TTPAL Promotes Colorectal Tumorigenesis

Published OnlineFirst April 24, 2019; DOI: 10.1158/0008-5472.CAN-18-2986

Cancer Molecular Cell Biology Research

TTPAL Promotes Colorectal Tumorigenesis by Stabilizing TRIP6 to Activate Wnt/b-Catenin Signaling Hongyan Gou1,2, Jessie Qiaoyi Liang2, Lijing Zhang2, Huarong Chen2, Yanquan Zhang2, Rui Li2, Xiaohong Wang3, Jiafu Ji3, Joanna H. Tong4, Ka-Fai To4, Joseph J.Y. Sung2, Francis K.L. Chan2, Jing-Yuan Fang1, and Jun Yu2

Abstract

Copy number alterations are crucial for the development of TTPAL. Depletion of TRIP6 significantly abolished the effects of colorectal cancer. Our whole-genome analysis identified TTPAL on cell proliferation and Wnt activation. Direct binding tocopherol alpha transfer protein-like (TTPAL) as preferentially of TTPAL with TRIP6 in the cytoplasm inhibited ubiquitin- amplified in colorectal cancer. Here we demonstrate that fre- mediated degradation of TRIP6 and, subsequently, increased quent copy number gain of TTPAL leads to gene overexpression levels of TRIP6 displaced b-catenin from the tumor suppressor in colorectal cancer from a Chinese cohort (n ¼ 102), whichwas MAGI1 via competitive binding. This sequence of events allows further validated by a The Cancer Genome Atlas (TCGA) cohort b-catenin to enter the nucleus and promotes oncogenic Wnt/ (n ¼ 376). High expression of TTPAL was significantly associ- b-catenin signaling. In conclusion, TTPAL is commonly over- ated with shortened survival in patients with colorectal cancer. expressed in colorectal cancer due to copy number gain, which TTPAL promoted cell viability and clonogenicity, accelerated promotes colorectal tumorigenesis by activating Wnt/b-catenin cell-cycle progression, inhibited cell apoptosis, increased cell signaling via stabilization of TRIP6. TTPAL overexpression may migration/invasion ability in vitro, and promoted tumorigenic- serve as an independent new biomarker for the prognosis of ity and cancer metastasis in vivo. TTPAL significantly activated patients with colorectal cancer. Wnt signaling and increased b-catenin activation and protein expression of cyclin D1 and c-Myc. Coimmunoprecipitation Significance: TTPAL, a gene preferentially amplified in followed by mass spectrometry identified thyroid receptor– colorectal cancer, promotes colon tumorigenesis via activation interacting protein 6 (TRIP6) as a direct downstream effector of of the Wnt/b-catenin pathway.

Introduction alterations (CNA) are common somatic changes in cancer fea- tured with gain or loss in copies of DNA sections (3). Deletions Colorectal cancer is the third most common cancer and the and copy number gains contribute to alterations in the expression second leading cause of cancer-related death worldwide (1, 2). of tumor suppressor genes and oncogenes, respectively. The The pathogenic mechanisms underlying colorectal cancer devel- stepwise accumulation of CNAs confers growth advantage and opment appear to be complex and heterogeneous. Copy number metastatic competence on cells, thus playing a crucial role in cancer initiation and progression. Recent studies have suggested that genes with CNAs are potential biomarkers and/or therapeutic 1Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenter- ology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes targets for colorectal cancer. Therefore, detection and mapping of and Related Genes, Shanghai Institute of Digestive Disease, Renji Hospital, copy number abnormalities provide an approach for associating School of Medicine, Shanghai Jiaotong University, Shanghai, China. 2Institute aberrations with disease phenotype and for localizing critical of Digestive Disease and Department of Medicine and Therapeutics, State Key genes (4, 5). It is importance to identify and functionally char- Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK acterize novel genes with CNAs that are associated with colorectal Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong. 3 cancer (4). Department of Surgery, Peking University Cancer Hospital and Institute, Beijing, fi China. 4Department of Anatomical and Cellular Pathology, State Key Laboratory Chromosome 20q ampli cation has been commonly found in of Oncology in South China, The Chinese University of Hong Kong, Hong Kong. colorectal cancer and is involved in transforming adenoma to carcinoma (5, 6). Chromosome 20q amplification is also a Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). potential indicator of poor prognosis in patients with colorectal cancer (7–9). By whole-genome sequencing (WGS), we identified H. Gou and J.Q. Liang are the co-first authors of this article. that TTPAL (tocopherol alpha transfer protein-like), located at Corresponding Author: Jun Yu, Institute of Digestive Disease and Department 20q13.12, was preferentially amplified in primary colorectal of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University tumor tissues. In keeping with our finding, analyses from The of Hong Kong, Hong Kong. Phone: 852-3763-6099; Fax: 852-2144-5330; E-mail: [email protected] Cancer Genome Atlas (TCGA) dataset showed that copy number gain of TTPAL occurs frequently in colorectal cancer and positively Cancer Res 2019;79:3332–46 correlates with its upregulated mRNA expression. Searching for doi: 10.1158/0008-5472.CAN-18-2986 the public protein datasets of Human Protein Altas, we found that 2019 American Association for Cancer Research. higher TTPAL protein expression was shown in colorectal cancer

