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. 2017 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 2017 American Association for Cancer Research. access of telomerase, which in turn elongates telomeres (14, 15).

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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 phosphorylation 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 g for 10 minutes at 4 C. Immunopre- cipitation was performed as previously described (20). The of 5% CO2. HTC75 cells were authenticated by Seimiya lab- oratory: these cells contain an exogenous hygromycin-resistant detailed description is given in Supplementary Materials and gene and therefore exhibit hygromycin-resistant growth, which Methods. has been routinely tested by cultivating the cells with the Fluorescence recovery after photobleaching assay medium containing 200 mg/mL hygromycin for a week. The HTC75 and PANC-1 cells expressing mWasabi-actin (Allele growth of HTC75 cells was not affected by this drug treatment, Biotechnology & Pharmaceuticals) were cultured on a poly-L- and we used the drug-resistant cells for further experiments in lysine–coated 35-mm glass-bottom dish (Matsunami Glass) the absence of hygromycin. PANC-1 and KLM-1 cells were and transfected with siRNAs for 48 hours. To monitor fluores- authenticated by short tandem repeat profiling analysis (BEX) cence recovery after photobleaching (FRAP), we used time- in 2016. lapse microscopy with a confocal laser scanning microscope Expression vectors and antibodies (Fluoview FV-1000, Olympus) equipped with a Plan Apochro- mat 60 oil objective lens (Olympus) and an incubation The detailed information about the expression vectors and antibodies used in this study is given in Supplementary Materials chamber to ensure a controlled atmosphere (37 C, 5% CO2). fl and Methods. To analyze uorescence recovery, cells were photobleached using a scanner (100% 488-nm laser transmission, 50 ms) siRNA transfection and imaged with a 488-nm laser for excitation and a 510-nm TNKS1BP1 and CapZA2 Stealth siRNAs were purchased from bandpass filter for emission. Three frames were taken every Invitrogen, Life Technologies: TNKS1BP1, 50-UAUCCAAGCG- 2 seconds before the photobleaching and then 60 frames CUCUUCCCAAACUCC-30 (#1) and 50-AAGACGAGGA- (for HTC75) and 40 frames (for PANC-1) were taken every GUAAUCUUCACCCUG-30 (#2);andCapZA2,50-GCAGCC- 2 seconds. The fluorescence intensity in the bleached area CAUGCAUUUGCACAGUAUA-30 (#6). As a control, Stealth was measured using ImageJ software (version 1.40g, NIH, RNAi negative control Med GC (#12935-300) was used. Cells Bethesda, MD).

