Author Manuscript Published OnlineFirst on December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-2309 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

GALNT1-mediated and activation of sonic hedgehog signaling

maintains the self-renewal and tumor-initiating capacity of bladder cancer stem

cells

Chong Li1,3, Ying Du1,3 , Zhao Yang1,3, Luyun He1, Yanying Wang1, Lu Hao1, Mingxia Ding2,

∗ ∗ Ruping Yan2, Jiansong Wang2, and Zusen Fan1,

1CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy

of Sciences, Beijing 100101, China

2Department of Urology, The Second Affiliated Hospital of Kunming Medical University,

Kunming 650101, China

∗ Corresponding author: Dr. Jiansong Wang, Email: [email protected]; Dr. Zusen

Fan, Email: [email protected]

3 These authors contributed equally to this work.

The authors disclose no potential conflicts of interest.

Running title: GALNT1-mediated SHH activation in BCSCs

1

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Abstract

The existence of bladder cancer stem cells (BCSCs) has been suggested to underlie

bladder tumor initiation and recurrence. Sonic hedgehog (Shh) signaling has been

implicated in promoting CSC self-renewal and is activated in bladder cancer, but its impact

on BCSC maintenance is unclear. In this study, we generated a monoclonal antibody

(BCMab1) against CD44+ bladder cancer cells that recognizes aberrantly

glycosylated integrin α3β1. The combination of BCMab1 with an anti-CD44 antibody

identified a BCMab1+CD44+ cell subpopulation as BCSCs with stem cell-like properties.

Gene expression analysis revealed that the hedgehog pathway was activated in the

BCMab1+CD44+ subpopulation, and was required for BCSC self-renewal. Furthermore,

the glycotransferase GALNT1 was highly expressed in BCMab1+CD44+ cells and

correlated with clinicopathologic features of bladder cancers. Mechanistically, GALNT1

mediated O-linked glycosylation of Shh to promote its activation, which was essential for

the self-renewal maintenance of BCSCs and bladder tumorigenesis. Finally, intravesical

instillation of GALNT1 siRNA and the Shh inhibitor cyclopamine exerted potent antitumor

activity against bladder tumor growth. Taken together, our findings identify a BCSC

subpopulation in human bladder tumors that appears to be responsive to inhibition of

GALNT1 and Shh signaling, and thus highlight a potential strategy for preventing the rapid

recurrence typical in bladder cancer patients.

Keywords: bladder cancer; cancer stem cell; GALNT1; Hedgehog signaling; glycosylation

2

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Introduction

Bladder cancer is the second most common urological malignancy with over 69,000

new cases and an estimated 150,000 deaths worldwide each year (1-3). The big problem

of bladder cancer is extremely easy recurrence after treatment and no recognized

second-line therapy (4). Thus, for new therapeutic strategies, it is of great importance to

delineate the mechanism of bladder cancer tumorigenesis.

Accumulating evidence from leukemias, germ cell tumors, and many solid tumors

support a cancer stem cell (CSC) model in which cancers are initiated by a rare

subpopulation of self-renewing and differentiating tumor-initiating cells (TICs) (5-7). The

cancer stem cell (CSC) population can self-renew limitlessly and differentiate into

heterogeneous cell populations, resulting in phenotypic and functional heterogeneity of

tumors (8-9). Quiescent CSCs may resist therapeutic intervention and confer to recurrence

after initial therapy (7). Chan et al identified a subpopulation (Lineage-CD44+CK5+CK20-)

from primary human bladder cancer as bladder cancer stem cells (BCSCs) (10), which

shared similar expression markers to normal bladder basal cells, suggesting the basal-like

property of BCSCs. A subpopulation of primary urothelial cancers, co-expressing the 67

kDa laminin receptor (67LR) and the basal cell-specific cytokeratin CK17, possesses more

carcinogenic ability (11). These subsets harbor high tumorigenicity, multipotency and

involvement in bladder tumor progression, suggesting that the CSCs exist in bladder

tumors.

Bladder cancer arises from the urothelium, a transitional epithelium lining the bladder

lumen. This multi-layered epithelium is composed of a luminal layer of fully differentiated

umbrella cells that overlie intermediate cells, and a basal layer of Sonic Hedgehog

(SHH)-expressing cells (12). The SHH-expressing basal cells, capable of regenerating all

cell types of the urothelium, have been defined as urothelial stem cells. The same group

reported that muscle-invasive bladder cancers originate exclusively from the

SHH-expressing basal stem cells of the basal urothelium in pro-carcinogen

N-butyl-N-4-hydroxybutyle nitrosamine (BBN)-induced mouse bladder cancer (13). These

SHH-expressing basal stem cells act as BCSCs to initiate bladder oncogenesis. Aberrant

activation of the Hedgehog signaling is implicated in many malignancies (14, 15). 3

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Emerging data support that Hh signaling plays a pivotal role in the self-renewal and

differentiation of CSCs (16, 17). Activation of Hh pathway causes CSC self-renewal and

expansion, while the SMO antagonist cyclopamine or the ligand-neutralizing antibody

induces terminal differentiation and loss of clonogenic growth capacity (16, 18). In this

study, we defined that Hedgehog signaling is essential for the self-renewal of BCSCs.

GALNT1-mediated glycosylation is required for SHH activation in BCSCs.

