TTPAL Promotes Colorectal Tumorigenesis by Stabilizing ...Molecular Cell Biology TTPAL Promotes...

Molecular Cell Biology

TTPAL Promotes Colorectal Tumorigenesis byStabilizing TRIP6 to Activate Wnt/b-CateninSignalingHongyan 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 ofcolorectal cancer. Our whole-genome analysis identifiedtocopherol alpha transfer protein-like (TTPAL) as preferentiallyamplified in colorectal cancer. Here we demonstrate that fre-quent copy number gain of TTPAL leads to gene overexpressionin colorectal cancer fromaChinese cohort (n¼102),whichwasfurther validated by a TheCancerGenomeAtlas (TCGA) cohort(n ¼ 376). High expression of TTPAL was significantly associ-ated with shortened survival in patients with colorectal cancer.TTPAL promoted cell viability and clonogenicity, acceleratedcell-cycle progression, inhibited cell apoptosis, increased cellmigration/invasion ability in vitro, and promoted tumorigenic-ity and cancer metastasis in vivo. TTPAL significantly activatedWnt signaling and increased b-catenin activation and proteinexpression of cyclin D1 and c-Myc. Coimmunoprecipitationfollowed by mass spectrometry identified thyroid receptor–interacting protein 6 (TRIP6) as a direct downstream effector of

TTPAL.Depletion of TRIP6 significantly abolished the effects ofTTPAL on cell proliferation andWnt activation. Direct bindingof TTPAL with TRIP6 in the cytoplasm inhibited ubiquitin-mediated degradation of TRIP6 and, subsequently, increasedlevels of TRIP6 displaced b-catenin from the tumor suppressorMAGI1 via competitive binding. This sequence of events allowsb-catenin to enter the nucleus and promotes oncogenic Wnt/b-catenin signaling. In conclusion, TTPAL is commonly over-expressed in colorectal cancer due to copy number gain, whichpromotes colorectal tumorigenesis by activatingWnt/b-cateninsignaling via stabilization of TRIP6. TTPAL overexpressionmayserve as an independent new biomarker for the prognosis ofpatients with colorectal cancer.

Significance: TTPAL, a gene preferentially amplified incolorectal cancer, promotes colon tumorigenesis via activationof the Wnt/b-catenin pathway.

IntroductionColorectal cancer is the third most common cancer and the

second leading cause of cancer-related death worldwide (1, 2).The pathogenic mechanisms underlying colorectal cancer devel-opment appear to be complex and heterogeneous. Copy number

alterations (CNA) are common somatic changes in cancer fea-tured with gain or loss in copies of DNA sections (3). Deletionsand copy number gains contribute to alterations in the expressionof tumor suppressor genes and oncogenes, respectively. Thestepwise accumulation of CNAs confers growth advantage andmetastatic competence on cells, thus playing a crucial role incancer initiation and progression. Recent studies have suggestedthat geneswith CNAs are potential biomarkers and/or therapeutictargets for colorectal cancer. Therefore, detection and mapping ofcopy number abnormalities provide an approach for associatingaberrations with disease phenotype and for localizing criticalgenes (4, 5). It is importance to identify and functionally char-acterize novel genes with CNAs that are associated with colorectalcancer (4).

Chromosome 20q amplification has been commonly found incolorectal cancer and is involved in transforming adenoma tocarcinoma (5, 6). Chromosome 20q amplification is also apotential indicator of poor prognosis in patients with colorectalcancer (7–9). By whole-genome sequencing (WGS), we identifiedthat TTPAL (tocopherol alpha transfer protein-like), located at20q13.12, was preferentially amplified in primary colorectaltumor tissues. In keeping with our finding, analyses from TheCancer Genome Atlas (TCGA) dataset showed that copy numbergain of TTPALoccurs frequently in colorectal cancer andpositivelycorrelates with its upregulated mRNA expression. Searching forthe public protein datasets of Human Protein Altas, we found thathigher TTPAL protein expression was shown in colorectal cancer

1Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenter-ology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenesand Related Genes, Shanghai Institute of Digestive Disease, Renji Hospital,School of Medicine, Shanghai Jiaotong University, Shanghai, China. 2Instituteof Digestive Disease and Department of Medicine and Therapeutics, State KeyLaboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHKShenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong.3Department of Surgery, PekingUniversity CancerHospital and Institute, Beijing,China. 4Department of Anatomical and Cellular Pathology, State Key Laboratoryof Oncology in South China, The Chinese University of Hong Kong, Hong Kong.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

H. Gou and J.Q. Liang are the co-first authors of this article.

Corresponding Author: Jun Yu, Institute of Digestive Disease and Departmentof Medicine and Therapeutics, Prince of Wales Hospital, The Chinese Universityof Hong Kong, Hong Kong. Phone: 852-3763-6099; Fax: 852-2144-5330; E-mail:[email protected]

Cancer Res 2019;79:3332–46

doi: 10.1158/0008-5472.CAN-18-2986

�2019 American Association for Cancer Research.

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tumor tissues compared with normal colon tissues from thedataset(http://www.proteinatlas.org). Moreover, high TTPAL mRNAexpression is significantly associated with poor survival inpatients with multiple cancer types (https://www.proteinatlas.org/ENSG00000124120-TTPAL/pathology#top; P < 0.001).Therefore, we hypothesize that TTPAL plays an oncogenic func-tion in colon carcinogenesis.

The role of TTPAL in human cancer remains uninvestigated. Inthis study, we identified the frequent overexpression of TTPAL incolorectal cancer due to its copy number gain. Further functionalstudies revealed that ectopic expression of TTPAL significantlypromoted colorectal cancer growth by enhancing G1–S cell-cycleprogression and reducing apoptosis. The tumor-promoting effectof TTPALwas revealed to be associatedwith the activation ofWnt/b-catenin signaling pathway by binding to thyroid receptor–interacting protein 6 (TRIP6). The direct binding of TTPAL withTRIP6 in cytoplasm inhibited TRIP6 ubiquitin degradation.Enhanced cytoplasm TRIP6 displaced b-catenin from the tumorsuppressor MAGI1 via competitive binding, which allowedb-catenin to enter into the nucleus and subsequently activatedthe oncogenic Wnt/b-catenin signaling. Moreover, TTPAL over-expression was significantly associated with poor survival ofpatients with colorectal cancer. Thus, this study revealed a noveloncogenicmechanism and the clinical application value of TTPALin colorectal cancer.

