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Molecular and Cellular Pathobiology

Tankyrase-Binding Protein TNKS1BP1 RegulatesActin Cytoskeleton Rearrangement and CancerCell InvasionTomokazu 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 andWnt/b-catenin signaling, has various binding partners, suggest-ing that it has as-yet unidentified functions. Here, we reportthat the tankyrase-binding protein TNKS1BP1 regulates actincytoskeleton and cancer cell invasion, which is closely associ-ated with cancer progression. TNKS1BP1 colocalized with actinfilaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion,and lamellipodia ruffling were increased with activation ofthe ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the

actin-capping protein CapZA2. TNKS1BP1 depletion dissociat-ed CapZA2 from the cytoskeleton, leading to cofilin phosphor-ylation and enhanced cell invasion. Tankyrase overexpressionincreased cofilin phosphorylation, dissociated CapZA2 fromcytoskeleton, and enhanced cell invasion in a PARP activity–dependent manner. In clinical samples of pancreatic cancer,TNKS1BP1 expression was reduced in invasive regions. Wepropose that the tankyrase-TNKS1BP1axis constitutes aposttrans-lational modulator of cell invasion whose aberration promotescancer malignancy. Cancer Res; 77(9); 2328–38. �2017 AACR.

IntroductionInvasion is a dynamic process that involves migration of cells

from their original location into depth of the tissue or outsideto disseminate to other organs. Enhanced cell invasion is linkedto cancer metastasis, the most prominent cause of the intrac-tability of the disease (1). Cell invasion essentially depends onthe mechanistic motility of the cell, which is regulated byinteractions and signaling from large macromolecular com-plexes called focal adhesions to the extracellular matrix (ECM).The cellular interface of focal adhesions consists of integrin-a/bheterodimers that bind ECM proteins (e.g., fibronectin, lami-

nin, and vitronectin) and adaptor complexes (e.g., talin, vin-culin, and tensin) via the extracellular and intracellulardomains, respectively (2). The adaptor complexes capture theretrograde flow of actin filaments (F-actin), and this interactionarray of ECM, integrin, adaptors, and F-actin generates tractiveforce for cell motility (3).

The Rho-associated protein kinases/LIM kinases/cofilin path-way (ROCK/LIMK/cofilin pathway) and CapZ-mediated regu-lation of actin filament dynamics play key roles in the actin/cytoskeleton network rearrangement (4, 5). ROCKs are serine/threonine kinases that promote actin organization throughphosphorylating several downstream targets, including LIMKs(6). Phosphorylated LIMKs then phosphorylate actin-depoly-merizing factor/cofilin on serine 3. While cofilin facilitatesactin depolymerization, phosphorylation of cofilin on serine3 (p-cofilin) attenuates its actin depolymerization activityand causes increased numbers of focal adhesion complexes,actin stress fiber formation, and enhanced cell motility (7, 8).Aberrant promotion of LIMK signaling (e.g., by increasedexpression of the upstream regulators RhoA and ROCK) isobserved in many cancers and is associated with cancer metas-tasis (9, 10). Therefore, LIMK inhibitors, which inhibit gener-ation of p-cofilin, are thought to be promising anti-invasiveagents (11).

Tankyrase is a member of the PARP family that catalyzesformation of long PAR chains onto acceptor proteins usingNADþ

(12). PARylation confers a drastic negative charge to the acceptorproteins andmodulates their functions (13). Tankyrase PARylatesthe telomeric protein TRF1, which is a negative regulatorof telomere elongation (12). PARylated TRF1 dissociates fromtelomeres and is degraded by the ubiquitin/proteasome system.The resulting telomeres exhibit an "open" state that allows easieraccess of telomerase, which in turn elongates telomeres (14, 15).

1Division of Molecular Biotherapy, Cancer Chemotherapy Center, JapaneseFoundation for Cancer Research, Koto-ku, Tokyo, Japan. 2Institute of MicrobialChemistry (BIKAKEN), Numazu, Numazu-shi, Shizuoka, Japan. 3Divison ofPathology, Cancer Institute, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan. 4Department of Gastroenterological Surgery, Cancer InstituteHospital, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan.5Molecular Profiling Research Center for Drug Discovery, National Institute ofAdvanced Industrial Science and Technology, Koto-ku, Tokyo, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for M. Katori: Musashimurayama Hospital, 1-1-5 Enoki, Musa-shimurayama, Tokyo 208-0022, Japan; and current address for S.-i. Iemura:Translational Research Center, FukushimaMedical University, 11-25 Sakaemachi,Fukushima City, Fukushima 960-8031, Japan.

Corresponding Author: Hiroyuki Seimiya, Division of Molecular Biotherapy,Cancer Chemotherapy Center, Japanese Foundation for Cancer Research,3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3570-0466; Fax:81-3-3570-0484; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1846

�2017 American Association for Cancer Research.

