DCLK1 promotes epithelial-mesenchymal transition via the · Tumor metastasis is a complicated...

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DCLK1 promotes epithelial-mesenchymal transition via thePI3K/Akt/NF-jB pathway in colorectal cancer

Weiying Liu, Shixing Wang, Qi Sun, Zhen Yang, Min Liu and Hua Tang

Tianjin Life Science Research Center and Department of Pathogen Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

Double cortin-like kinase 1 (DCLK1) plays important roles during the epithelial-mesenchymal transition (EMT) process in

human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this

study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF-

jB-dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF-jBp65 subunit expression

through the PI3K/Akt/Sp1 axis and activated NF-jBp65 through the PI3K/Akt/IjBa pathway during the EMT of CRC cells.

Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collec-

tively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential thera-

peutic target for CRC metastasis.

Introduction

Colorectal cancer (CRC) is still the third most common can-

cer in the world,1 and the overwhelming majority of patients

diagnosed with advanced stage are usually accompanied by

metastasis.2,3 Metastasis in patients with CRC is a major

cause of recurrence before radical surgery, and patients with

synchronous liver metastasis exhibit a poor 5-year survival

rate of only 3.3%.3 Although the extensive published litera-

ture has uncovered several molecular mechanisms that cause

the metastasis in CRC, a variety of molecular factors and sig-

nal transduction pathways involving tumor metastasis still

remain largely elusive.4

Tumor metastasis is a complicated process influenced by

several molecular factors. Epithelial-mesenchymal transition

(EMT) has been considered to be a key program associated

with the invasion and metastasis of several malignant tumors,

including CRC.4,5 EMT allows the epithelial cells to disrupt

cell–cell adherence, lose their apical-basal polarity and

acquire mesenchymal characteristics through cytoskeletal

remodeling.6 During EMT, epithelial cells gradually lose the

expression of E-cadherin, which serves as the most important

epithelial marker while concomitantly increasing the expres-

sion of mesenchymal markers, such as Vimentin and Fibro-

nectin.7 EMT can be induced through the upregulation of

several upstream transcriptional regulators such as SNAIL,

ZEB and TWIST.8,9 Several signaling molecules and pathways

have been demonstrated to be involved in this process.

Double cortin-like kinase 1 (DCLK1, formerly known as

DCAMKL1), a member of the protein kinase superfamily and

the double cortin family, was initially identified as a critical reg-

ulator of neurogenesis and neuronal migration.10 Recently,

DCLK1 has been identified as a tumor stem cell marker in

CRC, pancreatic cancer and other cancers.11–16 DCLK1 protein

is highly expressed in several types of cancers, including CRC,17

pancreatic cancer,18 hepatocellular carcinoma (HCC),19 and

renal clear cell carcinoma (RCC).16 Moreover, recent studies

have demonstrated that the DCLK1 gene promoter is hyper

methylated in several cancers.20–23 Subsequent studies have

shown that the DCLK1 gene can encode four different isoforms

through alternative promoters and splicing mechanism, and

DCLK1 isoform 2, originating from alternate 50 (b)-promoter,

is highly expressed in human CRC cell lines and patient sam-

ples.24 DCLK1 can promote pluripotency, angiogenesis and

EMT through microRNA-dependent mechanisms through the

downregulation of several key tumor suppressor microRNAs.

Forinstance, DCLK1 can promote pluripotency through the

downregulation of the expression of miR-143/145, which can

Key words: double cortin-like kinase 1, colorectal cancer, epithelial-

mesenchymal transition, NF-jB, PI3K/Akt pathway

Abbreviations: CRC: colorectal cancer; DCLK1: Double cortin-like

kinase 1; EMT: epithelial-mesenchymal transition; NF-jB: nuclear

factor kappa-light-chain- enhancer of activated B cells

Additional Supporting Information may be found in the online

version of this article.

Conflict of interest: The authors declare that they have no conflicts

of interest with the contents of this article.