Materials and MethodsColon cancer cell lines

The colon cancer cell lines (DLD-1, HCT116, HT29, LOVO,SW480, RKO, and SW1116) were obtained from the ATCCbetween 2014 and 2015 and cells' authentication were confirmedby short tandem repeat profiling. Cells were routinely culturedand maintained in DMEM supplemented with 10% FBS andantibiotics (Gibco BRL) according to the ATCC protocols, exceptHCT116 cells, which were cultured in complete McCoy 5A medi-um (Gibco BRL). Routine Mycoplasma testing was performed byPCR. Cells were grown for no more than 25 passages in total forany experiment.

Subjects and sample collectionPaired primary colorectal tumors and adjacent nontumor

tissues were collected immediately after surgical resection at thePrince of Wales Hospital (Hong Kong, China; 182 primarytumors and 18 nontumor tissues) and Peking University CancerHospital and Institute (Beijing, China; 102 primary tumors 50nontumor tissues). The specimens were snap-frozen in liquidnitrogen and stored at 80�C and were also fixed in 10%formalin and embedded in paraffin for routine histologicexamination. Biopsies from 3 cases of normal mucosa obtainedduring colonoscopy were recruited as healthy controls, whichwere confirmed by an experienced pathologist at the Prince ofWales Hospital and Peking University Cancer Hospital. Thestudy protocol was approved by the Clinical Research EthicsCommittee of The Chinese University of Hong Kong and theClinical Research Ethics Committee of Peking University Can-cer Hospital. All patients provided written informed consent forobtaining the study specimens. This study was carried out inaccordance with the Declaration of Helsinki of the WorldMedical Association.

PCR copy number analysisDNA was extracted from 102 frozen colorectal cancer samples

of Chinese Cohort using the AllPrep DNA/RNA/Protein Kit(Qiagen). The purified genomic DNA was amplified with a probespecific to target gene TTPAL (Mm00082375_cn) by TaqManCopy Number Assays (Applied Biosystems). The results wereanalyzed by Copy Caller software v2.0 (Applied Biosystems).

Subcutaneous xenograft and experimental metastasis mousemodels

HCT116andDLD1cells stably transfectedwithTTPALexpressionvector or empty vector (5 � 106 cells/0.1 mL PBS) were injectedsubcutaneously into the left and right dorsal flanks of 4- to 6-week-old femaleBalb/c nudemice (n¼ 8/group), respectively. Tumor sizewas measured every 2 days for 2–3 weeks using a digital caliper.Tumor volume (mm3) was estimated bymeasuring the longest andshortest diameters of the tumor and calculating as described previ-ously (10). At the endpoint, tumors were harvested and weighted.The excised tissues were either fixed in 10% neutral-buffered for-malin or snap frozen in liquid nitrogen. Tumor sections fromparaffin-embedded blocks were used for histologic examination.

For metastasis model, HCT116 cells stably transfected withTTPAL expression vector or empty vector (2 � 106 cells in 0.1 mLPBS) were injected intravenously via the tail vein (n ¼ 5).After 6–8 weeks, mice were sacrificed and their lungs were har-vested. The lungs were sectioned and stained with hematoxylinand eosin. The number of lung metastases were counted. Allexperimental procedures were approved by the Animal EthicsCommittee of the Chinese University of Hong Kong.

Coimmunoprecipitation and liquid chromatography–massspectrometry

Coimmunoprecipitation (co-IP) assays were carried out asdescribed previously (11, 12). Briefly, total protein fromHCT116cells (�5 � 106/reaction) stably transfected with TTPAL (Flag-tagged) expression vector or empty vector was extracted in radio-immunoprecipitation assay (RIPA) buffer supplemented withproteinase inhibitor (Novagen). Immunoprecipitation was per-formed using anti-FlagM2 antibody (A2220, Sigma-Aldrich). Theimmune complexes were precipitated by PureProteome ProteinA/GMix magnetic beads (LSKMAGAG02, Millipore) overnight at4�C. Beads with extracted proteins were washed three times by50 mmol/L ammonium bicarbonate buffer and subjected todigestion by trypsin at 37�C for 2 hours (Promega). Trypticpeptides were then extracted for liquid chromatography–massspectrometry (LC/MS) analysis.

Co-IP and Western blot analysesWhole lysate (150 mg protein) and co-IP precipitant by anti-

Flag-tag, anti-thyroid receptor–interacting protein 6 (TRIP6)antibody (ab70747, Abcam), anti-HA-tag (ab18181, Abcam),or IgG were immunoblotted with either anti-TRIP6, anti-HA, oranti-Flag (A2220, Sigma-Aldrich) antibody to confirm the inter-action between TTPAL and TRIP6 that was identified by LC/MS.The lysate (1% input, 10 mg protein) was also used as a control.The antibodies used are listed in Supplementary Table S1.

Ubiquitination assayHCT116 and DLD1 cells stably transfected with TTPAL expres-

sion vector or empty vector were cotransfectedwithUbiquitin-HAfor 24 hours. Then the cells were incubated in the presence or

Role of TTPAL in Colorectal Cancer

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absence of 10 mmol/L MG132 (M-7449, Sigma-Aldrich) for 12hours. Cells were lysed with RIPA buffer supplemented withprotease inhibitors. Immunoprecipitation was performed usinganti-TRIP6 or IgG, respectively. Immunoprecipitated proteinswere analyzed byWestern blot using anti-HA (ab18181, Abcam).

