ABSTRACT
TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.
Subject(s)
14-3-3 Proteins/genetics , Fetal Proteins/genetics , Lymphocytes/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein-Tyrosine Kinases/genetics , Ubiquitin/genetics , 14-3-3 Proteins/metabolism , A549 Cells , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Lymphocytes/drug effects , Lymphocytes/pathology , Mice , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction , Survival Analysis , Tumor Burden/drug effects , Ubiquitin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
As a newly emerged technology, PROTAC (proteolysis targeting chimera) is a promising therapeutic strategy for varieties of diseases. Unlike small molecule inhibitors, PROTACs catalytically induce target proteins degradation, including currently "undruggable" target proteins. In addition, PROTACs can be a potentially successful strategy to overcome drug resistance. IAPs can inhibit apoptosis by inhibiting caspase, and also exhibits the activity of E3 ubiquitin ligase. Specific and nongenetic IAP-based protein erasers (SNIPERs) are hybrid molecules that designed based on IAPs, and used to degrade the target proteins closely associated with diseases. Their structures consist of three parts, including target protein ligand, E3 ligase ligand and the linker between them. SNIPERs (PROTACs) degrade diseases-associated proteins through human inherent ubiquitin-proteasome system. So far, many SNIPERs have been developed to treat diseases that difficult to handle by traditional methods, such as radiotherapy, chemotherapy and small molecule inhibitors, and showed promising prospects in application. In this paper, the recent advances of SNIPERs were summarized, and the chances and challenges associated with this area were also highlighted.
Subject(s)
Inhibitor of Apoptosis Proteins/metabolism , Ligands , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , Humans , Huntingtin Protein/antagonists & inhibitors , Huntingtin Protein/metabolism , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Receptor, Notch1/antagonists & inhibitors , Receptor, Notch1/metabolism , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism , Receptors, Estrogen/metabolismABSTRACT
It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.
Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Fetal Proteins/antagonists & inhibitors , Tamoxifen/pharmacology , Antigens, CD/genetics , Antigens, CD/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Female , Fetal Proteins/genetics , Fetal Proteins/metabolism , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
New approaches to drug discovery are unlocking enormous therapeutic potential residing in cancer-specific molecules. Brachyury is emerging as an exciting new drug target for the rare bone cancer chordoma. Here, recent advances targeting Brachyury in chordoma are discussed and how these might open doors to the targeting of other, more common cancer types.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/antagonists & inhibitors , Bone Neoplasms/drug therapy , Chordoma/drug therapy , Fetal Proteins/antagonists & inhibitors , T-Box Domain Proteins/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Bone Neoplasms/diagnosis , Bone Neoplasms/pathology , Chordoma/diagnosis , Chordoma/pathology , Computer Simulation , Crystallography, X-Ray , Drug Discovery , Fetal Proteins/analysis , Fetal Proteins/metabolism , Fetal Proteins/ultrastructure , Humans , Models, Molecular , Protein Domains , T-Box Domain Proteins/analysis , T-Box Domain Proteins/metabolism , T-Box Domain Proteins/ultrastructureABSTRACT
Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.
Subject(s)
Antigens, CD/genetics , Cell Adhesion Molecules, Neuronal/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Fetal Proteins/genetics , Immunity , Lymphatic Vessels , Allografts , Animals , Antigens, CD/metabolism , Biopsy , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/metabolism , Cell Movement/genetics , Cell Movement/immunology , Corneal Transplantation , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Genetic Engineering , Graft Rejection/genetics , Graft Rejection/immunology , Lymphangiogenesis , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Skin/immunology , Skin/metabolism , Skin/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRß, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRß inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603-13. ©2017 AACR.
Subject(s)
Afatinib/therapeutic use , Bone Neoplasms/drug therapy , Chordoma/drug therapy , ErbB Receptors/antagonists & inhibitors , Fetal Proteins/antagonists & inhibitors , T-Box Domain Proteins/antagonists & inhibitors , Xenograft Model Antitumor Assays , Animals , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Chordoma/genetics , Chordoma/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Fetal Proteins/genetics , Fetal Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice, Nude , Phosphorylation/drug effects , Protein Kinase Inhibitors/therapeutic use , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses.
