Cancer cells' ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined ...migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.
The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery ...are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.
Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants ...generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.
Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in ...epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT’s nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth.
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•APC controls microtubule organization and NFAT-driven cytokine gene expression•APC silencing impairs NFAT nuclear localization and activation•NFAT associates with microtubules that control its localization and its activation•ApcMin/+ Tregs have intrinsically reduced differentiation and IL-10 production
Agüera-González et al. investigate the role of the polarity regulator and tumor suppressor Adenomatous polyposis coli (APC) in CD4 T cell activation and effector function. APC controls microtubule reorganization, NFAT transcription factor localization, and cytokine gene expression. In ApcMin/+ mutant mice, regulatory T cell (Treg) differentiation and anti-inflammatory function were intrinsically affected.
Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP ...family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.
The MHC class I-related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised ...immune response and progression of disease. Recently, thiol disulphide isomerases and members of the ADAM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MICA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression.
MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ...ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are ...less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
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•Deformed nuclei and DNA damage are enriched at micro-invasive foci of breast cancer•TREX1 causes DNA damage in deformed nuclei upon nuclear envelope rupture events•TREX1 drives senescence in normal cells and an invasive phenotype in cancer cells•Nuclear envelope rupture events and DNA damage are observed in human tumors
Extreme nuclear deformations in dense microenvironments lead to repeated nuclear envelope rupture followed by TREX1-dependent chronic DNA damage. This triggers a partial EMT in malignant cells which might underlie tumor progression.
Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 ...complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.
MICA and MICB (MHC‐class‐I‐related chain A/B) are transmembrane proteins expressed in pathological conditions that are ligands for NKG2D, an activating receptor found on cytotoxic lymphocytes. The ...recognition on target cells of NKG2D ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells. Besides being expressed at the cell surface, MICA/B can be released as soluble proteins. Soluble NKG2D ligands downmodulate expression of the NKG2D receptor on lymphocytes, leading to a diminished cytotoxic response. Prior studies suggested that recruitment of MICA/B molecules to cholesterol‐enriched microdomains was an important factor regulating the proteolytic release of these molecules. We now show that recruitment of MICA to these microdomains depends on palmitoylation of two cysteine residues that allow MICA molecules to reside in the membrane in the same domains as caveolin‐1. Compared with WT molecules, nonpalmitoylated mutant MICA molecules were shed to the supernatant with low efficiency; however, both WT and mutant MICA were able to trigger NK cell cytotoxicity. These data suggest that the presence of NKG2D ligands at the plasma membrane is sufficient to activate cytotoxicity and reflect the need of different ligands to exploit different cellular pathways to reach the cell surface upon different stress situations.
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