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Immunohistochemical analysis fiber formation, and enhanced cell motility (7). As expected, Human tumor tissue microarrays (401-2206, pancreas tumor, TNKS1BP1 depletion increased the level of p-cofilin compared matched normal tissue, and pancreatitis) were purchased from with the control cells (Fig. 2A, lanes 1–3 and Supplementary Provitro. A pathologist (T. Migita) assessed the data to determine Figs. S1A and S2A). This phenomenon occurred irrespective of the expression level of TNKS1BP1. Formalin-fixed, paraffin- the existence of EGF, an inducer of cofilin phosphorylation embedded tissues were collected from 48 pancreatic tumors, (Supplementary Fig. S2B; ref. 25). some of which contained noninvasive lesions such as intraepithe- Phosphorylation of cofilin on serine 3 is mediated by ROCK lial neoplasia [pancreatic intraepithelial neoplasia (PanIN), n ¼ and its downstream effector kinase LIMK (26, 27). To deter- 17], under informed consent at the Cancer Institute Hospital, mine whether the ROCK/LIMK pathway contributes to the JFCR (Tokyo, Japan). The tissue samples were chosen by two of increased p-cofilin level in TNKS1BP1-depleted HTC75 cells, the pathologists (M. Katori and Y. Ishikawa). Ethical clearance we used Y27632, a ROCK inhibitor. As expected, Y27632 was obtained in advance from the Institutional Review Board of attenuated the level of p-cofilin upregulation upon TNKS1BP1 JFCR. The detailed analysis procedure is given in Supplementary depletion (Fig. 2A, lanes 4–6). As controls, this attenuation Materials and Methods. was not observed when the cells were treated with either LY294002 (an inhibitor of PI3K) or U0126 [an inhibitor of PARP assay MAPK/ERK 1 and 2 (MEK1 and 2); Fig. 2A, lanes 7–12]. To In vitro PARP assay was performed as previously described further confirm the involvement of LIMK in p-cofilin upregu- (19, 22). The detailed description is given in Supplementary lation, we used a specific LIMK inhibitor (LIMKi). As predicted, Materials and Methods. LIMKi attenuated the increase in p-cofilin level in TNKS1BP1- depleted HTC75 cells (Fig. 2B). Similarly, Y27632 and LIMKi Statistical analysis also repressed p-cofilin upregulation in TNKS1BP1-depleted All data are representative of at least 3 independent experi- PANC-1 cells (Supplementary Fig. S3A). These observations ments. Statistical analysis was carried out using the Student t test. indicate that TNKS1BP1 depletion upregulates p-cofilin level through the ROCK/LIMK pathway, resulting in a reduced rate Results of actin depolymerization. TNKS1BP1 negatively regulates focal adhesion and cell To examine whether TNKS1BP1 affects the dynamics of actin invasion filaments, we performed FRAP assay of the fluorescent mWa- TNKS1BP1 colocalizes with actin filaments (18; Fig. 1A). sabi-actin–transfected HTC75 cells. In control siRNA–trans- Because dynamic remodeling of actin filaments is involved in fected cells, it took 130 seconds to recover 80% of mWasabi- cellular motility and invasion, we first examined whether actin signal after photobleaching (Fig. 3). In contrast, in TNKS1BP1 affects cellular invasion. As shown in Fig. 1B and TNKS1BP1-depleted cells, it took only 70 seconds to recover Supplementary Fig. S1A and S1B, siRNA-induced TNKS1BP1 80% of the signal, which was completely recovered by 120 depletion in HTC75 fibrosarcoma and PANC-1 and KLM-1 pan- seconds. TNKS1BP1 depletion in PANC-1 cells gave similar creatic cancer cells increased the invasion activity. As a control, results (Supplementary Fig. S3B). TNKS1BP1-depleted cells cytochalasin D, which disrupts actin polymerization, decreased exhibited a greater number of actin filaments and dynamic invasion activity, confirming that this activity depends on actin ruffling of the actin-containing lamellipodia (Fig. 3 and Sup- filament dynamics. In contrast, overexpression of TNKS1BP1 plementary Videos S1 and S2). These observations indicate that significantly decreased invasion activity (Fig. 1C). These results TNKS1BP1 depletion activates the ROCK/LIMK pathway, which suggest that TNKS1BP1 affects actin filaments and represses cell induces an inhibitory phosphorylation of cofilin and promotes invasion. F-actin dynamics. Cell invasion is promoted by the focal adhesion, a distinct site of cellular adhesion to the ECM (23). Because integrin-mediated Actin-capping proteins as functional binding partners for tethering of actin filaments forms the focal adhesion, we next TNKS1BP1 addressed whether TNKS1BP1 affects the focal adhesion. Figure To identify the factor that binds TNKS1BP1 and regulates actin 1D shows immunofluorescent staining with paxillin, a marker of reorganization, we performed liquid chromatography/mass spec- focal adhesion, coupled with phalloidin staining, which detects trometry analysis of anti-FLAG immunocomplexes from the the actin stress fibers (24). Focal adhesions were detected as bright lysates of FLAG-tagged TNKS1BP1–expressing cells. The identified foci at the tips of the actin filaments. TNKS1BP1 depletion proteins included the actin-capping protein subunits, such as increased the number of these foci and the signal intensity of the CapZA1, CapZA2, and CapZB, which regulate assembly and actin filaments, as compared with the control siRNA–treated dynamics of actin filaments (5). Coimmunoprecipitation assays HTC75 and PANC-1 cells (Fig. 1D and E and Supplementary Fig. confirmed that endogenous TNKS1BP1 interacts with all of the S1C). These observations suggest that TNKS1BP1 negatively reg- ectopically expressed capping proteins in intact cells (Fig. 4A). To ulates cell invasion by repressing the effective assembly of focal examine whether the endogenous capping proteins interact with adhesion complexes and actin stress fiber formation. TNKS1BP1, HTC75 cell lysates were immunoprecipitated with TNKS1BP1 antibody and subjected to Western blot analysis with TNKS1BP1 depletion activates the ROCK/LIMK/cofilin pathway each anti-CapZ antibody. Figure 4B shows that only CapZA2 was To elucidate the mechanism for the enhanced invasion coimmunoprecipitated with TNKS1BP1. Therefore, we focused activity of TNKS1BP1-depleted cells, we monitored the level on CapZA2 in the subsequent analyses. of p-cofilin. While cofilin facilitates actin depolymerization, Coimmunoprecipitation assays showed that CapZA2 directly p-cofilin attenuates actin depolymerization activity and causes binds to a C-terminal region (amino acids 1,543–1,635) of increased number of focal adhesion complexes, actin stress TNKS1BP1 (Fig. 4C). To further investigate the relationship

