TTPAL Promotes Colorectal Tumorigenesis by Stabilizing TRIP6 To

Author Manuscript Published OnlineFirst on April 24, 2019; DOI: 10.1158/0008-5472.CAN-18-2986 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TTPAL promotes colorectal tumorigenesis by stabilizing TRIP6 to

activate Wnt/β-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 JY

Sung2, Francis KL Chan2, Jing-Yuan Fang1, Jun Yu2*

1 Diversion of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology

and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related

Genes; Shanghai Institute of Digestive Disease; Renji Hospital, School of Medicine,

Shanghai Jiaotong University, Shanghai, China.

2Institute of Digestive Disease and Department of Medicine and Therapeutics, State

Key laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK

Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong

3Department of Surgery, Peking University Cancer Hospital and Institute, Beijing,

China

4Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology

in South China, The Chinese University of Hong Kong, Hong Kong

# Co-first author.

Running title: Role of TTPAL in Colorectal Cancer

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 24, 2019; DOI: 10.1158/0008-5472.CAN-18-2986 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abbreviations: CRC: colorectal cancer; TTPAL: Tocopherol alpha transfer protein-

like; PARP: poly (ADP-ribose) polymerase; 7-AAD: 7-aminoactinomycin D; TCGA:

The Cancer Genome Atlas; TNM: Tumor-Nodes-Metastasis; CHX: cyclohexamide;

TUNEL, terminal deoxynucleotidyl transferase; Co-IP: Co-Immunoprecipitation;

TRIP6: Thyroid receptor-interacting protein 6; MAGI1: Membrane-associated

guanylate kinase, WW and PDZ domain-containing protein 1.

*Corresponding author: Professor Jun Yu, Institute of Digestive Disease and

Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese

University of Hong Kong, Hong Kong. Tel: (852) 37636099; Fax: (852) 21445330;

Email: [email protected].

Author contributions: HG involved in study design, conducted experiments and

drafted manuscript; QL involved in study design and revised the paper; LZ, YZ, HC

and RL performed experiments; XW, JJ provided human samples; JJYS, FKLC and JF

commented on the study; JY designed, supervised the study and revised the manuscript.

Conflicts of interest: No potential conflicts of interest were disclosed.

Word count: 5,276 words.

Total number of figures/tables: 7 (6 figures and 1 table).

Abstract

Copy number alterations are crucial for the development of colorectal cancer (CRC).

Our whole genome analysis identified tocopherol alpha transfer protein-like (TTPAL)

as preferentially amplified in CRC. Here we demonstrate that frequent copy number

gain of TTPAL leads to gene overexpression in CRC from a Chinese cohort (n=102),

which was further validated by a TCGA cohort (n=376). High expression of TTPAL

was significantly associated with shortened survival in CRC patients. TTPAL promoted

cell viability and clonogenicity, accelerated cell cycle progression, inhibited cell

apoptosis, increased cell migration/invasion ability in vitro, and promoted

tumorigenicity and cancer metastasis in vivo. TTPAL significantly activated Wnt

signaling and increased β-catenin activation and protein expression of cyclin D1 and c-

Myc. Co-immunoprecipitation followed by mass spectrometry identified thyroid

receptor-interacting protein 6 (TRIP6) as a direct downstream effector of TTPAL.

Depletion of TRIP6 significantly abolished the effects of TTPAL on cell proliferation

and Wnt activation. Direct binding of TTPAL with TRIP6 in the cytoplasm inhibited

ubiquitin-mediated degradation of TRIP6; subsequently increased levels of TRIP6

displaced β-catenin from the tumor suppressor MAGI1 via competitive binding. This

sequence of events allows β-catenin to enter the nucleus and promote oncogenic Wnt/β-

catenin signaling. In conclusion, TTPAL is commonly overexpressed in CRC due to

copy number gain, which promotes colorectal tumorigenesis by activating Wnt/β-

catenin signaling via stabilization of TRIP6. TTPAL overexpression may serve as an

independent new biomarker for the prognosis of CRC patients.

Introduction

Colorectal cancer (CRC) is the third most common cancer and the second leading cause

of cancer-related death worldwide (1,2). The pathogenic mechanisms underlying CRC

development appear to be complex and heterogeneous. Copy number alterations (CNAs)

are common somatic changes in cancer featured with gain or loss in copies of DNA

sections (3). Deletions and copy number gains contribute to alterations in the expression

of tumor suppressor genes and oncogenes, respectively. The stepwise accumulation of

CNAs confers growth advantage and 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 targets for CRC.

Therefore, detection and mapping of copy number abnormalities provide an approach

for associating aberrations with disease phenotype and for localizing critical genes (4,5).

It is importance to identify and functionally characterize novel genes with CNAs that

are associated with CRC (4).

Chromosome 20q amplification has been commonly found in CRC and is involved in

transforming adenoma to carcinoma (5,6). Chromosome 20q amplification is also a

potential indicator of poor prognosis in CRC patients (7-9). By whole genome

sequencing, we identified that TTPAL (Tocopherol alpha transfer protein-like), located

at 20q13.12, was preferentially amplified in primary colorectal tumor tissues. In

keeping with our finding, analyses from TCGA dataset showed that copy number gain

of TTPAL occurs frequently in CRC and positively correlates with its upregulated

mRNA expression. Searching for the public protein datasets of Human Protein Altas,

we found that higher TTPAL protein expression was shown in CRC tumor tissues

compared to normal colon tissues from the dataset (http://www.proteinatlas.org).

Moreover, high TTPAL mRNA expression is significantly associated with poor survival

in patients with multiple cancer types

(https://www.proteinatlas.org/ENSG00000124120-TTPAL/pathology#top; P<0.001).

Therefore, we hypothesize that TTPAL plays an oncogenic function in colon

carcinogenesis.

The role of TTPAL in human cancer remains uninvestigated. In this study, we

identified the frequent overexpression of TTPAL in CRC due to its copy number gain.

Further functional studies revealed that ectopic expression of TTPAL significantly

promoted CRC growth by enhancing G1-S cell cycle progression and reducing

apoptosis. The tumor promoting effect of TTPAL was revealed to be associated with

the activation of Wnt/β-catenin signaling pathway by binding to tyroid receptor-

interacting protein 6 (TRIP6). The direct binding of TTPAL with TRIP6 in cytoplasm

inhibited TRIP6 ubiquitin degradation. Enhanced cytoplasm TRIP6 displaced β-catenin

from the tumor suppressor MAGI1 via competitive-binding, which allowed β-catenin

to enter into the nucleus and subsequently activated the oncogenic Wnt/β-catenin

signaling. Moreover, TTPAL overexpression was significantly associated with poor

survival of CRC patients. Thus, this study revealed a novel oncogenic mechanism and

the clinical application value of TTPAL in CRC.