Statistical analysisThe results were expressed as mean � SD. Statistical analysis

was performed using the Statistical Package for the Social Sciences

(SPSS; standard V.16.0; IBM Corporation). The Pearson correla-tion coefficient was used to evaluate the correlation betweenTTPAL gene amplification and expression in the clinical samples.The x2 test was used for comparison of patient characteristics anddistributions of expression and covariates by vital status. ROCcurve was used to estimate the cut-off value of the methylationpercentage. Cut-off value was analyzed by survival significanceanalysis using the tool Cutoff Finder (http://molpath.charite.de/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 ofTTPAL is preferentially amplified in colon and rectum cancers as compared with other cancer types. Data were from TCGA studies, with sample sizes indicatedunder each caner type. COAD, colon adenocarcinoma; READ, rectum adenocarcinoma; BRCA, breast invasive carcinoma; HNSC, head and neck squamous cellcarcinoma; 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, glioblastomamultiforme; 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, TTPALwas expressed in colon cancer cell lines, but not in normal colon tissues, as determined by RT-PCR andWestern blot analysis. D, TTPAL mRNA expression wasupregulated in colorectal cancer compared with paired adjacent normal tissues as shown by RT-PCR (top) and qRT-PCR (bottom left, Chinese cohorts 1 and 2from Beijing and Hong Kong, respectively). RNA-seq data from TCGA study also show upregulation of TTPAL in colorectal cancer as compared with adjacentnormal tissues (bottom right, paired and unpaired samples). E, TTPAL protein expression was upregulated in primary colorectal cancer as compared withadjacent normal tissues byWestern blot analysis. F, TTPAL protein expression was significantly higher in primary colorectal cancer as compared with adjacentnormal tissues as shown by IHC staining. G, Kaplan–Meier survival analysis showed patients with colorectal cancer with high TTPAL expression had poorersurvival than those with low TTPAL expression at both mRNA and protein levels from Chinese population. H, Prognostic value of TTPAL expression was validatedin patients with colorectal cancer from TCGA study.

Table 1. Univariate and multivariate Cox regression analyses of potential poor prognostic factors in colorectal cancer patients

Chinese cohort - mRNA levelUnivariate 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) 0.110Gender 0.696 0.703M 1.132 (0.608–2.107) 1.128 (0.606–2.101)F 1 1

Location 0.904Colon 0.964 (0.36–1.736)Rectum 1

Differentiation 0.586Moderate or high 1.216 (0.602–2.457)Low 1

TNM 0.067II 1.789 (0.961–3.330)III 1

TTPAL expression 0.028 0.034High 2.202(1.090–4.447) 2.146 (1.061–4.340)Low 1 1

Chinese cohort - protein levelUnivariate Multivariate

Variable RR (95% CI) P RR (95% CI) PAge 1.004 (0.987–1.021) 0.637 1.008 (0.992–1.025) 0.346Gender 0793 0.634M 1.949 (0.641–1.404) 1.103 (0.736–1.655)F 1 1

Location 0.279Colon 0.805 (0.544–1.192)Rectum 1

Differentiation 0.142Moderate or high 1.430 (0.887–2.306)Low 1

TNMEarly (I & II) 0.227 (0.137–0.374) <0.0001 0.202 (0.122–0.355) <0.0001Late (III & IV) 1 1

TTPAL expression <0.0001 <0.0001High 1.954 (1.258–3.036) 2.441 (1.544–3.858)Low 1 1





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TTPAL expression were estimated by univariate Cox proportionalhazards regression model first. Multivariate Cox model was thenconstructed to estimate the adjusted RR for TTPAL expression.Overall survival in relation to expression was evaluated bythe Kaplan–Meier survival curve and the log-rank test. Mann–Whitney U test or Student t test was performed to compare thevariables of two groups. mRNA expression z-scores (RNA Seq V2RSEM) from 382 colorectal cancer samples were downloadedfromTCGA via cBioPortal. Nonparametric Spearman correlationsin expression levels between TTPAL and Wnt target genes werethen computed. The difference in cell viability and tumor growthrate between two groups of nude mice was determined by repeat-ed-measures ANOVA. P values <0.05 were taken as statisticalsignificance.

ResultsTTPAL is frequently overexpressed in colorectal cancer due tocopy number gain

By WGS analysis to compare genetic alterations in primarycolorectal cancer tissues with corresponding peripheral bloodsamples, we identified TTPAL to be preferentially amplified incolorectal cancer tumor tissues by WGS (Supplementary Fig.S1A). TTPAL was also frequently amplified in colon and rectumcancers from TCGA cohort but less amplified in other cancers(Fig. 1A), demonstrating that copy number gain of TTPAL maybe specifically associated with colorectal tumorigenesis. Wefurther quantitated the copy number and mRNA expression ofTTPAL in colorectal cancer samples by TaqMan copy numberassay and qRT-PCR, respectively. In 102 patients with colorectalcancer, 48% (49/102) of primary colorectal cancer tissuesexhibited TTPAL copy number gain, which was positivelycorrelated with its mRNA expression (R2 ¼ 0.3537, P <0.0001; Fig. 1B). This finding was further validated in colorectalcancer samples from TCGA cohort, with copy number gaindetected in 70.2% of samples (�1.2 fold, 436/621) and mRNAexpression also positively correlated with copy number (n ¼376, R2 ¼ 0.5632, P < 0.0001; Fig. 1B). These results demon-strate that TTPAL is commonly overexpressed in colorectalcancer due to copy number gain.

TTPAL expression is upregulated in colon cancer cells andprimary colorectal cancer tumors

We then examined TTPAL expression in colon cancer cells andprimary colorectal cancer tissues. TTPAL expression at bothmRNAand protein levels was detected in all 7 colon cancer cells tested(DLD1, LOVO, RKO, HT29, HCT116, SW480, and SW1116), butnot in normal colon tissues (Fig. 1C). TTPAL mRNA expressionwas significantly upregulated in primary colorectal cancer tumorsas compared with their adjacent normal tissues as determined by

qRT-PCR (Fig. 1D). Specifically, the upregulation of TTPALmRNAin colorectal cancer was validated in three independent cohorts ofpaired tumor and adjacent normal samples including BejingChinese cohort (n ¼ 50; P < 0.0001), Hong Kong Chinese cohort(n ¼ 18; P < 0.0001), and the TCGA cohort (n ¼ 23; P <0.0001; Fig. 1D). Significant upregulation was also observed incolorectal cancer (n ¼ 286) compared with unpaired adjacentnormal tissues (n ¼ 39) from TCGA study (P < 0.0001; Fig. 1D).Western blot and IHC staining further confirmed the upregulationof TTPAL at the protein level in colorectal cancers as comparedwith adjacent normal tissues (Fig. 1E and F).