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tumor tissues compared with normal colon tissues from the PCR copy number analysis dataset DNA was extracted from 102 frozen colorectal cancer samples (http://www.proteinatlas.org). Moreover, high TTPAL mRNA of Chinese Cohort using the AllPrep DNA/RNA/Protein Kit expression is significantly associated with poor survival in (Qiagen). The purified genomic DNA was amplified with a probe patients with multiple cancer types (https://www.proteinatlas. specific to target gene TTPAL (Mm00082375_cn) by TaqMan org/ENSG00000124120-TTPAL/pathology#top; P < 0.001). Copy Number Assays (Applied Biosystems). The results were Therefore, we hypothesize that TTPAL plays an oncogenic func- analyzed by Copy Caller software v2.0 (Applied Biosystems). tion in colon carcinogenesis. The role of TTPAL in human cancer remains uninvestigated. In Subcutaneous xenograft and experimental metastasis mouse this study, we identified the frequent overexpression of TTPAL in models colorectal cancer due to its copy number gain. Further functional HCT116 and DLD1 cells stably transfected with TTPAL expression studies revealed that ectopic expression of TTPAL significantly vector or empty vector (5 106 cells/0.1 mL PBS) were injected promoted colorectal cancer growth by enhancing G1–S cell-cycle subcutaneously into the left and right dorsal flanks of 4- to 6-week- progression and reducing apoptosis. The tumor-promoting effect old female Balb/c nude mice (n ¼ 8/group), respectively. Tumor size of TTPAL was revealed to be associated with the activation of Wnt/ was measured every 2 days for 2–3 weeks using a digital caliper. b-catenin signaling pathway by binding to thyroid receptor– Tumor volume (mm3) was estimated by measuring the longest and interacting protein 6 (TRIP6). The direct binding of TTPAL with shortest diameters of the tumor and calculating as described previ- TRIP6 in cytoplasm inhibited TRIP6 ubiquitin degradation. ously (10). At the endpoint, tumors were harvested and weighted. Enhanced cytoplasm TRIP6 displaced b-catenin from the tumor The excised tissues were either fixed in 10% neutral-buffered for- suppressor MAGI1 via competitive binding, which allowed malin or snap frozen in liquid nitrogen. Tumor sections from b-catenin to enter into the nucleus and subsequently activated paraffin-embedded blocks were used for histologic examination. the oncogenic Wnt/b-catenin signaling. Moreover, TTPAL over- For metastasis model, HCT116 cells stably transfected with expression was significantly associated with poor survival of TTPAL expression vector or empty vector (2 106 cells in 0.1 mL patients with colorectal cancer. Thus, this study revealed a novel PBS) were injected intravenously via the tail vein (n ¼ 5). oncogenic mechanism and the clinical application value of TTPAL After 6–8 weeks, mice were sacrificed and their lungs were har- in colorectal cancer. vested. The lungs were sectioned and stained with hematoxylin and eosin. The number of lung metastases were counted. All experimental procedures were approved by the Animal Ethics Materials and Methods Committee of the Chinese University of Hong Kong. Colon cancer cell lines The colon cancer cell lines (DLD-1, HCT116, HT29, LOVO, Coimmunoprecipitation and liquid chromatography–mass SW480, RKO, and SW1116) were obtained from the ATCC spectrometry between 2014 and 2015 and cells' authentication were confirmed Coimmunoprecipitation (co-IP) assays were carried out as by short tandem repeat profiling. Cells were routinely cultured described previously (11, 12). Briefly, total protein from HCT116 and maintained in DMEM supplemented with 10% FBS and cells (5 106/reaction) stably transfected with TTPAL (Flag- antibiotics (Gibco BRL) according to the ATCC protocols, except tagged) expression vector or empty vector was extracted in radio- HCT116 cells, which were cultured in complete McCoy 5A medi- immunoprecipitation assay (RIPA) buffer supplemented with um (Gibco BRL). Routine Mycoplasma testing was performed by proteinase inhibitor (Novagen). Immunoprecipitation was per- PCR. Cells were grown for no more than 25 passages in total for formed using anti-Flag M2 antibody (A2220, Sigma-Aldrich). The any experiment. immune complexes were precipitated by PureProteome Protein A/G Mix magnetic beads (LSKMAGAG02, Millipore) overnight at Subjects and sample collection 4C. Beads with extracted proteins were washed three times by Paired primary colorectal tumors and adjacent nontumor 50 mmol/L ammonium bicarbonate buffer and subjected to tissues were collected immediately after surgical resection at the digestion by trypsin at 37C for 2 hours (Promega). Tryptic Prince of Wales Hospital (Hong Kong, China; 182 primary peptides were then extracted for liquid chromatography–mass tumors and 18 nontumor tissues) and Peking University Cancer spectrometry (LC/MS) analysis. Hospital and Institute (Beijing, China; 102 primary tumors 50 nontumor tissues). The specimens were snap-frozen in liquid Co-IP and Western blot analyses nitrogenandstoredat80Candwerealsofixed in 10% Whole lysate (150 mg protein) and co-IP precipitant by anti- formalin and embedded in paraffin for routine histologic Flag-tag, anti-thyroid receptor–interacting protein 6 (TRIP6) examination. Biopsies from 3 cases of normal mucosa obtained antibody (ab70747, Abcam), anti-HA-tag (ab18181, Abcam), during colonoscopy were recruited as healthy controls, which or IgG were immunoblotted with either anti-TRIP6, anti-HA, or were confirmed by an experienced pathologist at the Prince of anti-Flag (A2220, Sigma-Aldrich) antibody to confirm the inter- Wales Hospital and Peking University Cancer Hospital. The action between TTPAL and TRIP6 that was identified by LC/MS. study protocol was approved by the Clinical Research Ethics The lysate (1% input, 10 mg protein) was also used as a control. Committee of The Chinese University of Hong Kong and the The antibodies used are listed in Supplementary Table S1. Clinical Research Ethics Committee of Peking University Can- cer Hospital. All patients provided written informed consent for Ubiquitination assay obtaining the study specimens. This study was carried out in HCT116 and DLD1 cells stably transfected with TTPAL expres- accordance with the Declaration of Helsinki of the World sion vector or empty vector were cotransfected with Ubiquitin-HA Medical Association. for 24 hours. Then the cells were incubated in the presence or

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Table 1. Univariate and multivariate Cox regression analyses of potential poor prognostic factors in colorectal cancer patients Chinese cohort - mRNA level Univariate Multivariate Variable RR (95% CI) P RR (95% CI) P Age 1.021 (0.997–1.045) 0.091 1.020 (0.996–1.045) 0.110 Gender 0.696 0.703 M 1.132 (0.608–2.107) 1.128 (0.606–2.101) F1 1 Location 0.904 Colon 0.964 (0.36–1.736) Rectum 1 Differentiation 0.586 Moderate or high 1.216 (0.602–2.457) Low 1 TNM 0.067 II 1.789 (0.961–3.330) III 1 TTPAL expression 0.028 0.034 High 2.202(1.090–4.447) 2.146 (1.061–4.340) Low 1 1 Chinese cohort - protein level Univariate Multivariate Variable RR (95% CI) P RR (95% CI) P Age 1.004 (0.987–1.021) 0.637 1.008 (0.992–1.025) 0.346 Gender 0793 0.634 M 1.949 (0.641–1.404) 1.103 (0.736–1.655) F1 1 Location 0.279 Colon 0.805 (0.544–1.192) Rectum 1 Differentiation 0.142 Moderate or high 1.430 (0.887–2.306) Low 1 TNM Early (I & II) 0.227 (0.137–0.374) <0.0001 0.202 (0.122–0.355) <0.0001 Late (III & IV) 1 1 TTPAL expression <0.0001 <0.0001 High 1.954 (1.258–3.036) 2.441 (1.544–3.858) Low 1 1

absence of 10 mmol/L MG132 (M-7449, Sigma-Aldrich) for 12 (SPSS; standard V.16.0; IBM Corporation). The Pearson correla- hours. Cells were lysed with RIPA buffer supplemented with tion coefficient was used to evaluate the correlation between protease inhibitors. Immunoprecipitation was performed using TTPAL gene amplification and expression in the clinical samples. anti-TRIP6 or IgG, respectively. Immunoprecipitated proteins The x2 test was used for comparison of patient characteristics and were analyzed by Western blot using anti-HA (ab18181, Abcam). distributions of expression and covariates by vital status. ROC curve was used to estimate the cut-off value of the methylation Statistical analysis percentage. Cut-off value was analyzed by survival significance The results were expressed as mean SD. Statistical analysis analysis using the tool Cutoff Finder (http://molpath.charite.de/ was performed using the Statistical Package for the Social Sciences cutoff/; ref. 13). Crude relative risks (RR) of death associated with

Figure 1. Overexpression of TTPAL is due to copy number gain in colorectal cancer and predicts poor prognosis of patients with colorectal cancer. A, Copy number of TTPAL is preferentially amplified in colon and rectum cancers as compared with other cancer types. Data were from TCGA studies, with sample sizes indicated under each caner type. COAD, colon adenocarcinoma; READ, rectum adenocarcinoma; BRCA, breast invasive carcinoma; HNSC, head and neck squamous cell carcinoma; LUSC, lung squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; BLCA, bladder urothelial carcinoma; LUAD, lung adenocarcinoma; UCEC, uterine corpus endometrioid carcinoma; OV, ovarian serous cystadenocarcinoma; GBM, glioblastoma multiforme; STAD, stomach adenocarcinoma. B, TTPAL copy number was positively correlated with its mRNA expression in Chinese and TCGA cohorts by the Pearson correlation coefficient analysis. C, TTPAL was expressed in colon cancer cell lines, but not in normal colon tissues, as determined by RT-PCR and Western blot analysis. D, TTPAL mRNA expression was upregulated in colorectal cancer compared with paired adjacent normal tissues as shown by RT-PCR (top) and qRT-PCR (bottom left, Chinese cohorts 1 and2 from Beijing and Hong Kong, respectively). RNA-seq data from TCGA study also show upregulation of TTPAL in colorectal cancer as compared with adjacent normal tissues (bottom right, paired and unpaired samples). E, TTPAL protein expression was upregulated in primary colorectal cancer as compared with adjacent normal tissues by Western blot analysis. F, TTPAL protein expression was significantly higher in primary colorectal cancer as compared with adjacent normal tissues as shown by IHC staining. G, Kaplan–Meier survival analysis showed patients with colorectal cancer with high TTPAL expression had poorer survival than those with low TTPAL expression at both mRNA and protein levels from Chinese population. H, Prognostic value of TTPAL expression was validated in patients with colorectal cancer from TCGA study.