Materials and methods

Colon cancer cell lines

The colon cancer cell lines (DLD-1, HCT116, HT29, LOVO, SW480, RKO and

SW1116) were obtained from the American Type Culture Collection (ATCC, Manassas,

VA) between 2014 and 2015 and cells authentication were confirmed by short tandem

repeat profiling. Cells were routinely cultured and maintained in DMEM medium

supplemented with 10% FBS and antibiotics (Gibco BRL, Rockville, MD) according

to the American Type Culture Collection protocols, except HCT116 cells, which were

cultured in complete McCoy’s 5A medium (Gibco BRL). Routine Mycoplasma testing

was performed by PCR. Cells were grown for no more than 25 passages in total for any

experiment.

Subjects and sample collection

Paired primary colorectal tumors and adjacent non-tumor tissues were collected

immediately after surgical resection at the Prince of Wales Hospital (Hong Kong, China;

182 primary tumors and 18 non-tumor tissues) and Peking University Cancer Hospital

and Institute (Beijing, China; 102 primary tumors 50 non-tumor tissues). The specimens

were snap-frozen in liquid nitrogen and stored at 80C and were also fixed in 10%

formalin and embedded in paraffin for routine histologic examination. Biopsies from 3

cases of normal mucosa obtained during colonoscopy were recruited as healthy controls,

which were confirmed by an experienced pathologist at the Prince of Wales Hospital

and Peking University Cancer Hospital. The study protocol was approved by the

Clinical Research Ethics Committee of The Chinese University of Hong Kong and the

Clinical Research Ethics Committee of Peking University Cancer Hospital. All patients

provided written informed consent for obtaining the study specimens. This study was

carried out in accordance with the Declaration of Helsinki of the World Medical

Association.

PCR copy number analysis

DNA was extracted from 102 frozen CRC samples of Chinese Cohort using the AllPrep

DNA/RNA/Protein Kit (Qiagen, Hilden, Germany). The purified genomic DNA was

amplified with a probe specific to target gene TTPAL (Mm00082375_cn ) by TaqMan

Copy Number Assays (Applied Biosystems, Waltham, MA). The results were analyzed

by Copy Caller software v2.0 (Applied Biosystems).

Subcutaneous xenograft and experimental metastasis mouse models

HCT116 and DLD1 cells stably transfected with TTPAL expression vector or empty

vector (5×106 cells/0.1 mL PBS) were injected subcutaneously into the left and right

dorsal flanks of 4- to 6-week-old female Balb/c nude mice (n = 8/group), respectively.

Tumor size was measured every 2 days for 2-3 weeks using a digital caliper. Tumor

volume (mm3) was estimated by measuring the longest and shortest diameters of the

tumor and calculating as previously described (10). At the endpoint, tumors were

harvested and weighted. The excised tissues were either fixed in 10% neutral-buffered

formalin or snap frozen in liquid nitrogen. Tumor sections from paraffin-embedded

blocks were used for histologic examination.

For metastasis model, HCT116 cells stably transfected with TTPAL expression vector

or empty vector (2 ×106 cells in 0.1mL PBS) were injected intravenously via the tail

vein (n = 5). After 6-8 weeks, mice were sacrificed and their lungs were harvested. The

lungs were sectioned and stained with H&E. The number of lung metastases were

counted. All experimental procedures were approved by the Animal Ethics Committee

of the Chinese University of Hong Kong.

Co-immunoprecipitation (co-IP) and liquid chromatography-mass spectrometry

Co-IP assays were carried out as previously described (11,12). Briefly, total protein

from HCT116 cells (~5 × 106/reaction) stably transfected with TTPAL (Flag-tagged)

expression vector or empty vector was extracted in radioimmunoprecipitation assay

(RIPA) buffer supplemented with proteinase inhibitor (Novagen, Darmstadt, Germany).

Immunoprecipitation was performed using anti-Flag M2 antibody (A2220, Sigma-

Aldrich, St. Louis, MO). The immune complexes were precipitated by PureProteome™

Protein A/G Mix magnetic beads (LSKMAGAG02, Millipore, Burlington, MA)

overnight at 4 °C. Beads with extracted proteins were washed 3 times by 50 mM

ammonium bicarbonate buffer and subjected to digestion by trypsin at 37 °C for 2 h

(Promega, Madison, WI). Tryptic peptides were then extracted for liquid

chromatography-mass spectrometry (LC-MS) analysis.

Co-IP and western blot

Whole lysate (150 μg protein) and co-IP precipitant by anti-Flag-tag, anti-Thyroid

receptor interacting protein 6 (TRIP6) antibody (ab70747, Abcam, Cambridge, UK), anti-HA-tag (ab18181, Abcam) or IgG were immunoblotted with either anti-TRIP6, anti-HA or anti-Flag (A2220, Sigma-Aldrich) antibody to confirm the interaction between TTPAL and TRIP6 that was identified by LC-MS. The lysate (1% input, 10 μg protein) was also used as a control. The antibodies used are listed in Supplementary table 1.

Ubiquitination assay

HCT116 and DLD1 cells stably transfected with TTPAL expression vector or empty

vector were co-transfected with Ubiquitin-HA for 24 h. Then the cells were incubated

in the presence or absence of 10 µM MG132 (M-7449, Sigma-Aldrich) for 12 h. Cells

were lysed with RIPA buffer supplemented with protease inhibitors.

Immunoprecipitation was performed using anti-TRIP6 or IgG, respectively.

Immunoprecipitated proteins were analyzed by western blot using anti-HA (ab18181,

Abcam).

Statistical analysis

The 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,

New York, USA). The Pearson correlation coefficient was used to evaluate the

correlation between TTPAL gene amplification and expression in the clinical samples.

The Chi-square test was used for comparison of patient characteristics and distributions

of expression and covariates by vital status. ROC curve was used to estimate the cutoff

value of the methylation percentage. Cutoff value was analyzed by survival significance

analysis using the tool Cutoff Finder (http://molpath.charite.de/cutoff/) (13). Crude relative

risks (RRs) of death associated with TTPAL expression were estimated by univariate

Cox proportional hazards regression model first. Multivariate Cox model was then

constructed to estimate the adjusted RR for TTPAL expression. Overall survival in

relation to expression was evaluated by the Kaplan-Meier survival curve and the log-

rank test. Mann-Whitney U test or Student’s t test was performed to compare the

variables of two groups. mRNA expression z-Scores (RNA Seq V2 RSEM) from 382

CRC samples were downloaded from TCGA via cBioPortal. Nonparametric Spearman

correlations in expression levels between TTPAL and Wnt target genes were then

computed. The difference in cell viability and tumor growth rate between two groups

of nude mice was determined by repeated-measures analysis of variance. P values <0.05

were taken as statistical significance.