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Tankyrase also upregulates Wnt/b-catenin signaling by PARyla-tion and subsequent degradation of Axins, which are members ofthe b-catenin destruction complex that consists of Axins, adeno-matous polyposis coli (APC), and glycogen synthase kinase 3(GSK3b; ref. 16). Tankyrase inhibitors stabilize Axins, which inturn promote b-catenin degradation and inhibit the growth ofb-catenin–dependent colorectal cancer cells (16, 17). Given thattankyrase has a large protein–protein interaction platform, calledANK repeat clusters (ARC; refs. 18, 19), and is broadly distributedto various intracellular loci, including telomeres, nucleoplasm,nuclear pore complexes, cytoplasm, Golgi, and spindle poles,tankyrase likely possesses yet unidentified functions.

TNKS1BP1 (also called as TAB182) is a tankyrase-bindingprotein that was identified by a yeast two-hybrid screen (18).This filament-like protein binds to the ARCs of tankyrase andcolocalizes with the cortical actin network. However, its biologicfunction has remained uncharacterized. Here, we demonstratethat TNKS1BP1 interacts with the actin-capping proteins andplays a role in cell motility and invasion. Our observations thatTNKS1BP1 depletion facilitates F-actin dynamics and cell inva-sion through ROCK/LIMK–dependent cofilin phosphorylationestablish TNKS1BP1 as a negative regulator of cell motility andinvasion. Furthermore, tankyrase also modulates cofilin phos-phorylation and cell invasion in a PARP activity–dependentmanner, implicating PARylation as a novel posttranslationalmodulator of cell motility and invasion.

Materials and MethodsCell line authentication and culture

HTC75 cells derived from HT1080 fibrosarcoma cells wereobtained from Dr. Susan Smith (New York University Schoolof Medicine, New York, NY) in 2001. PANC-1 and KLM-1 cellswere provided by Cell Resource Center for BiomedicalResearch Institute of Development, Aging and Cancer, TohokuUniversity (Sendai, Japan) in 2009 and RIKEN BioResourceCenter in 2010, respectively. They were grown in DMEMsupplemented with 10% heat-inactivated calf serum and100 mg/mL of kanamycin at 37�C in a humidified atmosphereof 5% CO2. HTC75 cells were authenticated by Seimiya lab-oratory: these cells contain an exogenous hygromycin-resistantgene and therefore exhibit hygromycin-resistant growth, whichhas been routinely tested by cultivating the cells with themedium containing 200 mg/mL hygromycin for a week. Thegrowth of HTC75 cells was not affected by this drug treatment,and we used the drug-resistant cells for further experiments inthe absence of hygromycin. PANC-1 and KLM-1 cells wereauthenticated by short tandem repeat profiling analysis (BEX)in 2016.

Expression vectors and antibodiesThe detailed information about the expression vectors and

antibodies used in this study is given in Supplementary Materialsand Methods.

siRNA transfectionTNKS1BP1 and CapZA2 Stealth siRNAs were purchased from

Invitrogen, Life Technologies: TNKS1BP1, 50-UAUCCAAGCG-CUCUUCCCAAACUCC-30 (#1) and 50-AAGACGAGGA-GUAAUCUUCACCCUG-30 (#2); and CapZA2, 50-GCAGCC-CAUGCAUUUGCACAGUAUA-30 (#6). As a control, StealthRNAi negative control Med GC (#12935-300) was used. Cells

were transfected with the siRNAs using Lipofectamine RNAi-MAX (Invitrogen, Life Technologies).

Western blot analysisWestern blot analysis was performed as described (18, 20). Cell

lysates were separated by SDS-PAGE, blotted onto polyvinylidenedifluoride membranes, and subjected to Western blot analysiswith the primary antibodies listed in Supplementary Materialsand Methods.

Invasion assayInvasion assay was performed using CytoSelect 96-well Colla-

gen Cell Invasion Assay Kit (Cell Biolabs) according to themanufacturer's instruction. The detailed procedure is given inSupplementary Materials and Methods.

Liquid chromatography/mass spectrometryFLAG-tagged TNKS1BP1 was expressed in HEK293T cells, and

the cell lysate was immunoprecipitated with FLAG antibody. Theimmunoprecipitated proteins were analyzed by a direct nanoflowliquid chromatography/tandem mass spectrometry system, asdescribed previously (21).

Subcellular fractionationSubcellular fractions (cytosolic, membrane/organelle, nuclear,

and cytoskeletal fractions) were obtained using a ProteoExtractSubcellular Proteome Extraction Kit (Merck Millipore) accordingto the manufacturer's instruction. Purity of the fractions wasconfirmed by Western blot analysis with maker proteins: calpainI for cytosolic, histone H2AX for nuclear, and vimentin forcytoskeletal fractions.

Immunoprecipitation assayCells were washed with ice-cold PBS and lysed in TNE buffer,

containing 10mmol/L Tris-HCl, pH 7.8, 1%NP-40, 150mmol/LNaCl, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonylfluoride, on ice for 30 minutes. Cell lysates were collected aftercentrifugation at 20,400 � g for 10 minutes at 4�C. Immunopre-cipitation was performed as previously described (20). Thedetailed description is given in Supplementary Materials andMethods.