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TTPAL expression were estimated by univariate Cox proportional qRT-PCR (Fig. 1D). Specifically, the upregulation of TTPAL mRNA hazards regression model first. Multivariate Cox model was then in colorectal cancer was validated in three independent cohorts of constructed to estimate the adjusted RR for TTPAL expression. paired tumor and adjacent normal samples including Bejing Overall survival in relation to expression was evaluated by Chinese cohort (n ¼ 50; P < 0.0001), Hong Kong Chinese cohort the Kaplan–Meier survival curve and the log-rank test. Mann– (n ¼ 18; P < 0.0001), and the TCGA cohort (n ¼ 23; P < Whitney U test or Student t test was performed to compare the 0.0001; Fig. 1D). Significant upregulation was also observed in variables of two groups. mRNA expression z-scores (RNA Seq V2 colorectal cancer (n ¼ 286) compared with unpaired adjacent RSEM) from 382 colorectal cancer samples were downloaded normal tissues (n ¼ 39) from TCGA study (P < 0.0001; Fig. 1D). from TCGA via cBioPortal. Nonparametric Spearman correlations Western blot and IHC staining further confirmed the upregulation in expression levels between TTPAL and Wnt target genes were of TTPAL at the protein level in colorectal cancers as compared then computed. The difference in cell viability and tumor growth with adjacent normal tissues (Fig. 1E and F). rate between two groups of nude mice was determined by repeat- ed-measures ANOVA. P values <0.05 were taken as statistical Overexpression of TTPAL is an independent predictor of poor significance. survival in patients with colorectal cancer We further evaluated the clinicopathologic and prognostic significance of TTPAL expression in patients with colorectal can- Results cer. We evaluated TTPAL mRNA expression in primary colorectal TTPAL is frequently overexpressed in colorectal cancer due to cancer tissues from 102 Chinese patients. By classifying samples copy number gain into low and high expression groups using cut-off finder, TTPAL By WGS analysis to compare genetic alterations in primary overexpression was detected in 64.7% (66/102) of primary colo- colorectal cancer tissues with corresponding peripheral blood rectal cancers. No correlation was found between TTPAL expres- samples, we identified TTPAL to be preferentially amplified in sion and clinicopathologic features such as age, gender, location, colorectal cancer tumor tissues by WGS (Supplementary Fig. differentiation, and Tumor–Node–Metastasis (TNM) stage S1A). TTPAL was also frequently amplified in colon and rectum (Supplementary Table S2). However, TTPAL overexpression was cancers from TCGA cohort but less amplified in other cancers associated with an increased risk of cancer-related death by (Fig. 1A), demonstrating that copy number gain of TTPAL may univariate Cox regression (RR ¼ 2.202, 95% CI: 1.090–4.447, be specifically associated with colorectal tumorigenesis. We P ¼ 0.028; Table 1). In particular, after adjustment for potential further quantitated the copy number and mRNA expression of confounding factors, TTPAL overexpression was found to be an TTPAL in colorectal cancer samples by TaqMan copy number independent risk factor for shortened survival in patients with assay and qRT-PCR, respectively. In 102 patients with colorectal colorectal cancer by multivariate Cox regression analysis (RR ¼ cancer, 48% (49/102) of primary colorectal cancer tissues 2.146, 95% CI: 1.061–4.340, P ¼ 0.034; Table 1). Kaplan–Meier exhibited TTPAL copy number gain, which was positively survival curves showed that patients with colorectal cancer with correlated with its mRNA expression (R2 ¼ 0.3537, P < TTPAL overexpression had significantly poorer overall survival 0.0001; Fig. 1B). This finding was further validated in colorectal based on the log-rank test (P < 0.05; Fig. 1G). cancer samples from TCGA cohort, with copy number gain TTPAL protein expression was further evaluated in primary detected in 70.2% of samples (1.2 fold, 436/621) and mRNA colorectal cancer tissues from another cohort of Chinese patients expression also positively correlated with copy number (n ¼ (n ¼ 182) by IHC using tissue microarrays. No correlation was 376, R2 ¼ 0.5632, P < 0.0001; Fig. 1B). These results demon- found between TTPAL protein expression and clinicopathologic strate that TTPAL is commonly overexpressed in colorectal features such as age, gender, location, differentiation, and TNM cancer due to copy number gain. stage (Supplementary Table S2). Further univariate and multivariate Cox regression analyses showed that TTPAL protein expres- TTPAL expression is upregulated in colon cancer cells and sion was also an independent predictor of poor survival in primary colorectal cancer tumors patients with colorectal cancer (multivariate RR ¼ 2.441; 95% We then examined TTPAL expression in colon cancer cells and CI: 1.544–3.858, P < 0.0001) besides TNM stage (Table 1). As primary colorectal cancer tissues. TTPAL expression at both mRNA shown in the Kaplan–Meier survival curves, colorectal cancer and protein levels was detected in all 7 colon cancer cells tested patients with high TTPAL protein expression had significantly (DLD1, LOVO, RKO, HT29, HCT116, SW480, and SW1116), but shorter survival than those with low expression (P ¼ 0.002, log- not in normal colon tissues (Fig. 1C). TTPAL mRNA expression rank test; Fig. 1G). Moreover, the prognostic significance of TTPAL was significantly upregulated in primary colorectal cancer tumors expression was well validated in TCGA cohort (Fig. 1H). These as compared with their adjacent normal tissues as determined by findings collectively indicated that TTPAL may serve as an

Figure 2. TTPAL exerts oncogenic functions in vitro. A, Overexpression of TTPAL in HCT116 and DLD1 cells, confirmed by RT-PCR and Western blot analysis, significantly increased cell viability and colony formation ability. B, Knockdown of TTPAL by siTTPAL in SW480 and SW1116, confirmed by RT-PCR and Western blot analysis, significantly inhibited cell viability and colony formation ability. C, Overexpression of TTPAL significantly increased cells in S-phase, while knockdown of TTPAL showed the opposite effect. D, Western blot analysis showed TTPAL enhanced protein levels of cyclin-D1, CDK4, and PCNA, while knockdown of TTPAL had the opposite effect. E, Overexpression of TTPAL inhibited cell apoptosis by flow cytometry after Annexin V/7-AAD dual staining, while knockdown of TTPAL promoted cell apoptosis. F, Western blot analysis showed TTPAL expression reduced activation of caspases-8, -9, -7, -3, and PARP, while knockdown of TTPAL exhibited the opposite effect. G, Overexpression of TTPAL promoted cell migration as shown by wound-healing assays. H, Overexpression of TTPAL significantly increased cell invasion ability as shown by Matrigel invasion assay. I, Knockdown of TTPAL significantly inhibited migration ability. J, Knockdown of TTPAL significantly decreased cell invasion ability. K, TTPAL increased expression of mesenchymal marker (vimentin) and decreased expression of epithelial markers (E-cadherin and claudin-1), while knockdown of TTPAL showed the opposite effects.