Overexpression of TTPAL is an independent predictor of poorsurvival in patients with colorectal cancer

We further evaluated the clinicopathologic and prognosticsignificance of TTPAL expression in patients with colorectal can-cer. We evaluated TTPAL mRNA expression in primary colorectalcancer tissues from 102 Chinese patients. By classifying samplesinto low and high expression groups using cut-off finder, TTPALoverexpression was detected in 64.7% (66/102) of primary colo-rectal cancers. No correlation was found between TTPAL expres-sion and clinicopathologic features such as age, gender, location,differentiation, and Tumor–Node–Metastasis (TNM) stage(Supplementary Table S2). However, TTPAL overexpression wasassociated with an increased risk of cancer-related death byunivariate Cox regression (RR ¼ 2.202, 95% CI: 1.090–4.447,P ¼ 0.028; Table 1). In particular, after adjustment for potentialconfounding factors, TTPAL overexpression was found to be anindependent risk factor for shortened survival in patients withcolorectal cancer by multivariate Cox regression analysis (RR ¼2.146, 95% CI: 1.061–4.340, P ¼ 0.034; Table 1). Kaplan–Meiersurvival curves showed that patients with colorectal cancer withTTPAL overexpression had significantly poorer overall survivalbased on the log-rank test (P < 0.05; Fig. 1G).

TTPAL protein expression was further evaluated in primarycolorectal cancer tissues from another cohort of Chinese patients(n ¼ 182) by IHC using tissue microarrays. No correlation wasfound between TTPAL protein expression and clinicopathologicfeatures such as age, gender, location, differentiation, and TNMstage (Supplementary Table S2). Further univariate and multi-variateCox regression analyses showed that TTPALprotein expres-sion was also an independent predictor of poor survival inpatients with colorectal cancer (multivariate RR ¼ 2.441; 95%CI: 1.544–3.858, P < 0.0001) besides TNM stage (Table 1). Asshown in the Kaplan–Meier survival curves, colorectal cancerpatients with high TTPAL protein expression had significantlyshorter survival than those with low expression (P ¼ 0.002, log-rank test; Fig. 1G).Moreover, the prognostic significance of TTPALexpression was well validated in TCGA cohort (Fig. 1H). Thesefindings 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 andWestern blot analysis, significantlyincreased cell viability and colony formation ability. B, Knockdown of TTPAL by siTTPAL in SW480 and SW1116, confirmed by RT-PCR andWestern blot analysis,significantly inhibited cell viability and colony formation ability. C,Overexpression of TTPAL significantly increased cells in S-phase, while knockdown of TTPALshowed the opposite effect. D,Western blot analysis showed TTPAL enhanced protein levels of cyclin-D1, CDK4, and PCNA, while knockdown of TTPAL had theopposite effect. E,Overexpression of TTPAL inhibited cell apoptosis by flow cytometry after Annexin V/7-AAD dual staining, while knockdown of TTPALpromoted cell apoptosis. F,Western blot analysis showed TTPAL expression reduced activation of caspases-8, -9, -7, -3, and PARP, while knockdown of TTPALexhibited the opposite effect. G,Overexpression of TTPAL promoted cell migration as shown by wound-healing assays. H,Overexpression of TTPAL significantlyincreased cell invasion ability as shown by Matrigel invasion assay. I, Knockdown of TTPAL significantly inhibitedmigration ability. J, Knockdown of TTPALsignificantly 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.






independent new biomarker for the prognosis of patients withcolorectal cancer.

TTPAL expression promotes colon cancer cell growthThe frequent overexpression of TTPAL in colorectal cancer

prompted us to investigate its potential oncogenic role in colo-rectal cancer. To this end, we performed in vitro gain- and loss-of-function assays on TTPAL. Ectopic expression of TTPAL inHCT116 and DLD1 cells (Fig. 2A) significantly increased cellviability and clonogenicity (Fig. 2A). On the other hand, TTPALknockdown by RNA interference in SW480 and SW1116 cellsmarkedly inhibited cell viability and clonogenicity (Fig. 2B).These results indicated that TTPAL plays an oncogenic role incolon cancer.

TTPAL promotes cell-cycle progression and inhibits cellapoptosis

We analyzed the cytokinetic effect of TTPAL. Overexpression ofTTPAL led to a significant decrease in G1 phase cells and aconcomitantly increase in S-phase cells in both HCT116 andDLD1 cells by BrdU staining (all P < 0.01; Fig. 2C). The promotingeffect of TTPAL on cell-cycle progression was confirmed by theincreased protein expression of two master G1–S checkpointregulators (cyclin D1 and CDK4) and the proliferation markerproliferating cell nuclear antigen (PCNA; Fig. 2D). On the otherhand, knockdown of TTPAL significantly increased cells in the G1

phase and decreased cells in S-phase of both SW480 and SW1116cells (all P < 0.01; Fig. 2C), and concomitantly reduced theexpression of cyclin D1, CDK4, and PCNA (Fig. 2D). Apoptosisanalysis showed that overexpression of TTPAL led to a significantreduction in apoptotic cell population in HCT116 (P < 0.01) andDLD1 (P < 0.001) cells (Fig. 2E, 1), while knockdown of TTPALsignificantly increased apoptosis of SW480 and SW1116 cells(both P < 0.001; Fig. 2E, 2). TTPAL-induced reduction of apo-ptosis was confirmed by the elevated expression of key apoptosismarkers (cleaved forms of caspase-8, caspase-9, caspase-7, cas-pase-3, and PARP), while knockdown of TTPAL showed oppositeeffects (Fig. 2F). These results demonstrate the role of TTPAL onpromoting cell growth by regulating cell-cycle progression andapoptosis in colon cancer cells.

TTPAL promotes cell migration and invasionTo determine the effects of TTPAL on cell migration and

invasion, wound-healing and Matrigel invasion assays were per-formed. Ectopic expression of TTPAL significantly increased themigration abilities in HCT116 and DLD1 cells (both P <0.01; Fig. 2G), and significantly promoted invasiveness of bothcells (both P < 0.001; Fig. 2H), while knockdown of TTPALmarkedly suppressed migration and invasion abilities in SW480and SW1116 cells (Fig. 2I). In keeping with these, protein expres-sion of epithelial-to-mesenchymal transition (EMT) markers wasregulated by TTPAL. TTPAL overexpression upregulated the mes-enchymal marker vimentin and downregulated the epithelialmarkers (E-cadherin and claudin-1), while knockdown of TTPALshowed the opposite effects (Fig. 2K). These findings demonstrat-ed that TTPAL also functioned in regulating cell migration andinvasion of colorectal cancer cells.