Materials and Methods

Reagents and mice

Mouse monoclonal antibody BCMab1 was purified through A-Sepharose from

ascites. Corresponding species-specific fluorescein isothiocyanate (FITC)-conjugated

secondary antibodies were from Sigma. The commercial primary antibodies were

phycoerythrin (PE)-conjugated anti-CD44 (BD Pharmingen; 550989), goat anti-human

C-terminus of SHH (Santa Cruz, sc-33942), goat anti-human N-terminus of SHH (Santa

Cruz; sc-1194), rabbit anti-Gli1 (Santa Cruz, sc-20687), goat anti-GALNT1 (Santa Cruz,

sc-68491), mouse Anti-β-actin antibody (Sigma, A1978). NOD/SCID mice were obtained

−/− from Vital River Laboratory Animal Technology Co. Ltd., Beijing. GALNT1 mice were

obtained from Jackson Laboratories. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN),

benzyl-N-acetyl-D-galactosaminide (BG) (Sigma), and cyclopamine were from Sigma.

GALNT1 siRNA (human) and GALNT1 siRNA (mouse) were from Santa Cruz.

Xenograft establishment

Human bladder cancer specimens were obtained with patient’s consent, as approved

by the Research Ethics Board at the second Affiliated Hospital of Kunming Medical

University in Kunming. For generation of xenografts, cells were injected with Matrigel

subcutaneously into the backs NOD/SCID mice. Animal work was carried out in

compliance with the ethical regulations approved by the Animal Care Committee, Institute

of Biophysics, Chinese Academy of Sciences, Beijing, China.

Flow cytometry sorting

Primary tumor tissues were digested with 200U Type I collagenase, 20U Type IV

collagenase) (Sigma, C-0130, C-5138) and DNase at 37°C for 2 h to 6 h. Then cancer

cells were stained with FITC-conjugated BCMab1 and PE-conjugated anti-CD44 antibody 4

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or the same isotype FITC/PE-conjugated antibody for 30 min on ice. Labeled cells were

analyzed and sorted by flow cytometry (BD FACSAria III).

DNA methylation analysis

Methylation was assayed by using the procedure as described (19). Genomic DNA

was treated with sodium bisulfate by using the EZ DNA methylation direct kit (Zymo

Research). PCR products were analyzed with the MassArray EpiTYPER system

(Invitrogen).

Quantitative real-time PCR

Total RNA was isolated from primary sorted cells, human bladder cancer cell lines

and mouse tissues using the RNA isolation kit (Tiangen Biotech, CHN). RNA was then

subjected to cDNA synthesis using M-MLV Reverse Transcriptase (Promega). The cDNA

was then processed for an amplification step with the SYBR Green reaction system

(Tiangen Biotech, CHN) running on an ABI 7300 (Applied Biosystems). Primer

oligonucleotides for qPCR were listed in the Supplementary Table 1.

In vitro glycosylation assay

Recombinant human WT SHH and T282A-SHH mutant were expressed in E.

coli. WT SHH or T282A-SHH mutant was incubated with GALNT1 (R&D) plus

BCMab1-CD44- cell lysates at 37°C for 4 h. Reaction products were analyzed by

immunoblotting.

Immunohistochemical staining

Tumor specimens were fixed in 10% buffered formalin and embedded in paraffin as

described (20). The immunosignals were detected using the ABC kit.

Intravesical administration of siRNA/liposomes and cyclopamine for treatment

Female 6-week-old NOD/SCID mice with a body weight of approximately 15 g were

used and kept under specific pathogen-free conditions. BCMab1+CD44+ cells were

labeled with luciferase and transplanted into the murine bladder cavity via 24-gauge

angiocatheters (21). The mice were grouped (24 mice per group) and intravesically

applied with 6 μM siCtrl/liposomes or siGALNT1/liposomes with or without 100 μM

cyclopamine (CPM) next day.

Statistical analysis 5

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The relationship between the expression levels of GALNT1 and clinicopathological

features was analyzed using the χ2 or the Kruskal-Wallis test. Kaplan-Meier analysis was

used to estimate the cumulative cause-specific survival rates, and the log-rank test was

used to correlate differences in patient survival with staining intensity of GALNT1. The

influence of GALNT1 on the growth of bladder cancers was analyzed by the Student’s t

test.

Results

A BCMab1+CD44+ subpopulation of cancerous cells acts as bladder cancer stem

cells (BCSCs)

To identify unique biomarkers of BCSCs, we isolated CD44+ BCSCs from human

bladder cancer resection specimens (Fig. 1A), which were used as immunogen to

immunize mice to generate monoclonal antibodies. About 458 hybridomas were screened

to bind BCSCs. Among these hybridomas, we identified several antibodies, including

BCMab1 and BCMab37, which were determined to recognize the BCSC surface antigens,

aberrantly glycosylated integrin α3β1 and CD47, respectively. We previously showed that

the BCMab1 antibody was also generated by T24 cell immunization, recognizing the

aberrantly glycosylated Integrin α3β1 on bladder cancer cells (20). The aberrantly

glycosylated integrin α3β1 is highly expressed in bladder cancer tissues. CD47 was also

confirmed to be a surface marker of BCSCs (11).

