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Tankyrase-Binding Protein TNKS1BP1 Regulates Actin

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Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-1846 Cancer Molecular and Cellular Pathobiology Research Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Cytoskeleton Rearrangement and Cancer Cell Invasion Tomokazu Ohishi1,2, Haruka Yoshida1, Masamichi Katori3, Toshiro Migita1, Yukiko Muramatsu1, Mao Miyake1, Yuichi Ishikawa3, Akio Saiura4, Shun-ichiro Iemura5, Tohru Natsume5, and Hiroyuki Seimiya1 Abstract Tankyrase, a PARP that promotes telomere elongation and actin-capping protein CapZA2. TNKS1BP1 depletion dissociat- Wnt/b-catenin signaling, has various binding partners, suggest- ed CapZA2 from the cytoskeleton, leading to cofilin phosphor- ingthatithasas-yetunidentified functions. Here, we report ylation and enhanced cell invasion. Tankyrase overexpression that the tankyrase-binding protein TNKS1BP1 regulates actin increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton and cancer cell invasion, which is closely associ- cytoskeleton, and enhanced cell invasion in a PARP activity– ated with cancer progression. TNKS1BP1 colocalized with actin dependent manner. In clinical samples of pancreatic cancer, filaments and negatively regulated cell invasion. In TNKS1BP1- TNKS1BP1 expression was reduced in invasive regions. We depleted cells, actin filament dynamics, focal adhesion, propose that the tankyrase-TNKS1BP1 axis constitutes a posttrans- and lamellipodia ruffling were increased with activation of lational modulator of cell invasion whose aberration promotes the ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the cancer malignancy. Cancer Res; 77(9); 2328–38. Ó2017 AACR. Introduction nin, and vitronectin) and adaptor complexes (e.g., talin, vin- culin, and tensin) via the extracellular and intracellular Invasion is a dynamic process that involves migration of cells domains, respectively (2). The adaptor complexes capture the from their original location into depth of the tissue or outside retrograde flow of actin filaments (F-actin), and this interaction to disseminate to other organs. Enhanced cell invasion is linked array of ECM, integrin, adaptors, and F-actin generates tractive to cancer metastasis, the most prominent cause of the intrac- force for cell motility (3). tability of the disease (1). Cell invasion essentially depends on The Rho-associated protein kinases/LIM kinases/cofilin path- the mechanistic motility of the cell, which is regulated by way (ROCK/LIMK/cofilin pathway) and CapZ-mediated regu- interactions and signaling from large macromolecular com- lation of actin filament dynamics play key roles in the actin/ plexes called focal adhesions to the extracellular matrix (ECM). cytoskeleton network rearrangement (4, 5). ROCKs are serine/ The cellular interface of focal adhesions consists of integrin-a/b threonine kinases that promote actin organization through heterodimers that bind ECM proteins (e.g., fibronectin, lami- phosphorylating several downstream targets, including LIMKs (6). Phosphorylated LIMKs then phosphorylate actin-depoly- merizing factor/cofilin on serine 3. While cofilin facilitates 1 Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese actin depolymerization, phosphorylation of cofilin on serine 2 Foundation for Cancer Research, Koto-ku, Tokyo, Japan. Institute of Microbial 3(p-cofilin) attenuates its actin depolymerization activity Chemistry (BIKAKEN), Numazu, Numazu-shi, Shizuoka, Japan. 3Divison of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Koto- and causes increased numbers of focal adhesion complexes, fi ku, Tokyo, Japan. 4Department of Gastroenterological Surgery, Cancer Institute actin stress ber formation, and enhanced cell motility (7, 8). Hospital, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan. Aberrant promotion of LIMK signaling (e.g., by increased 5Molecular Profiling Research Center for Drug Discovery, National Institute of expression of the upstream regulators RhoA and ROCK) is Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan. observed in many cancers and is associated with cancer metas- Note: Supplementary data for this article are available at Cancer Research tasis (9, 10). Therefore, LIMK inhibitors, which inhibit gener- Online (http://cancerres.aacrjournals.org/). ation of p-cofilin, are thought to be promising anti-invasive Current address for M. Katori: Musashimurayama Hospital, 1-1-5 Enoki, Musa- agents (11). shimurayama, Tokyo 208-0022, Japan; and current address for S.