Subject(s)
Actins/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/toxicity , Bacterial Toxins/chemistry , Bacterial Toxins/toxicity , Fetal Proteins/antagonists & inhibitors , Microfilament Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Animals , Antigens, Bacterial/genetics , Bacterial Toxins/genetics , Cell Line , Formins , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Polymerization/drug effects , Protein Structure, Tertiary , RatsABSTRACT
Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal "actin hotspots" along axons-spaced â¼3-4 µm apart-where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons-a phenomenon we call "actin trails." Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal "actin rings" described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin-but not Arp2/3-dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable "actin rings" providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.
Subject(s)
Actins/metabolism , Axons/metabolism , Fetal Proteins/metabolism , Growth Cones/metabolism , Microfilament Proteins/metabolism , Nuclear Proteins/metabolism , Synaptic Vesicles/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actins/biosynthesis , Animals , Cell Membrane/physiology , Fetal Proteins/antagonists & inhibitors , Formins , Green Fluorescent Proteins , Mice , Microfilament Proteins/antagonists & inhibitors , Microtubules/metabolism , Nuclear Proteins/antagonists & inhibitors , Presynaptic Terminals/metabolismABSTRACT
The objective of the current study was to investigate the impact of Brachyury on epithelial-mesenchymal transitions and chemosensitivity in non-small cell lung cancer (NSCLC). In 115 archived NSCLC tissue samples, the expression of Brachyury was observed to be significantly higher than that in adjacent normal lung tissues. In addition, the current study demonstrated that the expression of Brachyury is closely associated with TNM staging, lymph node metastasis and the prognosis of NSCLC, although not with patient age, gender or tumor differentiation. Brachyury expression is also accompanied by the downregulation of E-cadherin and the upregulation of N-cadherin. Brachyury may promote lung cancer through induction of epithelial-mesenchymal transition, which leads to metastasis and consequent poor prognosis in patients with lung cancer. Furthermore, the present study observed that interfering with Brachyury increases the sensitivity of cells to chemotherapeutic treatment with cisplatin. These results, in combination with those of additional studies, suggest that Brachyury may be used as a novel target for the prevention and treatment of lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Epithelial-Mesenchymal Transition/genetics , Fetal Proteins/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , T-Box Domain Proteins/genetics , Aged , Aged, 80 and over , Antigens, CD/genetics , Antigens, CD/metabolism , Antineoplastic Agents/pharmacology , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Survival Analysis , T-Box Domain Proteins/antagonists & inhibitors , T-Box Domain Proteins/metabolismABSTRACT
The complement system is an important aspect of immune defense against microbial invasion. Eukaryotic cells express various complement regulatory proteins to protect them from uncontrolled complement activation. However, some eukaryotic cells possess constitutive complement system activation that does not require specific triggering factors, which is known to have unexpected effects on cell proliferation and survival. This area of research is still preliminary and a standard method to measure complement system activation in eukaryotic cells has yet to be identified. Here, we present a quantitative in vitro method to measure complement system activation in eukaryotic cells by detecting C5b-9, the membrane attack complex, on cell surfaces. The results obtained using this assay correlated with C3b deposition measured using flow cytometry and C5b-9 deposition detected using an immunofluorescence assay. Furthermore, we showed that various cancer cell lines displayed different levels of complement system activation by using this assay.
