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Significance of Polypyrimidine Tract–Binding Protein 1 Expression In Published OnlineFirst April 22, 2015; DOI: 10.1158/1535-7163.MCT-14-0142 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer Hidekazu Takahashi1, Junichi Nishimura1, Yoshinori Kagawa1,2, Yoshihiro Kano3, Yusuke Takahashi1,4, Xin Wu1, Masayuki Hiraki1, Atsushi Hamabe1, Masamitsu Konno3, Naotsugu Haraguchi1, Ichiro Takemasa1, Tsunekazu Mizushima1, Masaru Ishii2, Koshi Mimori4, Hideshi Ishii3, Yuichiro Doki1, Masaki Mori1, and Hirofumi Yamamoto1 Abstract Polypyrimidine tract–binding protein (PTBP1) is an RNA- observed that PTBP1 affects cell invasion, which was partially binding protein with various molecular functions related to correlated to CD44 splicing, and this correlation was also RNA metabolism and a major repressive regulator of alternative confirmed in clinical samples. PTBP1 expression also affected splicing, causing exon skipping in numerous alternatively anchorage-independent growth in colorectal cancer cell lines. spliced pre-mRNAs. Here, we have investigated the role of PTBP1 expression also affected cell proliferation. Using time- PTBP1 in colorectal cancer. PTBP1 expression levels were sig- lapse imaging analysis, PTBP1 was implicated in prolonged nificantly overexpressed in cancerous tissues compared with G2–M phase in HCT116 cells. As for the mechanism of pro- corresponding normal mucosal tissues. We also observed that longed G2–M phase in HCT116 siPTBP1 cells, Western blotting PTBP1 expression levels, c-MYC expression levels, and PKM2: revealed that PTBP1 expression level was correlated to PKM1 ratio were positively correlated in colorectal cancer CDK11p58 expression level, which was reported to play an specimens. Moreover, PTBP1 expression levels were positively important role on progression to complete mitosis. These correlated to poor prognosis and lymph node metastasis. In findings indicated that PTBP1 is a potential therapeutic target analyses of colorectal cancer cells using siRNA for PTBP1, we for colorectal cancer. Mol Cancer Ther; 14(7); 1705–16. Ó2015 AACR. Introduction of colorectal cancer is essential for developing novel therapeutic strategies. Colorectal cancer is a leading cause of cancer-related death in Alternative splicing greatly affects protein levels and functions. the western world and is estimated to be one of the most Cancer-specific abnormal pre-mRNA splicing can affect tumor frequently diagnosed cancers; estimated new cases of colorectal initiation and promotion (4). Alternative RNA splicing can greatly cancer were 142,820 and corresponding expected mortality was affect protein levels and functions. In cancer, abnormal splicing 50,830 in the United States in 2013 (1). Although monoclonal often leads to cancer-promoting splice variants that are translated antibodies, including bevacizumab, an inhibitor of vascular into oncogenes or aberrant tumor suppressors (5). Normal splic- endothelial growth factor, and cetuximab, an epidermal growth ing patterns can be disrupted by either cis-acting mutations of factor receptor inhibitor (2, 3), are currently feasible as novel splicing regulatory elements (5). molecular-based therapies, many patients with colorectal cancer Increases in the splicing factor polypyrimidine tract–binding still die from disease recurrence, mainly because of liver metas- protein (PTBP1, also known as hnRNPI) that are associated with tasis. Therefore, further elucidation of the molecular mechanisms glioma malignancy could have similar oncogenic effects (6). PTBP1 has been reported to play a key role in pre-mRNA splicing in cancer. Actually, PTBP1 have a critical effect on pyruvate kinase 1Department of Gastroenterological Surgery, Osaka University Grad- (PKM) alternative splicing in glioma, and this splicing strongly uate School of Medicine, Osaka, Japan. 2Laboratory of Cellular influences cancer progression (7, 8). PTBP1 also has multiple Dynamics,WPI-Immunology Frontier Research Center, Osaka Univer- sity, Osaka, Japan. 3Department of Frontier-Science for Cancer and functions other than pre-mRNA splicing and affects glioma cell Chemotherapy, Graduate School of Medicine, Osaka, Japan. 