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. 2015 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 2015 American Association for Cancer Research. reported in colorectal cancers.

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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 80 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 (200) 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 Dulbecco's modified Eagle medium supplemented with chambers (BD Biosciences) according to the manufacturer's pro- 10% fetal bovine serum at 37 C in a humidified incubator with tocols. Briefly, 5 105 cells were seeded in triplicate on the 5% CO2. Matrigel-coated membrane. After 48 hours, cells that had invaded the undersurface of the membrane were fixed with 100% meth- RNA preparation for reverse-transcription PCR anol and stained with 1% toluidine blue. Four microscopic fields Total RNA was isolated using a modified acid–guanidinium– were randomly selected for cell counting. phenol–chloroform procedure. Complementary DNA was syn- thesized from 1 mg of total RNA using random hexamer primers Proliferation assay and M-MLV reverse transcriptase (RT; Invitrogen). A total of 5 105 cells were seeded onto 6-well plates (BD). At 24 hours after seeding, cells were transfected with siRNAs using Evaluation of gene expression in clinical samples Lipofectamine (Invitrogen) according to the manufacturer's pro- For quantitative real-time RT (qRT)-PCR, the primer sequences tocol. At 24 hours after transfection, 3 103 cells were seeded used are listed in Supplementary Table S1. For confirmation of onto a 96-well plate (BD), and relative cell numbers were deter- RNA quality, the glyceraldehyde-3-phosphate dehydrogenase mined using the Cell Counting Kit-8 (Dojindo) and the indicated (GAPDH) gene served as an internal control. The amplification time course. protocol included initial denaturation at 95C for 10 minutes, followed by 45 cycles at 95 C for 10 seconds and at 60 C for 30 Western blotting seconds. PCR was performed in a LightCycler 480 System (Roche Antibodies were purchased as follows: anti-PTBP1 and anti- Applied Science) using the LightCycler 480 Probes Master Kit Fbwx7 from Abnova; p27, Skp2, cyclin A, cyclin B, cyclin E, CDK2, (Roche Applied Science). All concentrations were calculated rel- CDK4, CDC2, and CDC25A from Santa Cruz Biotechnology; p21 ative to the concentration of cDNA from Human Universal from Cell Signaling Technology; cMyc and cyclin D from Epi- Reference total RNA (Clontech). tomics; CDK11 from Abcam, and b-actin from Sigma-Aldrich. Western blotting was carried out as described previously (13). Immunohistochemistry Briefly, the protein samples (15 mg) were separated by 12.5% Immunohistochemical analyses of PTBP1 were performed polyacrylamide gel electrophoresis followed by blotting on a 0.4- using surgical specimens from selected patients with colorectal mm membrane. After the blocking, the membrane was incubated