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independent new biomarker for the prognosis of patients with protein-1 (AP-1), MAPK/ERK (SRE), Wnt/b-catenin, Akt (FHRE), colorectal cancer. and NFkB. Ectopic expression of TTPAL significantly increased the activity of Wnt signaling as demonstrated by TOPflash/FOPflash TTPAL expression promotes colon cancer cell growth luciferase reporter assay, and knockdown of TTPAL showed the The frequent overexpression of TTPAL in colorectal cancer opposite effect on Wnt activity (Fig. 3A; Supplementary Fig. S2A). prompted us to investigate its potential oncogenic role in colo- Consistently, overexpression of TTPAL significantly upregulated rectal cancer. To this end, we performed in vitro gain- and loss-of- the protein levels of cyclin D1, c-Myc, b-catenin, and active function assays on TTPAL. Ectopic expression of TTPAL in b-catenin, while TTPAL knockdown diminished expression of HCT116 and DLD1 cells (Fig. 2A) significantly increased cell these key Wnt signaling genes (Fig. 3B). Moreover, Wnt signaling viability and clonogenicity (Fig. 2A). On the other hand, TTPAL inhibitor IWP-2 significantly inhibited cell growth in DLD1 and knockdown by RNA interference in SW480 and SW1116 cells HCT116 cells stably transfected with TTPAL as compared with markedly inhibited cell viability and clonogenicity (Fig. 2B). DMSO treatment. IWP-2 also eliminated the growth-promoting These results indicated that TTPAL plays an oncogenic role in effect exerted by TTPAL overexpression (P > 0.05 for IWP-2-treated colon cancer. TTPAL-overexpressed cells vs. DMSO-treated vector-transfected cells; Fig. 3C). TTPAL knockdown showed weaker effect on TTPAL promotes cell-cycle progression and inhibits cell proliferation in RKO cells, which do not harbor mutation in APC apoptosis or CTNNB1 gene (Supplementary Fig. S2B). These results dem- We analyzed the cytokinetic effect of TTPAL. Overexpression of onstrate that the oncogenic role of TTPAL is largely mediated by TTPAL led to a significant decrease in G1 phase cells and a activation of Wnt/b-catenin signaling. concomitantly increase in S-phase cells in both HCT116 and DLD1 cells by BrdU staining (all P < 0.01; Fig. 2C). The promoting TRIP6 directly interacts with TTPAL effect of TTPAL on cell-cycle progression was confirmed by the To identify the direct interacting partners of TTPAL in colorectal increased protein expression of two master G1–S checkpoint cancer, we performed co-IP followed by LC/MS. TTPAL binding regulators (cyclin D1 and CDK4) and the proliferation marker candidates were then identified by comparing the anti-TTPAL-Flag proliferating cell nuclear antigen (PCNA; Fig. 2D). On the other IP products of TTPAL (Flag)-overexpressed cells with those of hand, knockdown of TTPAL significantly increased cells in the G1 control cells (Fig. 3D). Among the identified candidates with 3 phase and decreased cells in S-phase of both SW480 and SW1116 mapping peptides, 7 genes have been reported with functions that cells (all P < 0.01; Fig. 2C), and concomitantly reduced the may be associated with cancer development (Supplementary expression of cyclin D1, CDK4, and PCNA (Fig. 2D). Apoptosis Table S3). Among them, TRIP6 is of interest due to its involvement analysis showed that overexpression of TTPAL led to a significant in Wnt signaling pathway (14). To validate the interaction reduction in apoptotic cell population in HCT116 (P < 0.01) and between TTPAL and TRIP6, Flag-TTPAL and HA-TRIP6 (or TRIP6) DLD1 (P < 0.001) cells (Fig. 2E, 1), while knockdown of TTPAL were co-expressed in HCT116 and DLD1 cells, and total proteins significantly increased apoptosis of SW480 and SW1116 cells were immunoprecipitated with anti-Flag or anti-HA/anti-TRIP6 (both P < 0.001; Fig. 2E, 2). TTPAL-induced reduction of apo- antibodies. Western blot results showed that TTPAL and TRIP6 ptosis was confirmed by the elevated expression of key apoptosis could be coprecipitated by each other in both cells. Moreover, the markers (cleaved forms of caspase-8, caspase-9, caspase-7, cas- interaction of endogenous TTPAL and TRIP6 in SW480 and pase-3, and PARP), while knockdown of TTPAL showed opposite SW1116 cells was confirmed by immunoprecipitation–Western effects (Fig. 2F). These results demonstrate the role of TTPAL on blot analyses (Fig. 3E), indicating the direct interaction between promoting cell growth by regulating cell-cycle progression and TTPAL and TRIP6. We next examined the expression and intra- apoptosis in colon cancer cells. cellular distribution of TTPAL and TRIP6 by immunofluorescence. Confocal microscopy images showed that TTPAL colocalized with TTPAL promotes cell migration and invasion TRIP6 in the cytoplasm of HCT116 and DLD1 cells following To determine the effects of TTPAL on cell migration and transfection of TTPAL and HA-TRIP6 (Fig. 3F, 1). The localization invasion, wound-healing and Matrigel invasion assays were per- of the two proteins was further confirmed by Western blotting of formed. Ectopic expression of TTPAL significantly increased the membrane, cytoplasmic, and nuclear fractions, which showed migration abilities in HCT116 and DLD1 cells (both P < that TTPAL and TRIP6 was mainly localized in the cytoplasm of 0.01; Fig. 2G), and significantly promoted invasiveness of both HCT116 and DLD1 cells (Fig. 3F, 2). cells (both P < 0.001; Fig. 2H), while knockdown of TTPAL markedly suppressed migration and invasion abilities in SW480 TTPAL promotes tumorigenicity and metastasis by regulating and SW1116 cells (Fig. 2I). In keeping with these, protein expres- TRIP6 and Wnt/b-catenin signaling in vivo sion of epithelial-to-mesenchymal transition (EMT) markers was In light of our in vitro findings, we tested the effect of TTPAL regulated by TTPAL. TTPAL overexpression upregulated the mes- in vivo. Results showed that stable transfection of TTPAL expres- enchymal marker vimentin and downregulated the epithelial sion vector in HCT116 and DLD1 cells significantly promoted the markers (E-cadherin and claudin-1), while knockdown of TTPAL growth of tumor volume over the entire assay period and showed the opposite effects (Fig. 2K). These findings demonstrat- increased tumor weight at the end point in subcutaneous xeno- ed that TTPAL also functioned in regulating cell migration and graft models (Fig. 4A). Overexpression of TTPAL was confirmed invasion of colorectal cancer cells. by IHC staining in DLD1 and HCT116 xenografts (Fig. 4B). Cell proliferation was significantly promoted as determined by Ki-67 TTPAL activates Wnt/b-catenin signaling staining, and apoptosis was significantly reduced as evidenced by We performed luciferase reporter assays to assess the effect of the TUNEL staining, in both TTPAL-overexpressed xenografts as TTPAL on important cancer pathways including p53, activator compared with controls (Fig. 4B). Moreover, the expression of

