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1.
Oncol Lett ; 15(2): 2349-2363, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29434944

ABSTRACT

MicroRNAs (miRs) are expected to serve as prognostic tools for cancer. However, many miRs have been reported as prognostic markers of recurrence or metastasis in oral squamous cell carcinoma patients. We aimed to determine the prognostic markers in early-stage tongue squamous cell carcinoma (TSCC). Based on previous studies, we hypothesized that miR-10a, 10b, 196a-5p, 196a-3p, and 196b were prognostic markers and we retrospectively performed miR expression analyses using formalin-fixed paraffin-embedded sections of surgical specimens. Total RNA was isolated from cancer tissues and adjacent normal tissue as control, and samples were collected by laser-capture microdissection. After cDNA synthesis, reverse transcription-quantitative polymerase chain reaction was performed. Statistical analyses for patient clinicopathological characteristics, recurrence/metastasis, and survival rates were performed to discern their relationships with miR expression levels, and the 2-ΔΔCq method was used. miR-196a-5p levels were significantly upregulated in early-stage TSCC, particularly in the lymph node metastasis (LNM) group. The LNM-free survival rate in the low miR-196a-5p ΔΔCq value regulation group was found to be lower than that in the high ΔΔCq value regulation group (P=0.0079). Receiver operating characteristic analysis of ΔΔCq values revealed that miR-196a-5p had a P-value=0.0025, area under the curve=0.740, and a cut-off value=-0.875 for distinguishing LNM. To our knowledge, this is the first study to examine LNM-related miRs in early-stage TSCC as well as miRs and 'delayed LNM' in head and neck cancer. miR-196a-5p upregulation may predict delayed LNM. Our data serve as a foundation for future studies to evaluate miR levels and facilitate the prediction of delayed LNM during early-stage TSCC, which prevent metastasis when combined with close follow-up and aggressive adjuvant therapy or elective neck dissection. Moreover, our data will serve as a foundation for future studies to evaluate whether miR-196a-5p can serve as a therapeutic marker for preventing metastasis.

2.
Biochem Biophys Res Commun ; 467(2): 235-41, 2015 Nov 13.
Article in English | MEDLINE | ID: mdl-26435501

ABSTRACT

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1ß, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1ß, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.


Subject(s)
Angiopoietins/immunology , Macrophages, Peritoneal/immunology , Monocytes/immunology , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/genetics , Animals , Cyclooxygenase 2/genetics , Cyclooxygenase 2/immunology , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/immunology , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages, Peritoneal/pathology , Mice , Mice, Inbred C57BL , Monocytes/pathology , NF-kappa B/genetics , NF-kappa B/immunology , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/immunology , Primary Cell Culture , Signal Transduction , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/immunology
3.
Biochem Biophys Res Commun ; 399(3): 365-72, 2010 Aug 27.
Article in English | MEDLINE | ID: mdl-20659422

ABSTRACT

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.


Subject(s)
Carcinoma, Squamous Cell/pathology , Keratin-18/biosynthesis , Keratin-8/biosynthesis , Keratinocytes/pathology , Skin Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Movement , Humans , Keratinocytes/metabolism , Mice , Neoplasm Invasiveness
4.
Biochem Biophys Res Commun ; 378(4): 732-7, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-19061864

ABSTRACT

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.


Subject(s)
Endosomes/enzymology , Lipoylation , rap GTP-Binding Proteins/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Endoplasmic Reticulum/enzymology , Enzyme Activation , Germinal Center Kinases , Golgi Apparatus/enzymology , Humans , Molecular Sequence Data , Phenotype , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism
5.
Biochem Biophys Res Commun ; 377(2): 573-578, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-18930710

ABSTRACT

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.


Subject(s)
Brain/metabolism , Crotalid Venoms/metabolism , Lectins, C-Type/metabolism , Protein Serine-Threonine Kinases/metabolism , rap GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Chromosome Pairing , Crotalid Venoms/genetics , Germinal Center Kinases , Humans , Lectins, C-Type/genetics , Mice , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Rats
6.
Biochem Biophys Res Commun ; 329(3): 1046-52, 2005 Apr 15.
Article in English | MEDLINE | ID: mdl-15752761

ABSTRACT

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific signaling role is unclear. By yeast two-hybrid screening, we have found that the Caenorhabditis elegans ortholog of Rap2 interacts with a protein containing a Rho-GTPase-activating protein (RhoGAP) domain, ZK669.1a, whose human ortholog PARG1 exhibits RhoGAP activity in vitro. ZK669.1a and PARG1 share a homology region with previously unknown function, designated the ZK669.1a and PARG1 homology (ZPH) region. Here we show that the ZPH region of PARG1 mediates interaction with Rap2. PARG1 interacted with Rap2 in a GTP-dependent manner but not with Ras or Rap1. We also show that PARG1 and its mutant lacking the ZPH region induce typical cytoskeletal changes for Rho inactivation in fibroblasts. Rap2 suppressed this in vivo action of PARG1 but not that of the mutant PARG1. These results suggest that PARG1 is a putative specific effector of Rap2 to regulate Rho.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , Cytoskeleton/metabolism , GTPase-Activating Proteins/metabolism , Protein Interaction Mapping , Protein Tyrosine Phosphatases/metabolism , Signal Transduction/physiology , rap GTP-Binding Proteins/metabolism , Animals , Intracellular Signaling Peptides and Proteins , Mice , NIH 3T3 Cells , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 13 , Two-Hybrid System Techniques
8.
J Biol Chem ; 279(47): 49488-96, 2004 Nov 19.
Article in English | MEDLINE | ID: mdl-15342639

ABSTRACT

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific roles in cell signaling remain unknown. In the present study, we have affinity-purified from rat brain a Rap2-interacting protein of approximately 155 kDa, p155. By liquid chromatography tandem mass spectrometry, we have identified p155 as Traf2- and Nck-interacting kinase (TNIK). TNIK possesses an N-terminal kinase domain homologous to STE20, the Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase kinase, and a C-terminal regulatory domain termed the citron homology (CNH) domain. TNIK induces disruption of F-actin structure, thereby inhibiting cell spreading. In addition, TNIK specifically activates the c-Jun N-terminal kinase (JNK) pathway. Among our observations, TNIK interacted with Rap2 through its CNH domain but did not interact with Rap1 or Ras. TNIK interaction with Rap2 was dependent on the intact effector region and GTP-bound configuration of Rap2. When co-expressed in cultured cells, TNIK colocalized with Rap2, while a mutant TNIK lacking the CNH domain did not. Rap2 potently enhanced the inhibitory function of TNIK against cell spreading, but this was not observed for the mutant TNIK lacking the CNH domain. Rap2 did not significantly enhance TNIK-induced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergent-insoluble cytoskeletal fraction. These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Protein Serine-Threonine Kinases/physiology , rap GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Cell Line , Chromatography, Liquid , DNA, Complementary/metabolism , Detergents/pharmacology , Gene Deletion , Germinal Center Kinases , Glutathione Transferase/metabolism , Guanosine Triphosphate/chemistry , Humans , Insecta , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase Kinases , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Nuclear Pore Complex Proteins/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Protein Structure, Tertiary , Protein Transport , Rats , Saccharomyces cerevisiae Proteins/chemistry , Two-Hybrid System Techniques
9.
J Biol Chem ; 279(16): 15711-4, 2004 Apr 16.
Article in English | MEDLINE | ID: mdl-14966141

ABSTRACT

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , rap GTP-Binding Proteins/metabolism , Animals , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , Mice , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Protein Isoforms/metabolism , Protein Structure, Tertiary , Substrate Specificity
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