Published OnlineFirst December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-2309 Cancer Tumor and Stem Cell Biology Research GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self- Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells Chong Li1, Ying Du1, Zhao Yang1, Luyun He1, Yanying Wang1, Lu Hao1, Mingxia Ding2, Ruping Yan2, Jiansong Wang2, and Zusen Fan1 Abstract þ þ The existence of bladder cancer stem cells (BCSC) has been was highly expressed in BCMab1 CD44 cells and correlated suggested to underlie bladder tumor initiation and recurrence. with clinicopathologic features of bladder cancers. Mechanis- Sonic Hedgehog (SHH) signaling has been implicated in pro- tically, GALNT1 mediated O-linked glycosylation of SHH moting cancer stem cell (CSC) self-renewal and is activated in to promote its activation, which was essential for the self- bladder cancer, but its impact on BCSC maintenance is unclear. renewal maintenance of BCSCs and bladder tumorigenesis. þ In this study, we generated a mAb (BCMab1) against CD44 Finally, intravesical instillation of GALNT1 siRNA and the SHH human bladder cancer cells that recognizes aberrantly glycosy- inhibitor cyclopamine exerted potent antitumor activity against lated integrin a3b1. The combination of BCMab1 with an anti- bladder tumor growth. Taken together, our findings identify a þ þ CD44 antibody identified a BCMab1 CD44 cell subpopula- BCSC subpopulation in human bladder tumors that appears tion as BCSCs with stem cell–like properties. Gene expression to be responsive to the inhibition of GALNT1 and SHH sig- analysis revealed that the hedgehog pathway was activated in naling, and thus highlight a potential strategy for preventing þ þ the BCMab1 CD44 subpopulation and was required for the rapid recurrence typical in patients with bladder cancer. BCSC self-renewal. Furthermore, the glycotransferase GALNT1 Cancer Res; 76(5); 1–11. Ó2015 AACR. Introduction differentiate into heterogeneous cell populations, resulting in phenotypic and functional heterogeneity of tumors (8, 9). Qui- Bladder cancer is the second most common urological malig- escent CSCs may resist therapeutic intervention and confer to nancy with over 69,000 new cases and an estimated 150,000 recurrence after initial therapy (7). Chan and colleagues identified deaths worldwide each year (1–3). The big problem of bladder À þ þ À a subpopulation (Lineage CD44 CK5 CK20 ) from primary cancer is the extremely easy recurrence after treatment and no human bladder cancer as bladder cancer stem cells (BCSC) recognized second-line therapy (4). Thus, for new therapeutic (10), which shared similar expression markers to normal bladder strategies, it is of great importance to delineate the mechanism of basal cells, suggesting the basal-like property of BCSCs. A sub- bladder cancer tumorigenesis. population of primary urothelial cancers, coexpressing the 67- Accumulating evidence from leukemias, germ cell tumors, and kDa laminin receptor (67LR) and the basal cell–specific cytoker- many solid tumors support a cancer stem cell (CSC) model in atin CK17, possesses more carcinogenic ability (11). These subsets which cancers are initiated by a rare subpopulation of self-renew- harbor high tumorigenicity, multipotency, and involvement in ing and differentiating tumor-initiating cells (TIC; refs. 5–7). The bladder tumor progression, suggesting that the CSCs exist in cancer stem cell (CSC) population can self-renew limitlessly and bladder tumors. Bladder cancer arises from the urothelium, a transitional epi- thelium lining the bladder lumen. This multilayered epithelium is 1CAS Key Laboratory of Infection and Immunity, Institute of Biophys- composed of a luminal layer of fully differentiated umbrella cells ics, Chinese Academy of Sciences, Beijing, China. 2Department of that overlie intermediate cells, and a basal layer of Sonic Hedge- fi Urology, The Second Af liated Hospital of Kunming Medical Univer- hog (SHH)-expressing cells (12). The SHH-expressing basal cells, sity, Kunming, China. capable of regenerating all cell types of the urothelium, have been Note: Supplementary data for this article are available at Cancer Research defined as urothelial stem cells. The same group reported that Online (http://cancerres.aacrjournals.org/). muscle-invasive bladder cancers originate exclusively from the C. Li, Y. Du, Z. Yang contributed equally to this article. SHH-expressing basal stem cells of the basal urothelium in Corresponding Authors: Zusen Fan, Institute of Biophysics, Chinese Academy procarcinogen N-butyl-N-4-hydroxybutyle nitrosamine (BBN)- of Sciences, 15 Datun Road, Room 1611, Beijing 100101, China. Phone: 8610-6488- induced