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in the appropriately diluted primary antibody solution. After Results incubation with secondary antibody solution, protein bands were PTBP1 is preferentially overexpressed in clinical colorectal detected with the Amersham ECL Detection System (Amersham cancers Biosciences). A total of 178 paired normal mucosa and primary tumor samples were studied using qRT-PCR. The expression value of Establishment of Fucci-expressing HCT116 PTBP1 mRNA in tumor tissues was significantly higher than that mAG-hGem(1/110) and mKO2-hCdt1(30/120) (provided by for corresponding paired normal tissues (P < 0.0001, t test; Dr. Miyawaki, RIKEN-BSI, Japan; ref. 14) were cloned into the Supplementary Fig. S1A). Supplementary Fig. S1B shows the CSII-EF-MCS vector (provided by Dr. Miyoshi, RIKEN-BRC, results of immunohistochemical studies of PTBP1 expression in Japan) and transfected into HEK293T cells with packaging plas- representative clinical samples of normal mucosa (i), well-differ- mids (15). Stable transformants were selected by FACS Aria (BD). entiated adenocarcinoma (ii), moderately differentiated adeno- mAG and mKO2 were excited by 488-nm laser lines, and their carcinoma (iii), and poorly differentiated adenocarcinoma (iv). emission was detected with 530/30BP or 585/42BP filters, The majority of the PTBP1 expression was observed in cancer cells, respectively. the minority in stromal cells, and scarce positivity in normal colonic epithelium. Immunohistochemical studies revealed that Time-lapse imaging of Fucci-expressing HCT116 the staining was strong (n ¼ 7), moderate (n ¼ 9), or weak (n ¼ A total of 5.0 104 Fucci-expressing HCT116 cells treated 18) in the tumor cells but very weak or nonexistent in the normal with transfection of siRNA (control or anti-PTBP1) were spread cells in all 34 cases. Immuohistochemical staining intensity into each chamber of a 4-well chamber glass-bottom dish differed significantly between the tumor and the normal samples (CELLview, #627871, Greiner Bio One) on the day before (P < 0.01, data not shown), and the data were similar to those observation. Time-lapse imaging was taken with an inverted obtained from mRNA expression analysis. All 16 tumors with microscope (A1R; Nikon) with a dry objective lens (Plan-Apo strong or moderate immunohistochemical expression for which VC, 20X/N.A. 0.75, Nikon). Culture conditions were controlled RNA data also were available showed higher mRNA expression at 5% CO and 37Cwithanincubationchamber(INUB-TIZB- 2 values (Supplementary Fig. S2; P ¼ 0.012). The expression of F1; TOKAI HIT). mAG and mKO2 were excited by 488-nm and PTBP1 mRNA seemed to correlate with protein expression. 561-nm laser lines, and their emission detected with 525/25BP or 595/25BP filters, respectively. The images were taken every other hour for 48 hours at 20 points for each sample. Raw PTBP1 expression is correlated with c-MYC expression imaging data were processed with Imaris (Bitplane) with a PTBP1 is transcriptionally upregulated by the Myc oncoprotein Gaussian filter for noise reduction. Automatic object counting in glioma cells (8). To assess if this transcriptional regulation with Imaris Spots was aided by manual correction to retrieve exists in colorectal cancer, we performed qRT-PCR for Myc in 64 fi cell coordinates over time. clinical colorectal cancer specimens and identi ed a relationship between PTBP1 and MYC mRNA levels (Supplementary Fig. S3A; Colony formation assay P ¼ 0.018). In colorectal cancer cell lines, silencing Myc with Cells (2 105) were transfected with siRNAs in 6-well plates. siRNA reduced PTBP1 protein levels (Supplementary Fig. S3B). After 48-hour culture, cells were trypsinized and 5,000 cells were reseeded into each well of CytoSelect (Cell Biolab INC). After PTBP1 regulates PKM isotype switch in colorectal cancer additional 7 days of culture, cell dose was obtained, according to As reported in gliomas (8), we hypothesized that PTBP1 reg- the manufacturer's protocols, from absorbance (485/520 nm) ulates PKM isotype in colon cancer. To investigate our hypothesis, using standard multiplate reader (PerkinElmer INC). we analyzed human normal mucosa (n ¼ 3) and clinical cancer specimens (n ¼ 10). RT-PCR including exons 8 to 11 was per- Antitumor activity assay formed, followed by digestion with PstI. If exon 10 was included Seven-week-old BALB/cA nude mice were purchased from (PKM2), the PCR products were divided into two fragments (Fig. CLEA Japan. HCT116 (1.0 106 cells) were inoculated subcuta- 1A). Normal mucosa samples had both PKM1 and PKM2 iso- neously in both the left and right flanks of the mice to prepare the forms; by contrast, cancerous tissues were PKM2 dominant except solid tumor model. An antitumor activity study was performed in one case (Fig. 1B). Fluorescence coimmunohistochemistry for when the HCT116 tumors were 5 to 6 mm in diameter. Mice were PTBP1 and PKM2 of clinical specimen revealed that while in treated on days 0, 2, 4, 7, 9, and 11 with 20 mg of sApa-control- tumor surface both of them were slightly positive (Fig. 1C, left), in siRNA or sApa-PTBP1-siRNA. Antitumor activity was evaluated in invasive front both of them were ubiquitously positive (Fig. 1C, terms of tumor size, which was estimated using the following right) and the distribution of both molecule was tightly over- equation: V ¼ a b2/2, where a and b represent the major and lapped suggested a relationship between these molecules in minor axes of the tumor, respectively. clinical specimens. Next, we performed silencing of PTBP1 using transient siRNA in Statistical analysis four colorectal cancer cell lines (HCT116, DLD1, HT-29, and Statistical analyses were performed using JMP 8.0.1 for Win- SW480). As indicated in Fig. 2A, the inclusion ratio of PKM1 dows (SAS Institute). Possible differences between groups were went up with PTBP1 silencing. Immunohistochemistry showed analyzed using the Student t test, x2 test, Wilcoxon test, or that PTBP1-positive lesions had a tendency to be distributed in the repeated-measures ANOVA. Survival curves were obtained by the tumor invasive front (Fig. 1C). Next, we analyzed early-stage Kaplan–Meier method; comparison between curves was complet- cancers by immunohistochemistry, dividing samples into two ed with the log-rank test. A probability level of 0.05 was chosen to groups according to the grade of staining (Supplementary Fig. S4). indicate statistical significance. In the strongly positive group, lymphatic invasion, venous

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Normal mucosa Colon cancer tissues A B (n = 3) (n = 10) Negative control Marker Marker

PKM1 8 9 11 digestion Pre Pst I Figure 1. PKM2 8 10 11 M1 Distribution of PKM2 and PTBP1. A, PstI scheme of PKM1 and PKM2, exon 8–10. Arrows, primer pairs used to amplify digestion Post Pst I M2 PKM cDNA. Arrowhead, PstI digestion site, found only in the PKM2 isoform (CTGCAG). B, electrophoresis for PCR products of PKM exon 8–10 from clinical samples pre- and post-PstI Ptbp1/Pkm2/DAPI/Phase contrast Ptbp1/Pkm2/DAPI/Phase contrast digestion (normal mucosa, n ¼ 3; C colon cancer tissue, n ¼ 7). C, immunoﬂuoresence costaining of a V clinical colon cancer specimen for V PKM2 and PTBP1 indicates a relationship between PKM2 and PTBP1. Scale bar, 100 mm.