By using BCMab1 and anti-CD44 antibodies, we distinguished a BCMab1+CD44+

subset from the tumor cells of human bladder tumor specimens (Fig. 1A), comprising 3%

to 5% of the total cell population. BCMab1+CD44+ positive cells were further confirmed by

immunofluorescence staining (Fig. 1C). We next determined whether BCMab1+CD44+

cells were more tumorigenic than their BCMab1-CD44- counterparts. BCMab1+CD44+ cells

freshly isolated from bladder cancer specimens were inoculated subcutaneously into

NOD/SCID mice. As few as 10 BCMab1+CD44+ cells could form xenograft tumors in vivo

(Fig. 1D, E), whereas at least 1×105 BCMab1-CD44- cells were necessary to establish

xenografts. The xenograft tumors formed with BCMab1+CD44+ cells and BCMab1-CD44-

cells in Figure 1E were used for further analysis. Intriguingly, the tumors induced by

BCMab1+CD44+ cells were able to reconstitute a mixture of BCMab1+CD44+ cells and 6

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differentiated BCMab1-CD44- tumor cells (Fig. 1F), but the tumors grafted by

BCMab1-CD44- cells could not reconstitute the BCMab1+CD44+ cell subpopulation.

Additionally, the self-renewal capacity of BCMab1+CD44+ cells was evaluated by an in

vivo serial passage assay. BCMab1+CD44+ or BCMab1-CD44- cells were sorted from the

primary mouse xenografts induced by the BCMab1+CD44+ cells and implanted into

secondary recipient NOD/SCID mice accordingly. The same procedure was used for the

3rd and 4th passages. All the serially xenografted mice injected with BCMab1+CD44+ cells

were able to form tumors (Fig. 1G, H), which grew much faster with serial passages.

However, xenografted tumors injected with BCMab1-CD44- cells only formed with high cell

number inoculation, while the formed xenografts were smaller even with multiple passages.

We next examined the self-renewal property of BCMab1+CD44+ cells by an in vitro sphere

formation assay. BCMab1+CD44+ cells freshly isolated from human bladder cancer

specimens exhibited significantly higher oncosphere-forming efficiency compared with

their respective BCMab1-CD44- counterparts (Fig. 1I). Taken together, the BCMab1+CD44+

subpopulation is high tumorigenic, with self-renewal and differentiation properties. Thus

we defined this BCMab1+CD44+ subset as the bladder CSCs (BCSCs).

Hedgehog signaling is essential for self-renewal of BCSCs

SHH-expressing basal stem cells, acting as BCSCs, to initiate tumorigenesis in

BBN-induced mouse bladder cancer (15). These results suggest that Hh signaling may be

implicated in the stemness of BCSCs. To determine the Hh pathway of BCMab1+CD44+

cells, we performed transcriptome microarray analysis of BCMab1+CD44+ and

BCMab1-CD44- cells isolated from human bladder tumor specimens. Microarray analysis

revealed that several Hh signaling molecules, including SHH, Smo, Gli1, and Gli2, were

highly expressed in BCMab1+CD44+ cells compared with their BCMab1-CD44-

counterparts (Fig. 2A). High Gli1 mRNA levels in BCMab1+CD44+ cells were verified in

BCMab1+CD44+ cells derived from each specimen of 72 human bladder cancer samples

(Fig. 2B). Bladder cancer tissues displayed high expression of SHH and Gli1 by

immunohistochemical staining (Fig. 2C). Additionally, Gli1 was highly expressed in the

nuclei of BCMab1+CD44+ cells, but undetectable in BCMab1-CD44- cells (Fig. 2D). More

importantly, SHH was highly expressed in BCMab1+CD44+ cells and apparently hydrolyzed 7

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to form its catalyzed band (Fig. 2E), but undetectable in BCMab1-CD44- cells. These data

suggest that the Hh signaling pathway is activated in BCSCs.

To further confirm the involvement of Hh signaling in BCSCs, we used the SMO

antagonist cyclopamine (CPM) to treat BCMab1+CD44+ cells for an in vitro sphere

formation assay. We observed that cyclopamine treatment dramatically impaired

oncosphere formation (Fig. 2F). Additionally, by using a soft agar colony formation assay,

cyclopamine treatment also significantly repressed anchorage-independent tumor growth

(Fig. 2G). Overall, the Hh signaling pathway is essential for the maintenance of

self-renewal of BCSCs.

GALNT1 is highly expressed in BCSCs

We performed transcriptome microarray of BCMab1+CD44+ cells versus

BCMab1-CD44- cells derived from bladder cancer samples. Microarray analysis displayed

that the glycotransferase GALNT1 mRNA level in BCMab1+CD44+ was over 7 fold higher

than that in BCMab1-CD44- cells (Fig. 3A). However, other glycotransferases (GALNT4, 7,

8, 9, and 12) that the microarray detected had no significant changes in BCMab1+CD44+

cells compared with BCMab1-CD44- cells. Since the GALNT family have 12 members, we

then examined other undetected glycotransferase members in the microarray, including

GALNT2, 3, 5, 6, 10, and 11, in the BCMab1+CD44+ cells. We noticed that these

undetected glycotransferases were not expressed in the BCMab1+CD44+ cells (data not

shown). These results were verified in BCMab1+CD44+ cells isolated from 72 bladder

cancer specimens by using quantitative real time PCR (Fig. 3B, C). Moreover, high

expression of GALNT1 in pooled BCMab1+CD44+ cells from ten bladder cancer

specimens was further validated by immunoblotting (Fig. 3D). High expression of GALNT1

in bladder cancer tissues was confirmed by immunohistochemical staining (Fig. 3E). In

addition, GALNT1 positive staining in bladder cancer tissues was related to BCMab1 and

CD44 staining.