Fluorescence recovery after photobleaching assayHTC75 and PANC-1 cells expressing mWasabi-actin (Allele

Biotechnology & Pharmaceuticals) were cultured on a poly-L-lysine–coated 35-mm glass-bottom dish (Matsunami Glass)and transfected with siRNAs for 48 hours. To monitor fluores-cence recovery after photobleaching (FRAP), we used time-lapse microscopy with a confocal laser scanning microscope(Fluoview FV-1000, Olympus) equipped with a Plan Apochro-mat 60� oil objective lens (Olympus) and an incubationchamber to ensure a controlled atmosphere (37�C, 5% CO2).To analyze fluorescence recovery, cells were photobleachedusing a scanner (100% 488-nm laser transmission, 50 ms)and imaged with a 488-nm laser for excitation and a 510-nmbandpass filter for emission. Three frames were taken every2 seconds before the photobleaching and then 60 frames(for HTC75) and 40 frames (for PANC-1) were taken every2 seconds. The fluorescence intensity in the bleached areawas measured using ImageJ software (version 1.40g, NIH,Bethesda, MD).

TNKS1BP1 Regulates Cell Invasion

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Immunohistochemical analysisHuman tumor tissue microarrays (401-2206, pancreas tumor,

matched normal tissue, and pancreatitis) were purchased fromProvitro. A pathologist (T. Migita) assessed the data to determinethe expression level of TNKS1BP1. Formalin-fixed, paraffin-embedded tissues were collected from 48 pancreatic tumors,some of which contained noninvasive lesions such as intraepithe-lial neoplasia [pancreatic intraepithelial neoplasia (PanIN), n ¼17], under informed consent at the Cancer Institute Hospital,JFCR (Tokyo, Japan). The tissue samples were chosen by two ofthe pathologists (M. Katori and Y. Ishikawa). Ethical clearancewas obtained in advance from the Institutional Review Board ofJFCR. The detailed analysis procedure is given in SupplementaryMaterials and Methods.

PARP assayIn vitro PARP assay was performed as previously described

(19, 22). The detailed description is given in SupplementaryMaterials and Methods.

Statistical analysisAll data are representative of at least 3 independent experi-

ments. Statistical analysis was carried out using the Student t test.

ResultsTNKS1BP1 negatively regulates focal adhesion and cellinvasion

TNKS1BP1 colocalizes with actin filaments (18; Fig. 1A).Because dynamic remodeling of actin filaments is involved incellular motility and invasion, we first examined whetherTNKS1BP1 affects cellular invasion. As shown in Fig. 1B andSupplementary Fig. S1A and S1B, siRNA-induced TNKS1BP1depletion in HTC75 fibrosarcoma and PANC-1 and KLM-1 pan-creatic cancer cells increased the invasion activity. As a control,cytochalasin D, which disrupts actin polymerization, decreasedinvasion activity, confirming that this activity depends on actinfilament dynamics. In contrast, overexpression of TNKS1BP1significantly decreased invasion activity (Fig. 1C). These resultssuggest that TNKS1BP1 affects actin filaments and represses cellinvasion.

Cell invasion is promoted by the focal adhesion, a distinct siteof cellular adhesion to the ECM (23). Because integrin-mediatedtethering of actin filaments forms the focal adhesion, we nextaddressed whether TNKS1BP1 affects the focal adhesion. Figure1D shows immunofluorescent staining with paxillin, a marker offocal adhesion, coupled with phalloidin staining, which detectsthe actin stress fibers (24). Focal adhesionswere detected as brightfoci at the tips of the actin filaments. TNKS1BP1 depletionincreased the number of these foci and the signal intensity of theactin filaments, as compared with the control siRNA–treatedHTC75 and PANC-1 cells (Fig. 1D and E and Supplementary Fig.S1C). These observations suggest that TNKS1BP1 negatively reg-ulates cell invasion by repressing the effective assembly of focaladhesion complexes and actin stress fiber formation.

TNKS1BP1depletionactivates theROCK/LIMK/cofilinpathwayTo elucidate the mechanism for the enhanced invasion

activity of TNKS1BP1-depleted cells, we monitored the levelof p-cofilin. While cofilin facilitates actin depolymerization,p-cofilin attenuates actin depolymerization activity and causesincreased number of focal adhesion complexes, actin stress

fiber formation, and enhanced cell motility (7). As expected,TNKS1BP1 depletion increased the level of p-cofilin comparedwith the control cells (Fig. 2A, lanes 1–3 and SupplementaryFigs. S1A and S2A). This phenomenon occurred irrespective ofthe existence of EGF, an inducer of cofilin phosphorylation(Supplementary Fig. S2B; ref. 25).

Phosphorylation of cofilin on serine 3 is mediated by ROCKand its downstream effector kinase LIMK (26, 27). To deter-mine whether the ROCK/LIMK pathway contributes to theincreased p-cofilin level in TNKS1BP1-depleted HTC75 cells,we used Y27632, a ROCK inhibitor. As expected, Y27632attenuated the level of p-cofilin upregulation upon TNKS1BP1depletion (Fig. 2A, lanes 4–6). As controls, this attenuationwas not observed when the cells were treated with eitherLY294002 (an inhibitor of PI3K) or U0126 [an inhibitor ofMAPK/ERK 1 and 2 (MEK1 and 2); Fig. 2A, lanes 7–12]. Tofurther confirm the involvement of LIMK in p-cofilin upregu-lation, we used a specific LIMK inhibitor (LIMKi). As predicted,LIMKi attenuated the increase in p-cofilin level in TNKS1BP1-depleted HTC75 cells (Fig. 2B). Similarly, Y27632 and LIMKialso repressed p-cofilin upregulation in TNKS1BP1-depletedPANC-1 cells (Supplementary Fig. S3A). These observationsindicate that TNKS1BP1 depletion upregulates p-cofilin levelthrough the ROCK/LIMK pathway, resulting in a reduced rateof actin depolymerization.