V Invasive front Tumor surface

invasion, and number of buddings were significantly higher than CD44 splicing variants were detected in this analysis. Then, we the negative/weakly positive group (Table 1). Because hypoxia designed the CD44v8-10–specific primer pair (inside V8-9) with promotes lymph node metastasis (16, 17), we performed coim- which all reported CD44 splicing variants (total CD44) can be munostaining for PTBP1 and CAIX in the early-stage clinical detected (inside exons 2–3). By qRT-PCR, when silencing PTBP1, specimens. PTBP1-positive cases (Fig. 2B, left) had no CAIX- CD44V8-10 and total CD44 expression ratio were decreased in positive lesions in the surface or middle layer (Fig. 2B (a), (b)); HCT116 and DLD1 (Fig. 3B; Supplementary Fig. S5B). In the in however, focal CAIX-positive lesions were observed in the inva- vitro invasion assay, silencing PTBP1 led cells to be less invasive in sive front that PTBP1 was highly positive (Fig. 2B (c)); in contrast, HCT116, DLD1, HT29, and SW480 (Fig. 3C). Calculated invaded PTBP1-negative cases had no CAIX-positive lesions even in the cells ratio was also significantly reduced in silencing PTBP1 in each invasive front (Fig. 2B, right and Fig. 2B (d)). cell lines (Fig. 3D).

PTBP1 expression is associated with tumor invasion through PTBP1 promotes cell proliferation in vivo and in vitro CD44 splicing PTBP1 is reported to promote cell proliferation in embryonic Because CD44 splicing variants in colorectal cancer, especially stem cells (20) and glioblastoma cells (7). To assess the effect of variant v8-10, are reported to have a strong relationship with the PTBP1 in proliferation of colorectal cancer cells, we performed a tumor invasion (18, 19), we hypothesized that PTBP1 regulates proliferation assay. When silencing PTBP1, proliferation ability CD44 splicing in colorectal cancer. To assess this issue, we was significantly reduced in HCT116, DLD1, HT29, and SW480 performed qRT-PCR of 91 clinical specimens using a CD44v8-10 (Fig. 4A). To further investigate PTBP1 function for proliferation, –specific primer pair. The PTBP1 high-expression group also had we performed Western blotting for cell-cycle mediators. Although significantly higher expression of CD44V8-10 mRNA than did the it has been reported that PTBP1 modulates the G1-to-S transition PTBP1 low-expression group (Fig. 3A). through enhancement of internal ribosome entry site (IRES)- Next, we analyzed four cell lines using another primer pair dependent translation of p27kip1 in 293T cells (21), the protein (Supplementary Fig. S5A) for validation of CD44 variant expres- level of p27kip1 in PTBP1-silencing cancer cells was not different sion. By gel electrophoresis (Supplementary Fig. S5B) and from that in control siRNA cells except SW480 cells (Fig. 4B). sequence analysis (Supplementary Fig. S5C), CD44v8-10 mRNA Interestingly, other various cell-cycle mediators, including was positive in HCT116, DLD1, HT29, and SW480, and CD44v10 cyclin A, cyclin B, cyclin C, cyclin E, CDC2, and Skp2, were mRNA was positive in DLD1 and HT-29, respectively; no other increased with PTBP1 silencing (Fig. 4B). To assess this

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A HCT116 DLD1 HT29 SW480

csi RNA + – + – + – + – si PTBP1 – + – + – + – + Anti Ptbp1

Anti Beta-Actin I n Pre Pst digestio I n M1 M2 Figure 2. digestio Dominance of the PKM2 isoform is Post Pst regulated by PTBP1, and a correlation between PKM2 and PTBP1 was PTBP1-positive case PTBP1-negative case observed in colorectal cancer B specimens. A, downregulation of PTBP1 using siRNA causes PKM1 b upregulation in colorectal cancer cell a lines. B, immunohistochemical ﬁndings of early-stage cancer specimens. Left, PTBP1-positive case. In the invasive front, PTBP1 become d strongly positive and the CAIX- c positive lesion emerged. Right, PTBP1-negative case. No CAIX- positive lesion was observed. Scale m bar, 500 m. a, surface of the PTBP1- PTBP1 - DAB PTBP1 CAIX DAPI Merged positive case, almost no PTBP1- and CAIX-positive lesion was observed. b, middle layer of the PTBP1-positive case, PTBP1-positive lesion emerged (a) slightly. c, invasive front of the PTBP1-positive case, PTBP1-positive lesions emerged ubiquitously and partially made CAIX-positive lesions. d, invasive front of the PTBP1-negative case, neither PTBP1- nor CAXI-positive (b) lesion was observed. (c) (d)