Grant sponsor: National Natural Science Foundation of China;

Grant numbers: 81602512, 91629302, 81572790, 31270818,

91029714; Grant sponsor: China Postdoctoral Science Foundation;

Grant number: 2015M581307

DOI: 10.1002/ijc.31232

History: Received 29 Sep 2017; Accepted 18 Dec 2017; Online 26

Dec 2017

Correspondence to: Hua Tang, PhD., Tianjin Life Science Research

Center and Department of Pathogen Biology, School of Basic

Medical Sciences, Tianjin Medical University, 22 Qi-Xiang-Tai Road,

Tianjin 300070, China, E-mail: [email protected]; Tel: 186 22

23542503

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Int. J. Cancer: 142, 2068–2079 (2018) VC 2017 UICC

International Journal of Cancer

IJC

http://orcid.org/0000-0003-3074-3561

result in an increase in the expression of pluripotency mainte-

nance factors such as NANOG, OCT4, KLF4, SOX2, as well as

RREB1 and KRAS.13 DCLK1 can promote EMT through the

downregulation of the expression of miR-200a, thus, increasing

the expression of the EMT-related transcription factors ZEB1,

ZEB2, SNAIL and SLUG.13,18 Although studies have demon-

strated that DCLK1 can regulate EMT through microRNA-

dependent mechanism, it is still unclear whether the underlying

mechanism of EMT is regulated by DCLK1.

In this study, we demonstrated that DCLK1 promotes

EMT through the NF-jB mediated mechanism in CRC cells.

On the one hand, DCLK1 increases the expression of NF-

jBp65 expression through the PI3K/Akt/Sp1 axis during

EMT, and, on the other hand, DCLK1 activates NF-jBp65

through the PI3K/Akt pathway. Taken together, these find-

ings may provide novel insights into the EMT mechanism

regulated by DCLK1, and targeting DCLK1 may be a thera-

peutic option for CRC metastasis.

Materials and Methods

Tissue samples

A total of 20 CRC tissues and their adjacent nontumor tis-

sues were acquired from the Department of Surgery, Second-

ary Hospital of Tianjin Medical University (Tianjin, China).

All the tissues confirmed by pathological analysis were imme-

diately collected, frozen in liquid nitrogen and stored at

2808C.

Cell culture and transfection

HCT116 and SW480 cell lines were cultured in RPMI-

1640medium (Invitrogen, Carlsbad, CA) supplemented with

10% fetal bovine serum (FBS; Gibco, Grand Island, NY), and

1% PS (100 U/ml penicillin, 100 lg/ml streptomycin) at 378C

and 5% CO2. Cells were transfected with the control, knock-

down and overexpressed vectors using LipofectamineTM2000

(Thermo Fisher Scientific, Inc.) according to the manufac-

turer’s protocols.

Plasmid construction

DCLK1 gene fragment was amplified from the cDNA of the

HCT116 cells and cloned into pcDNA3 vector (Invitrogen). Rel-

atively oligo nucleotides of DCLK1, p65, Sp1 and PIK3R1 were

annealed and cloned into pSilencer 2.1-U6 neo vector (Ambion,

Austin, TX), generation of their knockdown plasmids. All the

primers and oligonucleotides used in this study are listed in Sup-

porting Information Table supplement 1.

Real-time quantitative PCR

Total RNA was isolated using TRIzol reagent (Invitrogen)

according to the manufacturer’s protocol. Real-time quantita-

tive PCR (RT-qPCR) methods were performed to detect

mRNA levels as described previously.25 The housekeeping

gene b-actin was used as an internal reference gene for nor-

malization of the relative expression levels of target genes.

The primers used for qPCR are listed in Supporting Informa-

tion Table supplement 1.

Transwell assays

HCT116 and SW480 cells were seeded into 24-well plates for

16–18 hr, and then the cells were transfected with relative

plasmids and the culture was continued for 18–24 hr. Trans-

well migration and invasion assays were carried out using

transwell chamber inserts (8 lm-pore size membranes, Milli-

pore) with Matrigel (for invasion) or without Matrigel (for

migration). The procedures were carried out according to the

literature as described previously.26 Briefly, after transfection,

CRC cells were seeded into the upper chamber of the insert

(6 3 104 cells for HCT116 or 8 3 104 cells for SW480) in

200 ll of serum-free RPMI 1640 medium, followed by the

addition of 600 ll of serum-free RPMI 1640 medium con-

taining 20% FBS into the bottom of the insert. The HCT116

cells were cultured for 36 hr for migration and for 48 hr for

invasion, whereas the SW480 cells were allowed to migrate

for 48 hr and invade for 72 hr. The migrated or invaded cells

were stained with crystal violet and photographed using a

microscope.

