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APC Mutations As a Potential Biomarker for Sensitivity To Published OnlineFirst February 8, 2017; DOI: 10.1158/1535-7163.MCT-16-0578 Companion Diagnostics and Cancer Biomarkers Molecular Cancer Therapeutics APC Mutations as a Potential Biomarker for Sensitivity to Tankyrase Inhibitors in Colorectal Cancer Noritaka Tanaka1,2, Tetsuo Mashima1, Anna Mizutani1, Ayana Sato1,3, Aki Aoyama3,4, Bo Gong3,4, Haruka Yoshida1, Yukiko Muramatsu1, Kento Nakata1,5, Masaaki Matsuura6, Ryohei Katayama4, Satoshi Nagayama7, Naoya Fujita3,4,5, Yoshikazu Sugimoto2, and Hiroyuki Seimiya1,3,5 Abstract In most colorectal cancers, Wnt/b-catenin signaling is acti- "short" truncated APCs lacking all seven b-catenin-binding vated by loss-of-function mutations in the adenomatous polyposis 20-amino acid repeats (20-AARs). In contrast, the drug-resistant coli (APC) gene and plays a critical role in tumorigenesis. cells possessed "long" APC retaining two or more 20-AARs. Knock- Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a neg- down of the long APCs with two 20-AARs increased b-catenin, ative regulator of b-catenin, and upregulate b-catenin signaling. Tcf/LEF transcriptional activity and its target gene AXIN2 expres- Tankyrase inhibitors downregulate b-catenin and are expected sion. Under these conditions, tankyrase inhibitors were able to to be promising therapeutics for colorectal cancer. However, downregulate b-catenin in the resistant cells. These results indicate colorectal cancer cells are not always sensitive to tankyrase that the long APCs are hypomorphic mutants, whereas they exert inhibitors, and predictive biomarkers for the drug sensitivity a dominant-negative effect on Axin-dependent b-catenin degra- remain elusive. Here we demonstrate that the short-form APC dation caused by tankyrase inhibitors. Finally, we established mutations predict the sensitivity of colorectal cancer cells to 16 patient-derived colorectal cancer cells and confirmed that tankyrase inhibitors. By using well-established colorectal cancer the tankyrase inhibitor–responsive cells harbor the short-form cell lines, we found that tankyrase inhibitors downregulated APC mutations. These observations exemplify the predictive b-catenin in the drug-sensitive, but not resistant, colorectal importance of APC mutations, the most common genetic alter- cancer cells. The drug-sensitive cells showed higher Tcf/LEF ation in colorectal cancers, for molecular targeted therapeutics. transcriptional activity than the resistant cells and possessed Mol Cancer Ther; 16(4); 752–62. Ó2017 AACR. Introduction those in KRAS, SMAD4, and TP53 (3, 4). APC functions as a functional scaffold for the b-catenin destruction complex, which Loss-of-function mutations in adenomatous polyposis coli (APC) is composed of b-catenin, Axin, casein kinase 1a and glycogen gene, a negative regulator of Wnt/b-catenin signaling, frequently synthase kinase 3b (GSK3b), and suppresses Wnt/b-catenin occur in colorectal cancer (1, 2). These APC mutations promote signaling (5–10). In the destruction complex, APC and Axin tumorigenesis together with several common mutations, such as promote the ubiquitin-dependent proteasomal degradation of b-catenin via casein kinase 1a- and GSK3b-mediated phosphory- 1Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese lation of b-catenin (8, 10). In genetically engineered mouse Foundation for Cancer Research, Tokyo, Japan. 2Division of Chemotherapy, models, APC mutation–derived activation of Wnt/b-catenin sig- 3 Faculty of Pharmacy, Keio University, Tokyo, Japan. Laboratory of Molecular naling was shown to be required not only for promotion of Target Therapy of Cancer, Department of Computational Biology and Medical colorectal cancer but also for tumor maintenance (11, 12). These Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, b Japan. 4Division of Experimental Chemotherapy, Cancer Chemotherapy Center, observations indicate that the Wnt/ -catenin signaling pathway Japanese Foundation for Cancer Research, Tokyo, Japan. 5Graduate School of is a rational therapeutic target for colorectal cancer. So far, how- Pharmaceutical Sciences, Meiji Pharmaceutical University, Tokyo, Japan. 6Grad- ever, this pathway lacks druggable molecular targets, which has uate School of Public Health, Teikyo University, Tokyo, Japan. 7Department of hampered the development of therapeutic drugs targeting Gastroenterological and Surgery, Cancer Institute Hospital, Japanese Founda- Wnt/b-catenin signaling. tion for Cancer Research, Tokyo, Japan. Tankyrases (tankyrase-1/PARP-5a and tankyrase-2/PARP-5b) Note: Supplementary data for this article are available at Molecular Cancer are members of the PARP family (13–15). This unique class of Therapeutics Online (http://mct.aacrjournals.org/). PARPs recognizes its binding partners via the multiple ankyrin Corresponding Author: Hiroyuki Seimiya, Division of Molecular Biotherapy, repeat cluster domain and poly(ADP-ribosyl)ates (PARylates) Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, them via the carboxyl terminal catalytic domain (13, 16, 17). 