discrepancy, we performed time-lapse imaging on HCT116/Fucci protein levels were downregulated (Fig. 5B). In clinical samples, p58 cells. Actually, the G2–M population (green cell) ratio was sig- PTBP1 expression was related to the expression of CDK11 niﬁcantly increased in siPTBP1 (Fig. 4C, P < 0.0001). To synchro- protein levels (Supplementary Fig. S6). In concert with these nize the cell cycle, we sorted red (G1–S) cells. In cell-cycle analysis, ﬁndings, PTBP1 might have an important role in progression to p58 G1–S cells changed into G2–M in a rapid manner with siPTBP1 complete mitosis of cells though CDK11 regulation; in other (Fig. 5A). For further analysis to explain the prolonged G2–Min words, silencing PTBP1 leads to prolonged G2–M phase and cell- HCT116 siPTBP1 cells, we performed immunoblotting of whole- cycle mediators might be upregulated by some feedback mech- cell lysate for CDK11p58 that is reported to play an important role anism in these settings. for maintenance of sister chromosome cohesion (22). As a result, On the basis of the fact that PTBP1 expression levels have in HCT116 and DLD1 cells, when silencing PTBP1, CDK11p58 relationship to cell invasion and proliferation, we hypothesized

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Table 1. The relationship between PTBP1 expression in the invasive front and ysis on lymph nodes, PTBP1-positive lesions were limited in clinicopathologic variables of early colorectal cancer specimens with lymph node with metastasis and no significant staining was submucosal invasion (n ¼ 70) observed in normal lymph node (Supplementary Fig. S7). In Clinicopathologic Negative/weakly Strongly positive PTBP1 n ¼ n ¼ P addition, mRNA expression was positively correlated with variables positive ( 31) ( 39) P ¼ Age (mean SD) 57.5 2.08 60.8 1.86 0.25 poor survival rate after surgery (Supplementary Fig. S8A; Sex 0.0138) and poor disease-free survival after curative surgery Male/female 25/6 29/10 0.53 (Supplementary Fig. S8B; P ¼ 0.0083). As for the CD44v8-10 Size expression, although poor prognosis was observed in high expres- <30 mm/31 mm 23/7 31/8 0.78 sion group (Supplementary Fig. S9), there was no significant co- a Tumor location relationship between any clinocopathologic variables and Right/left 6/25 7/32 0.88 Gross form CD44v8-10 expression in this series. Protrusion/flat 13/18 18/21 0.72 Histologic grade Well/othersb 20/11 16/23 0.050 Discussion Histologic grade of PTBP1, also known as HnRNP I, was first cloned and identified invasive front as playing a central role in a-tropomyosin alternative splicing Well/othersb 22/9 31/8 0.41 fi (23). PTBP1 serves as a repressor of alternative splicing in mam- Absolute in ltration 1,554.8 265.2 1,943.6 236.4 0.28 – distance (mm) malian cells (24 29) and contains RNA-binding domains, each of Lymphatic invasion which binds to CU-rich elements (30). It is involved in poly- 0 Absent/present 24/7 11/28 <0.0001c adenylation of the pre-mRNA 3 end (31–33) and also plays an Venous invasion important role in translational regulation of a subset of RNA c Absent/present 31/0 7/32 0.0030 transcription through internal ribosome entry sites (21, 34–37). Lymph node As for malignancies, PTBP1 is overexpressed in glioblastoma (7, metastasis Absent/present 30/1 36/3 0.41 8) and breast cancer (38), suppresses p27 expression, and con- Number of budding 1.10 0.43 2.08 0.38 0.047c tributes largely to cell proliferation (21, 39). In zebrafish, PTBP1 Abbreviation: well, well-differentiated adenocarcinoma. ablation leads to increased proliferation and cell apoptosis (40). aRelative to splenic flexure. Until now, the role of PTBP1 in colorectal cancer progression has bModerately and poorly differentiated adenocarcinoma. not clearly been identified. In this study, we report that PTBP1 is c Significant difference (P < 0.05). preferentially overexpressed in human colorectal cancer and related to invasion and proliferation in colorectal cancer cells. Moreover, our results indicate that PTBP1 expression level is that anchorage-independent cell growth depends on PTBP1 highly correlated with poor prognosis in colorectal cancer expression and we performed colony formation assay. As a result, patients. colony formation activity was downregulated by silencing PTBP1 In colorectal cancer, the PKM2 isoform plays a central role in in HCT116, DLD1, HT29, and SW480 cells (Fig. 5C). We further metabolism and growth (41). The important isoform switch tested the efficacy of PTBP1 silencing on tumor