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Figure 3. TTPAL activates Wnt signaling pathway and interacts with TRIP6. A, Overexpression of TTPAL significantly increased TOPflash/FOPflash luciferase reporter (Wnt) activity in DLD1 cells, while knockdown of TTPAL significantly suppressed Wnt activity in SW480 cells. B, Western blot analysis results showed that TTPAL expression enhanced the expression of Wnt signaling–related markers, while knockdown of TTPAL showed the opposite effect. C, Wnt signaling inhibitor IWP-2 treatment abolished the promoting effects of TTPAL on cell proliferation and clonogenicity in HCT116 and DLD1 cells. D, Co-IP followed by LC/MS identified TRIP6 to be a TTPAL-binding protein. E, Co-IP followed by Western blot analyses confirmed the binding between TTPAL and TRIP6 in ectopic and endogenous. F, TTPAL and TRIP6 are mainly colocalized in cytoplasm as demonstrated by confocal immunofluorescence analysis (F1) and Western blot of membrane, cytoplasmic, and nuclear fractions (F2) in HCT116 and DLD1cells cotransfected with TTPAL and TRIP6 expression vectors. Mem, membrane; Cyto, cytoplasm; Nuc, nucleus.

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Figure 4. TTPAL enhances tumorigenicity and metastasis in nude mice. A, HCT116 cells stably expressing TTPAL expression promoted subcutaneous tumor growth as compared with control vector transfection in both HCT116 (A1) and DLD1 (A2) cells, both in terms of tumor volume over the entire assay period and tumor weight at the end point. B, IHC staining confirmed TTPAL overexpression in HCT116 and DLD1 subcutaneous xenografts, which enhanced cell proliferation (by Ki-67 staining) and inhibited cell apoptosis (by TUNEL staining). IHC staining results also showed that TTPAL increased TRIP6 and b-catenin expression in xenografts. C, Western blot analysis further confirmed that TTPAL expression in HCT116 and DLD1 xenografts increased the expression of TRIP6 and b-catenin. D, Overexpression of TTPAL promoted experimental metastasis of HCT116 cells in vivo. Representative images of lungs and hematoxylin and eosin (H&E) staining of lung tissues from nude mice injected with TTPAL- or control vector–transfected HCT116 cells. Quantitative analysis showed that TTPAL expression significantly increased the number of metastatic lesions. E, Knockdown of TTPAL by stably expressing shTTPAL inhibited subcutaneous tumor growth as compared with shNC transfection in SW480. F, Knockdown of TTPAL significantly decreased the number of metastatic lesions in SW480 cells.

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Figure 5. TTPAL exerts its oncogenic function, partially depending on TRIP6 in colorectal cancer. A, Knockdown of TRIP6 in HCT116 and DLD1 cells with stable TTPAL overexpression was confirmed by RT-PCR and Western blot analysis. B, Knockdown of TRIP6 significantly abolished the promoting effect of TTPAL on colon cancer cell growth. C, Knockdown of TRIP6 significantly abolished the promoting effect of TTPAL on clonogenicity of colon cancer cells. D, Knockdown of TRIP6 significantly decreased migration ability of colon cancer cells that was promoted by TTPAL. E, Protein expression of key factors in Wnt signaling pathway in TTPAL-overexpressed HCT116 and DLD1 cells with or without transient knockdown of TRIP6. F, Knockdown of TRIP6 significantly abolished the TOPflash/ FOPflash luciferase activity induced by TTPAL. G–J, TRIP6 overexpression in TTPAL-inhibited SW480 cells rescued cell proliferation (G), clonogenicity (H), migration (I), and Wnt activation (J).

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3342 Cancer Res; 79(13) July 1, 2019 Cancer Research

Role of TTPAL in Colorectal Cancer

b-catenin and TRIP6 was dramatically increased in TTPAL-over- of TTPAL in HCT116 and DLD1 cells, and decreased after knock- expressed HCT116 and DLD1 xenografts by IHC, validating the down of the endogenous TTPAL in SW1116 cells (Fig. 6A, 1). molecular basis identified in vitro (Fig. 4B). TTPAL overexpression TRIP6 protein was also upregulated by TTPAL in xenografts of promoted lung metastasis in nude mice (P < 0.01; Fig. 4D). nude mice (Fig. 4C and D). However, mRNA level of TRIP6 Furthermore, stable knockdown of TTPAL in SW480 cells signif- was not changed by overexpression or knockdown of TTPAL icantly inhibited tumorigenicity (Fig. 4E) and metastasis in nude (Fig. 6A, 1). Moreover, the protein levels of TRIP6 in various mice (Fig. 4F). Collectively, these results in immunodeficient colorectal cancer cell lines were positively correlated with the nude mice suggest that TTPAL promoted colorectal tumorigenic- protein levels of TTPAL in these cell lines (Fig. 6A, 2), indicating ity and may also facilitate metastasis. that TTPAL may stabilize TRIP6 in colorectal cancer. We therefore assessed whether TTPAL regulated the stability of TRIP6 – The oncogenic role of TTPAL is dependent on TRIP6 protein. We treated TTPAL or control vector transfected cells We then examined the importance of TRIP6 in TTPAL-mediated with the protein synthesis inhibitor cycloheximide. As shown oncogenic function in colorectal cancer. TRIP6 mRNA expression in Fig. 6B, 1, TRIP6 was more stable in the presence of TTPAL was significantly increased in colorectal cancer tumor tissues as in HCT116 and DLD1 cells. To investigate whether TTPAL compared with adjacent normal tissues in TCGA cohort (P < increased the stability of TRIP6 by affecting its ubiquitination/ 0.001, Supplementary Fig. S3A). TRIP6 mRNA was readily degradation, we treated cells with the proteasome inhibitor expressed in all 7 colon cancer cells tested, but not in normal MG132 after cotransfecting ubiquitin-HA and TTPAL/control colon tissues (Supplementary Fig. S3B), TRIP6 expression pro- vectors in HCT116 and DLD1 cells, and then cellular lysates were moted cell proliferation and clonogenicity in DLD1 and HT-29 immunoprecipitated with anti-TRIP6 or IgG. As speculated, cells, whereas TRIP6 silence showed significant suppressive effect TTPAL decreased TRIP6 ubiquitination and increased its protein on cell proliferation and clonogenicity in SW480, SW1116, and level (Fig. 6B, 2), inferring that TTPAL binds to TRIP6 and inhibits HCT116, suggesting the oncogenic role of TRIP6 in colorectal the ubiquitin-mediated degradation of TRIP6 protein. cancer (Supplementary Fig. S4). To investigate the effect of TRIP6 on the TTPAL-mediated cell proliferation and metastasis, HCT116 TTPAL activates Wnt/b-catenin signaling by promoting TRIP6– and DLD1 cells stably transfected with TTPAL or control vectors MAGI1 interaction were cotransfected with siRNA against TRIP6 (Fig. 5A). TRIP6 TRIP6, containing the same PDZ-binding motif with b-cate- fi knockdown signi cantly abolished the promoting effect of TTPAL nin, can compete with b-catenin to bind to the PDZ domain of on cell viability (Fig. 5B), clonogenicity (Fig. 5C), and migration the tumor suppressor MAGI1(Supplementary Fig. S5; ref. 14). ability of both HCT116 and DLD1 cells (Fig. 5D). We next We therefore hypothesize that TRIP6 protein increased by examined whether TTPAL activated Wnt signaling pathway TTPAL binds to MAGI1 and subsequently release more b-cate- through mediating TRIP6. Indeed, TRIP6 knockdown abolished nin to ultimately activate Wnt/b-catenin signaling. To confirm the TTPAL-induced protein expression of Wnt effectors (cyclin D1, the interaction between TRIP6 and MAGI1 in a more physio- b b c-Myc, -catenin, and active -catenin) by Western blot analysis logic context, co-IP studies were carried out. HCT116 and DLD1 (Fig. 5E). TRIP6 knockdown also blunted the TTPAL-activated cells were transiently transfected with TRIP6, and cellular Wnt signaling as evidenced by luciferase reporter assay in HCT116 lysates were then immunoprecipitated with anti-TRIP6 or IgG. and DLD1 cells (Fig. 5F). These results suggested that the onco- Western blot analysis confirmed the presence of MAGI1 in anti- genic role of TTPAL and its effect on activating Wnt signaling were TRIP6 IP products of both cells (Fig. 6C). To assess the effect of dependent on TRIP6. Consistently, TRIP6 overexpression in TTPAL on TRIP6–MAGI1 interaction, TTPAL or control vectors TTPAL-inhibited SW480 cells can rescue cell proliferation were transfected in HCT116 and DLD1 cells, and cellular lysates (Fig. 5G), clonogenicity (Fig. 5G), migration (Fig. 5I), and Wnt were immunoprecipitated with anti-TRIP6 or IgG. Western blot activation (Fig. 5J). results showed that ectopic expression of TTPAL increased TRIP6 protein level and the binding of TRIP6–MAGI1 TTPAL inhibits ubiquitin-mediated degradation of TRIP6 (Fig.6C).ByoverexpressingTRIP6and/orMAGI1inHCT116 protein and DLD1 cells followed by co-IP and Western blot assays, we To gain insights into how TRIP6 mediates the role of TTPAL, we further showed the binding between MAGI1 and b-catenin was further evaluated the effect of TTPAL on TRIP6. We observed that reduced by the presence of TRIP6 (Fig. 6D). In line with these TRIP6 protein expression was upregulated by ectopic expression results, both TRIP6 overexpression and MAGI1 knockdown