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A DIC TNKS1BP1 F-Actin G-Actin Merge

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30 kDa Mock siRNA GAPDH Control siRNA (#2) siRNA (#1) TNKS1BP1 TNKS1BP1 No treatment Cytochalasin D Myc-TNKS1BP1

DEControl siRNA TNKS1BP1 siRNA (#1) P < 0.01 P < 0.01 㻟㻡 Paxillin 㻟㻜

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Figure 1. 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 cells were transfected with the indicated siRNAs and incubated for 48 hours. Left, whole-cell extracts were subjected to Western blot analysis. Right, siRNA-treated cells were analyzed by invasion assays. After 20-hour incubation, invaded cells were quantitated. As a positive control, cytochalasin D was added to the medium at a 2-mmol/L final concentration. P value indicates statistical significance (t test). C, Western blot analysis (left) and invasion assay (right) of HTC75 cells overexpressing Myc-tagged TNKS1BP1. D, Focal adhesion and actin stress fiber formation in TNKS1BP1-depleted cells. HTC75 cells were transfected with control or TNKS1BP1 siRNAs and stained with Alexa 350-phalloidin (F-actin/blue), TNKS1BP1 (red), and paxillin (green) antibodies. Scale bar, 10 mm. E, Numbers of focal adhesions (paxillin dots) per cell were quantified. The graph shows the averages of at least three experiments.

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A Y27632 LY294002 U0126 B Control TNKS1BP1 TNKS1BP1 TNKS1BP1 TNKS1BP1 TNKS1BP1 siRNA siRNA (#1) siRNA siRNA siRNA siRNA #1 #2 #1 #2 #1 #2 #1 #2 LIMKi (μmol/L) 033100 10 Control Control Control Control (kDa) (kDa) TNKS1BP1 250 TNKS1BP1 250 Phospho cofilin (Ser3) 15 Phospho cofilin (Ser3) Cofilin 15 15 Phospho Cofilin 60 Akt (Ser473) 15 Phospho 40 MEK1,2 40 GAPDH (Ser217/221) 40 GAPDH Lane 1 2 3 4 5 6 7 8 9 101112

Figure 2. 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 (PI3K inhibitor, lanes 7–9), or 10 mmol/L U0126 (MEK inhibitor, lanes 10–12) for 12 hours. Cells were examined by Western blot analysis. B, LIMK inhibitor (LIMKi3) decreased the cofilin phosphorylation induced by TNKS1BP1 depletion. After transfection with indicated siRNAs for 36 hours, LIMKi3 was added and incubated for 12 hours. The cell extracts were subjected to Western blot analysis.

