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Membrane and cytoskeleton dynamics

Group leader : Philippe Chavrier

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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

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

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.

Key publications

  • Year of publication : 2016

  • The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

  • Year of publication : 2015

  • Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

  • Year of publication : 2014

  • Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.

  • Year of publication : 2013

  • In most eukaryotic cells microtubules undergo post-translational modifications such as acetylation of α-tubulin on lysine 40, a widespread modification restricted to a subset of microtubules that turns over slowly. This subset of stable microtubules accumulates in cell protrusions and regulates cell polarization, migration and invasion. However, mechanisms restricting acetylation to these microtubules are unknown. Here we report that clathrin-coated pits (CCPs) control microtubule acetylation through a direct interaction of the α-tubulin acetyltransferase αTAT1 (refs 8, 9) with the clathrin adaptor AP2. We observe that about one-third of growing microtubule ends contact and pause at CCPs and that loss of CCPs decreases lysine 40 acetylation levels. We show that αTAT1 localizes to CCPs through a direct interaction with AP2 that is required for microtubule acetylation. In migrating cells, the polarized orientation of acetylated microtubules correlates with CCP accumulation at the leading edge, and interaction of αTAT1 with AP2 is required for directional migration. We conclude that microtubules contacting CCPs become acetylated by αTAT1. In migrating cells, this mechanism ensures the acetylation of microtubules oriented towards the leading edge, thus promoting directional cell locomotion and chemotaxis.

  • Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.

  • Year of publication : 2011

  • We present a model of cell motility generated by actomyosin contraction of the cell cortex. We identify, analytically, dynamical instabilities of the cortex and show that they yield steady-state cortical flows, which, in turn, can induce cell migration in three-dimensional environments. This mechanism relies on the regulation of contractility by myosin, whose transport is explicitly taken into account in the model. Theoretical predictions are compared to experimental data of tumor cells migrating in three-dimensional matrigel and suggest that this mechanism could be a general mode of cell migration in three-dimensional environments.

  • The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.

  • Year of publication : 2009

  • BACKGROUND: Recent work has highlighted the importance of the recycling of endocytic membranes to the intercellular bridge for completion of cytokinesis in animal cells. ADP-ribosylation factor 6 (ARF6), which localizes to the plasma membrane and endosomal compartments, regulates endocytic recycling to the bridge during cytokinesis and is required for abscission. RESULTS: Here, we report that the JNK-interacting proteins JIP3 and JIP4, two highly related scaffolding proteins for JNK signaling modules, also acting as binding partners of kinesin-1 and dynactin complex, can function as downstream effectors of ARF6. In vitro, binding of GTP-ARF6 to the second leucine zipper domain of JIP3 and JIP4 interferes with JIPs' association with kinesin-1, whereas it favors JIPs' interaction with the dynactin complex. With protein silencing by small interfering RNA and dominant inhibition approaches, we show that ARF6, JIP4, kinesin-1, and the dynactin complex control the trafficking of recycling endosomes in and out of the intercellular bridge and are necessary for abscission. CONCLUSION: Our findings reveal a novel function for ARF6 as a regulatory switch for motor proteins of opposing direction that controls trafficking of endocytic vesicles within the intercellular bridge in a mechanism required for abscission.

  • Year of publication : 2008

  • Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.