Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma Via Transcriptional Repression of Twist1

Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nkx2.8 inhibits epithelial-mesenchymal transition in bladder

urothelial carcinoma via transcriptional repression of Twist1

Chunping Yu 1,2,3§, Zhuowei Liu1,2,3§, Qiuhong Chen1,2,3§, Yonghong Li1,2,3, Lijuan

Jiang1,2,3, Zhiling Zhang1,2,3*, Fangjian Zhou1,2,3*

1 State Key Laboratory of Oncology in Southern China, Guangzhou, China

2 Department of Urology, Sun Yat-sen University Cancer Center, Guangzhou, China

3 Collaborative Innovation Center for Cancer Medicine, Guangzhou, China

§Yu CP, Liu ZW and Chen QH contributed equally to this work.

*To whom all correspondence should be addressed:

Zhang ZL, Department of Urology, Cancer Center, Sun Yat-sen University,

Guangzhou, China

Phone: 86-20-87343860; Fax: 86-20-87343656; E-mail: [email protected]

Or Zhou FJ, Department of Urology, Cancer Center, Sun Yat-sen University,

Guangzhou, China

Phone: 86-20-87343312; Fax: 86-20-87343656; E-mail: [email protected]

Running title: Nkx2.8 regulates EMT through Twist1

Keywords: Nkx2.8, EMT, bladder urothelial carcinoma, Twist1

The authors declare no potential conflicts of interest.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT Epithelial-to-mesenchymal transition (EMT) promotes metastasis which is the

main cause of bladder urothelial carcinoma (UC)-related death. Loss of the candidate

tumor suppressor gene Nkx2.8 has been associated with UC lymph node metastasis.

Here we show that enforced expression of Nkx2.8 is sufficient to inhibit EMT, reduce

motility and blunt invasiveness of UC cells. Mechanistic investigations showed that

Nkx2.8 negatively regulated expression of the EMT inducer Twist1 in UC cells, at

both the level of mRNA and protein accumulation. Nkx2.8 bound directly to the

promoter region of this gene and transcriptionally repressed its expression. Twist1

upregulation reversed EMT inhibition by Nkx2.8, restoring the invasive phenotype of

UC cells. In clinical UC specimens, expression of Nkx2.8 inversely correlated with

Twist1 expression, and UC patients with Nkx2.8 positivity and low Twist1 expression

displayed the best prognosis. Our findings highlight the Nkx2.8-Twist1 axis as

candidate target for therapeutic intervention in advanced UC.

INTRODUCTION

The prognosis of metastatic bladder urothelial carcinoma (UC) is extremely poor,

with a median survival time of less than 15 months, even with systematic therapy (1).

However, the mechanism underlying UC metastasis is not clear.

Epithelial-to-mesenchymal transition (EMT), a process of cell phenotypic change, is

characterized by decreased E-cadherin expression and weakened adhesive cell-cell or

cell-stroma attraction and subsequently facilitates cell migration (2-4). EMT has been

implicated in the conversion of early stage tumors into invasive malignancies (2-4),

which suggests that a better understanding of the mechanism underlying EMT may be

important with respect to the clinical management of UC.

Twist, a highly conserved transcriptional factor, has been well known as a key

EMT inducer (5). Twist down-regulates E-cadherin expression, up-regulates

fibronectin and vimentin expression, and subsequently facilitates EMT (5-7). Yang

and colleagues (6) has found that forced expression of Twist results in decrease of

E-cadherin-mediated adhesion and induction of cell motility, which suggests that

Twist1 plays a promoting role in EMT. It is also well known that Twist1 participates

in regulating the expression of Bmi-1, miR-200 and miR-205 (7-8), which are all

involved in EMT. In UC, Twist has been found to be associated with tumor grade and

progression and is inversely correlated with E-cadherin expression (9-11). Moreover,

in bladder cancer tissues Twist is always been found to be negatively associated with

the expression of E-cadherin (10-11). In addition, Twist is reported to be involved in

UC invasiveness (9-10, 12). However, how Twist expression levels are regulated in

UC remains a mystery.

Human Nk2 homeobox 8 (Nkx2.8), which acts as a transcription factor, is a

member of the NK-2 gene family (13). Nkx2.8 usually binds to DNA sequences

containing 5'-(C/T)AAG-3' motifs (14-16). The findings of several studies have

suggested that Nkx2.8 acts as a tumor suppressor in human carcinogenesis (17-19). In

lung cancer, Harris and colleagues (17) found most tumors had low expression of

Nkx2.8 and enforced expression of Nkx2.8 can inhibit proliferation of lung cancer

cells. Lin et al (18) reported that down-regulation of Nkx2.8 can activate NF-B and

promote angiogenesis in esophageal cancer cells. Qu and colleagues (19) also found a

tumor suppressive role of Nkx2.8 in human liver cancer. Our previous study

established that Nkx2.8 expression was markedly reduced in UC tissues and that

Nkx2.8 negativity was associated with lymph node metastasis and prognosis in UC

patients (20). However, the role of Nkx2.8 in metastasis and the regulatory

mechanisms underlying this phenomenon are largely unknown. In the current study,

we investigated the role of Nkx2.8 in EMT, the relationship between Nkx2.8 and

Twist1 in UC and the mechanisms underlying the effects of Nkx2.8 in UC.