suppression in vivo mechanism is regulated by HnRNP proteins, including PTBP1, using HCT116 xenografts and a drug delivery system of carbonate in brain tumor cells (8). Switching the pyruvate kinase isoform to apatite. Significant antitumor efficacy was observed in the siPTBP1 PKM1 leads to breakdown of the Warburg effect, which involves group at day 14 (Fig. 5D, P < 0.01). reduced lactate production and increased oxygen consumption (41). In this study, when PTBP1 was preferentially overexpressed, High PTBP1 mRNA expression correlates with poor the PKM2 isoform was dominant, and PTBP1 and PKM2 expres- clinicopathologic variables and prognosis sion correlated in clinical colorectal cancer specimens; thus, we The experimental samples were divided into two groups (the hypothesized that PTBP1 regulates PKM1/PKM2 splicing in colo- high-expression group with PTBP1 expression values > 0.089, n ¼ rectal cancer. In testing this hypothesis, we found that PTBP1 115, and the low-expression group, n ¼ 63) to investigate PTBP1 silencing in colorectal cancer cells leads to a shift in the prevalent expression in association with clinicopathologic variables (Table PKM1 isoform. 2). The cutoff between the two groups was defined by an upper Pursuing this finding further, we performed coimmunohisto- limit, including 95% of the expression values of the normal chemistry of PTBP1 and CAIX, a hypoxia marker (42), in early- samples. Significant between-group differences were observed in stage clinical specimens and found that PTBP-positive cases were venous invasion (P ¼ 0.045, x2 test), lymph node metastasis (P ¼ associated with hypoxic lesions while PTBP1-negative cases did 0.0020), distant metastasis (P ¼ 0.0016), and Dukes stage (P ¼ not contain CAIX-positive lesions. Moreover, PTBP1 was linked to 0.039). the hypoxic lesions through dysregulation of PKM2 expression in Using a logistic regression model for lymph node metastasis, in colorectal cancer. This finding indicates that PTBP1 expression univariate analysis, tumor size (P ¼ 0.018), histologic grade (P ¼ correlates positively with hypoxic lesions that are generally resis- 0.024), depth of tumor invasion (P ¼ 0.0016), lymphatic inva- tant to radiochemotherapy and creates conditions that promote sion (P < 0.0001), venous invasion (P < 0.0001), and overexpres- cancer progression (43). sion of PTBP1 mRNA (P ¼ 0.0016) were significant. In multivar- Although PKM2 itself promotes cell migration (44), we iate analysis (Table 3), overexpression of PTBP1 mRNA was focusedonCD44splicingthatincludesavariantthatinduces significantly associated with lymph node metastasis (P ¼ a metastatic phenotype in tumor cells (45). In colorectal cancer, 0.008) as well as with lymphatic invasion (P ¼ 0.0011) and CD44 splicing variants, especially v8-10, are reported to have a venous invasion (P ¼ 0.043). Using immunohistochemical anal- strong relationship with tumor invasion (18, 19). In clinical

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A Clinical specimens B HCT116 DLD1 HT29 SW480 *** *** N.S. N.S. mRNA levels relative to GAPDH 10 - v8 mRNA levels relative to GAPDH CD44

Whole CD44 CD44v8-10 C D Control siRNA si PTBP1 HCT116 DLD1 HT29 SW480

*** ***

HCT116 *** *** DLD1 HT29 Number of invaded cells/HPF SW480

Scale bar, 50 mm

Figure 3. PTBP1 deregulates CD44 alternative splicing and tumor invasion. A, qRT-PCR of clinical colorectal cancer specimens indicated correlation between CD44v8-10 and PTBP1 mRNA levels (n ¼ 91). , significant difference, P < 0.0001. B, effect of silencing PTBP1 for CD44 alternative splicing was validated by qRT-PCR in four colorectal cancer cell lines. , significant difference, P < 0.0001; n.s., not significant. C, effect of PTBP1 silencing for cancer invasion capacity validated by invasion assay in four cell lines. Scale bar, 50 mm. D, semiquantified invaded cells in four cell lines (n ¼ 3). , significant difference, P < 0.0001. specimens, PTBP1 expression correlated positively with SW480 did not respond to siPTBP1 on splicing of CD44. On the CD44v8-10 mRNA expression. PTBP1 silencing in colorectal basis of cell invasion assay results, the invasion ratio was cancer cells reduced the CD44v8-10 ratio in HCT116 and DLD1, reducedinallanalyzedcelllines.HnRNPA1isreportedto which are microsatellite-unstable cell lines that do not respond promote tumor invasion through upregulating CD44v6 in to TGFb-induced EMT (46). On the other hand, HT-29 and hepatocarcinoma cells (47). These findings suggest that PTBP1