To examine whether TNKS1BP1 affects the dynamics of actinfilaments, we performed FRAP assay of the fluorescent mWa-sabi-actin–transfected HTC75 cells. In control siRNA–trans-fected cells, it took 130 seconds to recover 80% of mWasabi-actin signal after photobleaching (Fig. 3). In contrast, inTNKS1BP1-depleted cells, it took only 70 seconds to recover80% of the signal, which was completely recovered by 120seconds. TNKS1BP1 depletion in PANC-1 cells gave similarresults (Supplementary Fig. S3B). TNKS1BP1-depleted cellsexhibited a greater number of actin filaments and dynamicruffling of the actin-containing lamellipodia (Fig. 3 and Sup-plementary Videos S1 and S2). These observations indicate thatTNKS1BP1 depletion activates the ROCK/LIMK pathway, whichinduces an inhibitory phosphorylation of cofilin and promotesF-actin dynamics.

Actin-capping proteins as functional binding partners forTNKS1BP1

To identify the factor that binds TNKS1BP1 and regulates actinreorganization, we performed liquid chromatography/mass spec-trometry analysis of anti-FLAG immunocomplexes from thelysates of FLAG-tagged TNKS1BP1–expressing cells. The identifiedproteins included the actin-capping protein subunits, such asCapZA1, CapZA2, and CapZB, which regulate assembly anddynamics of actin filaments (5). Coimmunoprecipitation assaysconfirmed that endogenous TNKS1BP1 interacts with all of theectopically expressed capping proteins in intact cells (Fig. 4A). Toexamine whether the endogenous capping proteins interact withTNKS1BP1, HTC75 cell lysates were immunoprecipitated withTNKS1BP1 antibody and subjected to Western blot analysis witheach anti-CapZ antibody. Figure 4B shows that only CapZA2 wascoimmunoprecipitated with TNKS1BP1. Therefore, we focusedon CapZA2 in the subsequent analyses.

Coimmunoprecipitation assays showed that CapZA2 directlybinds to a C-terminal region (amino acids 1,543–1,635) ofTNKS1BP1 (Fig. 4C). To further investigate the relationship

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TNKS1BP1 negatively regulates focal adhesion and cell invasion. A, TNKS1BP1 colocalizes with actin filaments. HTC75 cells were fixed and stained with Alexa 350-phalloidin (F-actin), Alexa 594-DNase I (G-actin), and anti-TNKS1BP1. Differential interference contrast (DIC) image is shown. Scale bar, 10 mm. B, HTC75 cellswere transfected with the indicated siRNAs and incubated for 48 hours. Left, whole-cell extracts were subjected toWestern blot analysis. Right, siRNA-treated cellswere analyzed by invasion assays. After 20-hour incubation, invaded cells were quantitated. As a positive control, cytochalasin D was added to the mediumat a 2-mmol/L final concentration. P value indicates statistical significance (t test). C, Western blot analysis (left) and invasion assay (right) of HTC75 cellsoverexpressing Myc-tagged TNKS1BP1. D, Focal adhesion and actin stress fiber formation in TNKS1BP1-depleted cells. HTC75 cells were transfected with control orTNKS1BP1 siRNAs and stained with Alexa 350-phalloidin (F-actin/blue), TNKS1BP1 (red), and paxillin (green) antibodies. Scale bar, 10 mm. E, Numbers of focaladhesions (paxillin dots) per cell were quantified. The graph shows the averages of at least three experiments.






between TNKS1BP1 and CapZA2, we fractionated the cells intocytosol, membrane/organelle, nucleus, and cytoskeleton frac-tions. While CapZA2 was enriched in the cytosol and mem-brane/organelle fractions, a small amount of the protein was alsodetected in the nucleus and cytoskeleton in control siRNA–treatedcells (Fig. 5A, lanes 1–4). Surprisingly, cytoskeletal CapZA2 dis-appeared in TNKS1BP1-depleted cells to the equivalent extent asin CapZA2-depleted cells (Fig. 5A, lanes 4, 8, 12, 16). UponTNKS1BP1 depletion, CapZA2 seemed to be released from thecytoskeleton, as the levels of cytosolic CapZA2 increased in thesecells. Elevated p-cofilin was observed not only in TNKS1BP1-depleted cells but also in CapZA2-depleted cells (Fig. 5A, lanes 1,5, 9, 13 and Supplementary Fig. S1A). These results suggest thatTNKS1BP1 works as a scaffold protein that stabilizes CapZA2 inthe cytoskeletal F-actin. Meanwhile, TNKS1BP1 was highly sen-sitive to the cytosolic preparation and extracted into the cytosolic,but not cytoskeletal, fraction. Supplementary Figure S4A con-firmed the interaction of TNKS1BP1 with CapZA2 even in asubcellular fraction.