Subject(s)
Cell Membrane/chemistry , Complement Activation , Complement Membrane Attack Complex/analysis , Enzyme-Linked Immunosorbent Assay/methods , Antibodies/pharmacology , Antigens, CD/genetics , Antigens, CD/immunology , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/immunology , Cell Line, Tumor , Cell Membrane/immunology , Complement C3b/pharmacology , Endoglin , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/genetics , Fetal Proteins/immunology , Flow Cytometry , Gene Expression , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/immunology , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/immunology , Organ Specificity , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunologyABSTRACT
Cluster of differentiation 166 (CD166) is critical for liver cancer cell survival. Our previously study demonstrated that CD166 exerts its anti-apoptotic role through interaction with YAP in liver cancer. However, the interaction between CD166 and other cell surface molecules remains unclear in liver cancer cells. In the current study, we found that both mRNA and protein of CD44 expression was significantly inhibited by knocking-down CD166. Moreover, CD166 affected-CD44 expression is dependent of transcription via blocking NF-κB pathway. On the contrary, CD44 promoted up-regulation of CD166 mRNA and protein. And it may be through E3 ubiquitin ligases COP1 and UBC3 to regulate CD166 protein degradation. Collectively, these results suggest that CD166 and CD44 play important roles in liver cancer development. Therefore, CD166 may develop as a potential therapeutic molecule target for the treatment of liver cancer.
Subject(s)
Antigens, CD/genetics , Antigens, CD/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Fetal Proteins/genetics , Fetal Proteins/metabolism , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , NF-kappa B/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Carcinoma, Hepatocellular/pathology , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation , Fetal Proteins/antagonists & inhibitors , Gene Knockdown Techniques , Humans , Liver Neoplasms/pathology , Phosphoproteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Signal Transduction , Transcription Factors , Up-Regulation , YAP-Signaling ProteinsABSTRACT
Recent evidence suggests that the expression of brachyury is necessary for chordoma growth. However, the mechanism associated with brachyury-regulated cell growth is poorly understood. Fibroblast growth factor (FGF), a regulator of brachyury expression in normal tissue, may also play an important role in chordoma pathophysiology. Using a panel of chordoma cell lines, we explored the role of FGF signaling and brachyury in cell growth and survival. Western blots showed that all chordoma cell lines expressed fibroblast growth factor receptor 2 (FGFR2), FGFR3, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), whereas no cell lines expressed FGFR1 and FGFR4. Results of enzyme-linked immunosorbent assay indicated that chordoma cells produced FGF2. Neutralization of FGF2 inhibited MEK/ERK phosphorylation, decreased brachyury expression and induced apoptosis while reducing cell growth. Activation of the FGFR/MEK/ERK/brachyury pathway by FGF2-initiated phosphorylation of FGFR substrate 2 (FRS2)-α (Tyr196) prevented apoptosis while promoting cell growth and epithelial-mesenchymal transition (EMT). Immunofluorescence staining showed that FGF2 promoted the translocation of phosphorylated ERK to the nucleus and increased brachyury expression. The selective inhibition of FGFR, MEK and ERK phosphorylation by PD173074, PD0325901 and PD184352, respectively, decreased brachyury expression, induced apoptosis, and inhibited cell growth and EMT. Moreover, knockdown of brachyury by small hairpin RNA reduced FGF2 secretion, inhibited FGFR/MEK/ERK phosphorylation and blocked the effects of FGF2 on cell growth, apoptosis and EMT. Those findings highlight that FGFR/MEK/ERK/brachyury pathway coordinately regulates chordoma cell growth and survival and may represent a novel chemotherapeutic target for chordoma.
Subject(s)
Apoptosis , Cell Proliferation , Chordoma/pathology , Fetal Proteins/metabolism , MAP Kinase Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , T-Box Domain Proteins/metabolism , Blotting, Western , Cell Movement , Cells, Cultured , Chordoma/genetics , Chordoma/metabolism , Epithelial-Mesenchymal Transition , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/genetics , Fibroblast Growth Factor 2/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Antibody Technique , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/genetics , Phosphorylation , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , T-Box Domain Proteins/antagonists & inhibitors , T-Box Domain Proteins/geneticsABSTRACT
Vaccinia virus dissemination relies on the N-WASP-ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP-ARP2/3 and Rac1-FHOD1 pathways.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Cell Movement/physiology , Fetal Proteins/metabolism , Nuclear Proteins/metabolism , Vaccinia virus/physiology , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/genetics , Fluorescent Antibody Technique , Formins , HeLa Cells , Humans , Image Processing, Computer-Assisted , Immunoblotting , Mice , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Protein Binding , RNA, Small Interfering/genetics , Vaccinia/metabolism , Vaccinia/virologyABSTRACT
Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development.