Western blotting analysis

Western blotting was performed using antibodies against

DCLK1 (diluted 1:200), Vimentin (diluted 1:500), GAPDH

(diluted 1:2,000), E-cadherin (diluted 1:100), p65 (diluted

1:500), p-p65S536 (diluted 1:500), p-IjBaS32/36 (diluted

1:100),IjBa (diluted 1:500), ZEB1 (diluted 1:500), TWIST1

(diluted 1:500), Sp1 (diluted 1:500), PIK3R1 (diluted 1:200),

Akt (diluted 1:500) and p-AktS473 (diluted 1:500). DCLK1,

Vimentin, E-cadherin, GAPDH, Sp1, PIK3R1 were purchased

from Tianjin Saier Biotech (Tianjin, China). H2A.X, ZEB1,

TWIST1, IjBa, p65, p-p65S536, Akt and p-AktS473 were pur-

chased from Wanlei bio Company (Shenyang, China). p-

IjBaS32/36 was purchased from ImmunoWay Biotech Com-

pany (United States). Secondary goat anti-rabbit antibody

was purchased from Sigma-Aldrich (St Louis, MO). Whole

cell lysate and cytoplasmic proteins were normalized with an

What’s new?

A change in tumor cells called “epithelial-mesenchymal transition” (EMT) helps certain types of tumors to metastasize. In this

study of colorectal cancer (CRC), the authors found that an enzyme called DCLK1 acts as a potent oncogene to drive EMT, via

NF-jBp65 induction. They, then, further identified the molecular pathways involved in this event regulated through PI3K/Akt

pathway. Finally, they found that silencing DCLK1 expression could inhibit metastasis of CRC cells in vivo. These results sug-

gest that DCLK1 may offer a potential therapeutic target for metastatic CRC.

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Liu et al. 2069


internal reference protein GAPDH, while nuclear proteins

were normalized with H2A.X. LabWorks Image Acquisition

and Analysis Software (UVP, Upland, CA) were performed

to quantify the band density.

Immunofluorescent staining

Standard immunofluorescence protocols were performed as

previously described.26 Specific primary antibodies against

Dclk1 (anti-rabbit, diluted 1:100) was obtained from Tianjin

Saier Biotech (Tianjin, China), and p65 (anti-mouse, diluted

1:200) was obtained from ImmunoWay, United States. Alexa

Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey

anti-mouse IgG were obtained from Invitrogen (United States).

Luciferase reporter assay

CRC cells were transfected with the indicated luciferase

reporter constructs and the luciferase activity was measured

by Dual luciferase assay system (Promega, Madison WI)

according to previously described.27,28

Immunohistochemical analysis

Standard immunohistochemistry (IHC) protocols were per-

formed with specific antibodies according to previously

reported.29

In vivo metastasis assay

Briefly, 1 3 106 cells of HCT116 stable cell strains carrying

empty vector with control shRNA or shDCLK1 plasmid were

injected into the lateral tail vein of 6-week-old female nude

mice. After 7 weeks of initial implantation, the mice were

euthanized and the lung tissues were isolated for further

study. Animal handling and procedures were carried out in

accordance with the National Institutes of Health guide for

the care and use of Laboratory animals (NIH Publications

No. 8023, revised 1978) and were approved by the Tianjin

Medical University Institutional Animal Care.

Statistical analysis

For statistical analyses, student’s t test was performed using

Graph Pad Prism 6, p values< 0.05 were considered statistically

significant (*p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001).

All experiments were performed independently at least three

times.

Results

DCLK1 promotes EMT in CRC cells

To investigate the role of DCLK1 in CRC, we first assessed

the results of DCLK1 (DCLK1 isoform 2, also termed as

DCLK1-S) expression levels by IHC and RT-qPCR methods.