To further verify the high expression of GALNT1 in BCMab1+CD44+ cells, we

examined the 5' CpG sites at the GALNT1 promoter of genomic DNAs by bisulfite

genomic sequencing analysis. Each group displayed ten independent sequencing clones.

The GALNT1 promoter region was over 90% unmethylated in BCMab1+CD44+ cells, 8

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whereas the same cytosine residues were about 60% methylated in BCMab1-CD44- cells

(Fig. 3F). Similar results were obtained with other three bladder cancer specimens. In sum,

these results suggest that the GALNT1 is indeed actively transcribed in BCMab1+CD44+

cells.

GALNT1 is required for the maintenance of stemness for BCSCs

We next wanted to determine whether GALNT1 takes part in the regulation of BCSCs.

We silenced GALNT1 in BCMab1+CD44+ cells derived from one human bladder cancer

specimen and established GALNT1 depleted cell lines (Fig. 4A). We found that GALNT1

knockdown significantly declined oncosphere formation and anchorage-independent

colony formation (Fig. 4B, C). Benzyl-N-acetyl-D- galactosaminide (BG) is a competitive

substrate inhibitor for all glycotransferases (22). BG treatment obtained similar results to

GALNT1 depletion (Fig. 4B, C). We detected mRNA levels of Gli1 and GALNT1 in

BCMab1+CD44+ cells derived from 72 bladder cancer samples by quantitative real time

PCR. Interestingly, Gli1 displayed a good correlation with GALNT1 expression (Pearson’s

correlation coefficient r = 0.945) (Fig. 4D).

Importantly, we observed that GALNT1 knockdown or BG treatment dramatically

reduced Gli1 expression in BCMab1+CD44+ cells (Fig. 4E). These results were verified by

immunofluorescence staining (Fig. 4F). We next overexpressed Gli1 and Gli2 in

BCMab1-CD44- cells, followed by oncosphere formation assays. We observed that Gli1

but not Gli2 overexpression could promote oncosphere formation (Supplementary Fig. 1A),

suggesting Gli1 was activated in BCSCs. Additionally, GALNT1 knockdown or BG

treatment significantly declined active SHH-N protein levels in BCMab1+CD44+ cells (Fig.

4G), suggesting that GALNT1 depletion impaired SHH processing. Consequently,

GALNT1 silenced BCMab1+CD44+ cells formed smaller tumor burden and displayed much

lower xenograft tumor formation rates compared with shCtrl treated cells (Fig. 4H, I).

We next rescued SHH and/or GALNT1 in GALNT1 silenced BCMab1-CD44- cells. We

found that rescue of both SHH and GALNT1 was able to process SHH and induced Gli1

expression (Supplementary Fig. 1B). By contrast, only rescue of SHH in GALNT1 silenced

BCMab1-CD44- cells neither hydrolyzed SHH nor triggered Gli1 expression

(Supplementary Fig. 1B). These data validate that GALNT1-mediated SHH processing can 9

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induce Gli1 expression. Finally, we examined expression levels of SHH and its receptor

PTCH1 in BCSCs from bladder cancer samples. We noticed that PTCH1 was expressed in

the BCMab1+CD44+ cells (Supplementary Fig. 1C), and SHH was processed in these

BCSCs. These results suggest that SHH processing in the BCSCs is directly related to its

own Hh signaling in the same type of cell. Thus we conclude that SHH is an autocrine

factor in the BCMab1+CD44+ cells. Altogether, these data suggest that GALNT1

contributes to the activation of Hh signaling and tumorigenesis of bladder cancer.

GALNT1-mediated glycosylation is required for SHH activation in BCSCs

To further test whether GALNT1 influences SHH activation, we detected the SHH

processing status in GALNT1 silenced or BG treated BCMab1+CD44+ cells. Interestingly,

we found that GALNT1 knockdown significantly impaired SHH hydrolysis in

BCMab1+CD44+ cells derived from bladder cancer specimens (Fig. 5A), whereas shCtrl

treated BCMab1+CD44+ cells exhibited apparent SHH processing. Moreover, the full

length SHH displayed a smaller size on the gel blot in GALNT1 depleted BCMab1+CD44+

cells, suggesting that SHH might not be glycosylated after depletion of GALNT1. BG

treatment showed similar results (Fig. 5A). Peanut agglutinin (PNA) can specifically

bind to the terminal galactose residues of O-glycans (23). To examine whether SHH

undergoes O-linked glycosylation, we used PNA to probe SHH immunoprecipitates

derived from GALNT1 silenced or shCtrl treated BCMab1+CD44+ cells. Interestingly, we

found that PNA could bind to SHH and its hydrolyzed SHH-C fragment (Fig. 5A),

suggesting the SHH underwent O-linked glycosylation in BCMab1+CD44+ cells. To further

confirm O-linked glycans, we used N-glycosidase F and O-glycosidase to treat

BCMab1+CD44+ cell lysates, respectively. PNA still bound to the SHH from

N-glycosidase-treated cell extracts, but not the SHH from O-glycosidase-treated cell

extracts (data not shown). These results verify that the O-linked glycosylation takes place

in SHH of BCSCs. We observed that immunofluorescence staining appeared in WT or

shCtrl treated BCMab1+CD44+ cells, but not in GALNT1 silenced or BG-treated

BCMab1+CD44+ cells (Fig. 5B). These data indicate that GALNT1 participates in the

processing activation of SHH.