Subject(s)
Cell Transformation, Neoplastic/genetics , ErbB Receptors/genetics , Gene Expression Regulation, Neoplastic , Interleukin-6/genetics , Receptors, Interleukin-6/genetics , TOR Serine-Threonine Kinases/genetics , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cellular Senescence/genetics , ErbB Receptors/metabolism , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/genetics , Fetal Proteins/metabolism , Humans , Interleukin-6/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Interleukin-6/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
The switch of tumor cells from an epithelial to a mesenchymal-like phenotype [designated as epithelial-to-mesenchymal transition (EMT)] is known to induce tumor cell motility and invasiveness, therefore promoting metastasis of solid carcinomas. Although multiple studies have focused on elucidating the signaling events that initiate this phenotypic switch, there has been so far no characterization of the pattern of soluble mediators released by tumor cells undergoing EMT, and the potential impact that this phenotypic switch could have on the remodeling of the tumor microenvironment. Here we show that induction of EMT in human carcinoma cells via overexpression of the transcription factor Brachyury is associated with enhanced secretion of multiple cytokines, chemokines, and angiogenic factors and, in particular, with the induction of the IL-8/IL-8R axis. Our results also indicate the essential role of interleukin 8 (IL-8) signaling for the acquisition and/or maintenance of the mesenchymal and invasive features of Brachyury-overexpressing tumor cells and show that IL-8 secreted by tumor cells undergoing EMT could potentiate tumor progression by inducing adjacent epithelial tumor cells into EMT. Altogether, our results emphasize the potential role of EMT in the modulation of the tumor microenvironment via secretion of multiple soluble mediators and suggest that IL-8 signaling blockade may provide a means of targeting mesenchymal-like, invasive tumor cells.
Subject(s)
Carcinoma/pathology , Epithelial-Mesenchymal Transition/physiology , Interleukin-8/physiology , Neoplasm Proteins/physiology , Tumor Microenvironment/physiology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Bystander Effect , Carcinoma/metabolism , Cell Line, Tumor/metabolism , Cell Line, Tumor/pathology , Cell Movement , Chemokines/metabolism , Culture Media, Conditioned/pharmacology , Culture Media, Serum-Free , Cytokines/metabolism , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/biosynthesis , Fetal Proteins/genetics , Fetal Proteins/physiology , Fibronectins/biosynthesis , Fibronectins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Neoplasm Invasiveness , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , RNA, Small Interfering/pharmacology , Receptors, Interleukin-8/biosynthesis , Receptors, Interleukin-8/genetics , Recombinant Fusion Proteins/antagonists & inhibitors , T-Box Domain Proteins/antagonists & inhibitors , T-Box Domain Proteins/biosynthesis , T-Box Domain Proteins/genetics , T-Box Domain Proteins/physiologyABSTRACT
Diaphanous-related formins (DRFs) are large multi-domain proteins that nucleate and assemble linear actin filaments. Binding of active Rho family proteins to the GTPase-binding domain (GBD) triggers localization at the membrane and the activation of most formins if not all. In recent years GTPase regulation of formins has been extensively studied, but other molecular mechanisms that determine subcellular distribution or regulate formin activity have remained poorly understood. Here, we provide evidence that the activity and localization of mouse formin mDia1 can be regulated through interactions with phospholipids. The phospholipid-binding sites of mDia1 are clusters of positively charged residues in the N-terminal basic domain (BD) and at the C-terminal region. Upon binding to the lipid bilayer the N-terminal region of mDia1 induces strong clustering of phosphatidylinositol-4,5-bisphosphate (PIP(2)) and subsequently inserts into the membrane bilayer thus anchoring mDia1 to the reconstituted plasma membrane. In addition, an interaction of phospholipids with the C-terminal region of mDia1 causes a drastic reduction of its actin filament assembly activity. Our data suggest that the N-terminal phospholipid-binding sites help to anchor formins at the plasma membrane, and the interaction with phospholipids in the C-terminus functions as a switch for transient inactivation.