TTPAL activates Wnt/b-catenin signalingWe performed luciferase reporter assays to assess the effect of

TTPAL on important cancer pathways including p53, activator

protein-1 (AP-1), MAPK/ERK (SRE), Wnt/b-catenin, Akt (FHRE),andNFkB. Ectopic expression of TTPAL significantly increased theactivity of Wnt signaling as demonstrated by TOPflash/FOPflashluciferase reporter assay, and knockdown of TTPAL showed theopposite effect onWnt activity (Fig. 3A; Supplementary Fig. S2A).Consistently, overexpression of TTPAL significantly upregulatedthe protein levels of cyclin D1, c-Myc, b-catenin, and activeb-catenin, while TTPAL knockdown diminished expression ofthese key Wnt signaling genes (Fig. 3B). Moreover, Wnt signalinginhibitor IWP-2 significantly inhibited cell growth in DLD1 andHCT116 cells stably transfected with TTPAL as compared withDMSO treatment. IWP-2 also eliminated the growth-promotingeffect exerted by TTPALoverexpression (P>0.05 for IWP-2-treatedTTPAL-overexpressed cells vs. DMSO-treated vector-transfectedcells; Fig. 3C). TTPAL knockdown showed weaker effect onproliferation in RKO cells, which do not harbor mutation in APCor CTNNB1 gene (Supplementary Fig. S2B). These results dem-onstrate that the oncogenic role of TTPAL is largely mediated byactivation of Wnt/b-catenin signaling.

TRIP6 directly interacts with TTPALTo identify the direct interacting partners of TTPAL in colorectal

cancer, we performed co-IP followed by LC/MS. TTPAL bindingcandidateswere then identifiedby comparing the anti-TTPAL-FlagIP products of TTPAL (Flag)-overexpressed cells with those ofcontrol cells (Fig. 3D). Among the identified candidates with �3mapping peptides, 7 genes have been reportedwith functions thatmay be associated with cancer development (SupplementaryTable S3). Among them, TRIP6 is of interest due to its involvementin Wnt signaling pathway (14). To validate the interactionbetween TTPAL and TRIP6, Flag-TTPAL andHA-TRIP6 (or TRIP6)were co-expressed in HCT116 and DLD1 cells, and total proteinswere immunoprecipitated with anti-Flag or anti-HA/anti-TRIP6antibodies. Western blot results showed that TTPAL and TRIP6could be coprecipitated by each other in both cells. Moreover, theinteraction of endogenous TTPAL and TRIP6 in SW480 andSW1116 cells was confirmed by immunoprecipitation–Westernblot analyses (Fig. 3E), indicating the direct interaction betweenTTPAL and TRIP6. We next examined the expression and intra-cellular distributionof TTPAL andTRIP6by immunofluorescence.Confocalmicroscopy images showed that TTPAL colocalizedwithTRIP6 in the cytoplasm of HCT116 and DLD1 cells followingtransfection of TTPAL and HA-TRIP6 (Fig. 3F, 1). The localizationof the two proteins was further confirmed by Western blotting ofmembrane, cytoplasmic, and nuclear fractions, which showedthat TTPAL and TRIP6 was mainly localized in the cytoplasm ofHCT116 and DLD1 cells (Fig. 3F, 2).

TTPAL promotes tumorigenicity and metastasis by regulatingTRIP6 and Wnt/b-catenin signaling in vivo

In light of our in vitro findings, we tested the effect of TTPALin vivo. Results showed that stable transfection of TTPAL expres-sion vector inHCT116 andDLD1 cells significantly promoted thegrowth of tumor volume over the entire assay period andincreased tumor weight at the end point in subcutaneous xeno-graft models (Fig. 4A). Overexpression of TTPAL was confirmedby IHC staining in DLD1 and HCT116 xenografts (Fig. 4B). Cellproliferation was significantly promoted as determined by Ki-67staining, and apoptosis was significantly reduced as evidenced bythe TUNEL staining, in both TTPAL-overexpressed xenografts ascompared with controls (Fig. 4B). Moreover, the expression of

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Figure 3.

TTPAL activatesWnt 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 suppressedWnt activity in SW480 cells. B,Western blot analysis results showed that TTPALexpression enhanced the expression ofWnt signaling–related markers, while knockdown of TTPAL showed the opposite effect. C,Wnt signaling inhibitor IWP-2treatment abolished the promoting effects of TTPAL on cell proliferation and clonogenicity in HCT116 and DLD1 cells. D, Co-IP followed by LC/MS identifiedTRIP6 to be a TTPAL-binding protein. E, Co-IP followed byWestern 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) andWestern 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.






Figure 4.

TTPAL enhances tumorigenicity and metastasis in nude mice. A, HCT116 cells stably expressing TTPAL expression promoted subcutaneous tumor growth ascompared 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 weightat the end point. B, IHC staining confirmed TTPAL overexpression in HCT116 and DLD1 subcutaneous xenografts, which enhanced cell proliferation (by Ki-67staining) 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) stainingof lung tissues from nudemice injected with TTPAL- or control vector–transfected HCT116 cells. Quantitative analysis showed that TTPAL expression significantlyincreased the number of metastatic lesions. E, Knockdown of TTPAL by stably expressing shTTPAL inhibited subcutaneous tumor growth as compared withshNC 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 TTPALoverexpression was confirmed by RT-PCR andWestern blot analysis. B, Knockdown of TRIP6 significantly abolished the promoting effect of TTPAL on coloncancer cell growth. C, Knockdown of TRIP6 significantly abolished the promoting effect of TTPAL on clonogenicity of colon cancer cells. D, Knockdown of TRIP6significantly decreased migration ability of colon cancer cells that was promoted by TTPAL. E, Protein expression of key factors inWnt signaling pathway inTTPAL-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), andWnt activation (J).






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b-catenin and TRIP6 was dramatically increased in TTPAL-over-expressed HCT116 and DLD1 xenografts by IHC, validating themolecular basis identified in vitro (Fig. 4B). TTPAL overexpressionpromoted lung metastasis in nude mice (P < 0.01; Fig. 4D).Furthermore, stable knockdown of TTPAL in SW480 cells signif-icantly inhibited tumorigenicity (Fig. 4E) and metastasis in nudemice (Fig. 4F). Collectively, these results in immunodeficientnude mice suggest that TTPAL promoted colorectal tumorigenic-ity and may also facilitate metastasis.