We next predicted O-linked glycosylation sites of SHH by using the NetOGlyc 3.1 10

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Server tool. Three potential candidate O-linked glycosylation sites of SHH protein,

including Thr282, Ser288 and Ser289, were selected out (Supplementary Fig. 2). To

identify the exact glycosylation sites, we generated three mutation construct vectors of

SHH (T282A-SHH, S288A-SHH, and S289A-SHH). We co-transfected GALNT1 and SHH

(WT) or its three respective mutants into BCMab1-CD44- cells. We noticed that only

T282A-SHH mutation abolished SHH hydrolysis, but WT SHH and other two mutants

(S288A-SHH and S289A-SHH) were still processed to generate its hydrolyzed forms (Fig.

5C). Furthermore, T282A-SHH mutant showed a smaller size of SHH on the blot,

suggesting T282A-SHH mutation could not undergo glycosylation. Additionally,

T282A-SHH mutant could not bind to PNA (Fig. 5C), whereas WT SHH and other two

mutants (S288A-SHH and S289A-SHH) were able to bind to PNA, indicating that the

O-linked glycosylation did not take place in T282A-SHH mutation. Overall, Thr282 was

defined as the glycosylation site for the O-linked glycosylation of SHH.

To further verify that the SHH hydrolysis is mediated by GALNT1 glycosylation,we

generated recombinant GALNT1, WT SHH and T282A-SHH mutant proteins for an in vitro

glycosylation assay. As expected, the T282A-SHH mutant incubated with GALNT1 was not

processed, and displayed a smaller size (Fig. 5D). By contrast, WT SHH incubated with

GALNT1 showed apparent hydrolysis and displayed a bigger size. Moreover, WT SHH

incubated with GALNT1 could bind to PNA, but not for the T282A-SHH mutant incubated

with GALNT1 (data not shown). SHH processing in BCMab1+CD44+ cells served as a

positive control. To further validate the glycosylation of SHH and its three mutants, we

constructed biotinylated for SHH and its mutants to analyze GALNT1-mediated

SHH glycosylation (Supplementary Fig. 3). We found that only the T282A-SHH mutant

peptide could not be glycosylated by GALNT1 (Fig. 5E), while WT SHH and the other two

mutants (S288A-SHH and S289A-SHH) indeed exhibited glycosylation. We conclude that

the GALNT1 mediated O-linked glycosylation of SHH is necessary for SHH processing.

We next examined rescue function of GALNT1 and SHH in co-transfected

BCMab1-CD44- cells from bladder cancer specimens. Notably, GALNT1 and SHH

co-transfection could induce formation of a subpopulation of BCMab1+CD44+ cells (Fig.

5F). However, GALNT1, SHH or T282A-SHH alone, or GALNT1 plus T282A-SHH mutant 11

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transfection failed to generate this subset. Additionally, GALNT1 and SHH co-transfection

were able to generate oncospheres efficiently (Fig. 5G). GALNT1 and SHH co-transfection

formed xenograft tumors rapidly after inoculation (Fig. 5H). By contrast, GALNT1, SHH or

T282A-SHH alone, or GALNT1 plus T282A-SHH mutant transfection had no such capacity.

Taken together, GALNT1-mediated activation of SHH is indispensable for the maintenance

of self-renewal of BCSCs.

GALNT1 deficiency inhibits bladder tumor growth

We induced bladder cancer in GALNT1 deficient mice or littermate control mice (WT)

by feeding BBN. At 4 months of BBN exposure, 23 WT mice out of 24 mice displayed

robust tumors in bladders, with bigger tumor sizes (Fig. 6A, B). By contrast, only 5

−/− GALNT1-deficient mice out of 24 GALNT1 mice showed bladder tumors with smaller

sizes. Tumors in WT or GALNT1 deficient mice were confirmed by histology staining (Fig.

−/− 6C). We prepared single tumor cells from WT or GALNT1 mice for an in vitro sphere

−/− formation assay. We observed that GALNT1 tumor cells formed much fewer

oncospheres than WT tumor cells (Fig. 6D). Expectedly, mRNA and protein levels of Gli1

−/− were undetectable in GALNT1 tumors (Fig. 6E, F), whereas Gli1 was highly expressed

in WT tumors. In sum, GALNT1 deletion in mice suppresses BBN-induced bladder tumors.

GALNT1 silencing or blockade of Hh signaling displays antitumor efficacy of

bladder cancer in vivo

The implanted mice were grouped (24 mice/group) and intravesically administrated

GALNT1 siRNA/liposomes, or GALNT1 siRNA/liposomes plus cyclopamine (CPM) next

day. Intriguingly, both GALNT1 siRNA and CPM could significantly suppressed tumor

growth (Fig. 7A, B). Importantly, GALNT1 siRNA plus CPM were able to abrogate tumor

formation. To detect the delivery efficiency of siRNA with liposome, we analyzed GALNT1

expression in bladder cancer tissues after 5 weeks of intravesical instillation of GALNT1

siRNA. GALNT1 siRNA was mixed with liposome for intravesical administration as

previously described (24). GALNT1 was almost undetectable in GALNT1

siRNA/liposome-treated bladder cancer tissues (Fig. 7C), whereas GALNT1 was

abundantly expressed in siCtrl-treated control mice. These data suggest that intravesical

instillation of GALNT1 siRNA/liposome could efficiently deliver into bladder tissues and 12

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successfully deplete GALNT1 expression. Notably, both GALNT1 siRNA and CPM

treatment were able to prolong survival rates of treated mice compared with siCtrl treated

mice. However, over 60% mice treated with GALNT1 siRNA plus CPM lived as long as WT

mice (Fig. 7D). Therefore, the therapeutic efficacy is due to elimination of the BCSCs.

