Hedgehog signaling restrains bladder cancer progression by eliciting stromal production of urothelial differentiation factors

Abstract

Hedgehog (Hh) pathway inhibitors are clinically effective in treatment of basal cell carcinoma and medulloblastoma, but fail therapeutically or accelerate progression in treatment of endodermally derived colon and pancreatic cancers. In bladder, another organ of endodermal origin, we find that despite its initial presence in the cancer cell of origin Sonic hedgehog (Shh) expression is invariably lost during progression to invasive urothelial carcinoma. Genetic blockade of stromal response to Shh furthermore dramatically accelerates progression and decreases survival time. This cancer-restraining effect of Hh pathway activity is associated with stromal expression of BMP signals, which stimulate urothelial differentiation. Progression is dramatically reduced by pharmacological activation of BMP pathway activity with low-dose FK506, suggesting an approach to management of human bladder cancer.

Figure 1. Absence of Shh expression in human invasive bladder carcinoma

(A) Realtime quantitative PCR measurement of SHH expression in benign urothelium and invasive urothelial carcinoma tissues. Data are presented as mean ± s.e.m. n = 3 technical replicates for each independent sample. (B) Benign human urothelium immunostained for Shh and counterstained with Hematoxylin. Arrowheads in the boxed area enlarged on the right indicate non-specific staining of dying cells being sloughed off into the lumen. (C) Human invasive urothelial carcinoma samples from four patients immunostained for Shh and counterstained with Hematoxylin. Arrowheads indicate regions of Shh reactivity within tumor vasculature. Scale bars represent 50 μm. Sample numbers correspond to those in Table S1. See also Figure S1/Table S1.

Figure 2. Genetic ablation of Hh response accelerates bladder carcinogenesis

(A) H&E stains of bladder sections from mice treated with tamoxifen (TM), then exposed to BBN for 3 months. Magnified views (lower panels) of boxed regions confirm the presence (Gli1^CreER/WT;Smo^flox/flox, mice with homozygous loss of Smo) or absence (Gli1^CreER/WT;Smo^flox/WT, heterozygous control mice) of invasive carcinoma. Scale bars represent 50 μm. (B) Kaplan-Meier survival curve of Gli1^CreER/WT;Smo^flox/flox and Gli1^CreER/WT;Smo^flox/WT mice (n=10 in each group) injected with TM, exposed to BBN, and euthanized when they became morbidly ill. Animals with homozygous loss of Smo survived a median of 140 days, as compared to 215 days for Smo heterozygous control animals (p<0.0001, as determined by the log-rank test).

Figure 3. Reduced expression of differentiation factors in Smo-ablated mice

(A) Comparison of gene expression profiles revealed 468 genes down-regulated by at least 2-fold in Gli1^CreER/WT;Smo^flox/flox as compared to Gli1^CreER/WT;Smo^flox/WT bladder samples (p<0.01). Gene ontology analysis indicated an enrichment of genes involved in cell differentiation. Additional functional annotation of the down-regulated genes showed enrichment in the Swiss-Prot and Protein Information Resource Keywords (SP_PIR_KEYWORD) “Signal” and “Secreted”. The Venn diagram shows the number of overlapping genes in each of the three categories, with 12 genes that encode secreted proteins involved in cell signaling and differentiation. (B) Expression in bladders of Gli1^CreER/WT;Smo^flox/flox as compared to Gli1^CreER/WT;Smo^flox/WT mice of Bmp4 (16 fold decrease, p<0.0001), Bmp5 (34 fold decrease, p<0.001), and Gli1 (3.2 fold decrease, p<0.0001). Mice were injected with TM just prior to 2 months of BBN exposure. (C and D) Expression in four bladders from BBN-exposed mice (6 months) with invasive carcinoma of Bmp4 (p<0.001) and Bmp5 (p<0.001) as compared to a control bladder (Ctrl) with no BBN exposure. Data are presented as mean ± s.e.m., and significance was calculated by an unpaired Student’s t-test. n = 3 technical replicates, and the entire experiment was repeated three times. See also Figure S2/Table S2.