The results showed that the expression level of DCLK1 was

markedly upregulated in human CRC tissues compared with

that in adjacent noncancerous tissues (Figs. 1a and 1b). Our

finding consisted with the recently reported finding that

DCLK1-S is highly expressed in human CRC cell lines and

patient samples.24 Then, the results of RT-qPCR and Western

blot analysis showed that overexpression of DCLK1 could

markedly increase the transcriptional level and protein level

of DCLK1 in both HCT116 and SW480 cells (Figs. 1c and 1d

and Supporting Information Fig. 1a). In contrast, the oppo-

site phenomenon was observed in HCT116 and SW480 cells

in which DCLK1-knockdown plasmid was stably expressed

(Figs. 1c and 1d and Supporting Information Fig. 1a). The

role of DCLK1 in cell migration and invasion in CRC cells

was determined by transwell assays, which showed that the

overexpression of DCLK1 markedly promoted the cell migra-

tion and invasion abilities; whereas, the downregulation of

DCLK1 exhibited relatively lower abilities (Figs. 1e and 1f).

To further investigate the underlying mechanisms of how

DCLK1 promotes cell migration and invasion, we detected

the expression of critical EMT markers, E-cadherin and

Vimentin. RT-qPCR and Western blot analysis revealed that

overexpression of DCLK1 led to a reduction of E-cadherin

expression, which is an epithelial marker, but it significantly

increased the expression of mesenchymal markers such as

Vimentin at both the mRNA and protein levels (Figs. 1g and

1h and Supporting Information Figs. 1b and 1c). In contrast,

the downregulation of DCLK1 could increase E-cadherin

expression but reduce Vimentin expression at both the

mRNA and protein levels (Figs. 1g and 1h and Supporting

Information Figs. 1b and 1c). In addition, we also detected

upstream transcriptional regulators (ZEB1and TWIST1) of

EMT, wherein the results showed that overexpression of

DCLK1 increased the expression of ZEB1and TWIST1, which

was reduced by the downregulation of DCLK1 at both the

mRNA and protein levels (Figs. 1g and 1h and Supporting

Information Figs. 1b and 1c).Taken together, our results indi-

cated that DCLK1 could promote the migration and invasion

of CRC cells through the induction of EMT.

DCLK1 promotes EMT through the upregulation

of p65 expression

To ourknowledge, the short form of DCLK1 (DCLK1 isoform

2) has been reported to be highly expressed in human CRC

tissues, and the high expressors had a markedly worse overall

survival compared to that of low expressors.24 However, in

mouse CRC tissues, it has been demonstrated that the long

form of DCLK1 (DCLK1 isoform1) was highly expressed.24

Previous reports have shown that p65 was highly expressed

in DCLK11 small intestinal epithelial tuft cells of mouse.30

Increasing evidence havs indicated that the NF-jB subunit

p65 playes a central role in the induction of EMT and the

promotion of tumor metastasis.31 To investigate whether p65

is involved in DCLK1-induced EMT, we analyzed the expres-

sion level of p65 in both HCT116 and SW480 cells. Our

results showed that p65 expression was significantly increased

in CRC tissues compared with that in the adjacent noncan-

cerous tissues (Figs. 2a and 2b), and the expression of p65

showed a positive correlation with the upregulation of

DCLK1 mRNA (Fig. 2c). Overexpression of DCLK1 in CRC

cells increased the p65 expression at both the mRNA and

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2070 DCLK1 regulates PI3K/Akt/NF-jB pathway


Figure 1. DCLK1 promotes migration and invasion of CRC cells via inducing EMT. (a) IHC staining of DCLK1 in normal tissues and CRC tis-

sues. (b) RT-qPCR analysis of DCLK1 mRNA levels in twenty pairs of human colorectal cancer tissues compared to adjacent noncancerous

tissues. (c) RT-qPCR analysis of DCLK1 mRNA levels in human CRC cell lines HCT116 and SW480 stably transfected with empty, DCLK1 over-

expression or knockdown plasmids. (d) Western blot analysis of DCLK1 expression levels in stable DCLK1 overexpressing or knockdown

CRC cells. (e and f) Migration and invasion experiments of CRC cells. (g) RT-qPCR analysis and (h) Western blot analysis of EMT-related

markers extracted from stably transfected cells. All of the experiments were repeated three times. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001. [Color figure can be viewed at wileyonlinelibrary.com]