We next examined whether GALNT1 affects metastasis of bladder cancer.

BCMab1+CD44+ cells injected via tail veins induced severe lung metastasis after 5 weeks,

whereas BCMab1-CD44- cells were unable to form apparent tumors in lungs or other

tissues (Fig. 7E). We established GALNT1 silenced BCMab1+CD44+ cell lines sorted from

human bladder cancer samples and injected them through tail veins. We observed that

GALNT1 silenced BCMab1+CD44+ cells significantly inhibited tumor metastasis (Fig. 7E),

while shCtrl treated cells still showed robust lung metastasis.

To further assess GALNT1 function in BBN-induced tumorigenesis of bladder cancer,

we induced mouse bladder cancer by feeding BBN and intravesically applied with GALNT1

siRNA every week. Then, we used ultrasound to observe tumor volumes every two weeks.

At 12 weeks, siCtrl treated mice displayed robust bladder tumors in most mice (Fig. 7F). By

contrast, GALNT1 siRNA administration effectively suppressed bladder tumor growth.

Additionally, GALNT1 siRNA treatment significantly declined tumor formation rates

compared with siCtrl treatment (Fig. 7G). Altogether, GALNT1 is implicated in the

tumorigenesis of bladder cancer.

Discussion

Bladder cancer stem cells (BCSCs) were considered as tumor-initiating cells (TICs)

(25), which could be responsible for bladder cancer initiation and recurrence. In this study,

we identified a BCMab1+CD44+ cell population as BCSCs. The BCMab1 antibody,

generating against CD44+ cells from human bladder cancer specimens, was defined to

recognize aberrantly glycosylated integrin α3β1 in our previous study (20), which is a novel

biomarker for BCSCs. We demonstrate that BCMab1+CD44+ cells have increased

tumorigenicity and self-renewal property in vivo and in vitro. We defined that the

GALNT1-mediated Hh signaling activation is essential for the maintenance of self-renewal

of BCSCs and bladder tumorigenesis.

The mammalian secreted Hh ligands include Sonic Hedgehog (SHH), Indian 13

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Hedgehog (IHH) and Desert Hedgehog (DHH), which initiate a signaling cascade via

engaging with their common receptor Patched1 (PTCH1) (17). Upon Hh engagement,

PTCH1 releases its inhibitory role in the positive regulatory transmembrane protein SMO,

resulting in activation of Gli family transcription factors (Gli1, Gli2 and Gli3) (26, 27). The

Hh pathway is largely inactive in the adult, except for its action in tissue repair and

maintenance (14, 28). The role of Hh signaling in the tumorigenesis of bladder cancer

remains controversial. Inconsistently, loss-of-function mutations of PCTH1 were rarely

identified in bladder cancer (29, 30). Recently, Robbins and his colleagues showed that the

Hh signaling is highly expressed in bladder cancer tissues and implicated in the

tumorgenesis of bladder cancer (31, 32). However, the role of Hh signaling pathway in

BCSCs is largely unknown.

Newly synthesized Hh proteins require a series of posttranslational processing

reactions to generate their respective active forms for signal transduction. Many properties

of Hh autoprocessing were defined from studies of the Drosophila protein (33). It is widely

considered that the Hh mature processing procedure is highly conserved in various

species. It has been revealed that the secreted Hh signaling domain is covalently modified

by cholesterol and palmitate (26). The N-terminal cysteine is catalyzed by the

acyl- Skinny hedgehog to form the amide linkage of palmitate (34, 35).

Hydrophobic modifications confer membrane affinity such that the signaling domain is

successfully associated with its membrane receptors leading to a signaling cascade (36,

37). Here we defined a novel glycosylation modification for SHH in BCSCs, which is

uniquely mediated by GALNT1.

Superficial tumors are usually treated by surgical resection and intravesical

chemotherapy and/or immunotherapy. The difficulty in managing bladder cancer is unable

to predict which tumors will recur or progress38. A previous report showed that intravesical

instillation of polo-like kinase (PLK-1) siRNA can effectively prevent bladder cancer growth

(24). siRNA mediated RNA interference is sequence-specific silencing and widely

used to silence (39, 40). Here we showed that intravesical administration

of GALNT1 siRNA can significantly suppress bladder tumor growth in orthotopic bladder

cancer mouse models and BBN-induced bladder tumors. The SMO antagonist 14

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cyclopamine and its derivatives have been reported to be effective agents against several

tumors (18, 41). One of cyclopamine derivative GDC-449 has been approved to treat

advanced unresectable basal cell carcinoma (BCC) as a first-line therapy (42). Of note,

intravesical administration of GALNT1 siRNA and cyclopamine can eradicate implanted

tumor formation without overt adverse effects. Finally, therapeutic use of GALNT1 siRNA,

the SMO inhibitor, and the BCMab1 antibody will provide a potent antitumor strategy

against bladder cancer.