Figure 6. TTPAL activates Wnt/b-catenin signaling pathway by promoting TRIP6–MAGI1 interaction. A1, TRIP6 expression was dependent on TTPAL at protein level but not at mRNA level as determined by Western blot analysis and RT-(q)PCR. A2, The protein level of TRIP6 was correlated with those of TTPAL in various of colon cancer cells. B, TTPAL stabilized TRIP6, as shown by treatment with the new protein synthesis inhibiter cyclohexamide (CHX; B1) and the proteasome inhibitor MG132 (B2) in HCT116 and DLD1 cells. C, TRIP6–MAGI1 interaction was determined by co-IP, followed by Western blot analysis in HCT116 and DLD1 cells. Overexpression of TTPAL increased the binding between TRIP6 and MAGI1 (right). D, MAGI1–b-catenin interaction was decreased by TRIP6 as shown by co-IP, followed by Western blot analysis. E, Western blot and TOPflash/FOPflash luciferase reporter assays showed that overexpression of TRIP6 and knockdown of MAGI1 significantly activated Wnt signaling pathway. F, Induction of TRIP6 promoted interaction between b-catenin and TCF4. G, PDZ binding–defective mutant of TRIP6 was incapable of binding to MAGI1. H, Wild-type TRIP6 induced Wnt activation as compared with PDZ-binding–defective mutant TRIP6 or vector transfection controls, which was indicated by the increased levels of active b-catenin and TCF4 (H1). Wild-type TRIP6 significantly promoted cell proliferation, clonogenicity, and migration in HCT116 and DLD1 cells, but not mutant TRIP6 (H2 and H3). I, TTPAL expression positively correlated with the expression of multiple downstream targets of Wnt signaling in primary colorectal cancer tissues from TCGA study. J, Schematic illustration of the molecular mechanism of TTPAL in Wnt signaling of colorectal cancer.