Results

TTPAL is frequently overexpressed in CRC due to copy number gain

By whole-genome sequencing analysis to compare genetic alterations in primary CRC

tissues with corresponding peripheral blood samples, we identified TTPAL to be

preferentially amplified in CRC tumor tissues by WGS (Supplementary Figure 1A).

TTPAL was also frequently amplified in colon and rectum cancers from TCGA cohort

but less amplified in other cancers (Figure 1A), demonstrating that copy number gain

of TTPAL may be specifically associated with colorectal tumorigenesis. We further

quantitated the copy number and mRNA expression of TTPAL in CRC samples by

TaqMan copy number assay and RT-qPCR, respectively. In 102 CRC patients, 48%

(49/102) of primary CRC tissues exhibited TTPAL copy number gain, which was

positively correlated with its mRNA expression (R2=0.3537, P<0.0001; Figure 1B).

This finding was further validated in CRC samples from TCGA cohort, with copy

number gain detected in 70.2% of samples (≥1.2 fold, 436/621) and mRNA expression

also positively correlated with copy number (n=376, R2=0.5632, P<0.0001; Figure 1B).

These results demonstrate that TTPAL is commonly overexpressed in CRC due to copy

number gain.

TTPAL expression is upregulated in colon cancer cells and primary CRC tumors

We then examined TTPAL expression in colon cancer cells and primary CRC tissues.

TTPAL expression at both mRNA and protein levels was detected in all 7 colon cancer

cells tested (DLD1, LOVO, RKO, HT29, HCT116, SW480 and SW1116), but not in

normal colon tissues (Figure 1C). TTPAL mRNA expression was significantly

upregulated in primary CRC tumors as compared with their adjacent normal tissues as

determined by RT-qPCR (Figure 1D). Specifically, the upregulation of TTPAL mRNA

in CRC was validated in three independent cohorts of paired tumor and adjacent normal

samples including Bejing Chinese cohort (n=50; P<0.0001), Hong Kong Chinese

cohort (n=18; P<0.0001) and the TCGA cohort (n=23; P<0.0001) (Figure 1D).

Significant upregulation was also observed in CRC (n=286) compared to unpaired

adjacent normal tissues (n=39) from TCGA study (P<0.0001) (Figure 1D). Western

blot and immunohistochemical (IHC) staining further confirmed the upregulation of

TTPAL at the protein level in CRCs as compared with adjacent normal tissues (Figures

1E&1F).

Overexpression of TTPAL is an independent predictor of poor survival in CRC

patients

We further evaluated the clinicopathological and prognostic significance of TTPAL

expression in patients with CRC. We evaluated TTPAL mRNA expression in primary

CRC tissues from 102 Chinese patients. By classifying samples into low- and high-

expression groups using cutoff finder, TTPAL overexpression was detected in 64.7%

(66/102) of primary CRCs. No correlation was found between TTPAL expression and

clinicopathological features such as age, gender, location, differentiation, and Tumor-

Nodes-Metastasis (TNM) stage (Supplementary Table 2). However, TTPAL

overexpression was associated with an increased risk of cancer-related death by

univariate Cox regression (relative risk (RR)=2.202, 95% CI: 1.090-4.447, P=0.028;

Table 1). In particular, after adjustment for potential confounding factors, TTPAL

overexpression was found to be an independent risk factor for shortened survival in

patients with CRC by multivariate Cox regression analysis (RR=2.146, 95% CI: 1.061-

4.340, P=0.034; Table 1). Kaplan-Meier survival curves showed that CRC patients with

TTPAL overexpression had significantly poorer overall survival based on the log-rank

test (P<0.05; Figure 1G).

TTPAL protein expression was further evaluated in primary CRC tissues from another

cohort of Chinese patients (n=182) by IHC using tissue microarrays. No correlation was

found between TTPAL protein expression and clinicopathological features such as age,

gender, location, differentiation, and TNM stage (Supplementary Table 2). Further

univariate and multivariate Cox regression analyses showed that TTPAL protein

expression was also an independent predictor of poor survival in CRC patients

(multivariate RR=2.441, 95% CI: 1.544 to 3.858, P<0.0001) besides TNM stage (Table

1). As shown in the Kaplan–Meier survival curves, CRC patients with high TTPAL

protein expression had significantly shorter survival than those with low expression

(P=0.002, log-rank test; Figure 1G). Moreover, the prognostic significance of TTPAL

expression was well validated in TCGA cohort (Figure 1H). These findings collectively

indicated that TTPAL may serve as an independent new biomarker for the prognosis of

CRC patients.

TTPAL expression promotes colon cancer cell growth

The frequent overexpression of TTPAL in CRC prompted us to investigate its potential

oncogenic role in CRC. To this end, we performed in vitro gain- and loss-of-function

assays on TTPAL. Ectopic expression of TTPAL in HCT116 and DLD1 cells (Figure

2A) significantly increased cell viability and clonogenicity (Figure 2A). On the other

hand, TTPAL knockdown by RNA interference in SW480 and SW1116 cells markedly

inhibited cell viability and clonogenicity (Figure 2B). These results indicated that

TTPAL plays an oncogenic role in colon cancer.

TTPAL promotes cell cycle progression and inhibits cell apoptosis

We analyzed the cytokinetic effect of TTPAL. Overexpression of TTPAL led to a

significant decrease in G1 phase cells and a concomitantly increase in S phase cells in

both HCT116 and DLD1 cells by BrdU staining (all P<0.01; Figure 2C). The

promoting effect of TTPAL on cell cycle progression was confirmed by the increased

protein expression of two master G1-S checkpoint regulators (cyclin D1 and CDK4)

and the proliferation marker proliferating cell nuclear antigen (PCNA) (Figure 2D). On

the other hand, knockdown of TTPAL significantly increased cells in the G1 phase and

decreased cells in S phase of both SW480 and SW1116 cells (all P<0.01; Figure 2C),

and concomitantly reduced the expression of cyclin D1, CDK4 and PCNA (Figure 2D).