Acknowledgments

We thank Drs. Junfeng Hao and Qiang Hou for their technical support. This work was

supported by the National Natural Science Foundation of China (81330047, 81472413);

The State Projects of Essential Drug Research and Development (2012ZX09103301-041);

973 Program of the MOST of China (2010CB911902), the Strategic Priority Research

Programs of the Chinese Academy of Sciences (XDA01010407).

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Figure legends:

Figure 1. A BCMab1+CD44+ subpopulation of cancerous cells is defined as BCSCs.

(A) Representative cystoscope of a typical bladder cancer. The asterisk indicates a

neoplasm. Asterisk denotes bladder tumor. (b) A subset of BCMab1+CD44+ cells was

fractionated in one human bladder cancer specimen. Single tumor cells were stained with

BCMab1 (B) and anti-CD44 (C) antibodies followed by flow cytometry. (C)

Immunofluorescence staining of BCMab1+CD44+ (left panel) and BCMab1-CD44- (right

panel) cells. (D) Tumor formation frequency of BCSCs from unsorted, BCMab1+CD44+,

and BCMab1-CD44- subpopulations derived from bladder cancer patient specimens. CI,

confidence interval. Six bladder cancer samples got similar results. (E) Representative

photographs demonstrating xenografted tumors (arrowheads). Bladder tumor cell

subpopulations (BCMab1+CD44+ or BCMab1-CD44-) with limiting dilutions were injected

subcutaneously into NOD/SCID mice with the indicated numbers of cells. (F) FACS

analysis of BCMab1 and CD44 expression in xenograft tumors generated from 1×105

BCMab1+CD44+ or BCMab1-CD44- cells. (G) Injection of 10, 100, 1000, 10,000 and

100,000 freshly isolated BCMab1+CD44+ (left panel) or BCMab1-CD44- cells (right panel)

derived from the primary graft tumors. Data represent means ± SD. n=6. (H) Serially

tumor formation assay. Tumor volumes were measured at the indicated time points and

calculated as means ± SD in BCMab1+CD44+ (left panel) or BCMab1-CD44- (right panel)

cell transplantations. n=6. (I) Oncospheres were counted in five separate fields after two

weeks and shown as means±SD.

Figure 2. Hedgehog signaling is essential for self-renewal of BCSCs. (A) Scatter plot

comparing global gene expression profiles between BCMab1+CD44+ and BCMab1-CD44-

cells. Black lines denote two-fold differences in either direction in gene expression levels.

Hedgehog signaling related genes were indicated with red arrowheads. (B) Real-time

PCR analysis of Gli1 mRNA levels in BCMab1+CD44+ cells derived from 72 bladder

cancer specimens. ∗∗, p<0.01. (C) Immunohistochemical staining of human bladder

cancer tissues and peri-carcinoma tissues with ant-SHH and anti-Gli1 antibodies. (D)

Immunofluorescence staining in BCMab1+CD44+ or BCMab1-CD44- cells by 18

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BCMab1-FITC, anti-CD44-PE and anti-Gli1-PE labeled antibodies. White arrowheads

indicate nuclear localization of Gli1. (E) Western blot analysis of SHH and Gli1 protein

levels in BCMab1+CD44+ cells pooled from bladder cancer samples. (F, G) Cyclopamine

significantly inhibits both oncosphere formation and anchorage-independent tumor growth

of BCMab1+CD44+ cells. BCMab1+CD44+ cells were treated with cyclopamine for 24 h

prior to oncosphere formation (F) or soft agar colony (G) assays. All data are

representative of four independent experiments and shown as means ± SD. ∗∗, p<0.01.

Figure 3. GALNT1 is highly expressed in BCSCs. (A) Heat map represents the relative

expression of GALNT1, Integrin α3β1, CD44 and Gli1 in BCMab1+CD44+ and

BCMab1-CD44- cells. (B) Real-time PCR analysis of mRNA levels of GALNTs in

BCMab1+CD44+ and BCMab1-CD44- cells derived from patient samples. ∗∗, p < 0.01. n=6.

(C, D) Real-time PCR (C) and Western blot (D) analysis of GALNT1 expression levels in

BCMab1+CD44+ and BCMab1-CD44- cells derived from 72 bladder cancer specimens. (E)

Immunohistochemical staining of human bladder cancer tissues with BCMab1, anti-CD44

and anti-GALNT1 antibodies. H&E, haematoxylin and eosin. (F) Bisulfite genomic

sequencing analysis of the 5’ CpG sites at the GALNT1 promoter. Each group includes ten

independent clones. Black dots denote methylated CpG, red dots indicate unmethylated

CpG.

Figure 4. GALNT1 is required for maintenance of stemness of BCSCs. (A) GALNT1

was silenced in BCMab1+CD44+ cells derived from one human bladder cancer specimen.