The oncogenic role of TTPAL is dependent on TRIP6We then examined the importance of TRIP6 in TTPAL-mediated

oncogenic function in colorectal cancer. TRIP6 mRNA expressionwas significantly increased in colorectal cancer tumor tissues ascompared with adjacent normal tissues in TCGA cohort (P <0.001, Supplementary Fig. S3A). TRIP6 mRNA was readilyexpressed in all 7 colon cancer cells tested, but not in normalcolon tissues (Supplementary Fig. S3B), TRIP6 expression pro-moted cell proliferation and clonogenicity in DLD1 and HT-29cells, whereas TRIP6 silence showed significant suppressive effecton cell proliferation and clonogenicity in SW480, SW1116, andHCT116, suggesting the oncogenic role of TRIP6 in colorectalcancer (Supplementary Fig. S4). To investigate the effect of TRIP6on the TTPAL-mediated cell proliferation andmetastasis, HCT116and DLD1 cells stably transfected with TTPAL or control vectorswere cotransfected with siRNA against TRIP6 (Fig. 5A). TRIP6knockdown significantly abolished the promoting effect of TTPALon cell viability (Fig. 5B), clonogenicity (Fig. 5C), and migrationability of both HCT116 and DLD1 cells (Fig. 5D). We nextexamined whether TTPAL activated Wnt signaling pathwaythrough mediating TRIP6. Indeed, TRIP6 knockdown abolishedthe TTPAL-induced protein expressionofWnt effectors (cyclinD1,c-Myc, b-catenin, and active b-catenin) by Western blot analysis(Fig. 5E). TRIP6 knockdown also blunted the TTPAL-activatedWnt signaling as evidenced by luciferase reporter assay inHCT116and DLD1 cells (Fig. 5F). These results suggested that the onco-genic role of TTPAL and its effect on activatingWnt signaling weredependent on TRIP6. Consistently, TRIP6 overexpression inTTPAL-inhibited SW480 cells can rescue cell proliferation(Fig. 5G), clonogenicity (Fig. 5G), migration (Fig. 5I), and Wntactivation (Fig. 5J).

TTPAL inhibits ubiquitin-mediated degradation of TRIP6protein

To gain insights into how TRIP6mediates the role of TTPAL, wefurther evaluated the effect of TTPAL on TRIP6. We observed thatTRIP6 protein expression was upregulated by ectopic expression

of TTPAL in HCT116 and DLD1 cells, and decreased after knock-down of the endogenous TTPAL in SW1116 cells (Fig. 6A, 1).TRIP6 protein was also upregulated by TTPAL in xenografts ofnude mice (Fig. 4C and D). However, mRNA level of TRIP6was not changed by overexpression or knockdown of TTPAL(Fig. 6A, 1). Moreover, the protein levels of TRIP6 in variouscolorectal cancer cell lines were positively correlated with theprotein levels of TTPAL in these cell lines (Fig. 6A, 2), indicatingthat TTPAL may stabilize TRIP6 in colorectal cancer. We there-fore assessed whether TTPAL regulated the stability of TRIP6protein. We treated TTPAL or control vector–transfected cellswith the protein synthesis inhibitor cycloheximide. As shownin Fig. 6B, 1, TRIP6 was more stable in the presence of TTPALin HCT116 and DLD1 cells. To investigate whether TTPALincreased the stability of TRIP6 by affecting its ubiquitination/degradation, we treated cells with the proteasome inhibitorMG132 after cotransfecting ubiquitin-HA and TTPAL/controlvectors in HCT116 and DLD1 cells, and then cellular lysates wereimmunoprecipitated with anti-TRIP6 or IgG. As speculated,TTPAL decreased TRIP6 ubiquitination and increased its proteinlevel (Fig. 6B, 2), inferring that TTPAL binds to TRIP6 and inhibitsthe ubiquitin-mediated degradation of TRIP6 protein.

TTPAL activates Wnt/b-catenin signaling by promoting TRIP6–MAGI1 interaction

TRIP6, containing the same PDZ-binding motif with b-cate-nin, can compete with b-catenin to bind to the PDZ domain ofthe tumor suppressor MAGI1(Supplementary Fig. S5; ref. 14).We therefore hypothesize that TRIP6 protein increased byTTPAL binds to MAGI1 and subsequently release more b-cate-nin to ultimately activate Wnt/b-catenin signaling. To confirmthe interaction between TRIP6 and MAGI1 in a more physio-logic context, co-IP studies were carried out. HCT116 and DLD1cells were transiently transfected with TRIP6, and cellularlysates were then immunoprecipitated with anti-TRIP6 or IgG.Western blot analysis confirmed the presence of MAGI1 in anti-TRIP6 IP products of both cells (Fig. 6C). To assess the effect ofTTPAL on TRIP6–MAGI1 interaction, TTPAL or control vectorswere transfected in HCT116 and DLD1 cells, and cellular lysateswere immunoprecipitated with anti-TRIP6 or IgG. Western blotresults showed that ectopic expression of TTPAL increasedTRIP6 protein level and the binding of TRIP6–MAGI1(Fig. 6C). By overexpressing TRIP6 and/or MAGI1 in HCT116and DLD1 cells followed by co-IP and Western blot assays, wefurther showed the binding between MAGI1 and b-catenin wasreduced by the presence of TRIP6 (Fig. 6D). In line with theseresults, both TRIP6 overexpression and MAGI1 knockdown

Figure 6.TTPAL activatesWnt/b-catenin signaling pathway by promoting TRIP6–MAGI1 interaction. A1, TRIP6 expression was dependent on TTPAL at protein level butnot at mRNA level as determined byWestern blot analysis and RT-(q)PCR. A2, The protein level of TRIP6 was correlated with those of TTPAL in various of coloncancer cells. B, TTPAL stabilized TRIP6, as shown by treatment with the new protein synthesis inhibiter cyclohexamide (CHX; B1) and the proteasome inhibitorMG132 (B2) in HCT116 and DLD1 cells. C, TRIP6–MAGI1 interaction was determined by co-IP, followed byWestern 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 byWestern blot analysis. E,Western blot and TOPflash/FOPflash luciferase reporter assays showed that overexpression of TRIP6 and knockdown ofMAGI1 significantly activatedWnt signaling pathway. F, Induction of TRIP6 promoted interaction between b-catenin and TCF4. G, PDZ binding–defective mutantof TRIP6 was incapable of binding to MAGI1. H,Wild-type TRIP6 inducedWnt activation as compared with PDZ-binding–defective mutant TRIP6 or vectortransfection 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 ofmultiple downstream targets ofWnt signaling in primary colorectal cancer tissues from TCGA study. J, Schematic illustration of the molecular mechanism ofTTPAL inWnt signaling of colorectal cancer.