Apoptosis analysis showed that overexpression of TTPAL led to a significant reduction

in apoptotic cell population in HCT116 (P<0.01) and DLD1 (P<0.001) cells (Figure

2E1), while knockdown of TTPAL significantly increased apoptosis of SW480 and

SW1116 cells (both P<0.001; Figure 2E2). TTPAL-induced reduction of apoptosis was

confirmed by the elevated expression of key apoptosis markers (cleaved forms of

caspase-8, caspase-9, caspase-7, caspase-3 and PARP), while knockdown of TTPAL

showed opposite effects (Figure 2F). These results demonstrate the role of TTPAL on

promoting cell growth by regulating cell cycle progression and apoptosis in colon

cancer cells.

TTPAL promotes cell migration and invasion

To determine the effects of TTPAL on cell migration and invasion, wound-healing and

Matrigel invasion assays were performed. Ectopic expression of TTPAL significantly

increased the migration abilities in HCT116 and DLD1 cells (both P<0.01; Figure 2G),

and significantly promoted invasiveness of both cells (both P<0.001; Figure 2H),

while knockdown of TTPAL markedly suppressed migration and invasion abilities in

SW480 and SW1116 cells (Figure 2I). In keeping with these, protein expression of

epithelial-mesenchymal transition (EMT) markers was regulated by TTPAL. TTPAL

overexpression upregulated the mesenchymal marker Vimentin and downregulated the

epithelial markers (E-cadherin and claudin-1), while knockdown of TTPAL showed the

opposite effects (Figure 2K). These findings demonstrated that TTPAL also functioned

in regulating cell migration and invasion of CRC cells.

TTPAL activates Wnt/β-catenin signaling

We 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/β-

catenin, Akt (FHRE) and NFB. Ectopic expression of TTPAL significantly increased

the activity of Wnt signaling as demonstrated by TOPflash/FOPflash luciferase reporter

assay, and knockdown of TTPAL showed the opposite effect on Wnt activity (Figure

3A and Supplementary Figure 2A). Consistently, overexpression of TTPAL

significantly upregulated the protein levels of cyclin D1, c-Myc, β-catenin and active

β-catenin, while TTPAL knockdown diminished expression of these key Wnt signaling

genes (Figure 3B). Moreover, Wnt signaling inhibitor IWP-2 significantly inhibited

cell growth in DLD1 and HCT116 cells stably transfected with TTPAL as compared to

DMSO treatment. IWP-2 also eliminated the growth promoting effect exerted by

TTPAL overexpression (P>0.05 for IWP-2-treated TTPAL-overexpressed cells vs.

DMSO-treated vector-transfected cells; Figure 3C). TTPAL knockdown showed

weaker effect on proliferation in RKO cells, which do not harbor mutation in APC or

CTNNB1 gene (Supplementary Figure 2B). These results demonstrate that the

oncogenic role of TTPAL is largely mediated by activation of Wnt/β-catenin signaling.

TRIP6 directly interacts with TTPAL

In order to identify the direct interacting partners of TTPAL in CRC, we performed Co-

IP followed by liquid chromatograph-mass spectrometry (LC-MS). TTPAL binding

candidates were then identified by comparing the anti-TTPAL-Flag IP products of

TTPAL (Flag)-overexpressed cells with those of control cells (Figure 3D). Among the

identified candidates with ≥3 mapping peptides, 7 genes have been reported with

functions that may be associated with cancer development (Supplementary Table 3).

Among them, TRIP6 is of interest due to its involvement in Wnt signaling pathway (14).

To validate the interaction between TTPAL and TRIP6, Flag-TTPAL and HA-TRIP6

(or TRIP6) were co-expressed in HCT116 and DLD1 cells, and total proteins were

immunoprecipitated with anti-Flag or anti-HA/anti-TRIP6 antibodies. Western blot

results showed that TTPAL and TRIP6 could be co-precipitated by each other in both

cells. Moreover, the interaction of endogenous TTPAL and TRIP6 in SW480 and

SW1116 cells was confirmed by IP-western analyses (Figure 3E), indicating the

directly interaction between TTPAL and TRIP6. We next examined the expression and

intracellular distribution of TTPAL and TRIP6 by immunofluorescence. Confocal

microscopy images showed that TTPAL co-localized with TRIP6 in the cytoplasm of

HCT116 and DLD1 cells following transfection of TTPAL and HA-TRIP6 (Figure

3F1). The localization of the two proteins was further confirmed by western blotting of

membrane, cytoplasmic and nuclear fractions, which showed that TTPAL and TRIP6

was mainly localized in the cytoplasm of HCT116 and DLD1 cells (Figure 3F2).

TTPAL promotes tumorigenicity and metastasis by regulating TRIP6 and Wnt/β-

catenin signaling in vivo

In light of our in vitro findings, we tested the effect of TTPAL in vivo. Results showed

that stable transfection of TTPAL expression vector in HCT116 and DLD1 cells

significantly promoted the growth of tumor volume over the entire assay period and

increased tumor weight at the end point in subcutaneous xenograft models (Figure 4A).

Overexpression of TTPAL was confirmed by immunohistochemistry staining in DLD1

and HCT116 xenografts (Figure 4B). Cell proliferation was significantly promoted as

determined by Ki-67 staining, and apoptosis was significantly reduced as evidenced by

the TUNEL staining, in both TTPAL-overexpressed xenografts as compared to controls

(Figure 4B). Moreover, the expression of β-catenin and TRIP6 was dramatically

increased in TTPAL-overexpressed HCT116 and DLD1 xenografts by IHC, validating

the molecular basis identified in vitro (Figure 4B). TTPAL overexpression promoted

lung metastasis in nude mice (P<0.01 Figure 4D). Furthermore, stable knockdown of

TTPAL in SW480 cells significantly inhibited tumorigenicity (Figure 4E) and

metastasis in nude mice (Figure 4F). Collectively, these results in immunodeficient

nude mice suggest that TTPAL promoted colorectal tumorigenicity and may also

facilitate metastasis.

The oncogenic role of TTPAL is dependent on TRIP6

We then examined the importance of TRIP6 in TTPAL-mediated oncogenic function in

CRC. TRIP6 mRNA expression was significantly increased in CRC tumor tissues as

compared with adjacent normal tissues in TCGA cohort (P<0.001, Supplementary

Figure 3A). TRIP6 mRNA was readily expressed in all 7 colon cancer cells tested, but

not in normal colon tissues (Supplementary Figure 3B), TRIP6 expression promoted

cell proliferation and clonogenicity in DLD1 and HT-29 cells, whilst TRIP6 silence

showed significant suppressive effect on cell proliferation and clonogenicity in SW480,

SW1116 and HCT116, suggesting the oncogenic role of TRIP6 in CRC

(Supplementary Figure 4). To investigate the effect of TRIP6 on the TTPAL-mediated

cell proliferation and metastasis, HCT116 and DLD1 cells stably transfected with

TTPAL or control vectors were co-transfected with siRNA against TRIP6 (Figure 5A).