Subject(s)
Actin Cytoskeleton/metabolism , Carrier Proteins/metabolism , Cell Membrane/metabolism , Fetal Proteins/metabolism , Membrane Lipids/physiology , Microfilament Proteins/metabolism , Nuclear Proteins/metabolism , Phospholipids/physiology , Actin Cytoskeleton/chemistry , Animals , Binding Sites/physiology , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Cell Membrane/chemistry , Enzyme Activation/physiology , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/chemistry , Formins , Membrane Lipids/antagonists & inhibitors , Membrane Lipids/chemistry , Mice , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/chemistry , NIH 3T3 Cells , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phospholipids/antagonists & inhibitors , Phospholipids/chemistry , Polymerization , Protein Binding/physiology , Protein Structure, Tertiary/physiologySubject(s)
Fetal Proteins/metabolism , Hodgkin Disease/enzymology , Hodgkin Disease/pathology , Protein-Tyrosine Kinases/metabolism , Adult , Blotting, Western , Bone Marrow/enzymology , Bone Marrow/pathology , Cell Proliferation , Female , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/genetics , Hodgkin Disease/genetics , Humans , Immunoprecipitation , Phosphopeptides/metabolism , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tyrosine/metabolismABSTRACT
Formins are potent actin assembly factors. Diaphanous formins, including mDia1, mDia2, and mDia3 in mammals, are implicated in mitosis and cytokinesis, but no chemical interactors have been reported. We developed an in vitro screen for inhibitors of actin assembly by mDia1 and identified an inhibitor of mDia1 and mDia2 that does not inhibit mDia3 at the concentrations tested. These results establish the druggability of mDia formins and introduce a first-generation inhibitor.
Subject(s)
Actins/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Carrier Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/antagonists & inhibitors , NADPH Dehydrogenase/antagonists & inhibitors , Actins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/physiology , Animals , Carrier Proteins/chemistry , Carrier Proteins/physiology , Fetal Proteins/antagonists & inhibitors , Formins , Humans , Mice , Microfilament Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/physiology , NADPH Dehydrogenase/chemistry , NADPH Dehydrogenase/physiology , Nuclear Proteins/antagonists & inhibitors , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/physiology , Protein Structure, Tertiary/physiology , Pyrenes/chemistry , Sequence Homology, Amino AcidABSTRACT
During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.
Subject(s)
Gastrula/physiology , Lytechinus/embryology , rhoA GTP-Binding Protein/physiology , Animals , Cloning, Molecular , Cytoskeleton/metabolism , Extracellular Matrix/metabolism , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/physiology , Lytechinus/genetics , Lytechinus/physiology , Molecular Sequence Data , Sequence Analysis, Protein , T-Box Domain Proteins/antagonists & inhibitors , T-Box Domain Proteins/physiology , rhoA GTP-Binding Protein/geneticsABSTRACT
Dehydroepiandrosterone (DHEA) can function to protect neural precursors and their progeny targeted with toxic insults; however, the molecular mechanisms underlying the neuroprotective effects of DHEA are not understood. We cultured neural precursors from the embryonic forebrain of rats and examined the effects of DHEA and its sulfated derivative (DHEAS) on the activation of the serine-threonine protein kinase Akt, which is widely implicated in cell survival signaling. We found that DHEA activated Akt in neural precursor culture, in association with a decrease in apoptosis. In contrast, DHEAS decreased activated Akt levels and increased apoptosis. The effects of DHEA on neural cell survival and activation of Akt were not blocked by the steroid hormone antagonists flutamide and tamoxifen, but both were blocked by a PI3-K inhibitor, LY294002. These findings suggest that during neurogenesis in the developing cortex, DHEA and DHEAS regulate the survival of neural precursors and progeny through the Akt signaling pathway.