significantly activated Wnt signaling, as evidenced by theincreased protein levels of active b-catenin and the increasedTOPflash/FOPflash reporter activities (all P < 0.01) in HCT116and DLD1 cells (Fig. 6E). TRIP6 promoted interaction betweenTCF4 and b-catenin (Fig. 6F). Moreover, PDZ binding–defectivemutant, the deletion of the 8 C-terminal aa residues of TRIP6,was conducted (14), while mutant TRIP6 could not bind toMAGI1 (Fig. 6G). Wild-type TRIP6 induced Wnt activation ascompared with mutant TRIP6 or vector transfection controls,which was indicated by the increased levels of active b-cateninand TCF4 (Fig. 6H, 1). In accordance with this, wild-type TRIP6significantly promoted cell proliferation, clonogenicity, andmigration in HCT116 and DLD1 cells (Fig. 6H, 2 and 3).Consistent with the in vitro and in vivo findings, TTPAL expres-sion positively correlated with multiple target genes of b-cate-nin in primary colorectal cancer tissues from TCGA cohort,including CMYC, CCND1, ASCL2, APCDD1, AXIN2, EDN1,FGF18, MET, NAV2, and VEGFA (Fig. 6I), further supportingthe activation of Wnt/b-catenin signaling by TTPAL. Collective-ly, our results suggest that upregulation of TTPAL increases thelevel of TRIP6 by inhibiting its ubiquitin-mediated degrada-tion, and subsequently TRIP6 competitively binds with MAGI1to release b-catenin; this sequence of events allows moreb-catenin to enter the nucleus and activates oncogenic Wnt/b-catenin signaling to promote colorectal tumorigenesis(Fig. 6J).

DiscussionIn this study, amplification of TTPAL was identified by WGS.

We revealed that the amplification of TTPAL was positivelyassociated with its mRNA overexpression in the 102 colorectalcancer cases in Chinese cohort (P < 0.001) and 376 colorectalcancer cases in TCGA cohort (P < 0.001), suggesting that increasedTTPAL copy number contributes to the upregulation of TTPAL.TTPALwas located on chromosome 20q13.12, which is one of themost frequently amplified regions in colorectal cancer and inother cancer types such as breast, ovarian, and gastric can-cers (7, 15, 16). Previously, we have reported amplification ofSLC12A5 (9), the same location with TTPAL, contributes tocolorectal tumor progression and metastasis. Analysis of copynumber of TTPAL in cancers from TCGA studies demonstratedthat TTPAL was preferentially and more frequently amplified incolorectal cancers as comparedwith other cancer types, suggestingits particular involvement in colorectal cancer. In keepingwithourfinding, TTPAL was expressed in 7 colon cancer cell lines, butsilenced in normal colon tissues. Upregulation of TTPAL wasdetected in primary colorectal tumors as compared with adjacentnormal epithelial tissues. Amplification of TTPAL is a commonevent due to copy number gain in colorectal cancer.

We further investigated the clinical importance of TTPALexpression in patientswith colorectal cancer atmRNAand proteinlevels in two independent cohorts, found that 64.8% of themshowed TTPAL overexpression. Both univariate and multivariateCox regression analyses showed high expression levels of TTPALwere correlated significantly with shortened overall survival ofpatients with colorectal cancer. The clinical outcome of colorectalcancer varies greatly depending on the aggressiveness of individ-ual tumors. Many patients experience disease recurrence follow-ing radical surgery. Thus, additional prognostic biomarkers mayprovide better risk assessment that can guide personalized che-

motherapy. Our results demonstrated that TTPAL may serve as anew independent prognostic marker for patients with colorectalcancer.

Because TTPAL was commonly upregulated in patients withcolorectal cancer, this implies important oncogenic function ofTTPAL during colorectal tumorigenesis. With this connection, weperformed in vitro gain- and loss-of-function assays on TTPAL.Overexpression of TTPAL inHCT116 andDLD1 cells significantlyenhanced cell viability and colony formation ability. Conversely,knockdown of TTPAL in SW480 and SW1116 cells significantlysuppressed cell growth. The mechanism by which TTPAL pro-moted colorectal cancer cell growth was mediated by promotingG1–S cell-cycle transition and inhibiting cell apoptosis. G1–Stransition promoted by TTPAL was associated with the upregula-tion of cyclin D1 and CDK4, which are the key regulators of thetransition through the G1 phase of the cell cycle (17). Concom-itantly, the growth enhancement effect of TTPAL was also relatedto inhibition of apoptosis, which was mediated by the caspase-dependent pathways including caspase-8, caspase-9, caspase-7,caspase-3, and PARP. Moreover, overexpression of TTPAL inHCT116 and DLD1 cells significantly promoted their migrationand invasion abilities. In accordance with the in vitro data, TTPALpromoted tumor growth in mouse subcutaneous xenograft mod-els and also promoted metastasis to the lung in tail vein injectionmodels. Collectively, these results indicate that TTPAL exertsoncogenic properties in colorectal carcinogenesis via promotingcell proliferation and cell-cycle progression, inhibiting apoptosisand increasing metastatic abilities. The effects of TTPAL in pro-moting migration and invasion abilities in vitro and in inducinglung metastasis in nude mouse models suggested the potentialrole of TTPAL in facilitating colorectal cancer metastasis in vivo.Additional investigation using TTPAL transgenic and orthotopicmouse models are essential for future investigating the signifi-cance of TTPAL in colorectal cancer metastasis.