TRIP6 knockdown significantly abolished the promoting effect of TTPAL on cell

viability (Figure 5B), clonogenicity (Figure 5C) and migration ability of both HCT116

and DLD1 cells (Figure 5D). We next examined if TTPAL activated Wnt signaling

pathway through mediating TRIP6. Indeed, TRIP6 knockdown abolished the TTPAL-

induced protein expression of Wnt effectors (cyclin D1, c-Myc, β-catenin and active β-

catenin) by Wesern blot (Figure 5E). TRIP6 knockdown also blunted the TTPAL-

activated Wnt signaling as evidenced by luciferase reporter assay in HCT116 and DLD1

cells (Figure 5F). These results suggested that the oncogenic role of TTPAL and its

effect on activating Wnt signaling were dependent on TRIP6. Consistently, TRIP6

overexpression in TTPAL-inhibited SW480 cells can rescue cell proliferation (Figure

5G), clonogenicity (Figure 5G), migration (Figure 5I), and Wnt activation (Figure 5J).

TTPAL inhibits ubiquitin-mediated degradation of TRIP6 protein

To gain insights into how TRIP6 mediates the role of TTPAL, we further evaluated the

effect of TTPAL on TRIP6. We observed that TRIP6 protein expression was

upregulated by ectopic expression of TTPAL in HCT116 and DLD1 cells, and

decreased after knockdown of the endogenous TTPAL in SW1116 cells (Figure 6A1).

TRIP6 protein was also upregulated by TTPAL in xenografts of nude mice (Figure

4C&D). However, mRNA level of TRIP6 was not changed by overexpression or

knockdown of TTPAL (Figure 6A1). Moreover, the protein levels of TRIP6 in various

CRC cell lines was positively correlated with the protein levels of TTPAL in these cell

lines (Figure 6A2), indicating that TTPAL may stabilize TRIP6 in CRC. We therefore

assessed whether TTPAL regulated the stability of TRIP6 protein. We treated TTPAL

or control vector transfected cells with the protein synthesis inhibitor cycloheximide

(CHX). As shown in Figure 6B1, TRIP6 was more stable in the presence of TTPAL in

HCT116 and DLD1 cells. To investigate whether TTPAL increased the stability of

TRIP6 by affecting its ubiquitination/degradation, we treated cells with the proteasome

inhibitor MG132 after co-transfecting ubiquitin-HA and TTPAL/control vectors in

HCT116 and DLD1 cells, and then cellular lysates were immunoprecipitated with anti-

TRIP6 or IgG. As speculated, TTPAL decreased TRIP6 ubiquitination and increased its

protein level (Figure 6B2), inferring that TTPAL binds to TRIP6 and inhibits the

ubiquitin-mediated degradation of TRIP6 protein.

TTPAL activates Wnt/β-catenin signaling by promoting TRIP6-MAGI1

interaction

TRIP6, containing the same PDZ binding motif with β-catenin, can compete with β-

catenin to bind to the PDZ domain of the tumor suppressor MAGI1(Supplementary

Figure 5) (14). We therefore hypothesize that TRIP6 protein increased by TTPAL binds

to MAGI1 and subsequently release more β-catenin to ultimately activate Wnt/β-

catenin signaling. To confirm the interaction between TRIP6 and MAGI1 in a more

physiological context, co-IP studies were carried out. HCT116 and DLD1 cells were

transiently transfected with TRIP6, and cellular lysates were then immunoprecipitated

with anti-TRIP6 or IgG. Western Blot analysis confirmed the presence of MAGI1 in

anti-TRIP6 IP products of both cells (Figure 6C). To assess the effect of TTPAL on

TRIP6-MAGI1 interaction, TTPAL or control vectors were transfected in HCT116 and

DLD1 cells, and cellular lysates were immunoprecipitated with anti-TRIP6 or IgG.

Western blot results showed that ectopic expression of TTPAL increased TRIP6 protein

level and the binding of TRIP6-MAGI1 (Figure 6C). By overexpressing TRIP6 and/or

MAGI1 in HCT116 and DLD1 cells followed by co-IP and western blot assays, we

further showed the binding between MAGI1 and β-catenin was reduced by the presence

of TRIP6 (Figure 6D). In line with these results, both TRIP6 overexpression and

MAGI1 knockdown significantly activated Wnt signaling, as evidenced by the

increased protein levels of active β-catenin and the increased TOPflash/FOPflash

reporter activities (all P<0.01) in HCT116 and DLD1 cells (Figure 6E). TRIP6

promoted interaction between TCF4 and β-catenin (Figure 6F). Moreover, PDZ

binding-defective mutant, the deletion of the 8 C-terminal aa residues of TRIP6, was

conducted (14), while mutant TRIP6 could not bind to MAGI1 (Figure 6G). Wild-type

TRIP6 induced Wnt activation as compared to mutant TRIP6 or vector transfection

controls, which was indicated by the increased levels of active β-catenin and TCF4

(Figure 6H1). In accordance with this, Wild-type TRIP6 significantly promoted cell

proliferation, clonogenicity and migration in HCT116 and DLD1 cells (Figures 6H2

and H3). Consistent with the in vitro and in vivo findings, TTPAL expression positively

correlated with multiple target genes of β-catenin in primary CRC tissues from TCGA

cohort, including CMYC, CCND1, ASCL2, APCDD1, AXIN2, EDN1, FGF18, MET,

NAV2, and VEGFA (Figure 6I), further supporting the activation of Wnt/β-catenin

signaling by TTPAL. Collectively, our results suggest that upregulation of TTPAL

increases the level of TRIP6 by inhibiting its ubiquitin-mediated degradation, and

subsequently TRIP6 competitively binds with MAGI1 to release β-catenin; this

sequence of events allows more β-catenin to enter the nucleus and activates oncogenic

Wnt/β-catenin signaling to promote colorectal tumorigenesis (Figure 6J).

