether such alterations are responsible for the invasive properties of glioblastomas and if they directly participate to gliomagenesis.
> We have recently demonstrated that perturbation of the expression level of the adherens junction molecule, N-cadherin, strongly perturb the establishment of polarity and leads to a faster and less persistent migration. N-cadherin protein levels appears decreased in gliomas. Downregulation of N-cadherin causes an increased velocity and a loss of cell polarity both in normal and tumorous astrocytes. Re-expression of N-cadherin in glioma cells drastically slows down cell speed and rescues cell polarity indicating that changes in N-cadherin expression level are likely to play a key role in glioma invasion.
Glioma cell invasion visualized in an in vitro 3D assay.
We plan to determine whether alterations of polarity pathways can be used as diagnostic markers of the invasive properties of glioblastomas. We thereby hope to identify new therapeutic targets to better control cellular migration. This project will bring new insights into the fundamental mechanisms responsible for loss of cell polarity in cancer and may help us define new therapeutic strategies to inhibit glioblastoma infiltration.