between TNKS1BP1 and CapZA2, we fractionated the cells into ment (GST-TNKS1BP1C) was PARylated by tankyrase, similarly cytosol, membrane/organelle, nucleus, and cytoskeleton frac- to the positive control GST-TRF1 (Supplementary Fig. S5A, tions. While CapZA2 was enriched in the cytosol and mem- lanes 1–6) but not by the PARP-dead mutant (H1184A/ brane/organelle fractions, a small amount of the protein was also E1291A: HE/A; lanes 7–9). To examine the role of the inter- detected in the nucleus and cytoskeleton in control siRNA–treated action between TNKS1BP1 and tankyrase, we first performed cells (Fig. 5A, lanes 1–4). Surprisingly, cytoskeletal CapZA2 dis- immunofluorescent staining of TNKS1BP1-depleted cells to appeared in TNKS1BP1-depleted cells to the equivalent extent as assess the PARP activity of tankyrase in an intact cell using in CapZA2-depleted cells (Fig. 5A, lanes 4, 8, 12, 16). Upon TRF1 as a marker. TRF1 is stripped out from telomeres by TNKS1BP1 depletion, CapZA2 seemed to be released from the tankyrase-mediated PARylation and subsequently degraded by cytoskeleton, as the levels of cytosolic CapZA2 increased in these the ubiquitin/proteasomal system (14, 15). TNKS1BP1 deple- cells. Elevated p-cofilin was observed not only in TNKS1BP1- tion did not enhance endogenous tankyrase activity, as assessed depleted cells but also in CapZA2-depleted cells (Fig. 5A, lanes 1, by the intensities of nuclear (telomeric) TRF1 dots (Supple- 5, 9, 13 and Supplementary Fig. S1A). These results suggest that mentary Fig. S5B). Moreover, while overexpression of tankyrase TNKS1BP1 works as a scaffold protein that stabilizes CapZA2 in in the nucleus [FN-tankyrase; tagged with FLAG and a nuclear the cytoskeletal F-actin. Meanwhile, TNKS1BP1 was highly sen- localization signal (NLS) at the amino terminus] diminished sitive to the cytosolic preparation and extracted into the cytosolic, TRF1 dots in the nucleus of the control cells, TNKS1BP1 but not cytoskeletal, fraction. Supplementary Figure S4A con- depletion did not change this activity (Supplementary Fig. firmed the interaction of TNKS1BP1 with CapZA2 even in a S5C). Thus, tankyrase PARP activity is not affected by subcellular fraction. TNKS1BP1 depletion. CapZA2-depleted cells showed significantly increased invasion Next, we examined the effects of tankyrase overexpression on activity (Fig. 5B). Because we did not detect any difference in cell TNKS1BP1 function. We overexpressed tankyrase either in the growth among control, TNKS1BP1, and CapZA2 siRNA–treated nucleus (FN-tankyrase: FLAG-tagged and an NLS at the amino cells, it is unlikely that the enhanced invasion activity was derived terminus) or in the cytoplasm (F-tankyrase: FLAG-tagged at the from the altered proliferative potential (Supplementary Fig. S4B). amino terminus; ref. 29). These tags did not significantly affect These observations indicate that TNKS1BP1 depletion leads to TNKS1BP1 protein level (Fig. 6A). Importantly, the level of p- CapZA2 release from the cytoskeletal actin filaments, which cofilin was increased when tankyrase was overexpressed in the results in enhanced cell invasion. cytoplasm where actin filaments polymerize (Fig. 6A). To confirm the importance of the cytoplasmic localization and the PARP Tankyrase PARylates TNKS1BP1 and upregulates p-cofilin and activity, we used tankyrase (HE/A)-overexpressing cells (29, 30). cell invasion Only the cytoplasmic and catalytically active tankyrase, but not TNKS1BP1 binds to tankyrase via the 6–aminoacidsequence the nuclear or the HE/A mutant, decreased cytoskeletal protein motif (RPQPDG) in TNKS1BP1 (28) with ARCs in tankyrase level of CapZA2 and increased p-cofilin (Fig. 6B). To further (18, 19). In agreement with our previous report (18), gluta- confirm the dependency of this phenotype on PARP activity of thione S-transferase (GST)-fused TNKS1BP1 C-terminal frag- tankyrase, we examined the effect of the tankyrase PARP inhibitor

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Control siRNA TNKS1BP1 siRNA TNKS1BP1 siRNA (n = 5) 1.4 Control siRNA (n = 5) –6 s 1.2

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Figure 3. 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.

XAV939 (16). As expected, XAV939 attenuated the elevated level TNKS1BP1 downregulation at pancreatic cancer invasion of p-cofilin led by tankyrase overexpression in the cytoplasm (Fig. We next examined TNKS1BP1 expression in clinical cancers 6C). Furthermore, only cells that overexpress wild-type tankyrase by immunohistochemistry of various tissue microarrays. We in the cytoplasm showed significantly enhanced invasion activity found that pancreatic cancers often show reduced levels of (Fig. 6D), which was inhibited by XAV939 (Supplementary Fig. TNKS1BP1 protein expression (Fig. 7A and B). Thus, we S5D). Neither the HE/A mutant nor overexpression in the nucleus focused on pancreatic cancer, which is highly aggressive and enhanced cell invasion, coincident with Fig. 6B. These observa- metastatic with a low survival rate (31). PanIN is a well-defined, tions suggest that tankyrase in the cytoplasm attenuates the common precursor of invasive pancreatic ductal adenocarci- function of TNKS1BP1 in a PARP activity–dependent manner to noma arising in the pancreatic duct. We selected patient tissue stabilize CapZA2 in the cytoskeletal F-actin, leading to enhanced samples in which we were able to find normal pancreatic ducts, invasion through rearrangement of the actin cytoskeleton. PanINs, and invasive adenocarcinoma in a single field, for easy