Discussion

In this study, amplification of TTPAL was identified by whole-genome sequencing. We

revealed that the amplification of TTPAL was positively associated with its mRNA

overexpression in the 102 CRC cases in Chinese cohort (P<0.001) and 376 CRC cases

in TCGA cohort (P<0.001), suggesting that increased TTPAL copy number contributes

to the upregulation of TTPAL. TTPAL was located on chromosome 20q13.12, which

is one of the most frequently amplified regions in CRC and in other cancer types such

as breast, ovarian and gastric cancers (7,15,16). Previously, we have reported

amplification of SLC12A5 (9), the same location with TTPAL, contributes to colorectal

tumor progression and metastasis. Analysis of copy number of TTPAL in cancers from

TCGA studies demonstrated that TTPAL was preferentially and more frequently

amplified in CRCs as compared to other cancer types, suggesting its particular

involvement in CRC. In keeping with our finding, TTPAL was expressed in 7 colon

cancer cell lines, but silenced in normal colon tissues. Upregulation of TTPAL was

detected in primary colorectal tumors as compared to adjacent normal epithelial tissues.

Amplification of TTPAL is a common event due to copy number gain in CRC. 22

We further investigated the clinical importance of TTPAL expression in CRC patients

at mRNA and protein levels in two independent cohorts, found that 64.8% of them

showed TTPAL overexpression. Both univariate and multivariate cox regression

analyses showed high expression levels of TTPAL were correlated significantly with

shortened overall survival of CRC patients. The clinical outcome of CRC varies greatly

depending on the aggressiveness of individual tumors. Many patients experience

disease recurrence following radical surgery. Thus, additional prognostic biomarkers

may provide better risk assessment that can guide personalized chemotherapy. Our

results demonstrated that TTPAL may serve as a new independent prognostic marker

for patients with CRC.

Since TTPAL was commonly upregulated in patients with CRC, this implies important

oncogenic function of TTPAL during colorectal tumorigenesis. With this connection,

we performed in vitro gain- and loss-of-function assays on TTPAL. Overexpression of

TTPAL in HCT116 and DLD1 cells significantly enhanced cell viability and colony

formation ability. Conversely, knockdown of TTPAL in SW480 and SW1116 cells

significantly suppressed cell growth. The mechanism by which TTPAL promoted CRC

cell growth was mediated by promoting G1-S cell cycle transition and inhibiting cell

apoptosis. G1-S transition promoted by TTPAL was associated with the upregulation of

cyclin D1 and CDK4, which are the key regulator of the transition through the G1 phase

of the cell cycle (17). Concomitantly, the growth-enhancement effect of TTPAL was

also related to inhibition of apoptosis, which was mediated by the caspase-dependent

pathways including casepase-8, casepase-9, casepase-7, casepase-3 and PARP.

Moreover, overexpression of TTPAL in HCT116 and DLD1 cells significantly

promoted their migration and invasion abilities. In accordance with the in vitro data,

TTPAL promoted tumor growth in mouse subcutaneous xenograft models and also

promoted metastasis to the lung in tail vein injection models. Collectively, these results

indicate that TTPAL exerts oncogenic properties in colorectal carcinogenesis via

promoting cell proliferation and cell cycle progression, inhibiting apoptosis and

increasing metastatic abilities. The effects of TTPAL in promoting migration and

invasion abilities in vitro and in inducing lung metastasis in nude mouse models

suggested the potential role of TTPAL in facilitating CRC metastasis in vivo. Additional

investigation using TTPAL transgenic and orthotopic mouse models are essential for

future investigating the significance of TTPAL in CRC metastasis.

We demonstrated that TTPAL could activate the TOPflash/FOPflash activity in colon

cancer cells. TTPAL-induced Wnt signaling activation was also evidenced by β-catenin

activation and increased protein levels of cyclin D1 and c-Myc. Furthermore, Wnt

signaling inhibitor abolished the oncogenic effect of TTPAL on cell proliferation and

clonogenicity. To understand the molecular basis of the role of TTPAL, Co-IP of

TTPAL followed by protein sequencing identified TRIP6 as an interacting partner of

TTPAL. The direct interaction between TTPAL and TRIP6 was confirmed by Western

blot analysis of Co-IP products. TTPAL was co-localized in the cytoplasm with TRIP6

by confocal immunofluorescence assay and Western blot analysis. In addition, the

expression of β-catenin and TRIP6 was dramatically increased in TTPAL expressing

xenografts tumors as compared with control xenografts. These results collectively

suggest that TTPAL function via activating Wnt signaling and interacting with TRIP6.

TRIP6 was reported to promote tumor growth in several cancer types and involve in

Wnt signaling (18-20). In keeping with this, we revealed that expression of TRIP6

significantly enhanced in CRC, signifying its oncogenic role in CRC. We then

uncovered that TRIP6 knockdown in colon cancer cells could significantly blunted the

effects of TTPAL on cell proliferation, colony formation, migration and Wnt signaling

activation. Conversely, TRIP6 expression can rescue Wnt activation and cancer-related

phenotypes in TTPAL-inhibited SW480 cells, inferring that the oncogenic role of

TTPAL in CRC is, at least in part, dependent on TRIP6.

To explore how TTPAL activates Wnt signaling through TRIP6, we assessed the

interplay between TTPAL and TRIP6. We found that the direct binding of TTPAL and

TRIP6 were existing in cytoplasm. TTPAL-TRIP6 complex may recruit lipid or

proteins which inhibited activity of ubiquitin ligases, in turn suppressed ubiquitin-

mediated degradation of TRIP6. Previous studies have shown that TRIP6 contains the

same PDZ-binding motif as β-catenin, and both TRIP6 and β-catenin are physiological

ligands of the PDZ domain-containing tumor suppressor MAGI1, therefore TRIP6

could bind to MAGI1 competitively with β-catenin (14,21-25). Our results

demonstrated that TTPAL expression promoted TRIP6-MAGI1 interaction, which in

turn inhibited the binding between MAGI and β-catenin. Meanwhile, both TRIP6

overexpression and MAGI1 silence dramatically induced Wnt signaling activation. In

addition, the impact of these findings was further strengthened by the observation that,

induction of TRIP6 promoted interaction between TCF4 and β-catenin. Whilst, PDZ

binding-defective mutant of TRIP6 was incapable of binding to MAGI1 and stimulating

cancer-related phenotypes. Moreover, TTPAL expression was positively correlated with

Wnt target genes in primary CRC tissues from TCGA cohort. Hence, these data imply

that, the direct binding of TTPAL with TRIP6 in cytoplasm inhibited ubiquitin-

mediated degradation of TRIP6. Then TRIP6, which contains the same PDZ-binding

motif as β-catenin, competitively bound to the PDZ domain-containing tumor

suppressor MAGI1 to release β-catenin. This sequence of events allowed β-catenin to

enter the nucleus and ultimately promoted the oncogenic Wnt/β-catenin signaling

cascade.