Materials and Methods

Cell lines

The bladder cancer cell lines T24, 5637 and J82 were obtained from ATCC in

2013. The BIU87 and EJ were obtained from the institute of urology at the first

affiliated hospital of Peking University as a gift in 2012. All cell lines were

maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum

(HyClone, Logan, UT), penicillin (100units/mL) , and streptomycin (100 units/mL)

and tested to ensure mycoplasma free. All cell lines used in this study were

authenticated three months before the beginning of the study (2013) based on viability,

recovery, growth, morphology, and isoenzymology by the supplier and all the cell

lines have not been in culture for greater than 2 months. The pBabe-Nkx2.8 and

pSuper-retro-Nkx2.8 RNAi(s) were generated as described previously (20).

Plasmids and retroviral infection

The wild type human TWIST1 promoter and the TWIST1 promoter with a deletion

or mutation of the Nkx2.8 binding sites were individually cloned into the pGL3

luciferase reporter plasmid (Promega). UC cells with endogenous silencing of Nkx2.8

and cells with the forced expression of exogenous Nkx2.8 were generated as

previously described. The T24 cells exhibited no expression of Nkx2.8 and were

infected with retroviruses carrying pBabe-Nkx2.8. The BIU87 cells showed high

expression levels of Nkx2.8 and were infected with retroviruses carrying

pSuper-retro-Nkx2.8-shRNAs. The 5637 cells showed moderate expression levels of

Nkx2.8 and thus were infected with retroviruses carrying either pBabe-Nkx2.8 or

pSuper-retro-Nkx2.8-shRNAs. Stable cell lines were selected with 0.5 g/ml

puromycin for 10 days after transfection. Cell lysates, which were prepared from

pooled populations of cells in sample buffer, were fractionated by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis to confirm Nkx2.8 protein levels.

RNA extraction, quantitative real-time PCR

Total RNA samples from cultured cells were extracted using Trizol reagent

(Invitrogen) according to the manufacturer’s instructions. The sequences of the

primers are listed in Supplemental Table 1. Quantitative real-time PCR (qRT-PCR)

was performed using an ABI PRISM 7500 Sequence Detection System (Applied

Biosystems). The housekeeping gene glyceraldehyde-3 phosphate dehydrogenase

(GAPDH) was used as an internal quantitative control.

Western blot and immunofluorescence analyses

Western blot and immunofluorescence analyses were performed according to

standard methods as described previously(20) using anti-Nkx2.8 (Santa Cruz

Boitechnology, Inc), anti-Twist1 (Abcam), anti-E-cadherin, anti-α-catenin,

anti-fibronectin and anti-vimentin (BD Transduction Laboratories) antibodies. For the

Western blot assays, anti-α-Tubulin antibody (Sigma) was used as a loading control.

For immunofluorescence analysis, the coverslips were counterstained with 4',

6-diamidino-2-phenylindole and imaged with a confocal laser-scanning microscope

(Olympus FV1000). The quantification of immunofluorescence staining was

measured using Olympus FV10-ASW software.

Patient information and immunohistochemistry

The study has been approved by Institutional Review Board of Sun Yat-sen

University Cancer Center and the study was performed in accordance with

Declaration of Helsinki. Written informed consent was obtained from the patients

before the study began. Twist1 expression in the paraffin-embedded bladder cancer

tissues of the previously studied 161 cases was detected by immunohistochemistry

(IHC) using an anti-Twist1 antibody. Patient information and the method for scoring

Nkx2.8 has been previously reported (20). For the Twist1 labeling index, we used the

following scoring system. In brief, the proportion of positive cells in the stained

sections was evaluated at ×200 magnification, and the mean value of 10

representative fields analyzed from each section was recorded. The proportion of

positive cells was scored as follows: <25%, 1; 25% to 50%, 2; 50% to 75%, 3;

and >75%, 4. We judged the intensity of Twist1 staining according to 4 categories:

negative, 0; weak, 1; moderate, 2; and strong, 3. We used the product of the staining

intensity score and the proportion of positive tumor cells as the staining index. Then,

the scores were divided into 2 groups (0-4, low expression; 5-12, high expression).

3D morphogenesis Matrigel culture

Cells infected with vector, Nkx2.8 or Nkx2.8 RNAi were seeded in 24-well

dishes coated with Growth Factor-Reduced Matrigel (BD Biosciences) at a density of

1×104 cells/well and were covered with growth medium supplemented with 2%

Matrigel. The medium was exchanged with 2% Matrigel every 3 to 4 days. Images

were captured via microscopy at 2-day intervals for 2-3 weeks. The quantification of

3D morphogenesis Matrigel culture was represented by mean spheroid area measured

using Olympus cellSens Standard 1.9.