A B IP:

WB: Mock FLAG-CapZA1 FLAG-CapZA2 FLAG-CapZB (kDa) Input (20%) Figure 4. WB: Normal IgG TNKS1BP1 TNKS1BP1 binds CapZ proteins. TNKS1 250 TNKS1BP1 Constructs BP1 CapZA1 A, HTC75 cells were transfected with CapZA2 Flag-tagged CapZA1, CapZA2, or 40 CapZB for 48 hours. Cell lysates were CapZB IP: FLAG FLAG immunoprecipitated with anti-FLAG 30 beads, and the immunocomplexes Δ1543–1635 Mock 1–1729 (WT) were analyzed by Western blot 1543–1729 1636–1729 1543–1635 analysis. B, HTC75 cell lysates were * (kDa) immunoprecipitated with normal IgG C TNKS1BP1 Constructs * 250 or anti-TNKS1BP1 antibody and Acidic Basic Acidic 100 160 subjected to Western blot analysis. C, 1–1729 (WT) HA-tagged TNKS1BP1 constructs were 1 200 1729 110 1543 WB: HA expressed in HTC75 cells. Cell lysates 1543–1729 (TNKS1 were subjected to immunoprecipitation 60 1636 BP1) with anti-HA antibodies, followed by 1636–1729 50

Western blot analysis with anti-HA or 1542 1636 IP: HA anti-CapZA2 antibodies. Δ1543–1635 * 30 1543–1635 1543 1635 * * 15 WB: CapZA2

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Control TNKS1BP1 TNKS1BP1 CapZA2 A siRNA siRNA (#1) siRNA (#2) siRNA (#6) B P < 0.05 㻟㻚㻡

㻟 Figure 5. Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton TNKS1BP1 regulates CapZA2 Lane 12345678 9 101112131415 16 㻞㻚㻡 WB: cytoskeletal localization. A, TNKS1BP1 㻞 depletion releases CapZA2 from the CapZA2 cytoskeleton. After transfection of HTC75 cells with the indicated siRNAs 㻝㻚㻡 Phospho for 72 hours, subcellular fractions were cofilin (Ser3) analyzed by Western blot analysis. B, 㻝 Invasion assay was performed as in Fig. Vimentin 1. P value indicates statistical

Invasion index (arbitrary unit) 㻜㻚㻡 signiﬁcance (t test).

Calpain I 㻜

Histone H2AX siRNA Control CapZA2 siRNA (#6)

evaluation of TNKS1BP1 expression changes in the process Discussion of cell invasion (32). The intensity of TNKS1BP1 expression TNKS1BP1 as a component of the actin cytoskeleton remains relatively high in PanINs (Fig. 7C, b) compared While tankyrase targets TRF1 and enhances telomere elonga- with the normal pancreatic ducts (Fig. 7C, a). In contrast, tion by telomerase (14), TNKS1BP1 does not bind telomeres or TNKS1BP1 expression was lower in the invasive adenocarcino- affect telomere length in human cancer cells (ref. 33 and H. ma than in the normal pancreatic ducts and PanINs (Fig. 7C, c). Seimiya, unpublished observations). MOTIF analysis (http:// Using 17 patient tissue samples, we found that the expression www.genome.jp/tools/motif/) indicates that there are no charac- levels of TNKS1BP1 in the invasive regions were signiﬁcantly teristic motifs or known functional domains in TNKS1BP1. lower than those in noninvasive regionsofthesametissue(Fig. According to the FASTA homology search (http://www. 7D). These observations indicate an inverse relationship genome.jp/tools/fasta/), however, this protein has a weak simi- between TNKS1BP1 expression and cancer invasion. larity to ECM proteins, such as collagens and proteoglycans,

A B FN- F- FN- Tankyrase F- Tankyrase F: FLAG Mock Tankyrase (HE/A) Tankyrase (HE/A) FN: FLAG-NLS

Figure 6. Cytoplasmic overexpression of Mock FN-Tankyrase F-Tankyrase (kDa) tankyrase upregulates coﬁlin

