Expression of sialyl-Tn sugar antigen in bladder cancer cells affects response to Bacillus Calmette Guérin (BCG) and to oxidative damage

Abstract

The sialyl-Tn (sTn) antigen is an O-linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patient's response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCR_sTn and MCR_Nc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCR_Nc, BCG-stimulated MCR_sTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCR_Nc and MCR_sTn revealed that ST6GALNAC1/sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCR_sTn cells displayed higher H₂O₂ sensitivity. In MCR_sTn,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1/sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.

Keywords: Bacillus Calmette-Guérin; ST6GALNAC1 sialyltransferase; bladder cancer; glycosylation; sialyl-Tn antigen.

Figure 1. Biosynthesis of sTn by ST6GALNAC1

The addition of α2,6-linked sialic acid on the Tn antigen (GalNAc-Ser/Thr), mediated by ST6GALNAC1 results in the biosynthesis of the sTn antigen, while the addition of α2,6-linked sialic acid on the T antigen (Galβ1,3GalNAc-Ser/Thr), resulting in the biosynthesis of the sialyl-6-T antigen, is catalyzed mainly by ST6GALNAC2.

Figure 2. Gene expression analysis of surgical specimens of bladder cancers and normal urothelium

Tissue specimens from 43 patients with nonmuscle-invasive bladder cancer eligible for BCG therapy, were collected, the RNA was extracted and the relative mRNA levels were analyzed by Real Time RT-PCR, as described in the Material and Methods section. Values indicate the number of mRNA molecules of ST6GALNAC1 or IL-6 genes per 1000 molecules of the reference gene (β-actin). (A) The expression of ST6GALNAC1 mRNA was compared in tumor tissue and matched normal urothelium and found to be significantly higher (p<0.05 according to Student's t test for paired samples) in tumor. (B) Patients were divided according to their follow up after BCG treatment. BCG Responders (Resp) were considered those without recurrence within a minimal period of 12 months following TURBT. BCG non responders (NON-Resp) were those that experienced disease recurrence within that period. High ST6GALNAC1 mRNA patients all belonged to the Responders group. (C) The expression of IL-6 mRNA was measured in the two groups and found to be higher in the Responders group. (D and E) correlation analysis indicated a significant (r= 0.375; p=0.04) relationship between ST6GALNAC1 and IL-6 mRNA expression in the responders group (D) but not in the non-responders group (E) (r= 0.281; p=0.33).

Figure 3. Cytokine secretion by BCG-challenged MCR cells

MCR_Nc (white bars) and MCR_sTn (black bars) cells were challenged or not with BCG and the concentration of ten cytokines secreted was determined in their conditioned media as described in the Materials and Methods section. Only IL-6 and IL-8 were detectable and showed BCG modulation, mainly in MCR_sTn cells. Data are the mean ± SD of three experiments. ***p<0.0001; **p<0.001; *p<0.05, according to two ways ANOVA, followed by Tukey multiple comparison test. p values are reported in Supplementary Table 1.

Figure 4. Cytokine secretion by macrophages treated with conditioned media of MCR cells

The secretion of ten cytokines was measured in the culture media of unstimulated macrophages or of macrophages stimulated with the conditioned media of MCR_Nc (white bars) or MCR_sTn (black bars) either BCG-challenged or unchallenged, analyzed in Figure 3. Bars indicate the concentrations of cytokines detected in the conditioned medium of unstimulated macrophages (MØ, gray bars) or macrophages stimulated with conditioned media of unchallenged MRC_Nc or MCR_sTn cells (MØ + unchallenged) or stimulated with conditioned media of BCG-challenged MRC_Nc or MCR_sTn cells (MØ + BCG-challenged). Macrophage stimulation by unchallenged cells was negligible. With the exception of IL-8, the conditioned medium of BCG-challenged MCR_sTn cells (black bars) potentiated cytokine release by macrophages more than that of MCR_Nc (white bars). Data are the mean ± SD of 3 experiments. ***p<0.0001; **p<0.001; *p<0.05, according to two ways ANOVA test, followed by Tukey multiple comparison test. p values are reported in Supplementary Table 1.

Figure 5. Cytotoxic effect of H₂O₂

(A) Cells were treated with H₂O₂ or mock-treated as described in Materials and Methods and photographed with a phase contrast microscope (original magnification 40 X). (B) Cell counting indicated a 20% reduction in MCR_Nc cells and a 68% reduction in MCR_sTn after H₂O₂ treatment. Data are the mean ± SD of at least three experiments performed in triplicate. * p< 0.05; *** p<0.0001 according to Student's t test.