-i. Iemura: Tankyrase is a member of the PARP family that catalyzes þ Translational Research Center, Fukushima Medical University, 11-25 Sakaemachi, formation of long PAR chains onto acceptor proteins using NAD Fukushima City, Fukushima 960-8031, Japan. (12). PARylation confers a drastic negative charge to the acceptor Corresponding Author: Hiroyuki Seimiya, Division of Molecular Biotherapy, proteins and modulates their functions (13). Tankyrase PARylates Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, the telomeric protein TRF1, which is a negative regulator 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3570-0466; Fax: of telomere elongation (12). PARylated TRF1 dissociates from 81-3-3570-0484; E-mail: [email protected] telomeres and is degraded by the ubiquitin/proteasome system. doi: 10.1158/0008-5472.CAN-16-1846 The resulting telomeres exhibit an "open" state that allows easier Ó2017 American Association for Cancer Research. access of telomerase, which in turn elongates telomeres (14, 15). 2328 Cancer Res; 77(9) May 1, 2017 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-1846 TNKS1BP1 Regulates Cell Invasion Tankyrase also upregulates Wnt/b-catenin signaling by PARyla- were transfected with the siRNAs using Lipofectamine RNAi- tion and subsequent degradation of Axins, which are members of MAX (Invitrogen, Life Technologies). the b-catenin destruction complex that consists of Axins, adeno- matous polyposis coli (APC), and glycogen synthase kinase 3 Western blot analysis (GSK3b; ref. 16). Tankyrase inhibitors stabilize Axins, which in Western blot analysis was performed as described (18, 20). Cell turn promote b-catenin degradation and inhibit the growth of lysates were separated by SDS-PAGE, blotted onto polyvinylidene b-catenin–dependent colorectal cancer cells (16, 17). Given that difluoride membranes, and subjected to Western blot analysis tankyrase has a large protein–protein interaction platform, called with the primary antibodies listed in Supplementary Materials ANK repeat clusters (ARC; refs. 18, 19), and is broadly distributed and Methods. to various intracellular loci, including telomeres, nucleoplasm, nuclear pore complexes, cytoplasm, Golgi, and spindle poles, Invasion assay tankyrase likely possesses yet unidentified functions. Invasion assay was performed using CytoSelect 96-well Colla- TNKS1BP1 (also called as TAB182) is a tankyrase-binding gen Cell Invasion Assay Kit (Cell Biolabs) according to the protein that was identified by a yeast two-hybrid screen (18). manufacturer's instruction. The detailed procedure is given in This filament-like protein binds to the ARCs of tankyrase and Supplementary Materials and Methods. colocalizes with the cortical actin network. However, its biologic function has remained uncharacterized. Here, we demonstrate Liquid chromatography/mass spectrometry that TNKS1BP1 interacts with the actin-capping proteins and FLAG-tagged TNKS1BP1 was expressed in HEK293T cells, and plays a role in cell motility and invasion. Our observations that the cell lysate was immunoprecipitated with FLAG antibody. The TNKS1BP1 depletion facilitates F-actin dynamics and cell inva- immunoprecipitated proteins were analyzed by a direct nanoflow sion through ROCK/LIMK–dependent cofilin phosphorylation liquid chromatography/tandem mass spectrometry system, as establish TNKS1BP1 as a negative regulator of cell motility and described previously (21). invasion. Furthermore, tankyrase also modulates cofilin phos- phorylation and cell invasion in a PARP activity–dependent Subcellular fractionation manner, implicating PARylation as a novel posttranslational Subcellular fractions (cytosolic, membrane/organelle, nuclear, modulator of cell motility and invasion. and cytoskeletal fractions) were obtained using a ProteoExtract Subcellular Proteome Extraction Kit (Merck Millipore) according Materials and Methods to the manufacturer's instruction. Purity of the fractions was confirmed by Western blot analysis with maker proteins: calpain Cell line authentication and culture I for cytosolic, histone H2AX for nuclear, and vimentin for HTC75cellsderivedfromHT1080fibrosarcoma cells were cytoskeletal fractions. obtained from Dr. Susan Smith (New York University School of Medicine, New York, NY) in 2001. PANC-1 and KLM-1 cells were provided by Cell Resource Center for Biomedical Immunoprecipitation assay Research Institute of Development, Aging and Cancer, Tohoku Cells were washed with ice-cold PBS and lysed in TNE buffer, University (Sendai, Japan) in 2009 and RIKEN BioResource containing 10 mmol/L Tris-HCl, pH 7.8, 1% NP-40, 150 mmol/L Center in 2010, respectively. They were grown in DMEM NaCl, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fl supplemented with 10% heat-inactivated calf serum and uoride, on ice for 30 minutes. Cell lysates were collected after  100 mg/mL of kanamycin at 37Cinahumidified atmosphere centrifugation at 20,400
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