Wound-healing assay

Cells infected with vector, Nkx2.8 or Nkx2.8 RNAi were seeded in 6-well plates

and grown under permissive conditions until reaching 90% confluence. The cells were

then serum starved for 24 h, and a linear wound was created in the confluent

monolayer using a pipette tip. Wounds were photographed immediately (time 0 h) and

thereafter at 10 and 20 h. Wound size was measured randomly at 10 sites

perpendicular to the wound. Each experiment was repeated at least three times.

Boyden chamber invasion assay

Cells infected with vector, Nkx2.8 or Nkx2.8 RNAi were serum starved for 24 h.

Then, 2×104 cells were plated into the upper chamber of a polycarbonate transwell

filter chamber coated with Matrigel (BD Biosciences) and incubated for 20 h in

serum-free medium. The lower chamber contained the usual serum-containing

medium as chemo-attractant. At the end of the 20-h incubation, cells inside the

chamber were removed with cotton swabs. The invaded cells that remained on the

lower surface of the filter were fixed in 1% paraformaldehyde, stained with

hematoxylin and counted (10 random 100× fields per well). Cell counts were

expressed as the mean number of cells per field of view. Three independent

experiments were performed, and the data were presented as the average±SEM.

Chromatin immunoprecipitation (ChIP) assays

The ChIP assay was performed according to the protocol described previously.

6 In brief, cells were plated at a concentration of 2×10 cells per 100-mm diameter dish

and cross-linked with 1% formaldehyde for 10 min. The cells were trypsinized and

resuspended in lysis buffer. The nuclei were then isolated and sonicated to shear the

DNA into 500-bp to 1-kb fragments, which was verified by agarose gel

electrophoresis.

Equal aliquots of chromatin supernatants were incubated with 1 g of

anti-Nkx2.8 antibody (Santa Cruz Biotechnology, Inc.) or an anti-IgG antibody

(Millipore) overnight at 4℃ with rotation. DNA was extracted, and the TWIST1

promoter was amplified with a quantitative PCR assay. All the ChIP assays were

performed three times, and representative results were presented. All the sequences of

the PCR primers are shown in Supplemental Table 2.

Luciferase assay

Twenty thousand cells were seeded in triplicate in 48-well plates and allowed to

settle for 24 h. Using the Lipofectamine 2000 reagent according to the manufacturer's

recommendations, 100 ng of luciferase reporter plasmids containing different

fragments of the TWIST1 promoter, or the control luciferase plasmid, plus 1 ng of

pRL-TK Renilla plasmid (Promega) was transfected into the cells. The luciferase and

Renilla signals were measured 24 h after transfection using the Dual Luciferase

Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer.

Xenografted tumor model and hematoxylin and eosin staining

BALB/c-nu mice (4-5 weeks of age, female, 18-20 g) were purchased from

Charles River Laboratories (Beijing, China). All the experimental procedures were

approved by the Institutional Animal Care and Use Committee of Sun Yat-sen

University. For the tail vein injection model, the BALB/c nude mice were randomly

divided into 5 groups (n=6/group). Each group of mice was intravenously injected in

the tail vein with 5637/vector cells, 5637/Nkx2.8 cells, 5637/scrambled cells or

5637/Nkx2.8 RNA interference cells (5×106) per mouse. After 8 weeks, the animals

were euthanized, and tumors were excised, weighed, and embedded in paraffin. For

the orthotopic xenograft bladder cancer model (21), the bladder was washed with

phosphate buffered saline (PBS) and then scratched with the catheter tip before

instilling 100L of 2×106 cells through a small catheter. The urethra was temporarily

closed with a single, sterile suture at the distal part of the urethra thus retaining the

cells in the bladder for 2 hours. The BALB/c nude mice were randomly divided into 5

groups (n=6/group). Each group of mice was instilled with T24/vector cells,

T24/Nkx2.8 cells, 5637/scrambled cells, 5637/Nkx2.8 RNAi cells or 5637/Nkx2.8

RNAi/Twist1 RNAi cells transfected with luciferase. Xenograft implantation was

confirmed by the presence of bioluminescence activity 1 week after cell implantation.

For bioluminescent evaluation each mouse received 150 mg luciferin/kg body weight

through an intraperitoneal injection. Imaging of the mice was then conducted in

anesthetized conditions with the IVIS® Lumins III (MA, USA). After 8 weeks, the

bioluminescence activity were detected again and the animals were sacrificed, and

bladder were excised, weighed, and embedded in paraffin. Serial 6.0-mm sections

were cut and subjected to H&E staining with Mayer's hematoxylin solution. Images

were captured using the AxioVision Rel.4.6 Computerized Image Analysis System

(Nikon Eclipse 80i).