TNKS1BP1 250 Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton Cytosol Membrane /organelle Nucleus Cytoskeleton phosphorylation and cell invasion. A, CapZA2 Whole-cell extracts of HTC75 cells that Tankyrase 160 stably overexpressed FLAG-tagged Phospho tankyrase constructs were subjected to cofilin (Ser3) Phospho Western blot analysis. F, FLAG epitope cofilin (Ser3) 15 Calpain I at the N-terminus; FN, FLAG epitope Cofilin and NLS. B, Subcellular fractionation Histone H2AX 15 and Western blot analysis of tankyrase- 40 GAPDH Vimentin overexpressing cells. C, Effect of XAV939, a tankyrase inhibitor, on Lane 1 2 3 4 5 6 7 8 91011121314151617181920 tankyrase-mediated phosphorylation of coﬁlin. Cells were treated with C Mock F-Tankyrase XAV939 for 48 hours, and Western blot D analysis was performed. Accumulation XAV939 (μmol/L) 0 5 30 0 5 Invasion index (arbitrary unit) (kDa) 0 12 of tankyrase protein in the mock cells is a pharmacodynamic marker of Tankyrase 160 Mock tankyrase inhibition. D, Invasion assay P < 0.01 FN-Tankyrase was performed as in Fig. 1. P value Phospho FN-Tankyrase (HE/A) indicates statistical signiﬁcance (t test). cofilin (Ser3) 15 F-Tankyrase Cofilin F-Tankyrase (HE/A) 15

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TNKS1BP1 Regulates Cell Invasion

ABDistribution (%) P < 0.01

300

250

200 Figure 7. Downregulation of TNKS1BP1 at Intensity 150 invasive sites of pancreatic cancer. A, Immunohistochemistry of tissue 100 microarrays with anti-TNKS1BP1 antibody. Representative photos 50 for three grades (low/medium/high) of

distribution and intensity patterns of Arbitrary unit (distribution×intensity) 0 TNKS1BP1 are shown. Scale bar, Normal Pancreatic cancer 500 mm. B, TNKS1BP1 tissue microarray n ( = 40) (n = 48) spots were classified according to the C grades in A, revealing lower expression in pancreatic cancers as compared with D the normal samples. C, Differential P < 0.01 expression of TNKS1BP1 in pancreatic cancer. Top, lower magnification. a Bottom, magnified images of the 㻝㻚㻤 squares in the top photo. a, normal 㻝㻚㻢 pancreatic duct region; b, PanIN; c, 㻝㻚㻠 invasive adenocarcinoma. Arrows, typical cells. Scale bar, 500 mm. D, b 㻝㻚㻞 fi Quanti cation of TNKS1BP1 intensity in 㻝㻚㻜 patients with pancreatic cancer 㻜㻚㻤 (n ¼ 17). Samples in C and D were c collected and analyzed under informed 㻜㻚㻢 consent at the Cancer Institute 㻜㻚㻠 Hospital, JFCR. abc 㻜㻚㻞 TNKS1BP1 intensity (% of control) 㻜 Noninvasive Invasive

suggesting a role in cell adhesion, motility, and/or invasion. This protein. Although the function of the short isoforms in the information and its intracellular colocalization with actin fila- nucleus remains to be determined, Zou and colleagues demon- ments support that TNKS1BP1 is a functional component of the strated that TNKS1BP1 functions in DNA double-strand break actin cytoskeleton. repair (34). In our original report, we also detected TNKS1BP1 in the nucleus, especially in the perinucleolar heterochromatic regions TNKS1BP1 and CapZ regulate ROCK/LIMK/cofilin pathway (18). We speculate that these nuclear TNKS1BP1 would be derived and cell invasion from alternative splicing, different start sites, or both of the We showed that TNKS1BP1 expression level is a determinant transcripts as observed in the H-InvDB Annotated Human Gene for the actin cytoskeletal rearrangement and the ability of the cell Database (http://www.h-invitational.jp), which give rise to to invade into the collagen matrix. Intriguingly, TNKS1BP1 deple- shorter forms (i.e., the C-terminal half of full-length protein). tion decreased the level of CapZA2, a newly identified TNKS1BP1- Consistent with this idea, the TNKS1BP1-truncated mutant that binding partner, in the cytoskeletal fraction. CapZA2 is a com- lacks the N-terminal 1220 amino acids accumulates in the nucleus ponent of the heterodimeric actin-capping protein, which consists when ectopically expressed in cells (H. Seimiya, unpublished of a (CapZA1 or CapZA2) and b (CapZB) subunits (35). They cap observation). Although TNKS1BP1 contains 2 NLS at the C- the barbed ends of growing actin filaments to block their elon- terminal basic region, their function appears to be recessive in gation (5). This effect is similar to those of cytochalasins, which the full-length protein because the full-length TNKS1BP1 colo- bind the barbed ends of growing actin filaments and inhibit cell calizes with actin filaments rather than in the nucleus. Nuclear invasion (ref. 36 and this study). Our results suggest that localization of the shorter isoforms is further supported by the fact TNKS1BP1 stabilizes the capping proteins at barbed ends and that antibodies raised against the N-terminal fragment of negatively regulates actin polymerization. TNKS1BP1 detect only cytoskeletal TNKS1BP1 but not nuclear What, then, is the molecular mechanism for CapZA2 dissoci- TNKS1BP1 by immunofluorescent staining (H. Seimiya, unpub- ation from actin filaments upon TNKS1BP1 depletion? Because lished observation). These facts and functional analyses in the there is a significant mismatch, that is, different symmetries present study establish full-length TNKS1BP1 as a cytoskeletal between 3-dimensional structures of CapZ and actin (37), it is