CapZA2-depleted cells showed significantly increased invasionactivity (Fig. 5B). Because we did not detect any difference in cellgrowth among control, TNKS1BP1, and CapZA2 siRNA–treatedcells, it is unlikely that the enhanced invasion activity was derivedfrom the altered proliferative potential (Supplementary Fig. S4B).These observations indicate that TNKS1BP1 depletion leads toCapZA2 release from the cytoskeletal actin filaments, whichresults in enhanced cell invasion.

Tankyrase PARylates TNKS1BP1 and upregulates p-cofilin andcell invasion

TNKS1BP1 binds to tankyrase via the 6–amino acid sequencemotif (RPQPDG) in TNKS1BP1 (28) with ARCs in tankyrase(18, 19). In agreement with our previous report (18), gluta-thione S-transferase (GST)-fused TNKS1BP1 C-terminal frag-

ment (GST-TNKS1BP1C) was PARylated by tankyrase, similarlyto the positive control GST-TRF1 (Supplementary Fig. S5A,lanes 1–6) but not by the PARP-dead mutant (H1184A/E1291A: HE/A; lanes 7–9). To examine the role of the inter-action between TNKS1BP1 and tankyrase, we first performedimmunofluorescent staining of TNKS1BP1-depleted cells toassess the PARP activity of tankyrase in an intact cell usingTRF1 as a marker. TRF1 is stripped out from telomeres bytankyrase-mediated PARylation and subsequently degraded bythe ubiquitin/proteasomal system (14, 15). TNKS1BP1 deple-tion did not enhance endogenous tankyrase activity, as assessedby the intensities of nuclear (telomeric) TRF1 dots (Supple-mentary Fig. S5B). Moreover, while overexpression of tankyrasein the nucleus [FN-tankyrase; tagged with FLAG and a nuclearlocalization signal (NLS) at the amino terminus] diminishedTRF1 dots in the nucleus of the control cells, TNKS1BP1depletion did not change this activity (Supplementary Fig.S5C). Thus, tankyrase PARP activity is not affected byTNKS1BP1 depletion.

Next, we examined the effects of tankyrase overexpression onTNKS1BP1 function. We overexpressed tankyrase either in thenucleus (FN-tankyrase: FLAG-tagged and an NLS at the aminoterminus) or in the cytoplasm (F-tankyrase: FLAG-tagged at theamino terminus; ref. 29). These tags did not significantly affectTNKS1BP1 protein level (Fig. 6A). Importantly, the level of p-cofilin was increased when tankyrase was overexpressed in thecytoplasmwhere actin filaments polymerize (Fig. 6A). To confirmthe importance of the cytoplasmic localization and the PARPactivity, we used tankyrase (HE/A)-overexpressing cells (29, 30).Only the cytoplasmic and catalytically active tankyrase, but notthe nuclear or the HE/A mutant, decreased cytoskeletal proteinlevel of CapZA2 and increased p-cofilin (Fig. 6B). To furtherconfirm the dependency of this phenotype on PARP activity oftankyrase, we examined the effect of the tankyrase PARP inhibitor

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TNKS1BP1 depletion upregulates cofilin phosphorylation via the ROCK/LIMK pathway. A, ROCK-dependent phosphorylation of cofilin by TNKS1BP1 depletion.HTC75 cells were transfected with the siRNAs for 36 hours and then treated with 10 mmol/L Y27632 (ROCK inhibitor, lanes 4–6), 10 mmol/L LY294002 (PI3Kinhibitor, lanes 7–9), or 10 mmol/L U0126 (MEK inhibitor, lanes 10–12) for 12 hours. Cells were examined byWestern blot analysis.B, LIMK inhibitor (LIMKi3) decreasedthe cofilin phosphorylation induced by TNKS1BP1 depletion. After transfection with indicated siRNAs for 36 hours, LIMKi3 was added and incubated for12 hours. The cell extracts were subjected to Western blot analysis.

Ohishi et al.





XAV939 (16). As expected, XAV939 attenuated the elevated levelof p-cofilin led by tankyrase overexpression in the cytoplasm (Fig.6C). Furthermore, only cells that overexpress wild-type tankyrasein the cytoplasm showed significantly enhanced invasion activity(Fig. 6D), which was inhibited by XAV939 (Supplementary Fig.S5D). Neither theHE/Amutant nor overexpression in the nucleusenhanced cell invasion, coincident with Fig. 6B. These observa-tions suggest that tankyrase in the cytoplasm attenuates thefunction of TNKS1BP1 in a PARP activity–dependent manner tostabilize CapZA2 in the cytoskeletal F-actin, leading to enhancedinvasion through rearrangement of the actin cytoskeleton.