A scrambled sequence (shCtrl) was used as a control. (B, C) GALNT1 silencing or

Benzyl-N-acetyl-D-galactosaminide (BG) treatment declines oncosphere formation (B)

and anchorage-independent tumor growth (C) of BCMab1+CD44+ cells. Oncospheres or

colonies were counted in five separate fields after two weeks. ∗∗, p< 0.01. (D) mRNA

levels of GALNT1 and Gli1 were detected by RT-PCR from 72 patient samples. p =

0.001, r = 0.945, Pearson’s correlation. (E) Real-time PCR analysis of Gli1 mRNA levels in

GALNT1 silenced or shCtrl BCMab1+CD44+ cells. BG treated BCMab1+CD44+ cells

were also analyzed. Data were normalized to β-actin and shown as means±SD. n=12. ∗∗, 19

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p<0.01. (F) Immunofluorescence staining of GALNT1 and Gli1 in shCtrl or GALNT1

silenced BCMab1+CD44+ cells. GALNT1 was labeled with FITC (green), Gli1 was labeled

with PE (red). (G) ELISA analysis of SHH-N levels shCtrl or GALNT1 depleted

BCMab1+CD44+ cells. BG treated cells were also analyzed. ∗∗, p<0.01. (H) Tumor

volumes were measured at the indicated time points and calculated as means ± SD (left

panel). Representative tumors at week 8 were shown in the right panel. ∗∗, p<0.01. n=12.

(I) Xenograft BCSCs sorted by FACS and infected with retrovirus expressing either

scramble vector (shCtrl) or shGALNT1, followed by transplantation into mice 24 h after

infection (n=10 for each group).

Figure 5. GALNT1-mediated glycosylation of SHH is needed for SHH activation in

BCSCs. (A) GALNT1 silenced or BG-treated BCMab1+CD44+ cell lysates were probed

with anti-C-terminal SHH, anti-GALNT1, and anti-Gli1 antibodies. IP, immunoprecipitation;

IB, immunoblotting. (B) Immunofluorescence staining with C- or N-terminal SHH antibody

in GALNT1 silenced or BG-treated BCMab1+CD44+ cells. The C-terminal of SHH antibody

was labeled with FITC, the N-terminal of SHH antibody was labeled with PE. (C). SHH

and its three mutations (T282A, S288A, S289A) were constructed into vectors and

co-transfected with GALNT1 into BCMab1-CD44- cells for 48 h, respectively. (D) WT SHH

or T282A-SHH mutant was incubated with GALNT1 plus BCMab1-CD44- cell lysates.

Reaction products were analyzed by immunoblotting. (E) Biotinylated peptides were

incubated with GALNT1 and UDP-GalNAc followed by detection of absorbance (450 nm).

(F, G) Co-transfection of SHH and GALNT1 can rescue formation of BCMab1+CD44+ cells

with self-renewing ability. BCMab1-CD44- cells were co-transfected with the indicated

vectors for 48 h followed by FACS analysis (F) and a sphere formation assay (G).

Oncospheres were counted in five separate fields after two weeks and shown as means

±SD. ∗∗, p < 0.01. (H) Tumors with the indicated treatment were subcutaneously injected

into the backs of NOD/SCID mice (n=12).

Figure 6. GALNT1 deficiency inhibits BBN-induced bladder tumor growth. (A, B)

−/− GALNT1 mice show a dramatic decrease in tumor formation rates (A) and sizes (B) of 20

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BBN-induced bladder cancer. Tumor formation rates of BBN-induced bladder cancer in

mice were calculated after four months with BBN exposure. Then the mice were sacrificed

for measurement of tumor volumes. (n=24). ∗∗, p < 0.01. (C) H&E counterstaining for

+/+ −/− GALNT1 and GALNT1 mice treated with BBN. Asterisk indicates bladder tumor. (D)

−/− Tumors induced from GALNT1 mice suppress oncosphere formation. Tumor cells

+/+ −/− induced from GALNT1 and GALNT1 mice were performed for sphere formation

+/+ assays. (E, F) GALNT1 and Gli1 expression levels in bladder tumors from GALNT1 or

−/− GALNT1 mice analyzed by PCR (E) and immunoblotting (F).

Figure 7. Intravesical administration of GALNT1 siRNA and cyclopamine eradicates

bladder tumor in vivo. (A) Intravesical instillation of GALNT1 siRNA or cyclopamine

significantly suppresses bladder tumor growth. Bioluminescence images were obtained by

IVIS at 8 week after BCMab1+CD44+ cells transplantation with intravesical instillation of

the indicated agents. CPM, cyclopamine. (B) Photon counts of each mouse are indicated

by pseudo-color scales. Growth curves of orthotopical tumors were measured by IVIS

(n=24 per group). ∗∗, p<0.01. (C) GALNT1 expression levels were analyzed by

immunoblotting. (D) Comparison of survival rates with the indicated treatment. n=24 per

group. (E) Representative bioluminescence images showing bladder cancer metastasis.

Mice were implanted with the indicated human bladder cancer cells via tail vein injection

for 5 weeks. (F) Imaging by stereoscope (middle panel) and H&E counterstaining (lower

panel) at 12 week after intravesical administration of GALNT1 siRNA with BBN exposure

(upper panel). (G) Tumor formation rates of GALNT1 siRNA or siCtrl treated mice with

exposure of BBN.

21

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GALNT1-mediated glycosylation and activation of sonic hedgehog signaling maintains the self-renewal and tumor-initiating capacity of bladder cancer stem cells

Chong Li, Ying Du, Zhao Yang, et al.

Cancer Res Published OnlineFirst December 16, 2015.

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