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possible that TNKS1BP1 is required for efficient interaction activity and cytoplasmic localization. According to Tian and between these two proteins. CapZ interaction with actin is also colleague, tankyrase inhibition by a small compound or by RNA inhibited by phosphatidylinositol 4,5-bisphosphate, myotro- interference reduces the invasive activity of neuroblastoma cells, phin/V-1 and CARMIL (38–40). Whether TNKS1BP1 knockdown depending on telomere shortening (45). Because the effect of may affect the intracellular levels, distributions, or both of these tankyrase inhibition on telomere function emerges rather slowly factors remains to be investigated. due to gradual shortening of telomeres (33, 46), we prefer a non- We found that knockdown of either TNKS1BP1 or CapZA2 telomeric mechanism for the altered invasive activity. increases the level of p-cofilin in a ROCK/LIMK pathway–depen- Tankyrase-mediated PARylation of the Wnt suppressor Axins dent manner, leading to an increased number of focal adhesions leads to ubiquitination of the PARylated Axins followed by that were accompanied by F-actin polymerization. So far, the proteasomal degradation (16). This is a unique signaling relay precise mechanism underlying why CapZA2 dissociation from the from tankyrase-mediated PARylation to RNF146-mediated ubi- actin filaments activates the ROCK/LIMK pathway remains elu- quitination: the ubiquitin E3 ligase RNF146 in its resting state sive. Given that ROCK inhibition enhances the protein levels of binds tankyrase, and PAR chains produced by tankyrase bind the CapZ (41) and strain-induced stimulation of CapZ dynamics WWE domain of RNF146, which causes allosteric activation of the depends on the RhoA/ROCK pathway (42), there might be a ubiquitin E3 ligase activity (47). Tankyrase-mediated PARylation feedback loop system in the RhoA/ROCK/LIMK pathway and the followed by ubiquitination is also observed in other tankyrase CapZ-mediated regulation machinery of actin reorganization. substrates, such as TRF1 and 3BP2 (15, 48). In contrast, tankyrase inhibitors did not upregulate the level of TNKS1BP1 protein TNKS1BP1 as a possible regulator of cancer invasion (T. Ohishi & H. Seimiya, unpublished observation), suggesting We demonstrated that TNKS1BP1 is downregulated in invasive that PARylated TNKS1BP1 may not be an efficient substrate for pancreatic adenocarcinoma. This observation suggests that ubiquitin-dependent proteasomal degradation. TNKS1BP1 may act as a suppressor of pancreatic cancer invasion The question regarding the mechanism by which tankyrase via negative regulation of actin cytoskeleton rearrangement. Giv- regulates the function of TNKS1BP1 remains unanswered. Tan- en that TNKS1BP1 downregulation is observed exclusively at the kyrase has 5 ARCs as a platform for protein–protein interactions, invasive area of the cancerous lesions, it is possible that its protein and ARCs 1, 2, 4, and 5 can bind TNKS1BP1 (19, 49). Existence of expression is regulated in a microenvironment-dependent, tran- multiple ARCs suggests a role for tankyrase as a molecular lattice, sient manner. In fact, the Oncomine database (https://www. in which TNKS1BP1 may be an essential component for func- oncomine.org) shows that the level of TNKS1BP1 transcript is tional linkage to the CapZ actin filaments. Formation of these not significantly altered in the bulk of pancreatic cancer cells. This protein complexes would depend on the specific interaction would be reminiscent of the epithelial–mesenchymal transition between tankyrase and TNKS1BP1 because TNKS1BP1 binds (EMT), through which an epithelial cancer cell converts to a tankyrase ARCs but not the conventional ankyrin G (18). Con- mesenchymal cell type with less cell-to-cell adhesion and higher sistent with this idea, efficient Wnt/b-catenin signaling requires motility (43). Microenvironmental factors, such as TGFb, pro- the lattice-like scaffolding of tankyrase, which is mediated by the mote EMT of metastasizing cancer cells. Furthermore, it has been sterile a motif–dependent polymerization and