Statistical analysis

Statistical analyses were conducted using the SPSS 11.0 statistical software

package. Statistical tests for the data analysis included Fisher’s exact test, the log-rank

test, , and Student’s two-tailed t test. The data were presented as the means±SD. A

value of p≤0.05 was considered statistically significant.

RESULTS

Nkx2.8 represses UC cell EMT

To determine the role of Nkx2.8 in UC metastasis, we evaluated whether Nkx2.8

can influence the EMT phenotype in UC cells. We established forced exogenous

Nkx2.8-expression cells and Nkx2.8-silenced cells and examined the expression

levels of several EMT-related proteins. Our Western blot results showed that the

expression levels of E-cadherin and α-catenin, which mediate cell-cell and cell-stroma

adhesion, were much higher in Nkx2.8-overexpression cells than in vector cells.

Accordingly, the expression levels of the mesenchymal markers fibronectin and

vimentin were markedly reduced in Nkx2.8-overexpression cells compared to vector

cells (Figure 1A). Immunofluorescence analysis in 2D and 3D cultured cells

confirmed the above findings (Figure 1B). Consistent with the above results,

Nkx2.8-silenced cells displayed decreased E-cadherin and α-catenin expression and

enhanced fibronectin and vimentin expression (Figures 1A and S1). Our data showed

that Nkx2.8 inhibits the EMT phenotype in UC cells.

Overexpression of Nkx2.8 inhibits the invasion and metastatic potential of UC

cells

EMT can augment tumor cell motility and lead to tumor cell invasion into the

basement membrane, which leads to advanced metastasis. Thus, we investigated the

effect of Nkx2.8 on UC cell invasion and metastatic potential. Matrigel-coated

Boyden chamber invasion assay, whose results were represented as the number of

migrated cells, showed that the invasiveness of Nkx2.8-overexpression cells was

much weaker than that of vector cells (Figure 2A). 3D morphogenesis cultures

revealed that Nkx2.8 overexpression reduced the numbers of irregular branched

structures that characterize the invasive phonotype. Mean spheroid area was

significantly smaller in Nkx2.8-overexpression cells compared with vector cells

(Figure 2B). Furthermore, wound-healing assay showed that cell motility was

dramatically hampered by Nkx2.8 overexpression (Figure 2C). These findings

suggested that Nkx2.8 overexpression inhibited UC cell motility and invasion.

We next evaluated the in vivo effects of Nkx2.8 on invasion and metastasis using

an experimental metastasis assay, in which we injected cells transfected with Nkx2.8

or vector into the lateral tail veins of nude mice and evaluated cell growth in the lungs

after 8 weeks. Fewer metastatic nodes were found on the lung surfaces of the

Nkx2.8-overexpression group than on the lung surfaces of the vector group. In

addition, H&E staining showed that there were smaller and fewer microscopic

metastatic nodules in the Nkx2.8-overexpression group than in the vector group

(Figure 2D). Furthermore, orthotopic xenograft bladder cancer model also provided

data about Nkx2.8's role on bladder cancer cells invasiveness. As shown in Figure S2

A, B and C, mice bladder implanted with T24/Nkx2.8 cells showed submucosa

infiltration lesion, while those with T24/vector cells showed muscle invasive disease.

These data indicate that Nkx2.8 acts as a negative regulator of the aggressive

metastasis of UC cells.

Silencing endogenous Nkx2.8 promoted the invasiveness and facilitated the

metastatic potential of UC cells

To investigate the impact of Nkx2.8 on EMT of UC cells further, we assessed the

invasiveness and metastatic potential of Nkx2.8-silenced cells. Both Matrigel-coated

Boyden chamber invasion assay and 3D morphogenesis cultures indicated that

ablation of endogenous Nkx2.8 induced UC cell invasiveness (Figure 3A and B).

Wound-healing assay also revealed that UC cells with Nkx2.8 ablation exhibited

significantly enhanced mobility compared to control cells (Figure S3A). Taken

together, these results suggested that silencing Nkx2.8 dramatically promoted UC cell

motility and invasiveness.

To determine the role of Nkx2.8 in UC cell metastasis in vivo, we constructed a

lung metastasis model by injecting cells with silenced endogenous Nkx2.8 or control

cells into the tail veins of 6-week-old nude mice. After 8 weeks, many more

metastatic nodes were observed on the lung surfaces of the Nkx2.8-silenced group

than on the lung surfaces of the control group. H&E staining confirmed that both the

numbers and the volumes of microscopic metastatic lesions were markedly increased

in the lungs of mice injected with Nkx2.8-silenced cells compared to the lungs of

control mice (Figure 3C). Moreover, as shown in Figure S3B, C and D, orthotopic

xenograft bladder cancer model, mice bladder implanted using 5637/Nkx2.8

shRNA#2 cells showed cancer cells infiltrated into submucosa, whereas those with

5637/scrambled cells showed dysplastic cells with nuclear hyperchromatism within

the mucosa. These results confirmed that Nkx2.8 exerts negative regulatory effects on

UC cell metastasis.