www.aacrjournals.org Cancer Res; 79(13) July 1, 2019 3343

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significantly activated Wnt signaling, as evidenced by the motherapy. Our results demonstrated that TTPAL may serve as a increased protein levels of active b-catenin and the increased new independent prognostic marker for patients with colorectal TOPflash/FOPflash reporter activities (all P < 0.01) in HCT116 cancer. and DLD1 cells (Fig. 6E). TRIP6 promoted interaction between Because TTPAL was commonly upregulated in patients with TCF4 and b-catenin (Fig. 6F). Moreover, PDZ binding–defective colorectal cancer, this implies important oncogenic function of mutant, the deletion of the 8 C-terminal aa residues of TRIP6, TTPAL during colorectal tumorigenesis. With this connection, we was conducted (14), while mutant TRIP6 could not bind to performed in vitro gain- and loss-of-function assays on TTPAL. MAGI1 (Fig. 6G). Wild-type TRIP6 induced Wnt activation as Overexpression of TTPAL in HCT116 and DLD1 cells significantly compared with mutant TRIP6 or vector transfection controls, enhanced cell viability and colony formation ability. Conversely, which was indicated by the increased levels of active b-catenin knockdown of TTPAL in SW480 and SW1116 cells significantly and TCF4 (Fig. 6H, 1). In accordance with this, wild-type TRIP6 suppressed cell growth. The mechanism by which TTPAL pro- significantly promoted cell proliferation, clonogenicity, and moted colorectal cancer cell growth was mediated by promoting migration in HCT116 and DLD1 cells (Fig. 6H, 2 and 3). G1–S cell-cycle transition and inhibiting cell apoptosis. G1–S Consistent with the in vitro and in vivo findings, TTPAL expres- transition promoted by TTPAL was associated with the upregula- sion positively correlated with multiple target genes of b-cate- tion of cyclin D1 and CDK4, which are the key regulators of the nin in primary colorectal cancer tissues from TCGA cohort, transition through the G1 phase of the cell cycle (17). Concom- including CMYC, CCND1, ASCL2, APCDD1, AXIN2, EDN1, itantly, the growth enhancement effect of TTPAL was also related FGF18, MET, NAV2, and VEGFA (Fig. 6I), further supporting to inhibition of apoptosis, which was mediated by the caspase- the activation of Wnt/b-catenin signaling by TTPAL. Collective- dependent pathways including caspase-8, caspase-9, caspase-7, ly, our results suggest that upregulation of TTPAL increases the caspase-3, and PARP. Moreover, overexpression of TTPAL in level of TRIP6 by inhibiting its ubiquitin-mediated degrada- HCT116 and DLD1 cells significantly promoted their migration tion, and subsequently TRIP6 competitively binds with MAGI1 and invasion abilities. In accordance with the in vitro data, TTPAL to release b-catenin; this sequence of events allows more promoted tumor growth in mouse subcutaneous xenograft mod- b-catenin to enter the nucleus and activates oncogenic Wnt/ els and also promoted metastasis to the lung in tail vein injection b-catenin signaling to promote colorectal tumorigenesis models. Collectively, these results indicate that TTPAL exerts (Fig. 6J). oncogenic properties in colorectal carcinogenesis via promoting cell proliferation and cell-cycle progression, inhibiting apoptosis and increasing metastatic abilities. The effects of TTPAL in pro- Discussion moting migration and invasion abilities in vitro and in inducing In this study, amplification of TTPAL was identified by WGS. lung metastasis in nude mouse models suggested the potential We revealed that the amplification of TTPAL was positively role of TTPAL in facilitating colorectal cancer metastasis in vivo. associated with its mRNA overexpression in the 102 colorectal Additional investigation using TTPAL transgenic and orthotopic cancer cases in Chinese cohort (P < 0.001) and 376 colorectal mouse models are essential for future investigating the signifi- cancer cases in TCGA cohort (P < 0.001), suggesting that increased cance of TTPAL in colorectal cancer metastasis. TTPAL copy number contributes to the upregulation of TTPAL. We demonstrated that TTPAL could activate the TOPflash/ TTPAL was located on chromosome 20q13.12, which is one of the FOPflash activity in colon cancer cells. TTPAL-induced Wnt sig- most frequently amplified regions in colorectal cancer and in naling activation was also evidenced by b-catenin activation and other cancer types such as breast, ovarian, and gastric can- increased protein levels of cyclin D1 and c-Myc. Furthermore, Wnt cers (7, 15, 16). Previously, we have reported amplification of signaling inhibitor abolished the oncogenic effect of TTPAL on SLC12A5 (9), the same location with TTPAL, contributes to cell proliferation and clonogenicity. To understand the molecular colorectal tumor progression and metastasis. Analysis of copy basis of the role of TTPAL, co-IP of TTPAL followed by protein number of TTPAL in cancers from TCGA studies demonstrated sequencing identified TRIP6 as an interacting partner of TTPAL. that TTPAL was preferentially and more frequently amplified in The direct interaction between TTPAL and TRIP6 was confirmed colorectal cancers as compared with other cancer types, suggesting by Western blot analysis of co-IP products. TTPAL was colocalized its particular involvement in colorectal cancer. In keeping with our in the cytoplasm with TRIP6 by confocal immunofluorescence finding, TTPAL was expressed in 7 colon cancer cell lines, but assay and Western blot analysis. In addition, the expression of silenced in normal colon tissues. Upregulation of TTPAL was b-catenin and TRIP6 was dramatically increased in TTPAL- detected in primary colorectal tumors as compared with adjacent expressing xenografts tumors as compared with control xeno- normal epithelial tissues. Amplification of TTPAL is a common grafts. These results collectively suggest that TTPAL function via event due to copy number gain in colorectal cancer. activating Wnt signaling and interacting with TRIP6. We further investigated the clinical importance of TTPAL TRIP6 was reported to promote tumor growth in several cancer expression in patients with colorectal cancer at mRNA and protein types and involve in Wnt signaling (18–20). In keeping with this, levels in two independent cohorts, found that 64.8% of them we revealed that expression of TRIP6 significantly enhanced in showed TTPAL overexpression. Both univariate and multivariate colorectal cancer, signifying its oncogenic role in colorectal cancer. Cox regression analyses showed high expression levels of TTPAL We then uncovered that TRIP6 knockdown in colon cancer cells were correlated significantly with shortened overall survival of could significantly blunted the effects of TTPAL on cell prolifer- patients with colorectal cancer. The clinical outcome of colorectal ation, colony formation, migration, and Wnt signaling activation. cancer varies greatly depending on the aggressiveness of individ- Conversely, TRIP6 expression can rescue Wnt activation and ual tumors. Many patients experience disease recurrence follow- cancer-related phenotypes in TTPAL-inhibited SW480 cells, infer- ing radical surgery. Thus, additional prognostic biomarkers may ring that the oncogenic role of TTPAL in colorectal cancer is, at provide better risk assessment that can guide personalized che- least in part, dependent on TRIP6.

3344 Cancer Res; 79(13) July 1, 2019 Cancer Research

Role of TTPAL in Colorectal Cancer

To explore how TTPAL activates Wnt signaling through TRIP6, plays a pivotal oncogenic role by activating Wnt/b-catenin sig- we assessed the interplay between TTPAL and TRIP6. We found naling pathway through binding to and stabilizing TRIP6 in that the direct binding of TTPAL and TRIP6 were existing in colorectal carcinogenesis. Therapeutic intervention of TTPAL cytoplasm. TTPAL–TRIP6 complex may recruit lipid or proteins might be a novel strategy for blunting uncontrolled growth and that inhibited activity of ubiquitin ligases, in turn, suppressed metastasis in Wnt/b-catenin–addicted colorectal cancer. More- ubiquitin-mediated degradation of TRIP6. Previous studies have over, TTPAL may serve as an independent prognostic marker for shown that TRIP6 contains the same PDZ-binding motif as patients with colorectal cancer. b-catenin, and both TRIP6 and b-catenin are physiologic ligands of the PDZ domain–containing tumor suppressor MAGI1, there- fl fore TRIP6 could bind to MAGI1 competitively with b-catenin (14, Disclosure of Potential Con icts of Interest – F.K.L. Chan reports receiving speakers bureau honoraria from Pfizer, Takeda, 21 25). Our results demonstrated that TTPAL expression pro- fl – and AstraZeneca. No potential con icts of interest were disclosed by the other moted TRIP6 MAGI1 interaction, which in turn inhibited the authors. binding between MAGI and b-catenin. Meanwhile, both TRIP6 overexpression and MAGI1 silence dramatically induced Wnt signaling activation. In addition, the impact of these findings Authors' Contributions was further strengthened by the observation that induction of Conception and design: H. Gou, J.Q. Liang, J.-Y. Fang, J. Yu TRIP6 promoted interaction between TCF4 and b-catenin. While Development of methodology: L. Zhang, Y. Zhang, J.-Y. Fang, J. Yu PDZ binding–defective mutant of TRIP6 was incapable of binding Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Li, X. Wang, J.H.M. Tong, K.-F. To, J. Yu to MAGI1 and stimulating cancer-related phenotypes. Moreover, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, TTPAL expression was positively correlated with Wnt target genes computational analysis): J.Q. Liang, X. Wang, K.-F. To in primary colorectal cancer tissues from TCGA cohort. Hence, Writing, review, and/or revision of the manuscript: J.Q. Liang, K.-F. To, these data imply that the direct binding of TTPAL with TRIP6 in J.J.Y. Sung, J. Yu cytoplasm inhibited ubiquitin-mediated degradation of TRIP6. Administrative, technical, or material support (i.e., reporting or organizing Then TRIP6, which contains the same PDZ-binding motif as data, constructing databases): J.Q. Liang, H. Chen, Y. Zhang, J. Ji, J.J.Y. Sung, b – J. Yu -catenin, competitively bound to the PDZ domain containing Study supervision: J.Q. Liang, J. Ji, J.J.Y. Sung, F.K.L. Chan, J. Yu tumor suppressor MAGI1 to release b-catenin. This sequence of events allowed b-catenin to enter the nucleus and ultimately promoted the oncogenic Wnt/b-catenin signaling cascade. Acknowledgments However, enhanced cell proliferation was seen even in the This project was supported by National Key R&D Program of China presence of a Wnt inhibitor. Moreover, TTPAL knockdown (2016YFC1303200, 2017YFE0190700); Science and Technology Program Grant Shenzhen (JCYJ20180307151253271, JCYJ20170413161534162); showed weak effect on inhibiting cell growth in RKO cells, whose Postdoctoral Natural Science Foundation of China (2017M622802); Wnt signaling is not hyperactivated. These results inferred the RGC-GRF Hong Kong (14111216, 14163817); National Natural Science potential existence of Wnt-independent mechanism of cell pro- Foundation of China (NSFC; 81773000); Vice-Chancellor's Discretionary liferation induced by TTPAL in colorectal cancer. In fact, onco- Fund CUHK and CUHK direct grant; Shenzhen Municipal Science and genic signaling pathways including NFkB, Akt, JNK-AP-1, Technology Shenzhen Virtual University Park Support Scheme to CUHK Hippo have been reported mediated by TRIP6 in cancer (20, Shenzhen Research Institute. 26, 27), the direct downstream effector of TTPAL. In this connec- The costs of publication of this article were defrayed in part by the payment of tion, TTPAL-induced cell proliferation should be a complex page charges. This article must therefore be hereby marked advertisement in mechanism in which multiple factors and signaling pathways in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. addition to Wnt signaling are involved. In summary, we demonstrate that TTPAL overexpression due to Received September 27, 2018; revised March 16, 2019; accepted April 19, gene amplification is a common event in colorectal cancer. TTPAL 2019; published first April 24, 2019.

