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 glycosylation 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. 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. 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+ human 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. 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. 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. 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. 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 protein 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. 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. 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 proteins 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.