Nkx2.8 down-regulated the expression of Twist1 in UC cells

To explore the underlying mechanism of Nkx2.8 inhibiting metastasis, we

detected the relationship between Nkx2.8 and EMT-inducing transcription factors,

including TWIST1, TWIST2, SNAIL, SLUG, ZEB1 and ZEB2, using quantitative PCR

(22). We discovered that Nkx2.8 down-regulated TWIST1 mRNA expression but had

no significant effect on other above mentioned gene mRNA expression. Twist1 is a

key regulator of EMT and plays an important role in UC metastasis (5-11). Further

study revealed an inverse relationship between Nkx2.8 and Twist1 expression in

cultured UC cell lines (Figure 4A). Western blotting analysis also revealed that

Twist1 expression was significantly decreased in cells forced to express exogenous

Nkx2.8 compared to vector control cells; however, Twist1 expression was markedly

up-regulated in Nkx2.8-silenced cells compared to vector control cells (Figures 4B

and 4C). Twist1, which also acted as transcriptional factor, was reported to be a direct

regulator of BMI1 and E-cadherin, two important elements in EMT occurrence (7).

Therefore, we detected the effect of Nkx2.8 on BMI1 and E-cadherin. As shown in

Figure 4D, BMI1 was down-regulated, while E-cadherin was up-regulated in

Nkx2.8-overexpression cells. Consistent with these findings, BMI1 was up-regulated,

and E-cadherin was down-regulated in Nkx2.8-silenced cells. These results are in

accordance with the results pertaining to the effects of Twist1 on Bmi1 and

E-cadherin. Our findings indicated that Nkx2.8 inhibited Twist1 expression.

Nkx2.8 binds to the TWIST1 promoter locus and transcriptionally represses

TWIST1

As a transcriptional factor, Nkx2.8 binds to special DNA sequences in promoters.

Interestingly, we identified two potential binding sites for Nkx2.8 in the TWIST1

promoter, each of which included three adjacent core sequences (Figure 5A).

Therefore, we speculated that Nkx2.8 binds to the promoter locus and regulates

TWIST1 transcription. We conducted ChIP assay to verify this speculation. As shown

in Figure 5B, we detected 13 TWIST1 promoter loci. As expected, Nkx2.8 bound to

the 2nd and 10th loci of the TWIST1 promoter (Figure 5B). These loci extend from

-1510 bp to -1472 bp and from +774 bp to + 801 bp, respectively (Figure 5A). This

binding was restrained when Nkx2.8 was silenced (Figure 5C), which confirmed that

Nkx2.8 directly targets the TWIST1 promoter.

Furthermore, we found that overexpressing Nkx2.8 decreased the luciferase

activity driven by the wild type TWIST1 promoter, while silencing Nkx2.8 increased

the luciferase activity driven by the TWIST1 promoter. However, neither

overexpression nor knockdown of Nkx2.8 had any effect on the luciferase activity

levels of TWIST1 promoters containing a deleted or mutated 2nd or 10th locus (Figure

5D), indicating that both the 2nd and the 10th locus are needed for Nkx2.8 to target

the TWIST1 promoter.

Ablation of Twist1 restores the inhibitory effect of Nkx2.8 on the invasiveness

and metastatic potential of Nkx2.8-silenced UC cells

To further analyze the functional correlation between Twist1 and Nkx2.8, we

tested whether the ablation of Twist1 expression in Nkx2.8-silenced UC cells could

restore the expression of factors downstream of Twist1 and the invasive phenotype of

these cells. As expected, silencing Twist1 expression with TWIST1 shRNA in

Nkx2.8-silenced cells decreased Bmi1 expression and increased E-cadherin

expression (Figure 6A). Moreover, the Boyden chamber invasion assay results

suggested that Twist1 knockdown markedly restrained the restored invasiveness of

the Nkx2.8-silenced cells (Figure 6B). The results of 3D Matrigel cultures and a

wound-healing assay confirmed the restoration of the motility of Nkx2.8-silenced

cells via the expression of TWIST1 shRNA (Figures S4 and S5).

To decipher the functional correlation between Nkx2.8 and Twist1 in vivo, we

performed xenograft tumor experiments. As shown in Figure 6C, Nkx2.8-silenced

cells caused mice to exhibit many more metastatic nodes on their lung surfaces, while

Twist1 knockdown in Nkx2.8-silenced cells caused mice to exhibit greatly reduced

numbers of metastatic nodes on their lung surfaces. H&E staining confirmed that the

tissues comprising Nkx2.8-silenced cells in which Twist1 was knocked down

displayed significantly less and smaller microscopic metastatic lesions than the tissues

comprising Nkx2.8-silenced cells expressing Twist1. Additionally, in orthotopic

xenograft bladder cancer model, mice bladder implanted using 5637/Nkx2.8

shRNA#2/Twist1 shRNA#2 cells illustrated atypical hyperplasia cells within the

mucosa, whilst those with 5637/Nkx2.8 shRNA#2 cells showed cancer cells

infiltrated into submucosa (Figure S6 A, B and C). These results suggest that Twist1

ablation could restore the inhibitory effect of Nkx2.8 on EMT and the metastatic

potential of Nkx2.8-silenced UC cells.