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A B HCT116 DLD1 HT29 SW480

15 30 csiRNA + – + – + – + – HCT116 DLD1 si PTBP1 +– +– +– +– *** *** p27 csiRNA csiRNA 10 siPTBP1 20 siPTBP1 Skp2

cMyc ** ** 5 10 Fbxw7 *** ** p21

0 0 Cyclin A 24 48 72 96 24 48 72 96 10 15 Cyclin B HT29 *** SW480 Cyclin D 8

Relative proliferation *** csiRNA csiRNA Cyclin E siPTBP1 10 siPTBP1 6 *** CDK2 *** 4 CDK4 *** 5 Figure 4. ** CDC2 Silencing PTBP1 regulates cell 2 CDC25A proliferation through a prolonged G2–M phase. A, proliferation assay in β-Actin 0 0 HCT116, DLD1, HT-29, and SW480 cells. 24 48 72 96 24 48 72 96 , significant difference, P < 0.001; , significant difference, P < 0.0001. Time after seeding (h) C B, Western blot analysis of cell-cycle 0 h 8 h 16 h 24 h 32 h 40 h 48 h mediators in control siRNA and silencing PTBP1 cells. b-Actin was used as a control. C, time-lapse imaging of HCT116/Fucci cells with and without PTBP1 silencing. Quantified ratios of csiRNA green/red cells described graphically. , significant difference, P < 0.0001, repeated-measures ANOVA.

PTBP1 si

1.0 csiRNA siPTBP1

0.8

0.6

*** 0.4 Green cells/Red cells 0.2

0.0 0 8 16 24 32 40 48 Time (hour)

might play an important role in cell invasion and that these cancer specimens. Recently, a CD44 variant form was found invasive properties arise partially through CD44 splicing. More- to play an important role in promotion of intestinal cancer over, these results fit with the presence identified here of PTBP1- formation in APC (min) mice (48). In concert with these positive cells in the invasive front of early-stage colorectal findings, we show here that PTBP1 affects cancer promotion

1712 Mol Cancer Ther; 14(7) July 2015 Molecular Cancer Therapeutics

Signiﬁcance of PTBP1 in Colorectal Cancer

A B 0 h 4 h 8 h 12 h 4.2% 17.3% 36.2% 0.1%

HCT116 DLD1 csiRNA 0.04% 1.5% 1.51% 6.3% csiRNA + – + –

PE siPTBP1 – + – +

0.3% 14.8% 20.8% 23.4% CDK11p58

PTBP1 β-Actin

Figure 5. si Silencing PTBP1 resulted in attenuated 0.04% 0.8% 6.2% 14.6% colony formation and tumor progression. A, tracing of cell cycle FITC after synchronization to G0–G1 phase in HCT116/Fucci cells with control C D 600 siRNA and siPTBP1. B, Western blot saline p58 150 csiRNA analysis for CDK11 on HCT116 and sApa-csiRNA siPTBP1 sApa-siPTBP1 DLD1 whole-cell lysate. C, colony 500 formation assay. Relative cell titers ) were normalized to csiRNA cells. 3 , signiﬁcant difference, P < 0.001. 100 400 D, antitumor activity assay in vivo using carbonate apatite. Arrows, * *** 300 administration of carbonate apatite including control siRNA or siPTBP1.

to csiRNA (%) 50 *** , signiﬁcant difference, P < 0.01, 200 repeated-measures ANOVA. E, (mm Tumor volume

Relative colony formation colony Relative *** proposed schema of this study. *** 100 0 1 9 0 2 8 0 D T 4 DL H W 0 1 2 3 4 5 6 7 8 9 101112131415 CT116 S H Days after start of treatment

E Anchorage- PKM2 Cell independent Invasion growth PTBP1 CD44v8-10 CDK11p58 Effective Cell-cycle cell mitosis mediators