TNKS1BP1 downregulation at pancreatic cancer invasionWe next examined TNKS1BP1 expression in clinical cancers

by immunohistochemistry of various tissue microarrays. Wefound that pancreatic cancers often show reduced levels ofTNKS1BP1 protein expression (Fig. 7A and B). Thus, wefocused on pancreatic cancer, which is highly aggressive andmetastatic with a low survival rate (31). PanIN is a well-defined,common precursor of invasive pancreatic ductal adenocarci-noma arising in the pancreatic duct. We selected patient tissuesamples in which we were able to find normal pancreatic ducts,PanINs, and invasive adenocarcinoma in a single field, for easy

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TNKS1BP1 binds CapZ proteins.A, HTC75 cells were transfected withFlag-tagged CapZA1, CapZA2, orCapZB for 48 hours. Cell lysates wereimmunoprecipitated with anti-FLAGbeads, and the immunocomplexeswere analyzed by Western blotanalysis. B, HTC75 cell lysates wereimmunoprecipitated with normal IgGor anti-TNKS1BP1 antibody andsubjected to Western blot analysis. C,HA-tagged TNKS1BP1 constructs wereexpressed in HTC75 cells. Cell lysateswere subjected to immunoprecipitationwith anti-HA antibodies, followed byWestern blot analysis with anti-HA oranti-CapZA2 antibodies.

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TNKS1BP1 depletion promotes actin dynamics. Left, FRAP assay of mWasabi-actin in HTC75 cells. Cells were treated with the indicated siRNAs for 48 hours.FRAP assay was performed with time-lapse microscopy. For each cell, a magnified view of photobleaching is shown in the right. Arrows, area of photobleaching.Scale bar, 20 mm. Right, fluorescence intensity in the bleached area was quantitated.






evaluation of TNKS1BP1 expression changes in the processof cell invasion (32). The intensity of TNKS1BP1 expressionremains relatively high in PanINs (Fig. 7C, b) comparedwith the normal pancreatic ducts (Fig. 7C, a). In contrast,TNKS1BP1 expression was lower in the invasive adenocarcino-ma than in the normal pancreatic ducts and PanINs (Fig. 7C, c).Using 17 patient tissue samples, we found that the expressionlevels of TNKS1BP1 in the invasive regions were significantlylower than those in noninvasive regions of the same tissue (Fig.7D). These observations indicate an inverse relationshipbetween TNKS1BP1 expression and cancer invasion.

DiscussionTNKS1BP1 as a component of the actin cytoskeleton

While tankyrase targets TRF1 and enhances telomere elonga-tion by telomerase (14), TNKS1BP1 does not bind telomeres oraffect telomere length in human cancer cells (ref. 33 and H.Seimiya, unpublished observations). MOTIF analysis (http://www.genome.jp/tools/motif/) indicates that there are no charac-teristic motifs or known functional domains in TNKS1BP1.According to the FASTA homology search (http://www.genome.jp/tools/fasta/), however, this protein has a weak simi-larity to ECM proteins, such as collagens and proteoglycans,

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Cytoplasmic overexpression oftankyrase upregulates cofilinphosphorylation and cell invasion. A,Whole-cell extracts of HTC75 cells thatstably overexpressed FLAG-taggedtankyrase constructs were subjected toWestern blot analysis. F, FLAG epitopeat the N-terminus; FN, FLAG epitopeand NLS. B, Subcellular fractionationandWestern blot analysis of tankyrase-overexpressing cells. C, Effect ofXAV939, a tankyrase inhibitor, ontankyrase-mediated phosphorylationof cofilin. Cells were treated withXAV939 for 48 hours, andWestern blotanalysis was performed. Accumulationof tankyrase protein in the mock cells isa pharmacodynamic marker oftankyrase inhibition. D, Invasion assaywas performed as in Fig. 1. P valueindicates statistical significance (t test).

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TNKS1BP1 regulates CapZA2cytoskeletal localization. A, TNKS1BP1depletion releases CapZA2 from thecytoskeleton. After transfection ofHTC75 cells with the indicated siRNAsfor 72 hours, subcellular fractions wereanalyzed by Western blot analysis. B,Invasion assay was performed as in Fig.1. P value indicates statisticalsignificance (t test).

Ohishi et al.




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suggesting a role in cell adhesion, motility, and/or invasion. Thisinformation and its intracellular colocalization with actin fila-ments support that TNKS1BP1 is a functional component of theactin cytoskeleton.

In our original report, we also detected TNKS1BP1 in thenucleus, especially in the perinucleolar heterochromatic regions(18).We speculate that these nuclear TNKS1BP1wouldbederivedfrom alternative splicing, different start sites, or both of thetranscripts as observed in the H-InvDB Annotated Human GeneDatabase (http://www.h-invitational.jp), which give rise toshorter forms (i.e., the C-terminal half of full-length protein).Consistent with this idea, the TNKS1BP1-truncated mutant thatlacks theN-terminal 1220 amino acids accumulates in the nucleuswhen ectopically expressed in cells (H. Seimiya, unpublishedobservation). Although TNKS1BP1 contains 2 NLS at the C-terminal basic region, their function appears to be recessive inthe full-length protein because the full-length TNKS1BP1 colo-calizes with actin filaments rather than in the nucleus. Nuclearlocalization of the shorter isoforms is further supported by the factthat antibodies raised against the N-terminal fragment ofTNKS1BP1 detect only cytoskeletal TNKS1BP1 but not nuclearTNKS1BP1 by immunofluorescent staining (H. Seimiya, unpub-lished observation). These facts and functional analyses in thepresent study establish full-length TNKS1BP1 as a cytoskeletal

protein. Although the function of the short isoforms in thenucleus remains to be determined, Zou and colleagues demon-strated that TNKS1BP1 functions in DNA double-strand breakrepair (34).