Clinical relevance of Nkx2.8 and Twist1 in human UC

To confirm the findings derived from the above in vitro and animal experiments,

we analyzed the levels of Nkx2.8 and Twist1 in 15 freshly collected clinical UC

samples. Real-time RT-PCR analyses revealed that NKX2.8 mRNA levels were

inversely correlated with TWIST1 expression levels (r2=0.589, p=0.0008; Figure 7A).

Immunohistochemical (IHC) analysis of 161 tissue specimens also showed that

Nkx2.8 expression was inversely correlated with Twist1 expression (p<0.0001; Figure

7B and C). The data pertaining to these cases have been published previously.

Patients with high Twist1 expression levels have much worse survival than patients

with low Twist1 expression (Figure S7). Further analysis showed that patients with

both Nkx2.8 positivity and low Twist1 expression had the best survival rate (Figure

7D and 7E). In summary, our results reveal that an inverse relationship exists between

Nkx2.8 and Twist1 expression and that Nkx2.8 positivity and low Twist1 expression

lead to better outcomes in UC patients than Nkx2.8 negativity or high Twist1

expression.

DISCUSSION

EMT is an initial event in cancer cell invasion and metastasis characterized by

repressed E-cadherin expression and subsequently facilitates cell migration and

invasion (2-4). Non-muscle-invasive UC carries a high risk of progressing to

muscle-invasive UC. This process is accompanied by decreased E-cadherin

expression, suggesting that EMT is involved in the process (23). Muscle-invasive UC

has a high incidence of lymph node or distal metastasis, again suggesting that EMT is

involved in its development (24). Nkx2.8 has been reported to be involved in the

carcinogenesis and progression of several human cancers (17-19). Our previous study

showed that Nkx2.8 acted as a tumor suppressor in UC by inhibiting cancer cell

proliferation through the MEK/ERK pathway. Moreover, we found that negative

Nkx2.8 expression was associated with lymphatic metastasis (20). In this study we

explored the correlation between Nkx2.8 expression and EMT in UC. We found that

Nkx2.8 overexpression inhibited UC cell EMT, while Nkx2.8 silencing promoted UC

cell EMT in vitro and UC cell metastatic potential in vivo. Thus, our present study has

demonstrated Nkx2.8 can function as a novel UC EMT inhibitor.

Twist1, which suppresses E-cadherin expression, has been considered an

important promoter in EMT (5-8). Numerous studies have shown that Twist1 plays an

important role in UC (9-11). However, the upstream regulatory mechanism

underlying the effects of Twist1 in EMT is not well illuminated. Twist1 has been

found to be a downstream target of NF-B-like transcription factor in both Drosophila

and vertebrates (25-27). Tan and colleagues (28) reported that Twist1 expression is

induced by the embryonic factor high mobility group A2 (HMGA2), which causes

mesenchymal transition, in mouse mammary epithelial cells. It has also been reported

that Twist1 stability can be regulated by the ubiquitin-proteasome system in embryos.

(29). However, little is known regarding the mechanism underlying Twist1 regulation

in EMT in bladder cancer. Shiota and colleagues (12) found that Foxo3a can

negatively regulate Twist1 and positively regulate E-cadherin in bladder cancer cells.

Our study has provided evidence that Nkx2.8 can transcriptionally repress Twist1

expression by directly binding to the Twist1 promoter and has thus also provided

strong evidence that this process represents the mechanism by which Nkx2.8 regulates

Twist1 in EMT. Furthermore, our study has demonstrated the existence of a novel

pathway (Nkx2.8-Twist1-E-cadherin) that participates in UC EMT.

Nk2 family proteins are characterized by three highly conserved domains, one of

which is Homeodomain, a region for specific DNA binding that usually binds to DNA

sequences containing 5'CTTG3' or 5'CAAG3' (14, 16). Kajiyama and colleagues

found that Nkx2.8 bound to the active AFP promoter and that antisense inhibition of

Nkx2.8 mRNA translation selectively reduced endogenous human AFP gene

expression (30). Nkx2.8 can also repress AKIP1 expression by directly targeting the

AKIP1 promoter and then inhibiting NF-B activation in esophageal cancer (18).