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Liu et al. 2071


http://wileyonlinelibrary.com

protein levels, whereas the mRNA and protein levels of p65

expression were decreased by the knockdown of DCLK1

(Figs. 2d and 2e and Supporting Information Fig. 2a). To

confirm whether DCLK1 promotes EMT through the p65-

mediated mechanism, we detected the effect of p65 expres-

sion on the DCLK1-induced cell migration and invasion in

Figure 2. DCLK1 promotes EMT via increasing p65 expression. (a) IHC staining of p65 in normal tissues and CRC tissues. (b) RT-qPCR analy-

sis of p65 mRNA levels in twenty pairs of human colorectal cancer tissues compared to adjacent noncancerous tissues. (c) Positive correla-

tion between the expression of DCLK1 and p65 was performed by Pearson’s correlation analysis (R50.72 ***, p<0.001). (d) RT-qPCR

analysis of p65 mRNA levels in human CRC cell lines that stably transfected with empty, DCLK1 overexpression or knockdown plasmids. (e)

Western blot analysis of p65 expression levels in stable DCLK1 overexpressing or knockdown CRC cells. (f and g) Migration and invasion

experiments of CRC cells showed that knockdown of p65 could partly rescue the migration and invasion abilities. (h) RT-qPCR analysis and

(i) Western blot analysis showed that knockdown of p65 could partly rescue the expression levels of EMT-related markers induced by

DCLK1. All of the experiments were repeated three times. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; NS, not significant. [Color

figure can be viewed at wileyonlinelibrary.com]

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CRC cells. The knockdown of p65 partly blocked the

DCLK1-induced cell migration and invasion in CRC cells

(Figs. 2f and 2g), as well as the expression of ZEB1, TWIST1,

E-cadherin and Vimentin (Figs. 2h and 2i and Supporting

Information Figs. 2b and 2c). These results indicated that

DCLK1-induced EMT is dependent on the expression of p65.

DCLK1 upregulates p65 expression by increasing Sp1

expression during EMT

It has been reported that the transcription factor Sp1 is

involved in the upregulation of the NF-jB subunit p65 by

binding the p65 promoter and increasing the p65 promoter

activity.32,33 To assess whether Sp1 is required for the upre-

gulation of p65 expression in DCLK1-induced EMT, we first

observed that Sp1 expression was highly expressed in CRC

tissues (Figs. 3a and 3b), which also showed a positive corre-

lation with the upregulation of DCLK1 (Fig. 3c). Sp1 was sig-

nificantly increased in DCLK1-overexpressed CRC cells,

whereas the expression of Sp1 was suppressed by the knock-

down of DCLK1 (Figs. 3d and 3e and Supporting Informa-

tion Fig. 3a). The knockdown of Sp1 partly rescued DCLK1-

induced changes in the EMT phenotype, including the migra-

tion and invasion (Figs. 3f and 3g), and the associated EMT

markers (E-cadherin and Vimentin) (Figs. 3h and 3i and

Supporting Information Figs. 3b and 3c). More importantly,

the knockdown of Sp1 was able to block the DCLK1-induced

upregulation of p65 expression (Figs. 3h and 3i and Support-

ing Information Figs. 3b and 3c). Therefore, our results indi-

cated that the induction of EMT and the upregulation of p65

expression by DCLK1 are dependent on Sp1 expression.

DCLK1 upregulates Sp1 expression via the PI3K/Akt

pathway

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling path-

way can increase the transcriptional expression of Sp1, and

the PI3K/Akt/Sp1 pathway is also positively involved in sev-

eral cellular functions.34–36 PI3Ks exist as heterodimers con-

taining a unique catalytic subunit (p110a, b or d) along with

one of a number of shared regulatory subunits (p85a, p85b

or p55d),37 and the p85a regulatory subunit of PI3K

(PIK3R1) is an oncogene involved in the tumorigenesis of

human colon cancer and ovarian tumors.38 To investigate the

role of PIK3R1 in DCLK1-induced EMT, we first determined

the expression level of PIK3R1 in CRC tissues and normal

tissues, and the results showed that PIK3R1 was upregulated

in CRC tissues compared with that in the adjacent noncan-

cerous tissues (Figs. 4a and 4b), which also displayed a posi-

tive correlation with the upregulation of DCLK1 (Fig. 4c).