4Depart- invasion (7). In ovarian cancer, PTBP1 levels correlate with the ment of Molecular and Cellular Biology, Division of Molecular and degree of malignancy (9). Higher amounts of PTBP1 occur in Surgical Oncology, Kyushu University, Medical Institute of Bioregula- advanced, as compared with benign, ovarian tumors, and PTBP1 tion, Ohita, Japan. increases when ovarian epithelial cells are immortalized (9). Note: Supplementary data for this article are available at Molecular Cancer Removal of PTBP1 from ovarian tumor cells makes cell less Therapeutics Online (http://mct.aacrjournals.org/). proliferated, anchorage-independent growth, and cell invasion. H. Takahashi and J. Nishimura share first authorship of this article. Moreover, PTBP1 can form complexes with focal adhesion-encod- Corresponding Author: Hirofumi Yamamoto, Department of Gastroenterolog- ing transcripts at the cell membrane, which might affect cell ical Surgery, Osaka University Graduate School of Medicine, Yamadaoka 2-2, spreading (10). When taken together, deregulation of PTBP1 Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3251; Fax: 81-6-6879-3259; could cause multiple changes in gene expression and translation E-mail: [email protected] to promote cancer and targeting PTBP1 might be a potential doi: 10.1158/1535-7163.MCT-14-0142 therapeutic target. However, few findings about PTBP1 have been Ó2015 American Association for Cancer Research. reported in colorectal cancers. www.aacrjournals.org 1705 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 22, 2015; DOI: 10.1158/1535-7163.MCT-14-0142 Takahashi et al. Here, we studied the clinicopathologic significance of PTBP1 cancer at Osaka and Kyushu University as previously described expression and found that it was associated with lymph node (11). Briefly, after deparaffinization, antigen retrieval in citrate metastasis and invasion as well as with disease-free survival. We buffer (pH 6.0), and blocking according to standard protocols, evaluated the significance of other related factors, including the antigen–antibody reaction was carried out overnight at tumor invasion and proliferation abilities, which are directly 4C. Mouse monoclonal antibody against human PTBP1 involved in tumor malignant potentials. (H00005725-M01; Abnova) was used at 3 mg/mL concentration, as was anti-human carbonic anhydrase 9 (CAIX) antibody (3829- Materials and Methods 1; Epitomics) at a 1:250 dilution. For the enzyme antibody technique, the avidin–biotin–peroxidase method (Vectastain Patients and sample collection Elite ABC reagent kit; Vector) was used according to standard A total of 178 patients with colorectal cancer who underwent protocols. Nuclei were stained with hematoxylin. For immuo- surgical treatment at the Kyushu University (Beppu, Japan) and fluorescence evaluation, Alexa Fluor 488–conjugated goat anti- affiliated hospitals between 1992 and 2002 were enrolled in this mouse IgG (1:500) and Alexa Fluor 555–conjugated goat anti- study. Resected tumors and paired non–tumor tissue specimens rabbit IgG (Invitrogen; 1:1,000) were used as secondary antibo- were immediately taken from resected colons and placed in RNA dies. Nuclei were stained with 40,6-diamidino-2-phenylindole– later (Takara), embedded in Tissue Tek OCT medium (Sakura), or containing mounting medium (Invitrogen). frozen in liquid nitrogen and kept at À80 C until RNA extraction. The median follow-up period was 2.93 years. All data pertaining Assessment of tumor budding to the samples, including age, sex, tumor size and depth, lym- Tumor budding was estimated according to the definition phatic invasion, lymph node metastasis, vascular invasion, liver proposed by Ueno and colleagues (12). An isolated cancer cell metastasis, peritoneal dissemination, distant metastasis, clinical or a cluster composed of fewer than five cancer cells was defined as stage, and histologic grade, were taken from clinical and patho- tumor budding. The number of buddings was counted under a logic records. Written informed consent was obtained from all high-power field (Â200) in the invasive front area. patients in accordance with the guidelines approved by the Institutional Research Board. This study was conducted under the supervision of the ethical board of Kyushu University. Transfection of small interfering RNA The small interfering RNA (siRNA) for PTBP1 (Stealth Select RNAi, HSS143520) and negative control siRNA (Negative Control Cell culture Hi GC) were purchased from Invitrogen. siRNA for cMYC and The human colon cancer cell lines HCT116, DLD1, SW480, and negative control (siGENOME nontargeting siRNA) were pur- HT29 were obtained from the American Type Culture Collection chased from Thermo Scientific. Cells were transfected with siRNA in 2001. Stocks were prepared after passage 2 and stored in liquid in 20 nmol/L concentration using Lipofectamine RNAiMAX (Invi- nitrogen. All experiments were performed with cells of passage of trogen) according to the manufacturer's protocols. <8. These cell lines were authenticated by morphologic inspec- tion, short tandem repeat profiling, and Mycoplasma testing by the Invasion assay ATCC. Mycoplasma testing was done also by the authors. Cells were The invasion assay was performed using Transwell cell culture grown in
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