Interestingly, we found that the promoter region of TWIST1 contains two potential

binding sites for Nkx2.8. This finding indicates that Nkx2.8 binds directly to the

TWIST1 promoter. Our ChIP analysis and luciferase assay showed that Nkx2.8 binds

to the two suspected regions mentioned above and represses activation of the TWIST1

promoter. Interestingly, Nkx2.8 must bind both sites to repress TWIST1 promoter

activation, as a TWIST1 promoter featuring deletions or mutations of the 2nd or 10th

locus is not affected by Nkx2.8. These two loci, which each contain three adjacent

core sequences, are located on either side of the transcription start site (TSS). Thus,

the TWIST1 promoter differs from the other known Nkx2.8 targets, which contain

only one site for Nkx2.8 (18, 30). The above result is illustrative of a new mechanism

by which Nkx2.8 exerts its regulatory effects and has thus provided us with important

information that can be used to identify new targets of Nkx2.8. However, the precise

mechanism by which Nkx2.8 regulates TWIST1 transcription warrants further

investigation.

Previous studies have demonstrated the prognostic significance of Nkx2.8 and

Twist1 in UC (9-11, 20), but no study has explored the relationship between Nkx2.8

and Twist1. Here, we showed that an inverse relationship exists between Nkx2.8 and

Twist1 expression in UC patients, a result consistent with those of previous studies

showing that Nkx2.8 represses Twist1 expression in vitro. This inverse relationship

between Nkx2.8 and Twist1 expression has an impact on the prognoses of UC

patients, as patients displaying Nkx2.8 positivity and low Twist1 expression have a

better prognosis than patients displaying Nkx2.8 negativity or high Twist1 expression.

In our study, Nkx2.8 failed to repress Twist1 expression in approximately 45% of

cases with positive Nkx2.8 expression, a finding that may be attributable to the fact

that Nkx2.8 is universally expressed at low levels in UC. The prognosis of these

patients was not significantly different from that of patients with negative Nkx2.8

expression, which indicates that the Twist1 repression is the main mechanism by

which Nkx2.8 exerts its effects. Fifteen percent of cases with negative Nkx2.8

expression showed low Twist1 expression, implying that Twist1 inhibition occurs

independently of Nkx2.8 expression. Thus, additional studies are needed to identify

other upstream regulators of Twist1.

In conclusion, the findings of our study serve as an extensive explanation of the

mechanism underlying the effects of Nkx2.8 on UC EMT, as we elucidated the

crucial role of the Nkx2.8-Twist1 pathway in UC EMT and may have thus identified

novel therapeutic targets for the treatment of UC.

Acknowledgments

This study was supported by grants from the National Natural Science

Foundation of China (Nos. 81272810, 81402114, 81300597, 81672530, 81472385,

81772726 and 81772716)and Natural Science Foundation o f Guangdong Province

(No. 2016A030 310213).

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Figures legends

Figure 1. Nkx2.8 represses UC cell EMT. (A) Expression of the epithelial proteins

E-cadherin and α-catenin and the mesenchymal proteins fibronectin and vimentin in

T24 and 5637 cells expressing the control vector pBabe or pBabe/Nkx2.8 and in 5637

and BIU87 cells expressing scrambled shRNA or Super-retro-Nkx2.8-shRNAs was

detected by Western blot analysis; α-Tubulin was used as a loading control. (B)

Immunofluorescence analysis was used to detect the expression of E-cadherin,

α-catenin, fibronectin and vimentin in 2D and 3D cultured T24 and 5637 cells

expressing the control vector pBabe or pBabe/Nkx2.8. The green signal represents

staining for the corresponding protein, while the red signal signifies nuclear DNA

staining with DAPI. Quantification of immunofluorescence staining represented by

average fluorescence intensity (lower). The data presented are the means±SD of 3

independent experiments; ***, p<0.0001 (Student’s t test).

Figure 2. Forced expression of exogenous Nkx2.8 inhibits the invasion and

metastatic potential of UC cells. (A) The invasive properties of T24 and 5637 cells

expressing the control vector pBabe or pBabe/Nkx2.8 were analyzed using a Boyden

chamber invasion assay. Migrated cells are plotted as the average number of cells per

field of view from 3 different experiments (×200). Error bars represent the SD of the

means from three independent experiments. (B) The acini formation of T24 and 5637

cells expressing the control vector pBabe or pBabe/Nkx2.8 was evaluated in 3D

morphogenesis Matrigel culture (upper) (×400). Quantification of 3D culture

represented by mean spheroid area (lower). The data presented are the means±SD of 3

independent experiments; *, p<0.01, **, p<0.001 (Student’s t test). (C) A wound was

introduced on a subconfluent culture of cells, and the rate of wound closure was

monitored at 0 and 20 h. A representative photograph (×200) from three independent

experiments(left) and quantification (right) of the wound healing assay is shown. (D)

Upper panels: Representative images of macroscopic lung metastases; arrowheads

indicate the metastatic nodes. Lower panels: Representative images of H&E staining

(×100). Right: Quantification of the average number of macroscopic metastatic nodes

formed on the lung surface (upper) or based on the pathological analysis of the

H&E-stained sections (lower). The data presented are the means±SD of 3 independent

experiments; ***, p<0.0001 (Student’s t test).