However, enhanced cell proliferation was seen even in the presence of a Wnt inhibitor.

Moreover, TTPAL knockdown showed weak effect on inhibiting cell growth in RKO

cells, whose Wnt signaling is not hyperactivated. These results inferred the potential

existence of Wnt-independent mechanism of cell proliferation induced by TTPAL in

CRC. In fact, oncogenic signaling pathways including NF-kB, Akt, JNK-AP-1, Hippo

have been reported mediated by TRIP6 in cancer (20,26,27), the direct downstream

effector of TTPAL. In this connection, TTPAL induced cell proliferation should

be a complex mechanism, in which multiple factors and signaling pathways in

additional to Wnt signaling being involved.

In summary, we demonstrate that TTPAL overexpression due to gene amplification is

a common event in CRC. TTPAL plays a pivotal oncogenic role by activating Wnt/β-

catenin signaling pathway through binding to and stabilizing TRIP6 in colorectal

carcinogenesis. Therapeutic intervention of TTPAL might be a novel strategy for

blunting uncontrolled growth and metastasis in Wnt/β-catenin addicted CRC. Moreover,

TTPAL may serve as an independent prognostic marker for CRC patients.

Acknowledgments: This project was supported by National Key R&D Program of

China (2016YFC1303200, 2017YFE0190700); Science and Technology Program

Grant Shenzhen (JCYJ20180307151253271, JCYJ20170413161534162); Postdoctoral

natural science foundation of China (2017M622802); RGC-GRF Hong Kong

(14111216, 14163817); National Natural Science Foundation of China (NSFC)

(81773000);Vice-Chancellor's Discretionary Fund CUHK and CUHK direct grant;

Shenzhen Municipal Science and Technology Shenzhen Virtual University Park

Support Scheme to CUHK Shenzhen Research Institute.

