Project description:The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and β3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.
Project description:Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.
Project description:Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target.
Project description:Hepcidin is a key hormone that induces the degradation of ferroportin (FPN), a protein that exports iron from reticuloendothelial macrophages and enterocytes. The aim of the present study was to experimentally evaluate if the obesity induced by a high-fat diet (HFD) modifies the expression of FPN in macrophages and enterocytes, thus altering the iron bioavailability. In order to directly examine changes associated with iron metabolism in vivo, C57BL/6J mice were fed either a control or a HFD. Serum leptin levels were evaluated. The hepcidin, divalent metal transporter-1 (DMT1), FPN and ferritin genes were analyzed by real-time polymerase chain reaction. The amount of iron present in both the liver and spleen was determined by flame atomic absorption spectrometry. Ferroportin localization within reticuloendothelial macrophages was observed by immunofluorescence microscopy. Obese animals were found to exhibit increased hepcidin gene expression, while iron accumulated in the spleen and liver. They also exhibited changes in the sublocation of splenic cellular FPN and a reduction in the FPN expression in the liver and the spleen, while no changes were observed in enterocytes. Possible explanations for the increased hepcidin expression observed in HFD animals may include: increased leptin levels, the liver iron accumulation or endoplasmic reticulum (ER) stress. Together, the results indicated that obesity promotes changes in iron bioavailability, since it altered the iron recycling function.
Project description:PurposeIntegrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFβ activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs).MethodsPrimary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry.ResultsUSP10 overexpression increased α5β1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5β1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN.ConclusionsOur data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.
Project description:The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.
Project description:Osteosarcoma (OS) commonly metastasizes to the lung, yet the underlying molecular mechanisms remain poorly understood. Exosomes play a crucial role in tumor migration, including OS lung migration. However, the underlying mechanism by which exosome-derived long non-coding RNAs (lncRNAs) contribute to lung migration in osteosarcoma (OS) remains unclear. This study presents a newly discovered lncRNA, linc00881, derived from OS exosomes. Our study shows that linc00881 promotes the migration of OS cells to the lung and induces the conversion of normal lung fibroblasts into cancer-associated fibroblasts (CAFs). Subsequently, we found that exosomal linc00881 secreted by OS cells can regulate the expression of matrix metalloproteinase 2 (MMP2) in HFL-1 cells by sponging miR-29c-3p, thereby activating the NF-κB signaling in lung fibroblasts. Finally, we discovered that pro-inflammatory cytokines, namely IL-1β, IL-6, and IL-8, were secreted through the linc00881/miR-29c-3p/MMP2 axis. These results suggest that OS-derived exosomes can mediate the intercellular crosstalk between OS cells and lung fibroblasts, ultimately impacting OS lung migration. Our study provides a potential target for the treatment of OS lung migration.
Project description:During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.
Project description:Tumor acquired radioresistance remains as the major limit in cancer radiotherapy (RT). Rab25, a receptor recycling protein, has been reported to be enhanced in tumors with aggressive phenotype and chemotherapy resistance. In this study, elevated Rab25 expression was identified in an array of radioresistant human cancer cell lines, in vivo radioresistant xenograft tumors. Clinical investigation confirmed that Rab25 expression was also associated with a worse prognosis in patients with lung adenocarcinoma (LUAD) and nasopharyngeal carcinoma (NPC). Enhanced activities of EGFR were observed in both NPC and LUAD radioresistant cells. Rab25 interacts with EGFR to enhance EGFR recycling to cell surface and to decrease degradation in cytoplasm. Inhibition of Rab25 showed synergized radiosensitivity with reduced aggressive phenotype. This study provides the clinical and experimental evidence that Rab25 is a potential therapeutic target to alleviate the hyperactive EGFR signaling and to prevent RT-acquired tumor resistance in patients with LUAD and NPC.
Project description:SecA2 is an ATPase present in some pathogenic Gram-positive bacteria, is required for translocation of a limited set of proteins across the cytosolic membrane, and plays an important role in virulence in several bacteria, including mycobacteria that cause diseases such as tuberculosis and leprosy. However, the mechanisms by which SecA2 affects virulence are incompletely understood. To investigate whether SecA2 modulates host immune responses in vivo, we studied Mycobacterium marinum infection in two different hosts: an established zebrafish model and a recently described mouse model. Here we show that M. marinum ?secA2 was attenuated for virulence in both host species and SecA2 was needed for normal granuloma numbers and for optimal tumor necrosis factor alpha response in both zebrafish and mice. M. marinum ?secA2 was more sensitive to SDS and had unique protrusions from its cell envelope when examined by cryo-electron tomography, suggesting that SecA2 is important for bacterial cell wall integrity. These results provide evidence that SecA2 induces granulomas and is required for bacterial modulation of the host response because it affects the mycobacterial cell envelope.