Figure 3. Silencing endogenous Nkx2.8 promoted the invasiveness and facilitated

the metastatic potential of UC cells. (A) The invasive properties of 5637 and BIU87

cells that expressed scrambled shRNA or Super-retro-Nkx2.8-shRNAs were analyzed

using a Boyden chamber invasion assay. Migrated cells are plotted as the average

number of cells per field of view from 3 different experiments (×200). Error bars

represent the SD of the means from three independent experiments. (B) The

three-dimensional morphology of 5637 and BIU87 cells expressing scrambled shRNA

or Super-retro-Nkx2.8-shRNAs was analyzed by culturing the cells in Matrigel (left)

(×400). Quantification of 3D culture represented by mean spheroid area (right). The

data presented are the means±SD of 3 independent experiments; **, p<0.001

(Student’s t test). (C) Left: Representative images of macroscopic lung metastases;

arrowheads indicate the metastatic nodes. Right: Representative images of H&E

staining (×100). Lower panels: Quantification of the average number of macroscopic

metastatic nodes formed on the lung surface (left) or based on the pathological

analysis of the H&E-stained sections (right). The data presented are the means±SD of

3 independent experiments; ***, p < 0.0001 (Student’s t test).

Figure 4. Nkx2.8 down-regulated Twist1 expression in UC cells. (A) Western blot

analysis of Nkx2.8 and Twist1 expression in the indicated cells; α-Tubulin was used

as a loading control. (B) Real-time PCR analysis of TWIST1 mRNA expression in the

indicated cells. Transcript levels were normalized to GAPDH. Error bars represent the

SD of the means from 3 independent experiments. (C) Western blot analysis of

Twist1 expression in the indicated cells; α-Tubulin was used as a loading control. (D)

Real-time PCR analysis of BMI1 and E-cadherin mRNA expression in the indicated

cells. Transcript levels were normalized to GAPDH; **, p<0.001 (Student’s t test).

Figure 5. Nkx2.8 binds to the TWIST1 promoter locus and transcriptionally

represses TWIST1. (A) Schematic representation of the promoter region of TWIST1.

Precipitated DNA was amplified in a PCR assay using primers specific for regions

1-13. The arrow indicates the transcriptional start site. (B) ChIP analysis was

performed using anti-Nkx2.8 antibody or IgG antibody to identify Nkx2.8 binding

sites on the TWIST1 promoter in 5637 cells. (C) ChIP analysis of Nkx2.8 binding

efficiency in 5637 cells expressing the scrambled shRNA or NKX2.8 shRNA. (D)

Transactivities of Nkx2.8 on serial TWIST1 promoter fragments as indicated in 5637

cells. Each bar represents the mean±SD of 3 independent experiments; *, p<0.05

(Student’s t test). TSS: Transcriptional start site.

Figure 6. Ablating Twist1 restores the inhibitory effect of Nkx2.8 on the

invasiveness, EMT and metastatic potential of Nkx2.8-silenced UC cells. (A)

Western blot analysis of Twist1, Bmi-1, and E-cadherin expression in the indicated

cells; α-Tubulin was used as a loading control. (B) Boyden chamber invasion assay of

the indicated cells. Migrated cells are plotted as the average number of cells per field

of view from 3 different experiments (×200). Error bars represent the SD of the means

from three independent experiments. (C) Upper panels: Representative images of

macroscopic lung metastases; arrowheads indicate the metastatic nodes. Lower panels:

Representative images of H&E staining (×100). Right: Quantification of the average

number of macroscopic metastatic nodes formed on the lung surface (upper) or based

on the pathological analysis of the H&E-stained sections (lower). The data presented

are the means±SD of 3 independent experiments; ***p<0.0001 (Student’s t test).

Figure 7. Clinical relevance of Nkx2.8 and Twist1 in human UC. (A) Correlation

between Nkx2.8 expression and TWIST1 mRNA expression in 15 freshly collected

human UC samples. (B) Nkx2.8 levels were negatively associated with Twist1

expression in 161 primary human UC specimens. Two representative cases are shown

(×200). (C) Percentage of UC specimens showing low or high Nkx2.8 expression

relative to the level of Twist1. (D) Left: comparison of the overall survival times of

patients with different levels of Nkx2.8 and Twist1. The p values of the comparisons

between each group are shown in the inset (log-rank test). (E) Prognostic significance

of Nkx2.8 positivity and low Twist1 expression in UC cases.

Nkx2.8 inhibits epithelial-mesenchymal transition in bladder urothelial carcinoma via transcriptional repression of Twist1

Chunping Yu, Zhuowei Liu, Qiuhong Chen, et al.

Cancer Res Published OnlineFirst January 8, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-1545

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