and invasion through upregulating a CD44 variant form and of cell-cycle mediators would be a plausible explanation for this that the CD44 alternative splicing to the CD44v8-10 isoform effect. However, in this study, actual cell proliferation was reduced regulated by PTBP1 is important, especially in microsatellite- in colorectal cancer cells. To clarify this discrepancy, we performed unstable colorectal cancer s. Unfortunately, less invasive abil- a time-lapse imaging study. In embryonic stem cells, silencing ities with silencing PTBP1 in MSS cell lines were still elusive, PTBP1 causes a prolonged G2–M phase (49). Here, we found that further experiments were required. silencing PTBP1 led to an increased G2–M population, based on Cell proliferation ability is also directly related to tumor pro- time-lapse tracing of HCT116 cells. With a synchronized cell cycle, gression. To test the effect of PTBP1 on the cell-cycle and cell PTBP1-silenced cells were entered into the cell cycle in a rapid proliferation, we performed a proliferation assay in four cell lines. manner; however, the percentage of cells with an active cell cycle In all analyzed cases, silencing PTBP1 led to reduced proliferation. was low. Analysis on CDK11p58, critical regulator of cell mitosis Next, we hypothesized that the effect of PTBP1 on proliferation (22), partially clariﬁed this discrepancy. Reduced expression of increased because of increased cell-cycle mediators or decreased PTBP1 downregulate CDK11p58, in turn, various cell-cycle med- cell-cycle suppressors. Interestingly, however, although PTBP1 is iators would be upregulated. As a result, cells go to G2–M phase in reported to promote p27/Kip1 protein expression (21), silencing rapid manner; however, no effective mitosis was occurred, PTBP1 had almost no effect on p27 protein levels in colorectal resulted in prolonged G2–M phase. In other words, several cell- cancer cells except SW480 cells. Moreover, other cell-cycle med- cycle mediators, such as cyclin A, cyclin B, cyclin C, cyclin D, cyclin iators, including Myc, Skp2, cyclin A, cyclin B, cyclin D, cyclin E, E, CDC2, and Skp2, would be upregulated reactively to the and CDC2, were overexpressed with PTBP1 suppression. Previous downregulation of CDK11p58. Although CDK11p58 was reported work has shown that in zebraﬁsh, silencing PTBP1 leads to to inhibit metastasis in ER-positive breast cancer, in androgen- proliferation of intestinal epithelial cells (40), and upregulation independent prostate cancer (50), CDK11p58 was reported to have

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Takahashi et al.

Table 2. The relationship between PTBP1 mRNA expression and needed to clarify the relationship between PTBP1 expression and clinicopathologic variables in an advanced case series (n ¼ 178) cancer stem cells. PTBP1 mRNA expression Another important finding of this study is the correlation Clinicopathologic Low High between PTBP1 expression and clinicopathologic features. Of variables (n ¼ 63) (n ¼ 115) P Age (mean SD) 66.9 1.40 66.7 1.37 0.92 note, high PTBP1 expression was positively correlated with Sex poor prognosis. In early-stage cancer cases, high PTBP1 expres- Male/female 38/25 67/48 0.79 sion levels were positively correlated with lymphatic invasion, Tumor size venous invasion, and number of buddings. Because the number <30 mm/31 mm 12/51 35/80 0.26 of cases with lymph node metastasis in this series was very low a Tumor location (4 of 70 cases), no positive correlation was evaluable. In the Right/left 23/40 37/78 0.34 Histologic grade advanced cancer series, high PTBP1 expression was positively Well/othersb 27/36 35/80 0.098 correlated with lymph node metastasis, distant metastasis, and Depth of tumor invasion tumor stage. Multivariate analysis showed that lymphatic inva- mp/ss 10/43 28/81 0.33 sion, venous invasion, and PTBP1 expression were indepen- Lymphatic invasion dently significant for lymph node metastasis. These finding also Absent/present 44/19 65/50 0.085 indicate that PTBP1 expression might be a useful marker for Venous invasion Absent/present 56/7 88/27 0.045 lymph node metastasis in addition to well-known markers such Lymph node metastasis as lymphatic or venous invasion. Our results also suggested that Absent/present 45/18 53/62 0.0020d PTBP1-targeting therapies could possibly lead to suppression of Distant metastasisc lymphnodemetastasisinpatientswithcolorectalcancer. d Absent/present 62/1 98/17 0.0016 DevelopmentofPTBP1inhibitors might be a novel therapeutic Dukes strategy for colorectal cancer. A, B/C, D 45/18 54/61 0.0029d In conclusion, PTBP1 is positively associated with cancer Abbreviations: mp, mucosa propria; ss, subserosa; well, well-differentiated progression properties, such as invasion or proliferation, in adenocarcinoma. aRelative to splenic flexure. colorectal cancer through upregulation of PKM2 and CD44 bModerately and poorly differentiated adenocarcinoma. variants and cell-cycle progression (Fig. 5E). PTBP1 might serve cLiver metastasis, 9 cases; lung metastasis, 2 cases; peritoneal dissemination, 4 as a marker for lymph node metastasis in colorectal cancer. As cases; simultaneous liver and lung metastasis, 2 cases; simultaneous liver regarding CDK11p58 expression levels, there is some discrep- metastasis and peritoneal dissemination, 1 case. ancy. Co-relationship between PTBP1, CDK11p58, and cell dSignificant difference (P < 0.05). behavior is specifically dependent on cell types, for example, in ER-positive breast cancer, CDK11p58 have negative effects on positive relationship to tumor progression through small leucine cancer progression (50), whereas in AR-independent prostate zipper protein (sLZIP), indicating that CDK11p58 would have cancer, CDK11p58 have a positive relationship to the cancer distinct role on cancer progression and it would be dependent on progression through sLZIP (51), and, in ES cells, the protein cancer type. Further study will clarify this discrepancy (51). We expression and IRES activity of CDK11p58 in PTBP1 / ES cells obtained similar results in the in vivo assay. In agreement with is higher than that of wild-type ES cells, indicating that PTBP1 is these findings, silencing PTBP1 led to less cell growth and an involved in the repression of CDK11p58 expression through increased G2–M population. Because almost all conventional IRES-dependent translation (49). However, our data indicate p58 anticancer drugs target cell division in the G2–M population, that PTBP1 have a positive effect on CKD11 expression. anti-PTBP1 therapy and conventional chemotherapy should be Moreover, the previous report indicates that MYC might play an a promising combination for colorectal cancer patients. In other important role and positive effect on IRES-dependent transla- words, because chemoresistant cell properties play an important tion (54). Further study might reveal this discrepancy. Targeting role in cancer stem cell theory (52, 53), PTBP1 might affect PTBP1 would be promising strategy for radical treatment for maintenance of cancer stem cell properties. Further studies are colorectal cancer patients.