References

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer

statistics, 2012. CA Cancer J Clin 2015;65:87-108

2. Siegel DF, Hilsenroth MJ. Process and technique factors associated with patient

ratings of session safety during psychodynamic psychotherapy. Am J

Psychother 2013;67:257-76

3. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global

variation in copy number in the human genome. Nature 2006;444:444-54

4. Sokolova V, Crippa E, Gariboldi M. Integration of genome scale data for

identifying new players in colorectal cancer. World J Gastroenterol

2016;22:534-45

5. Ptashkin RN, Pagan C, Yaeger R, Middha S, Shia J, O'Rourke KP, et al.

Chromosome 20q Amplification Defines a Subtype of Microsatellite Stable,

Left-Sided Colon Cancers with Wild-type RAS/RAF and Better Overall

Survival. Mol Cancer Res 2017;15:708-13

6. Wang H, Liang L, Fang JY, Xu J. Somatic gene copy number alterations in

colorectal cancer: new quest for cancer drivers and biomarkers. Oncogene

2016;35:2011-9

7. Tanikawa C, Kamatani Y, Takahashi A, Momozawa Y, Leveque K, Nagayama

S, et al. GWAS Identifies Two Novel Colorectal Cancer Loci at 16q24.1 and

20q13.12. Carcinogenesis 2018

8. Houlston RS, Cheadle J, Dobbins SE, Tenesa A, Jones AM, Howarth K, et al.

Meta-analysis of three genome-wide association studies identifies susceptibility

loci for colorectal cancer at 1q41, 3q26.2, 12q13.13 and 20q13.33. Nat Genet

2010;42:973-7

9. Xu L, Li X, Cai M, Chen J, Li X, Wu WK, et al. Increased expression of Solute

carrier family 12 member 5 via gene amplification contributes to tumour

progression and metastasis and associates with poor survival in colorectal

cancer. Gut 2016;65:635-46

10. Wong CC, Qian Y, Li X, Xu J, Kang W, Tong JH, et al. SLC25A22 Promotes

Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations

and Xenograft Tumor Progression in Mice via Intracellular Synthesis of

Aspartate. Gastroenterology 2016;151:945-60 e6

11. Salasova A, Yokota C, Potesil D, Zdrahal Z, Bryja V, Arenas E. A proteomic

analysis of LRRK2 binding partners reveals interactions with multiple signaling

components of the WNT/PCP pathway. Mol Neurodegener 2017;12:54

12. Wang K, Liang Q, Li X, Tsoi H, Zhang J, Wang H, et al. MDGA2 is a novel

tumour suppressor cooperating with DMAP1 in gastric cancer and is associated

with disease outcome. Gut 2016;65:1619-31

13. Budczies J, Klauschen F, Sinn BV, Gyorffy B, Schmitt WD, Darb-Esfahani S,

et al. Cutoff Finder: a comprehensive and straightforward Web application

enabling rapid biomarker cutoff optimization. PloS one 2012;7:e51862

14. Chastre E, Abdessamad M, Kruglov A, Bruyneel E, Bracke M, Di Gioia Y, et

al. TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule,

promotes invasiveness. FASEB J 2009;23:916-28

15. Rooney PH, Murray GI, Stevenson DA, Haites NE, Cassidy J, McLeod HL.

Comparative genomic hybridization and chromosomal instability in solid

tumours. Br J Cancer 1999;80:862-73

16. Weiss MM, Snijders AM, Kuipers EJ, Ylstra B, Pinkel D, Meuwissen SG, et al.

Determination of amplicon boundaries at 20q13.2 in tissue samples of human

gastric adenocarcinomas by high-resolution microarray comparative genomic

hybridization. J Pathol 2003;200:320-6

17. Blain SW. Switching cyclin D-Cdk4 kinase activity on and off. Cell Cycle

2008;7:892-8

18. Gur'ianova OA, Sablina AA, Chumakov PM, Frolova EI. Down-regulation of

TRIP6 expression induces actin cytoskeleton rearrangements in human

carcinoma cell lines. Mol Biol (Mosk) 2005;39:905-9

19. Zhao W, Dai Y, Dai T, Xie T, Su X, Li J, et al. TRIP6 promotes cell proliferation

in hepatocellular carcinoma via suppression of FOXO3a. Biochem Biophys Res

Commun 2017;494:594-601

20. Lin VT, Lin FT. TRIP6: an adaptor protein that regulates cell motility,

antiapoptotic signaling and transcriptional activity. Cell Signal 2011;23:1691-7

21. Yi J, Beckerle MC. The human TRIP6 gene encodes a LIM domain protein and

maps to chromosome 7q22, a region associated with tumorigenesis. Genomics

1998;49:314-6

22. Zaric J, Joseph JM, Tercier S, Sengstag T, Ponsonnet L, Delorenzi M, et al.

Identification of MAGI1 as a tumor-suppressor protein induced by

cyclooxygenase-2 inhibitors in colorectal cancer cells. Oncogene 2012;31:48-

23. Jia S, Lu J, Qu T, Feng Y, Wang X, Liu C, et al. MAGI1 inhibits migration and

invasion via blocking MAPK/ERK signaling pathway in gastric cancer. Chin J

Cancer Res 2017;29:25-35

24. Gujral TS, Karp ES, Chan M, Chang BH, MacBeath G. Family-wide

investigation of PDZ domain-mediated protein-protein interactions implicates

beta-catenin in maintaining the integrity of tight junctions. Chem Biol

2013;20:816-27

25. Dobrosotskaya IY, James GL. MAGI-1 interacts with beta-catenin and is

associated with cell-cell adhesion structures. Biochem Biophys Res Commun

2000;270:903-9

26. Dutta S, Mana-Capelli S, Paramasivam M, Dasgupta I, Cirka H, Billiar K, et al.

TRIP6 inhibits Hippo signaling in response to tension at adherens junctions.

EMBO Rep 2018;19:337-50

27. Li L, Bin LH, Li F, Liu Y, Chen D, Zhai Z, et al. TRIP6 is a RIP2-associated

common signaling component of multiple NF-kappaB activation pathways. J

Cell Sci 2005;118:555-63

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 value RR (95% CI) P value Age 1.021 (0.997 to 1.045) 0.091 1.020 (0.996 to 1.045) 0.110 Gender 0.696 0.703 M 1.132 (0.608 to 2.107) 1.128 (0.606 to 2.101) F 1 1

Location 0.904

Colon 0.964 (0.36 to 1.736)

Rectum 1

Differentiation 0.586

Moderate or high 1.216 (0.602 to 2.457)

Low 1

TNM 0.067

II 1.789 (0.961 to 3.330)

III 1 TTPAL expression 0.028 0.034 High 2.202(1.090 to 4.447) 2.146 (1.061 to 4.340) Low 1 1 Chinese cohort - protein level Univariate Multivariate Variable RR (95% CI) P value RR (95% CI) P value Age 1.004 (0.987 to 1.021) 0.637 1.008 (0.992 to 1.025) 0.346 Gender 0793 0.634 M 1.949 (0.641 to 1.404) 1.103 (0.736 to 1.655) F 1 1 Location 0.279 Colon 0.805 (0.544 to 1.192) Rectum 1 Differentiation 0.142 Moderate or high 1.430 (0.887 to 2.306) Low 1 TNM Early (I & II) 0.227 (0.137 to 0.374) <0.0001 0.202 (0.122 to 0.355) <0.0001 Late (III & IV) 1 1 TTPAL expression <0.0001 <0.0001 High 1.954 (1.258 to 3.036) 2.441 (1.544 to 3.858) Low 1 1

RR, relative risk; 95% CI, 95% confidence interval.

Figure Legends

Figure 1. Overexpression of TTPAL is due to copy number gain in CRC and

predicts poor prognosis of CRC patients. (A) Copy number of TTPAL is

preferentially amplified in colon and rectum cancers as compared to 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. (D) TTPAL

mRNA expression was upregulated in CRC compared to paired adjacent normal tissues

as shown by RT-PCR (top panel) and qRT-PCR (lower left panels, Chinese cohorts 1

and 2 from Beijing and Hong Kong respectively). RNA-seq data from TCGA study also

upregulation of TTPAL in CRC as compared to adjacent normal tissues (lower right

panels, paired and unpaired samples). (E) TTPAL protein expression was upregulated

in primary CRC as compared to adjacent normal tissues by western blot. (F) TTPAL

protein expression was significantly higher in primary CRC as compared to adjacent

normal tissues as shown by IHC staining. (G) Kaplan–Meier survival analysis showed

CRC patients 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 CRC patients from TCGA study.

Figure 2. TTPAL exerts oncogenic functions in vitro. (A) Overexpression of TTPAL

in HCT116 and DLD1 cells, confirmed by RT-PCR and western blot, 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, 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 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 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.

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 SW1116 cells. (B) Western blot 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-immunoprecipitation (Co-IP)

followed by liquid chromatography-mass spectrometry (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 co-localized in cytoplasm as demonstrated by confocal

immunofluorescence analysis (F1) and western blot of membrane, cytoplasmic and

nuclear fractions (F2) in HCT116 and DLD1cells co-transfected 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 as compared to 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 β-catenin expression in xenografts. (C) Western blot

analysis further confirmed that TTPAL expression in HCT116 and DLD1 xenografts

increased the expression of TRIP6 and β-catenin. (D) Overexpression of TTPAL

promoted experimental metastasis of HCT116 cells in vivo. Representative images of

lungs and 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 to shNC

transfection in SW480. (F) Knockdown of TTPAL significantly decreased the number

of metastatic lesions in SW480 cells.

Figure 5. TTPAL exerts its oncogenic function partially depending on TRIP6 in

CRC. (A) Knockdown of TRIP6 in HCT116 and DLD1 cells with stable TTPAL

overexpression was confirmed by RT-PCR and western blot. (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 DLD1cells 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).

Figure 6. TTPAL activates Wnt/β-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 and RT-(q)PCR. (A2) The protein

level of TRIP6 was corelated 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 in HCT116 and DLD1 cells. Overexpression of TTPAL increased the

binding between TRIP6 and MAGI1 (right panel). (D) MAGI1-β-catenin interaction

was decreased by TRIP6 as shown by co-IP followed by western blot. (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 β-catenin and TCF4. (G) PDZ

binding-defective mutant of TRIP6 was incapable of binding to MAGI. (H) Wild-type

TRIP6 induced Wnt activation as compared to PDZ binding-defective mutant TRIP6

or vector transfection controls, which was indicated by the increased levels of active β-

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 CRC tissues from TCGA study. (J)

Schematic illustration of the molecular mechanism of TTPAL in Wnt signaling of CRC.

TTPAL promotes colorectal tumorigenesis by stabilizing TRIP6 to activate Wnt/ β-catenin signaling

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

Cancer Res Published OnlineFirst April 24, 2019.

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

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/04/24/0008-5472.CAN-18-2986.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2019/04/24/0008-5472.CAN-18-2986. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.