Figure 4. Stromal Hh response regulates expression of human BMP4 and BMP5 genes

(A) Expression of GLI1 in primary human bladder stromal cells treated with SAG (5.4 fold increase, p<0.001), ShhN (1.6 fold increase, p<0.05), and ShhN + cyclopamine (Cyc) (no statistically significant change) as compared to unstimulated control (Ctrl) stromal cells or in primary human bladder urothelial cells as compared to Ctrl urothelial cells (no statistically significant change with any treatment). (B) BMP4 and BMP5 expression in primary human bladder stromal cells treated with SAG (BMP4: 5.8 fold increase, p<0.001; BMP5: 2 fold increase, p<0.01), and ShhN (BMP4: 3.9 fold increase, p<0.001; BMP5: 1.6 fold increase, p<0.05), and ShhN + Cyc (no statistically significant change) as compared to unstimulated control (Ctrl) cells. (C) Expression of BMP target gene ID1 in cells treated with BMP agonist FK506 as compared to the DMSO vehicle control (Ctrl) (2.4 fold increase in urothelial cells, p<0.01; 2 fold increase in J82 urothelial carcinoma cells, p<0.01) or as compared to cells treated with FK506 after pre-incubation with the BMP inhibitor LDN-193189 (LDN)(4.5 fold increase in urothelial cells, p<0.0001; 10 fold increase in J82 cells, p<0.001). (D) Expression of the urothelial differentiation marker UPK1B in cells treated with FK506 as compared to DMSO Ctrl (3.2 fold increase in urothelial cells, p<0.01; 2.3 fold increase in J82 cells, p<0.01) or as compared to cells treated with FK506 after pre-incubation with LDN (2.7 fold increase in urothelial cells, p<0.01; 1.8 fold increase in J82 cells, p<0.05). (E) Expression in human invasive urothelial carcinoma as compared to benign bladder tissues of BMP4 (11 fold decrease, p<0.001) and BMP5 (7 fold decrease, p<0.001). Data are presented as mean ± s.e.m., and significance was calculated by an unpaired Student’s t-test. n = 3 technical replicates, and the entire experiment was repeated three times. See also Figure S3.

Figure 5. Pharmacological activation of BMP signaling in vivo impedes tumor progression

(A) Schematic diagram of experimental strategy used to analyze the effect of BMP pathway activation on bladder cancer progression. Mice exposed to BBN for 4 months, were treated with the BMP agonist FK506 or a vehicle control for a month with continued BBN exposure before histopathological analysis of the bladders. (B) Bladder sections from mice exposed to BBN for 5 months with 1 month of treatment with FK506 or a vehicle control were stained with Hematoxylin and Eosin (H&E). Lower panels show magnified views of the regions highlighted by boxes in upper panels. Scale bars represent 50 μm. (C) Results obtained from the treatment of mice exposed to BBN with FK506 or a vehicle control are summarized. See also Figure S4.

Figure 6. Reduced expression of Hh and BMP pathway ligands and targets in human samples of invasive carcinoma as compared to normal urothelium

RNA-seq data from The Cancer Genome Atlas (TCGA) database with annotation of samples based on published TCGA clusters (Cancer Genome Atlas Research Network, 2014). The y-axis indicates log₂-transformed FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values normalized using the RSEM algorithm (Li and Dewey, 2011). If no reads map to the corresponding gene (FPKM=0), the y-axis value is set to -10. Note that for SHH, FPKM=0 for 17/31 cluster 3 (basal) samples. Data are presented as mean and 95% confidence interval; normal: n=19, cluster 1: n=41, cluster 2: n=42, cluster 3: n=31, cluster 4: n=15; significance was calculated by Student’s t-test. See also Figure S5.

Figure 7. Model of progression to invasive urothelial carcinoma through epithelial loss of Hh expression and consequent reduced stromal expression of urothelial differentiation factors (BMPs)

Carcinoma in situ (CIS) precursor lesion containing cells that express or do not express Shh (basal red and supra-basal pink cells, respectively) progresses to invasive carcinoma through clonal loss of Shh expression (brown cells) and expansion of a resulting invasive event. See text.