Table 3. Univariate and multivariate analyses for lymph node metastasis (logistic regression model) Univariate analysis Multivariate analysis RR (95% CI) P RR (95% CI) P Age (<65/66) 0.69 (0.35–1.39) 0.305 Sex (Male/female) 0.99 (0.51–2.01) 0.983 Tumor locationa (right/left) 0.74 (0.38–1.42) 0.49 Tumor size (<30 mm/31 mm) 2.49 (1.17–5.63) 0.018c 1.77 (0.68–4.79) 0.24 Histologic grade (well/othersb) 2.14 (1.11–4.19) 0.024c 1.55 (0.70–3.48) 0.28 Depth of tumor invasion (mp/ss) 3.53 (1.59–8.49) 0.0016c 2.3 (0.91–6.17) 0.078 Lymphatic invasion (negative/positive) 5.72 (2.94–11.6) <0.0001c 3.58 (1.67–7.86) 0.0011c Venous invasion (negative/positive) 5.81 (2.46–15.5) <0.0001c 2.77 (1.03–8.07) 0.043c PTBP1 mRNA expression (low/high) 2.9 (1.49–5.84) 0.0016c 2.89 (1.32–6.55) 0.008c Abbreviations: CI, confidence interval; mp, mucosa propia; RR, relative risk; ss, subserosa; well, well-differentiated adenocarcinoma. aRelative to splenic flexure. bModerately and poorly differentiated adenocarcinoma. cSignificant difference (P < 0.05).

1714 Mol Cancer Ther; 14(7) July 2015 Molecular Cancer Therapeutics

Signiﬁcance of PTBP1 in Colorectal Cancer

Disclosure of Potential Conﬂicts of Interest Analysis and interpretation of data (e.g., statistical analysis, biostatistics, H. Ishii reports receiving commercial research support from computational analysis): H. Takahashi, J. Nishimura, Y. Kagawa, M. Konno, Taiho Co. No potential conﬂicts of interest were disclosed by the other H. Ishii, H. Yamamoto authors. Writing, review, and/or revision of the manuscript: H. Takahashi, J. Nishi- mura, T. Mizushima, H. Ishii, M. Mori Administrative, technical, or material support (i.e., reporting or organizing Authors' Contributions data, constructing databases): H. Takahashi, T. Mizushima, M. Ishii, H. Ishii Conception and design: H. Takahashi, J. Nishimura, N. Haraguchi, H. Ishii, Study supervision: H. Takahashi, H. Ishii, Y. Doki, M. Mori, H. Yamamoto M. Mori, H. Yamamoto Development of methodology: H. Takahashi, J. Nishimura, Y. Kano, The costs of publication of this article were defrayed in part by the payment of N. Haraguchi, H. Ishii page charges. This article must therefore be hereby marked advertisement in Acquisition of data (provided animals, acquired and managed patients, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided facilities, etc.): H. Takahashi, Y. Kagawa, Y. Kano, Y. Takahashi, X. Wu, M. Hiraki, A. Hamabe, N. Haraguchi, I. Takemasa, K. Mimori, H. Ishii, Received February 20, 2014; revised April 10, 2015; accepted April 12, 2015; H. Yamamoto published OnlineFirst April 22, 2015.

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1716 Mol Cancer Ther; 14(7) July 2015 Molecular Cancer Therapeutics

Significance of Polypyrimidine Tract−Binding Protein 1 Expression in Colorectal Cancer

Hidekazu Takahashi, Junichi Nishimura, Yoshinori Kagawa, et al.

Mol Cancer Ther 2015;14:1705-1716. Published OnlineFirst April 22, 2015.

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