References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer 7. Tanikawa C, Kamatani Y, Takahashi A, Momozawa Y, Leveque K, statistics, 2012. CA Cancer J Clin 2015;65:87–108. Nagayama S, et al. GWAS identifies two novel colorectal cancer loci at 2. Siegel DF, Hilsenroth MJ. Process and technique factors associated with 16q24.1 and 20q13.12. Carcinogenesis 2018;39:652–60. patient ratings of session safety during psychodynamic psychotherapy. 8. Houlston RS, Cheadle J, Dobbins SE, Tenesa A, Jones AM, Howarth K, et al. Am J Psychother 2013;67:257–76. Meta-analysis of three genome-wide association studies identifies suscep- 3. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global tibility loci for colorectal cancer at 1q41, 3q26.2, 12q13.13 and 20q13.33. variation in copy number in the human genome. Nature 2006;444:444–54. Nat Genet 2010;42:973–7. 4. Sokolova V, Crippa E, Gariboldi M. Integration of genome scale data for 9. Xu L, Li X, Cai M, Chen J, Li X, Wu WK, et al. Increased expression of Solute identifying new players in colorectal cancer. World J Gastroenterol 2016; carrier family 12 member 5 via gene amplification contributes to tumour 22:534–45. progression and metastasis and associates with poor survival in colorectal 5.PtashkinRN,PaganC,YaegerR,MiddhaS,ShiaJ,O'RourkeKP,etal. cancer. Gut 2016;65:635–46. Chromosome 20q amplification defines a subtype of microsatellite 10. Wong CC, Qian Y, Li X, Xu J, Kang W, Tong JH, et al. SLC25A22 promotes stable, left-sided colon cancers with wild-type RAS/RAF and better proliferation and survival of colorectal cancer cells with KRAS mutations overall survival. Mol Cancer Res 2017;15:708–13. and xenograft tumor progression in mice via intracellular synthesis of 6. Wang H, Liang L, Fang JY, Xu J. Somatic gene copy number alterations in aspartate. Gastroenterology 2016;151:945–60. colorectal cancer: new quest for cancer drivers and biomarkers. Oncogene 11. Salasova A, Yokota C, Potesil D, Zdrahal Z, Bryja V, Arenas E. A proteomic 2016;35:2011–9. analysis of LRRK2 binding partners reveals interactions with multiple

www.aacrjournals.org Cancer Res; 79(13) July 1, 2019 3345

Gou et al.

signaling components of the WNT/PCP pathway. Mol Neurodegener 2017; 20. Lin VT, Lin FT. TRIP6: an adaptor protein that regulates cell motility, 12:54. antiapoptotic signaling and transcriptional activity. Cell Signal 2011;23: 12. Wang K, Liang Q, Li X, Tsoi H, Zhang J, Wang H, et al. MDGA2 is a novel 1691–7. tumour suppressor cooperating with DMAP1 in gastric cancer and is 21. Yi J, Beckerle MC. The human TRIP6 gene encodes a LIM domain protein associated with disease outcome. Gut 2016;65:1619–31. and maps to chromosome 7q22, a region associated with tumorigenesis. 13. Budczies J, Klauschen F, Sinn BV, Gyorffy B, Schmitt WD, Darb-Esfahani S, Genomics 1998;49:314–6. et al. Cutoff Finder: a comprehensive and straightforward Web application 22. Zaric J, Joseph JM, Tercier S, Sengstag T, Ponsonnet L, Delorenzi M, et al. enabling rapid biomarker cutoff optimization. PLoS ONE 2012;7:e51862. Identiﬁcation of MAGI1 as a tumor-suppressor protein induced by cyclo- 14. Chastre E, Abdessamad M, Kruglov A, Bruyneel E, Bracke M, Di Gioia Y, oxygenase-2 inhibitors in colorectal cancer cells. Oncogene 2012;31: et al. TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, 48–59. promotes invasiveness. FASEB J 2009;23:916–28. 23. Jia S, Lu J, Qu T, Feng Y, Wang X, Liu C, et al. MAGI1 inhibits migration and 15. Rooney PH, Murray GI, Stevenson DA, Haites NE, Cassidy J, McLeod HL. invasion via blocking MAPK/ERK signaling pathway in gastric cancer. Comparative genomic hybridization and chromosomal instability in solid Chin J Cancer Res 2017;29:25–35. tumours. Br J Cancer 1999;80:862–73. 24. Gujral TS, Karp ES, Chan M, Chang BH, MacBeath G. Family-wide inves- 16. Weiss MM, Snijders AM, Kuipers EJ, Ylstra B, Pinkel D, Meuwissen SG, et al. tigation of PDZ domain-mediated protein-protein interactions implicates Determination of amplicon boundaries at 20q13.2 in tissue samples of beta-catenin in maintaining the integrity of tight junctions. Chem Biol human gastric adenocarcinomas by high-resolution microarray compar- 2013;20:816–27. ative genomic hybridization. J Pathol 2003;200:320–6. 25. Dobrosotskaya IY, James GL. MAGI-1 interacts with beta-catenin and is 17. Blain SW. Switching cyclin D-Cdk4 kinase activity on and off. Cell Cycle associated with cell-cell adhesion structures. Biochem Biophys Res Com- 2008;7:892–8. mun 2000;270:903–9. 18. Gur'ianova OA, Sablina AA, Chumakov PM, Frolova EI. Down-regulation 26. Dutta S, Mana-Capelli S, Paramasivam M, Dasgupta I, Cirka H, Billiar K, of TRIP6 expression induces actin cytoskeleton rearrangements in human et al. TRIP6 inhibits Hippo signaling in response to tension at adherens carcinoma cell lines. Mol Biol 2005;39:905–9. junctions. EMBO Rep 2018;19:337–50. 19. Zhao W, Dai Y, Dai T, Xie T, Su X, Li J, et al. TRIP6 promotes cell 27. Li L, Bin LH, Li F, Liu Y, Chen D, Zhai Z, et al. TRIP6 is a RIP2-associated proliferation in hepatocellular carcinoma via suppression of FOXO3a. common signaling component of multiple NF-kappaB activation path- Biochem Biophys Res Commun 2017;494:594–601. ways. J Cell Sci 2005;118:555–63.

3346 Cancer Res; 79(13) July 1, 2019 Cancer Research

TTPAL Promotes Colorectal Tumorigenesis by Stabilizing TRIP6 to Activate Wnt/ β-Catenin Signaling

Hongyan Gou, Jessie Qiaoyi Liang, Lijing Zhang, et al.

Cancer Res 2019;79:3332-3346. Published OnlineFirst April 24, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-2986

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