We demonstrated that TTPAL could activate the TOPflash/FOPflash activity in colon cancer cells. TTPAL-induced Wnt sig-naling activation was also evidenced by b-catenin activation andincreased protein levels of cyclinD1 and c-Myc. Furthermore,Wntsignaling inhibitor abolished the oncogenic effect of TTPAL oncell proliferation and clonogenicity. To understand themolecularbasis of the role of TTPAL, co-IP of TTPAL followed by proteinsequencing identified TRIP6 as an interacting partner of TTPAL.The direct interaction between TTPAL and TRIP6 was confirmedbyWestern blot analysis of co-IP products. TTPALwas colocalizedin the cytoplasm with TRIP6 by confocal immunofluorescenceassay and Western blot analysis. In addition, the expression ofb-catenin and TRIP6 was dramatically increased in TTPAL-expressing xenografts tumors as compared with control xeno-grafts. These results collectively suggest that TTPAL function viaactivating Wnt signaling and interacting with TRIP6.

TRIP6 was reported to promote tumor growth in several cancertypes and involve in Wnt signaling (18–20). In keeping with this,we revealed that expression of TRIP6 significantly enhanced incolorectal cancer, signifying its oncogenic role in colorectal cancer.We then uncovered that TRIP6 knockdown in colon cancer cellscould significantly blunted the effects of TTPAL on cell prolifer-ation, colony formation,migration, andWnt signaling activation.Conversely, TRIP6 expression can rescue Wnt activation andcancer-related phenotypes in TTPAL-inhibited SW480 cells, infer-ring that the oncogenic role of TTPAL in colorectal cancer is, atleast in part, dependent on TRIP6.

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To explore how TTPAL activates Wnt signaling through TRIP6,we assessed the interplay between TTPAL and TRIP6. We foundthat the direct binding of TTPAL and TRIP6 were existing incytoplasm. TTPAL–TRIP6 complex may recruit lipid or proteinsthat inhibited activity of ubiquitin ligases, in turn, suppressedubiquitin-mediated degradation of TRIP6. Previous studies haveshown that TRIP6 contains the same PDZ-binding motif asb-catenin, and both TRIP6 and b-catenin are physiologic ligandsof the PDZ domain–containing tumor suppressor MAGI1, there-fore TRIP6 couldbind toMAGI1 competitivelywithb-catenin (14,21–25). Our results demonstrated that TTPAL expression pro-moted TRIP6–MAGI1 interaction, which in turn inhibited thebinding between MAGI and b-catenin. Meanwhile, both TRIP6overexpression and MAGI1 silence dramatically induced Wntsignaling activation. In addition, the impact of these findingswas further strengthened by the observation that induction ofTRIP6 promoted interaction between TCF4 and b-catenin. WhilePDZbinding–defectivemutant of TRIP6was incapable of bindingto MAGI1 and stimulating cancer-related phenotypes. Moreover,TTPAL expression was positively correlated with Wnt target genesin primary colorectal cancer tissues from TCGA cohort. Hence,these data imply that the direct binding of TTPAL with TRIP6 incytoplasm inhibited ubiquitin-mediated degradation of TRIP6.Then TRIP6, which contains the same PDZ-binding motif asb-catenin, competitively bound to the PDZ domain–containingtumor suppressor MAGI1 to release b-catenin. This sequence ofevents allowed b-catenin to enter the nucleus and ultimatelypromoted the oncogenic Wnt/b-catenin signaling cascade.

However, enhanced cell proliferation was seen even in thepresence of a Wnt inhibitor. Moreover, TTPAL knockdownshowed weak effect on inhibiting cell growth in RKO cells, whoseWnt signaling is not hyperactivated. These results inferred thepotential existence of Wnt-independent mechanism of cell pro-liferation induced by TTPAL in colorectal cancer. In fact, onco-genic signaling pathways including NFkB, Akt, JNK-AP-1,Hippo have been reported mediated by TRIP6 in cancer (20,26, 27), the direct downstream effector of TTPAL. In this connec-tion, TTPAL-induced cell proliferation should be a complexmechanism in which multiple factors and signaling pathways inaddition to Wnt signaling are involved.

In summary, we demonstrate that TTPAL overexpression due togene amplification is a common event in colorectal cancer. TTPAL

plays a pivotal oncogenic role by activating Wnt/b-catenin sig-naling pathway through binding to and stabilizing TRIP6 incolorectal carcinogenesis. Therapeutic intervention of TTPALmight be a novel strategy for blunting uncontrolled growth andmetastasis in Wnt/b-catenin–addicted colorectal cancer. More-over, TTPAL may serve as an independent prognostic marker forpatients with colorectal cancer.

Disclosure of Potential Conflicts of InterestF.K.L. Chan reports receiving speakers bureau honoraria from Pfizer, Takeda,

and AstraZeneca. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: H. Gou, J.Q. Liang, J.-Y. Fang, J. YuDevelopment of methodology: L. Zhang, Y. Zhang, J.-Y. Fang, J. YuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Li, X. Wang, J.H.M. Tong, K.-F. To, J. YuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.Q. Liang, X. Wang, K.-F. ToWriting, review, and/or revision of the manuscript: J.Q. Liang, K.-F. To,J.J.Y. Sung, J. YuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.Q. Liang, H. Chen, Y. Zhang, J. Ji, J.J.Y. Sung,J. YuStudy supervision: J.Q. Liang, J. Ji, J.J.Y. Sung, F.K.L. Chan, J. Yu

AcknowledgmentsThis project was supported by National Key R&D Program of China

(2016YFC1303200, 2017YFE0190700); Science and Technology ProgramGrant Shenzhen (JCYJ20180307151253271, JCYJ20170413161534162);Postdoctoral Natural Science Foundation of China (2017M622802);RGC-GRF Hong Kong (14111216, 14163817); National Natural ScienceFoundation of China (NSFC; 81773000); Vice-Chancellor's DiscretionaryFund CUHK and CUHK direct grant; Shenzhen Municipal Science andTechnology Shenzhen Virtual University Park Support Scheme to CUHKShenzhen Research Institute.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 27, 2018; revised March 16, 2019; accepted April 19,2019; published first April 24, 2019.

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2019;79:3332-3346. Published OnlineFirst April 24, 2019.Cancer Res Hongyan Gou, Jessie Qiaoyi Liang, Lijing Zhang, et al.

-Catenin SignalingβActivate Wnt/TTPAL Promotes Colorectal Tumorigenesis by Stabilizing TRIP6 to

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