TNKS1BP1 and CapZ regulate ROCK/LIMK/cofilin pathwayand cell invasion

We showed that TNKS1BP1 expression level is a determinantfor the actin cytoskeletal rearrangement and the ability of the cellto invade into the collagenmatrix. Intriguingly, TNKS1BP1 deple-tion decreased the level of CapZA2, a newly identified TNKS1BP1-binding partner, in the cytoskeletal fraction. CapZA2 is a com-ponent of the heterodimeric actin-capping protein,which consistsofa (CapZA1 or CapZA2) and b (CapZB) subunits (35). They capthe barbed ends of growing actin filaments to block their elon-gation (5). This effect is similar to those of cytochalasins, whichbind the barbed ends of growing actin filaments and inhibit cellinvasion (ref. 36 and this study). Our results suggest thatTNKS1BP1 stabilizes the capping proteins at barbed ends andnegatively regulates actin polymerization.

What, then, is the molecular mechanism for CapZA2 dissoci-ation from actin filaments upon TNKS1BP1 depletion? Becausethere is a significant mismatch, that is, different symmetriesbetween 3-dimensional structures of CapZ and actin (37), it is

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Downregulation of TNKS1BP1 atinvasive sites of pancreatic cancer.A, Immunohistochemistry of tissuemicroarrays with anti-TNKS1BP1antibody. Representative photosfor three grades (low/medium/high) ofdistribution and intensity patterns ofTNKS1BP1 are shown. Scale bar,500 mm. B, TNKS1BP1 tissue microarrayspots were classified according to thegrades in A, revealing lower expressionin pancreatic cancers as compared withthe normal samples. C, Differentialexpression of TNKS1BP1 in pancreaticcancer. Top, lower magnification.Bottom, magnified images of thesquares in the top photo. a, normalpancreatic duct region; b, PanIN; c,invasive adenocarcinoma. Arrows,typical cells. Scale bar, 500 mm. D,Quantification of TNKS1BP1 intensity inpatients with pancreatic cancer(n ¼ 17). Samples in C and D werecollected and analyzed under informedconsent at the Cancer InstituteHospital, JFCR.





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possible that TNKS1BP1 is required for efficient interactionbetween these two proteins. CapZ interaction with actin is alsoinhibited by phosphatidylinositol 4,5-bisphosphate, myotro-phin/V-1 and CARMIL (38–40). Whether TNKS1BP1 knockdownmay affect the intracellular levels, distributions, or both of thesefactors remains to be investigated.

We found that knockdown of either TNKS1BP1 or CapZA2increases the level of p-cofilin in a ROCK/LIMK pathway–depen-dent manner, leading to an increased number of focal adhesionsthat were accompanied by F-actin polymerization. So far, theprecisemechanismunderlyingwhyCapZA2dissociation from theactin filaments activates the ROCK/LIMK pathway remains elu-sive. Given that ROCK inhibition enhances the protein levels ofCapZ (41) and strain-induced stimulation of CapZ dynamicsdepends on the RhoA/ROCK pathway (42), there might be afeedback loop system in the RhoA/ROCK/LIMK pathway and theCapZ-mediated regulation machinery of actin reorganization.

TNKS1BP1 as a possible regulator of cancer invasionWe demonstrated that TNKS1BP1 is downregulated in invasive

pancreatic adenocarcinoma. This observation suggests thatTNKS1BP1 may act as a suppressor of pancreatic cancer invasionvia negative regulation of actin cytoskeleton rearrangement. Giv-en that TNKS1BP1 downregulation is observed exclusively at theinvasive area of the cancerous lesions, it is possible that its proteinexpression is regulated in a microenvironment-dependent, tran-sient manner. In fact, the Oncomine database (https://www.oncomine.org) shows that the level of TNKS1BP1 transcript isnot significantly altered in the bulk of pancreatic cancer cells. Thiswould be reminiscent of the epithelial–mesenchymal transition(EMT), through which an epithelial cancer cell converts to amesenchymal cell type with less cell-to-cell adhesion and highermotility (43). Microenvironmental factors, such as TGFb, pro-mote EMT of metastasizing cancer cells. Furthermore, it has beenpostulated that cancer cells having settled at the metastatic siteoften undergo the reverse event, mesenchymal–epithelial transi-tion (MET). Thus, EMT and MET are mutually reversible, and thebalance between these two events regulates cell motility andinvasion. We monitored epithelial and mesenchymal markerproteins, such as E-cadherin and N-cadherin, respectively,and found no evidence that TNKS1BP1 directly regulates EMT(T. Ohishi & H. Seimiya, unpublished observation).