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3570-0466; Fax: Tankyrases enhance Wnt/b-catenin signaling by PARylation and 81-3-3570-0484; E-mail: [email protected] subsequent destabilization of Axins (18, 19). Pharmacologic doi: 10.1158/1535-7163.MCT-16-0578 inhibition of tankyrases causes accumulation of Axins, which in Ó2017 American Association for Cancer Research. turn facilitates degradation of b-catenin and suppresses the 752 Mol Cancer Ther; 16(4) April 2017 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 8, 2017; DOI: 10.1158/1535-7163.MCT-16-0578 APC as a Predictive Biomarker in Colorectal Cancer Therapy growth of several APC-mutated colorectal cancer cell lines antibodies were anti-tankyrase-1/2 (H350; Santa Cruz Biotech- (18, 20–23). These observations suggest that tankyrase inhibitors nology), anti-active b-catenin (8E7; Millipore), anti-phospho- are promising drugs for colorectal cancer treatment. However, b-catenin (Ser33/37/Thr41; Cell Signaling Technology), anti- colorectal cancer cells are not equally sensitive to tankyrase Axin1 (C76H11; Cell Signaling Technology), anti-Axin2 (76G6; inhibitors (22). Although appropriate stratification of patients Cell Signaling Technology), anti-GAPDH (6C5; Fitzgerald), anti- has profound implications for new drug development, predictive APC (ALi 12-28; Santa Cruz Biotechnology) for detecting the biomarkers for sensitivity to tankyrase inhibitors remain elusive. short APC (aa 1–811), and anti-APC (2504; Cell Signaling In this study, we demonstrate that a class of APC mutations Technology) for APC larger than 811 aa. predicts the sensitivity of colorectal cancer cells to tankyrase inhibitors. siRNA transfection ON-TARGETplus (human CTNNB1 and nontargeting control) APC Materials and Methods and Silencer Select [ (s1433: siAPC#1; s1434: siAPC#2; s1435: siAPC#3) and negative control no. 1] siRNAs were pur- Cell culture and proliferation assay chased from GE Healthcare Dharmacon and Thermo Fisher Human colorectal cancer cell lines COLO-320DM, SW403, Scientific, respectively. siRNAs were introduced into cells with SW480, and DLD-1 were maintained in RPMI1640 medium reverse transfection method using Lipofectamine RNAiMAX (Gibco, Life Technologies) supplemented with 10% FBS. RKO Transfection Reagent (Thermo Fisher Scientific). cells were maintained in DMEM low glucose (Nacalai Tesque) with 10% FBS. These cell lines were obtained from ATCC. HCT-15, qRT-PCR HCC2998, HT-29, KM12, and HCT-116 cells were maintained as Total RNAs were purified with the RNeasy Mini Kit (Qiagen) and described previously (24). All cell lines were authenticated by cDNA were synthesized with the SuperScript III First-Strand Syn- short tandem repeat profiling analysis (BEX) in 2016. Cell pro- thesis SuperMix for qRT-PCR (Thermo Fisher Scientific). The liferation was evaluated by the MTT assay (5-day drug treatment) expression levels of AXIN2 or Naked1 (NKD1)mRNAwerequan- as previously described (25) or by colony formation assay (10-day tified by real-time PCR analysis with the LightCycler 480 Real-Time drug treatment). Normalization was done to cells treated with PCR System with Universal ProbeLibrary Probe #36 (Roche). 0 DMSO as vehicle, which were defined as 100%. Final concentra- The primers were as follows: AXIN2,5-CACACCCTTCTCCAATC- 0 0 0 tion of DMSO was constant and the same for all the treatments. CAA-3 (forward) and 5 -TGCCAGTTTCTTTGGCTCTT-3 (reverse); and NKD1,50-TCTCGCCGGGATAGAAAAC-30 (forward) and 50- 0 Plasmid construction TCTCGCCGGGATAGAAAAC-3 (reverse). Total RNA was purified from COLO-320DM cells using the Immunofluorescence staining RNeasy Kit (Qiagen) and used as a template to amplify the Cells on glass coverslips were fixed with 2% formaldehyde/PBS truncated APC [amino acid (aa) 1–811] fragment by reverse and permeabilized with 0.5% Nonidet P-40/PBS. Immunofluo- transcription-PCR. The primer sets were: 50-ATCGGATCCGCT- rescence staining was performed as previously described (17) with GCCACCATGGAACAAAAGCTGATTTCTGAAGAAGATCTGGAA- antibody against nonphosphorylated (i.e., active) b-catenin TTCGCTGCAGCTTCATATGATCAGTTGTTA-30 (forward) and 50- 0 (D13A1; Cell Signaling Technology). The nuclei were stained ACACTCGAGATATGGTGAAAGGACAGTCATGTTGC-3 (reverse). 0 with VECTASHIELD Antifade Mounting Medium with 4 ,6-dia- The forward and reverse primers contain BamHI and XhoIsites, midino-2-phenylindole
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