PIK3R1 expression was markedly upregulated in DCLK1-

overexpressed CRC cells, whereas the expression of PIK3R1

was reduced by the silencing of DCLK1 (Figs. 4d and 4e and

Supporting Information Fig. 4a). The silencing of PIK3R1

partly blocked DCLK1-induced changes in EMT phenotype

(Figs. 4f and 4g), and associated EMT markers (E-cadherin

and Vimentin) (Figs. 4h and 4i and Supporting Information

Figs. 4b and 4c). Moreover, downregulation of PIK3R1 could

abrogate the DCLK1-induced upregulation of Sp1 and p65

expression (Figs. 4h and 4i and Supporting Information Figs.

4b and 4c), indicating that blockage of the PI3K/Akt pathway

reversed DCLK1-induced EMT.

DCLK1 promotes EMT through the activation of NF-jB

signaling

NF-jBp65 performs its function requiring the translocation

of thep65 subunit from the cytoplasm to the nucleus. To fur-

ther investigate the effects of DCLK1 on NF-jB activation,

immunofluorescence analysis showed that the nuclear form

of p65 was markedly higher in DCLK1-overexpressed CRC

cells than that in empty vector- transfected cells, and the

reverse results were obtained in DCLK1-downexpressed CRC

cells (Fig. 5a). Furthermore, Western blot analysis demon-

strated that DCLK1 could activate p65 expression in CRC

cells (Fig. 5b and Supporting Information Figs. 5a and 5b).

Luciferase reporter assays demonstrated that NF-jB tran-

scriptional activity was significantly higher in stably DCLK1-

transfected cells than that in empty vector-transfected cells,

and NF-jB transcriptional activity was relatively lower in sta-

bly DCLK1-downregulated cells than in empty vector-

transfected cells (Fig. 5c). Taken together, these results indi-

cated that NF-jB signaling pathway was activated in

DCLK1-induced EMT.

DCLK1 activates NF-jB through the PI3K/Akt pathway

during EMT

Our results have demonstrated that the DCLK1/PI3K/Akt/

Sp1 axis could increase the expression of p65. However, it is

known that the PI3K/Akt pathway can also activate NF-jB

signaling.39 Therefore, we hypothesized that DCLK1 activated

NF-jB through the PI3K/Akt pathway in CRC cells. To clar-

ify this hypothesis, we first detected the expression levels of

PIK3R1, p-Akt, p-IjB and p-p65 in stably DCLK1-

overexpressed CRC cells and found that PIK3R1, p-Akt, p-

IjB, p-p65 and downstream signal molecules were all upre-

gulated in such cells (Supporting Information Figs. 6a and

6b, Fig. 6a and Supporting Information Figs. 7a and 7b). In

addition, PIK3R1, p-Akt, p-IjB, p-p65 and downstream sig-

nal molecules were all downregulated while DCLK1 was

knocked down in CRC cells (Supporting Information Figs. 6a

and 6b, Fig. 6a and Supporting Information Figs. 7a and 7b).

To further validate the involvement of PI3K/Akt in NF-jB

activation, we found that the knockdown of PIK3R1 abro-

gated the expression levels of p-Akt, p-IjB, p-p65 and down-

stream signal molecules in DCLK1-induced cells (Supporting

Information Figs. 6c and 6d, Fig. 6b and Supporting Informa-

tion Figs. 7c and 7d). Moreover, in vivo animal metastasis

experiments showed that silencing DCLK1 inhibited lung

metastasis of CRC cells (Fig. 6c). Taken together, these results

indicated that DCLK1 exactly activates NF-jB through the

PI3K/Akt pathway during EMT.

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Discussion

DCLK1 is known to be a critical regulator involved in the promo-

tion of pluripotency, angiogenesis and EMT through microRNA-

dependent mechanisms by downregulating several key tumor

suppressor microRNAs. Except for the microRNA-dependent

mechanisms, the underlying mechanism of EMT induced by

DCLK1 is still unclear.