multivalent ARC postulated that cancer cells having settled at the metastatic site interaction with Axins (50). often undergo the reverse event, mesenchymal–epithelial transi- In conclusion, we have shown that TNKS1BP1 interacts with tion (MET). Thus, EMT and MET are mutually reversible, and the actin-capping proteins and regulates the ROCK/LIMK/cofilin balance between these two events regulates cell motility and pathway for actin reorganization. These observations give a new invasion. We monitored epithelial and mesenchymal marker insight into the molecular mechanism for actin-regulated cell proteins, such as E-cadherin and N-cadherin, respectively, motility and how its perturbation could contribute to cancer and found no evidence that TNKS1BP1 directly regulates EMT invasion. (T. Ohishi & H. Seimiya, unpublished observation). Our data suggest that TNKS1BP1 regulates CapZ recruitment to Disclosure of Potential Conflicts of Interest actin cytoskeleton. Recently, Lee and colleagues reported that No potential conflicts of interest were disclosed. CapZA1 expression is associated with decreased cancer cell migration and invasion and could be used as a good prognostic marker Authors' Contributions of gastric cancer (44). The authors also showed that CapZA1 Conception and design: T. Ohishi, H. Seimiya depletion causes a significant increase in gastric cancer cell migra- Development of methodology: T. Migita, H. Seimiya tion and invasion, whereas CapZA1 overexpression shows the Acquisition of data (provided animals, acquired and managed patients, opposite effects. These results are in good agreement with our provided facilities, etc.): T. Ohishi, H. Yoshida, M. Katori, Y. Muramatsu, M. Miyake, Y. Ishikawa, A. Saiura, S.-i. Iemura, H. Seimiya observation that CapZA2 depletion increases the invasive activity Analysis and interpretation of data (e.g., statistical analysis, biostatistics, of cancer cells. Together, these observations suggest that computational analysis): T. Ohishi, M. Katori, T. Natsume, H. Seimiya TNKS1BP1-CapZ interaction with the actin cytoskeleton plays a Writing, review, and/or revision of the manuscript: T. Ohishi, H. Seimiya negative role in cancer cell invasion. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Seimiya A new role for tankyrase in cell invasion Study supervision: H. Seimiya Our result that tankyrase-overexpressing cells phenocopy Acknowledgments TNKS1BP1-depleted cells in terms of (i) CapZA2 release from fi fi The authors thank Dr. Sho Isoyama for technical assistance to quantify the actin lament, (ii) p-co lin upregulation, and (iii) enhanced cell immunohistochemical staining, Drs. Kazuhiro Katayama and Yoshikazu Sugi- invasion suggest that tankyrase is the upstream repressor of moto for the 3HA plasmid, and Dr. Toru Hirota for the pcDNA3-FLAG TNKS1BP1 function. These effects of tankyrase require its PARP destination vector.

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TNKS1BP1 Regulates Cell Invasion

Grant Support The costs of publication of this article were defrayed in part by the This work was supported in part by a Grant-in-Aid for Young Scientists (B), payment of page charges. This article must therefore be hereby marked Japan Society for the Promotion of Science (JSPS; no. 23701068, 25871074 to T. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Ohishi), a Grant-in-Aid for Scientiﬁc Research on Innovative Areas, Ministry of this fact. Education, Culture, Sports, Science and Technology (no. 23117527 to H. Seimiya), and a Grant-in-Aid for Challenging Exploratory Research, JSPS (no. Received July 22, 2016; revised September 16, 2016; accepted January 29, 26640109 to H. Seimiya). 2017; published OnlineFirst February 15, 2017.

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2338 Cancer Res; 77(9) May 1, 2017 Cancer Research

Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Cytoskeleton Rearrangement and Cancer Cell Invasion

Tomokazu Ohishi, Haruka Yoshida, Masamichi Katori, et al.

Cancer Res 2017;77:2328-2338. Published OnlineFirst February 15, 2017.

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