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. 2019 Apr 30;8:e43024. doi: 10.7554/eLife.43024

Figure 6. Increased methylation of the SHH gene induces the basal subtype of human urothelial carcinoma through decreased activity of Hh/BMP signaling feedback between cancer cells and the tumor stroma.

(A) The methylation status of the CpG island and CpG shore regions of the human SHH gene was analyzed by bisulfite sequencing in three human invasive urothelial carcinoma cell lines, J82, T24, and TCC-SUP with or without 5’-azacitidine treatment. Each circle represents one of 117 CpG sites, and the average degree of methylation is indicated by the black portion of the white circle. (B) The results obtained from bisulfite sequencing analysis of (A) are summarized. (C) Expression of SHH in J82, T24, and TCC-SUP treated with 5’-azacitidine (J82, 6-fold increase; T24, 7-fold increase; TCC-SUP, 3-fold increase) compared to that of untreated controls. Data are presented as the mean ± SEM, and significance was calculated with an unpaired Student’s t test (**, p<0.01; ***, p<0.001). n = 3 technical replicates, and the entire experiment was repeated three times. (D) Orthotopic xenograft of J82 cells in immunocompromised (NOD/SCID/IL2Rgnull) mice (14 animals in total), followed by 5’-azacitidine treatment for 1 month. (E) H and E staining of orthotopic xenograft sections from mice treated with the vehicle control (left panels, seven animals) or 5’-azaciditine (right panels, seven animals). The lower panels show magnified views of the boxed regions in the upper panels. Scale bars represent 300 µm. (F) Expression of the luminal markers, FOXA1 (1.8-fold increase) and GATA3 (1.8-fold increase), and the basal markers, CDH3 (6-fold decrease) and KRT6A (9-fold decrease) in tumor xenografts from mice treated with 5’-azacitidine compared to those of the vehicle control. Data are presented as the mean ± SEM, and significance was calculated with an unpaired Student’s t test (*, p<0.05). n = 3 technical replicates, and the entire experiment was repeated six times. (G) The J28 cell line was infected using a lentivirus containing shRNA targeting SHH or BMPR1A. The resulting cell line was orthotopically xenografted into the dome of the bladder, and the mice were treated with 5’-azacitidine for 1 month. (H) Expression of SHH or BMPR1A in tumor xenografts from mice injected with J82 carrying shRNA targeting SHH or BMPR1A, respectively. Data are presented as the mean ± SEM, and significance was calculated with an unpaired Student’s t test (*, p<0.05; **, p<0.01). (I) Expression of the luminal markers, FOXA1 (shRNA for SHH, 2.5-fold decrease; shRNA for BMPR1A, 3-fold decrease) and GATA3 (shRNA for SHH, 2-fold decrease; shRNA for BMPR1A, 2-fold decrease), and the basal markers, CDH3 (shRNA for SHH, 2.3-fold increase; shRNA for BMPR1A, 4-fold increase) and KRT6A (shRNA for SHH, 2.5-fold increase; shRNA for BMPR1A, 7.2-fold increase) in tumor xenografts from mice injected with control J82 or J82 carrying shRNA targeting SHH or BMPR1A. n = 3 technical replicates, and the entire experiment was repeated six times. Data are presented as the mean ± SEM, and significance was calculated with an unpaired Student’s t test (*, p<0.05; **, p<0.01; ***, p<0.001). See also Figure 6—figure supplement 1A,B,C and Figure 6—source data 1.

Figure 6—source data 1. Expression of SHH, luminal and basal markers in bladder tumor cell lines and in tumor xenografts from mice treated with 5’-azacitidine, or expressing shRNA for SHH or BMPR1A.
elife-43024-fig6-data1.xlsx (18.8KB, xlsx)
DOI: 10.7554/eLife.43024.020

Figure 6.

Figure 6—figure supplement 1. Expression analyses of patient samples and large-scale transcriptional data suggest that the decreased methylation of the CpG shore of the human SHH promoter region impedes the growth of human urothelial carcinoma by inducing a luminal-like subtype through increased Hh/BMP signaling feedback.

Figure 6—figure supplement 1.

(A) Schematic diagram of the CpG island and flanking shore regions of human SHH. A total of 117 CpG sites were analyzed for methylation status. TSS, transcription start site; CGs, CpG sites; blue areas, CpG-rich region. (B, C) Orthotopic xenograft of J82 cells in immunocompromised (NOD/SCID/IL2Rgnull) mice, followed by 5’-azacitidine treatment for 1 month. H and E staining of orthotopic xenograft sections from mice treated with the vehicle control (B, bladder #1–6) or 5’-azaciditine (C, bladder #7–12). Scale bars represent 300 µm. (D) Stratification of gene expression in RNA-seq data from the TCGA database of muscle-invasive urothelial carcinoma. Heatmap showing the expression levels of SHH, BMP4, BMP5, ID2, ID3 and ID4. Based on unsupervised hierarchical clustering, two subgroups were identified; low SHH (red, n = 147) and high SHH (blue, n = 246). (E) Heatmaps showing the expression of basal and luminal makers in two clusters stratified from (D). (F) Survival analysis of two groups clustered in (D). Low SHH and high SHH are indicated in red and blue, respectively. The low-SHH group exhibited lower median survival (24 months) than the high-SHH group (48 months). Note that the low-SHH group exhibited a significantly shorter life-span than the high SHH group (log rank p<0.0001). (G) Expression of SHH and BMP pathway ligands and targets in five distinct subtypes of muscle-invasive urothelial carcinoma. Data are presented as the median and lower to upper quartile values of the gene expression data: luminal-papillary, n = 142; luminal-infiltrated, n = 78; luminal, n = 26; basal, n = 142; and neuronal, n = 20. Significance was calculated by the Mann-Whitney U test.