mouse bladder cancer (13). These SHH-expressing basal 8457; Fax: 8610-6487-1293; E-mail: [email protected]; and Jiansong Wang, stem cells act as BCSCs to initiate bladder oncogenesis. Aberrant E-mail: [email protected] activation of the Hedgehog signaling is implicated in many doi: 10.1158/0008-5472.CAN-15-2309 malignancies (14, 15). Emerging data support that Hh signaling Ó2015 American Association for Cancer Research. plays a pivotal role in the self-renewal and differentiation of CSCs www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-2309 Li et al. (16, 17). Activation of Hh pathway causes CSC self-renewal and In vitro glycosylation assay expansion, while the SMO antagonist cyclopamine or the ligand- Recombinant human wild-type (WT) SHH and T282A-SHH neutralizing antibody induces terminal differentiation and loss of mutant proteins were expressed in E. coli. WT SHH or T282A-SHH clonogenic growth capacity (16, 18). In this study, we defined that mutant was incubated with GALNT1 (R&D Systems) plus À À Hh signaling is essential for the self-renewal of BCSCs. GALNT1- BCMab1 CD44 cell lysates at 37C for 4 hours. Reaction pro- mediated glycosylation is required for SHH activation in BCSCs. ducts were analyzed by immunoblotting. Materials and Methods Immunohistochemical staining fi Reagents and mice Tumor specimens were xed in 10% buffered formalin and fi Mouse mAb BCMab1 was purified through protein A-Sephar- embedded in paraf n as described (20). The immunosignals were ose from ascites. Corresponding species-specific FITC-conjugated detected using the ABC kit. secondary antibodies were purchased from Sigma. The commer- cial primary antibodies were phycoerythrin (PE)-conjugated anti- Intravesical administration of siRNA/liposomes and CD44 (BD Pharmingen; 550989), goat anti-human C-terminus of cyclopamine for treatment SHH (Santa Cruz Biotechnology, sc-33942), goat anti-human N- Female 6-week-old NOD/SCID mice with a body weight of terminus of SHH (Santa Cruz Biotechnology; sc-1194), rabbit approximately 15 g were used and kept under specific pathogen- þ þ anti-Gli1 (Santa Cruz Biotechnology, sc-20687), goat anti- free conditions. BCMab1 CD44 cells were labeled with lucifer- GALNT1 (Santa Cruz Biotechnology, sc-68491), and mouse ase and transplanted into the murine bladder cavity via 24-gauge anti-b-actin antibody (Sigma, A1978). NOD/SCID mice were angiocatheters (21). The mice were grouped (24 mice per group) obtained from Vital River Laboratory Animal Technology Co. and intravesically applied with 6 mmol/L siCtrl/liposomes or À/À Ltd.. GALNT1 mice were obtained from Jackson Laboratories. siGALNT1/liposomes with or without 100 mmol/L cyclopamine BBN, benzyl-N-acetyl-D-galactosaminide (BG), and cyclopamine next day. were from Sigma. GALNT1 siRNA (human) and GALNT1 siRNA (mouse) were from Santa Cruz Biotechnology. Statistical analysis The relationship between the expression levels of GALNT1 and Xenograft establishment clinicopathologic features was analyzed using the c2 or the Human bladder cancer specimens were obtained with patient's Kruskal–Wallis test. Kaplan–Meier analysis was used to estimate consent, as approved by the Research Ethics Board at The Second the cumulative cause-specific survival rates, and the log-rank test Affiliated Hospital of Kunming Medical University (Kunming, was used to correlate differences in patient survival with staining China). For the generation of xenografts, cells were injected with intensity of GALNT1. The influence of GALNT1 on the growth of Matrigel subcutaneously into the backs of NOD/SCID mice. bladder cancers was analyzed by the Student t test. Animal work was carried out in compliance with the ethical regulations approved by the Animal Care Committee, Institute Results þ þ of Biophysics, Chinese Academy of Sciences (Beijing, China). A BCMab1 CD44 subpopulation of cancerous cells acts as BCSCs þ Flow cytometry sorting To identify unique biomarkers of BCSCs, we isolated CD44 Primary tumor tissues were digested with 200U type I colla- BCSCs from human bladder cancer resection specimens (Fig. 1A), genase, 20U type IV collagenase (Sigma, C-0130, C-5138), and which were used as immunogen to immunize mice to generate DNase at 37 C for 2 to 6 hours. Then, the cancer cells were stained mAbs. About 458 hybridomas were screened to bind BCSCs. with FITC-conjugated BCMab1 and PE-conjugated anti-CD44 Among these hybridomas, we identified several antibodies, – antibody or the same isotype FITC/PE conjugated antibody for including BCMab1 and BCMab37, which were determined to fl 30 minutes on ice. Labeled cells were analyzed and sorted by ow recognize the BCSC surface antigens, aberrantly glycosylated