The current study provides novel insights into how

DCLK1 promotes EMT through the NF-jB-mediated mecha-

nism in CRC cells. Due to the fact that p65 was highly

Figure 3. DCLK1 increaes p65 expression via upregulation of Sp1 expression. (a) IHC staining of Sp1 in normal tissues and CRC tissues. (b)

RT-qPCR analysis of Sp1 mRNA levels in twenty pairs of human colorectal cancer tissues compared to adjacent noncancerous tissues. (c)

Positive correlation between the expression of DCLK1 and Sp1 was performed by Pearson’s correlation analysis (R50.70 ***, p<0.001).

(d) RT-qPCR analysis of Sp1 mRNA levels in human CRC cell lines that stably transfected with empty, DCLK1 overexpression or knockdown

plasmids. (e) Western blot analysis of Sp1 expression levels in stable DCLK1 overexpressing or knockdown CRC cells. (f and g) Migration

and invasion experiments of CRC cells showed that knockdown of Sp1 could partly rescue the migration and invasion abilities. (h) RT-qPCR

analysis and (i) Western blot analysis showed that knockdown of Sp1 could partly rescue the expression levels of EMT-related markers

induced by DCLK1. All of the experiments were repeated three times. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; NS, not sig-

nificant. [Color figure can be viewed at wileyonlinelibrary.com]

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Figure 4. DCLK1 increases p65 expression via upregulation of PIK3R1 expression. (a) IHC staining of PIK3R1 in normal tissues and CRC tis-

sues. (b) RT-qPCR analysis of PIK3R1 mRNA levels in twenty pairs of human colorectal cancer tissues compared to adjacent noncancerous

tissues. (c) Positive correlation between the expression of DCLK1 and PIK3R1 was performed by Pearson’s correlation analysis (R50.70

***, p<0.001). (d) RT-qPCR analysis of PIK3R1 mRNA levels in human CRC cell lines that stably transfected with empty, DCLK1 overexpres-

sion or knockdown plasmids. (e) Western blot analysis of PIK3R1 expression levels in stable DCLK1 overexpressing or knockdown CRC cells.

(f and g) Migration and invasion experiments of CRC cells showed that knockdown of PIK3R1 could partly rescue the migration and invasion

abilities. (h) RT-qPCR analysis and (i) Western blot analysis showed that knockdown of PIK3R1 could partly rescue the expression levels

of EMT-related markers induced by DCLK1. All of the experiments were repeated three times. * p<0.05; ** p<0.01; *** p<0.001;

**** p<0.0001; NS, not significant. [Color figure can be viewed at wileyonlinelibrary.com]

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expressed in DCLK11 small intestinal epithelial tuft cells of

mouse,30 we hypothesized that DCLK1 also promotes the

expression of p65 in human CRC. Our results showed that

DCLK1 exactly promoted the mRNA and protein levels of

p65, which was involved in the DCLK1-induced EMT pro-

cess. Previous studies have shown that the p65 promoter

region contains Sp1 binding sites and the increase of Sp1

expression can increase the p65 promoter activity and upre-

gulate NF-jB subunit p65 expression.32,33 To investigate

whether Sp1 involving in upregulating p65 expression, we

first observed whether Sp1 was regulated by DCLK1. We

found that DCLK1 promoted Sp1 expression, subsequently

increasing the expression of p65. Knockdown of Sp1 partly

rescued the DCLK1-induced changes in the EMT phenotype

and the associated EMT markers (E-cadherin and Vimentin).

Given that the PI3K/Akt/Sp1 pathway is positively involved

in several cellular functions,34–36 we analyzed the role of

PI3K/Akt in Sp1 expression during DCLK1-induced EMT.

PIK3R1 is the regulatory subunit of PI3Ks and has proven to

be an oncogene involved in the tumorigenesis of human

colon cancer and ovarian tumors.38 We observed that

PIK3R1 was markedly upregulated in DCLK1-overexpressed

CRC cells, and the silencing of PIK3R1 partly blocked the

DCLK1-induced changes in the EMT phenotype and the

associated EMT markers (E-cadherin and Vimentin). Fur-

thermore, the downregulation of PIK3R1 was able to abro-

gate the DCLK1-induced upregulation of Sp1 and p65

expression. Accordingly, p65 is involved in the DCLK1-

induced EMT through the PI3K/Akt/Sp1 pathway in CRC

cells.