Our data suggest that TNKS1BP1 regulates CapZ recruitment toactin cytoskeleton. Recently, Lee and colleagues reported thatCapZA1 expression is associated with decreased cancer cell migra-tion and invasion and could be used as a good prognostic markerof gastric cancer (44). The authors also showed that CapZA1depletion causes a significant increase in gastric cancer cell migra-tion and invasion, whereas CapZA1 overexpression shows theopposite effects. These results are in good agreement with ourobservation that CapZA2 depletion increases the invasive activityof cancer cells. Together, these observations suggest thatTNKS1BP1-CapZ interaction with the actin cytoskeleton plays anegative role in cancer cell invasion.

A new role for tankyrase in cell invasionOur result that tankyrase-overexpressing cells phenocopy

TNKS1BP1-depleted cells in terms of (i) CapZA2 release fromactin filament, (ii) p-cofilin upregulation, and (iii) enhanced cellinvasion suggest that tankyrase is the upstream repressor ofTNKS1BP1 function. These effects of tankyrase require its PARP

activity and cytoplasmic localization. According to Tian andcolleague, tankyrase inhibition by a small compound or by RNAinterference reduces the invasive activity of neuroblastoma cells,depending on telomere shortening (45). Because the effect oftankyrase inhibition on telomere function emerges rather slowlydue to gradual shortening of telomeres (33, 46), we prefer a non-telomeric mechanism for the altered invasive activity.

Tankyrase-mediated PARylation of the Wnt suppressor Axinsleads to ubiquitination of the PARylated Axins followed byproteasomal degradation (16). This is a unique signaling relayfrom tankyrase-mediated PARylation to RNF146-mediated ubi-quitination: the ubiquitin E3 ligase RNF146 in its resting statebinds tankyrase, and PAR chains produced by tankyrase bind theWWEdomain of RNF146,which causes allosteric activation of theubiquitin E3 ligase activity (47). Tankyrase-mediated PARylationfollowed by ubiquitination is also observed in other tankyrasesubstrates, such as TRF1 and 3BP2 (15, 48). In contrast, tankyraseinhibitors did not upregulate the level of TNKS1BP1 protein(T. Ohishi & H. Seimiya, unpublished observation), suggestingthat PARylated TNKS1BP1 may not be an efficient substrate forubiquitin-dependent proteasomal degradation.

The question regarding the mechanism by which tankyraseregulates the function of TNKS1BP1 remains unanswered. Tan-kyrase has 5 ARCs as a platform for protein–protein interactions,and ARCs 1, 2, 4, and 5 can bind TNKS1BP1 (19, 49). Existence ofmultiple ARCs suggests a role for tankyrase as a molecular lattice,in which TNKS1BP1 may be an essential component for func-tional linkage to the CapZ actin filaments. Formation of theseprotein complexes would depend on the specific interactionbetween tankyrase and TNKS1BP1 because TNKS1BP1 bindstankyrase ARCs but not the conventional ankyrin G (18). Con-sistent with this idea, efficient Wnt/b-catenin signaling requiresthe lattice-like scaffolding of tankyrase, which is mediated by thesterile a motif–dependent polymerization and multivalent ARCinteraction with Axins (50).

In conclusion, we have shown that TNKS1BP1 interacts withactin-capping proteins and regulates the ROCK/LIMK/cofilinpathway for actin reorganization. These observations give a newinsight into the molecular mechanism for actin-regulated cellmotility and how its perturbation could contribute to cancerinvasion.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Ohishi, H. SeimiyaDevelopment of methodology: T. Migita, H. SeimiyaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Ohishi, H. Yoshida, M. Katori, Y. Muramatsu, M.Miyake, Y. Ishikawa, A. Saiura, S.-i. Iemura, H. SeimiyaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Ohishi, M. Katori, T. Natsume, H. SeimiyaWriting, review, and/or revision of the manuscript: T. Ohishi, H. SeimiyaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. SeimiyaStudy supervision: H. Seimiya

AcknowledgmentsThe authors thank Dr. Sho Isoyama for technical assistance to quantify the

immunohistochemical staining, Drs. Kazuhiro Katayama and Yoshikazu Sugi-moto for the 3�HA plasmid, and Dr. Toru Hirota for the pcDNA3-FLAGdestination vector.

Ohishi et al.




https://www.oncomine.org

https://www.oncomine.org


Grant SupportThis work was supported in part by a Grant-in-Aid for Young Scientists (B),

Japan Society for the Promotion of Science (JSPS; no. 23701068, 25871074 to T.Ohishi), a Grant-in-Aid for Scientific Research on Innovative Areas, Ministry ofEducation, Culture, Sports, Science and Technology (no. 23117527 to H.Seimiya), and a Grant-in-Aid for Challenging Exploratory Research, JSPS (no.26640109 to H. Seimiya).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 22, 2016; revised September 16, 2016; accepted January 29,2017; published OnlineFirst February 15, 2017.

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2017;77:2328-2338. Published OnlineFirst February 15, 2017.Cancer Res Tomokazu Ohishi, Haruka Yoshida, Masamichi Katori, et al. Cytoskeleton Rearrangement and Cancer Cell InvasionTankyrase-Binding Protein TNKS1BP1 Regulates Actin

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