Group leader position
The Cell Biology department at the Institut Curie (UMR 144 Institut Curie/CNRS) in Paris is inviting applications from outstanding candidates interested in normal epithelium and cancer stem cells biology. Priority will be given to projects focused on mammary gland development, mammary stem cell biology and the mechanisms of breast cancer.
Membrane and cytoskeleton dynamics
Group leader : Philippe Chavrier
Breast cancer, the most frequent cancer in women, represents a wide spectrum of diseases, which can be recognized at the histological and molecular levels based on gene expression profiling. Ductal carcinoma in situ (DCIS) are characterized by the proliferation of tumor cells within the ductal-lobular system that spare the myoepithelium and the basement membrane and are precursors of more aggressive invasive carcinomatous lesions. Our aim is to understand the molecular and cellular mechanisms underlying the transition of DCIS to invasive carcinoma with the long-term goal of finding new avenues for therapeutic intervention.
The capacity of breast carcinoma cells to invade across tissue boundaries and to metastasize depends on their ability to breach the basement membrane and then migrate through the stromal environment consisting of interstitial collagen. One major route of invasion requires cancer cells to proteolytically cleave extracellular matrix components via a mechanism based upon the formation of invadopodia. These specialized structures of invasive cells correspond to sites of actin filament assembly and accumulation of MT1-MMP, a trans-membrane metalloproteinase (MMP) that is crucial for matrix degradation and invasion. How the mechanisms of actin assembly, signaling and MT1-MMP trafficking are integrated to produce the ECM degrading machine of invasive tumor cells is poorly understood and is the focus of our work.
Figure 1. Exocytosis of MT1-MMP at contact sites with collagen fibers. MDA-MB-231 cells expressing mCherry-tagged WASH (shown in red in left panel) together with MT1-MMP tagged with pH-sensitive pHluorin GFP variant (green) and were plated on a layer of fluorescently-labeled collagen I fibers (blue) for 30 min and imaged by confocal spinning disk microscopy. Arrows point to pHluorin flashes corresponding to exocytic events of MT1-MMP-, WASH-positive endosomes occurring at the plasma membrane in association with collagen fibers. The left panel is a merge of the three channels, the right panel shows only MT1-MMPpHluorin and the collagen fibers.
Our most recent data support a model whereby MT1-MMP-positive secretory endosomes establish dynamic tubular connections with the invadopodial plasma membrane through which the protease is transferred to the surface. Formation of these endosomal tubular membrane extensions requires WASH-Arp2/3-dependent actin assembly on MT1-MMP-positive endosomes, and then docking and fusion of the tubular extensions with the invadopodial plasma membrane. The exocyst complex and the SNARE protein VAMP7 mediate these latter steps, respectively. We also found that the master polarity atypical protein kinase C iota is up-regulated together with MT1-MMP and co-up-regulation represents a predictor of negative outcome in human cancers (increased metastasis risk). We unraveled an unprecedented role for aPKCiota in the regulation of MT1-MMP transport to invadopodia. Another aspect of our work was to investigate the function of microtubules during the polarized trafficking of MT1-MMP, focusing on the role of tubulin post-translational modifications and microtubule-based motors. Our data revealed that the microtubule network and molecular motors are essential for polarized traffic of MT1-MMP-positive endosomes to the cell periphery. This polarizing function of microtubules is positively regulated by microtubule acetylation and by the small GTP-binding protein ARF6 together with its downstream effectors JIP3/4.
Figure 2. The intraductal xenograft model recapitulates the transition from in situ to invasive breast carcinoma. Tissue sections of ductal carcinoma in situ generated by intraductal injection of MCF10DCIS.com human breast cancer cells in the primary mammary ducts of 6-8-week old SCID mouse are shown. (A) The section is stained with Picrosirious red, a collagen specific dye and shows tumor cells initiating a basement membrane-invasive program (see higher magnification inset). (B) The image shows tumor cells (stained for human specific Ku70, in green) transmigrating through the myoepithelium (myoepithelial cells are stained for alpha- smooth muscle actin, in red). Scale bars, 50 um.
Changes in MT1-MMP levels during breast cancer progression and association with clinicopathological parameters were analyzed using a breast cancer tissue microarray comprising ~600 DCIS and invasive breast cancers. We observed that MT1-MMP expression increased from a low level in normal breast epithelium, to medium in DCIS up to a high level in IDC and was strongly up-regulated in high grade hormone receptor-negative breast tumors and associated with higher risk of metastasis. The role of MT1-MMP during breast cancer progression was directly investigated using the intraductal xenograft model. Human breast DCIS.com cells were injected in the primary ducts through the nipple of mammary glands of SCID mice; they generated DCIS lesions after 5-6 weeks that spontaneously progressed in IDC after 10-12 weeks. In contrast, progression of tumors formed by MT1-MMP-depleted DCIS.com cells was blocked at the DCIS stage, demonstrating requirement for MT1-MMP during the DCIS-to-IDC transition in vivo. Our data support the conclusion that MT1-MMP is a key component of the basement membrane-invasive program of breast carcinoma cells and that several polarity factors are diverted from their physiological functions in normal breast epithelial cells, and contribute to the invasive capacity of breast tumor cells by controlling the trafficking and exocytosis of MT1-MMP at invadopodia.