Figure 5. NF-jB is activated in the regulation of EMT induced by DCLK1. (a) Confocal image of DCLK1 (red) and p65 (green) in HCT116 and

SW480 cells, Cell nuclei were stained with DAPI (blue). Scale bars, 20 lm. (b) DCLK1 and p65 protein levels in whole cell lysate, cytosol

and nucleus were analyzed by Western blot. (c). NF-jB reporter vector and indicated plasmids were cotransfected into HCT116 and SW480

cells, respectively, after which luciferase activity was measured. All of the experiments were repeated three times. ** p<0.01. [Color figure

can be viewed at wileyonlinelibrary.com]

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Although DCLK1 can promote EMT by increasing the

expression of p65, the activation of NF-jB has been demon-

strated to be a recognized event in the EMT process.31

Immunofluorescence analysis, Western blot analysis and

luciferase reporter assays were performed to demonstrate

whether the NF-jB signaling pathway was activated in

DCLK1-induced EMT. It is known that the PI3K/Akt path-

way not only increases the p65 expression through the upre-

gulation of Sp1 but also activates NF-jB signaling.39 To

clarify whether the PI3K/Akt pathway is also involved in the

activation of p65 during the DCLK1-induced EMT process,

the downstream molecules of PI3K were detected by Western

blotting. The expression levels of p-Akt, p-IjB and p-p65

were all upregulated in stably DCLK1-overexpressed CRC

cells, indicating the possible activation of NF-jB by the

PI3K/Akt pathway. PIK3R1 is the p85a regulatory subunit of

PI3K and has been proven to be an oncogene involved in

human tumorigenesis. However, the role of PIK3R1 in NF-

Figure 6. DCLK1 mediates regulation of NF-jB via PI3K-AKT pathway during EMT. (a) Western blot analysis showed that DCLK1 could upregu-

lation the expression levels of p65 and the downstream EMT-related markers via PI3K-AKT pathway. (b) Western blot analysis showed that

knockdown of PIK3R1 could partly rescue the expression levels of p65 and the downstream EMT-related markers. (c) Knockdown of DCLK1

inhibits the lung metastasis abilities of CRC cells in vivo; relative H&E staining results of metastatic nodules in the lung are shown.

(d) Proposed model of DCLK1-induced EMT via upregulation and activation of NF-jB pathway in CRC cells. All of the experiments

were repeated three times. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; NS, not significant. [Color figure can be viewed at

wileyonlinelibrary.com]

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Liu et al. 2077



jB activation is still unknown.38 To further validate whether

the activation of NF-jB is dependent on the increased

expression of PIK3R1 in DCLK1-induced EMT, we observed

that the downregulation of PIK3R1 blocked the expression

levels of p-Akt, p-IjB and p-p65 in DCLK1-induced cells.

Being the important transcription factors and the major regu-

lators in the EMT, ZEB1 and TWIST1 are directly regulated

by NF-jB.40,41 The expression levels of ZEB1 and TWIST1,

as well as their downstream EMT markers E-cadherin and

Vimentin, were also regulated by PIK3R1 in DCLK1-induced

EMT. Moreover, it has been reported that NF-jB can directly

bind the promoter of Vimentin to directly regulate its expres-

sion.42 Taken together, these results indicated that DCLK1

exactly activates NF-jB through the PI3K/Akt pathway dur-

ing EMT.

In summary, our study demonstrated the involvement of

NF-jB in the DCLK1-induced EMT process in CRC cells.

DCLK1 can promote EMT through the upregulation of NF-

jBp65 expression by the PI3K/Akt/Sp1 axis, and NF-jBp65

is also activated through the PI3K/Akt pathway (Fig. 6d).

Our findings may provide a new insight into the DCLK1-

mediated EMT mechanism, thus, implicating DCLK1 as a

potential therapeutic target for CRC metastasis.

Author Contributions

W. Y. L. and H. T. designed the study and wrote the paper. W.

Y. L, S. X. W., Q. S. and Z. Y. performed the experiments. M. L.

provided technical assistance and contributed to the preparation

of the figures and manuscript. All authors reviewed the results

and approved the final version of the manuscript.

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