Jagged1 upregulation in prostate epithelial cells promotes formation of reactive stroma in the Pten null mouse model for prostate cancer

Abstract

The role of Notch signaling in prostate cancer has not been defined definitively. Several large scale tissue microarray studies have revealed that the expression of some Notch signaling components including the Jagged1 ligand are upregulated in advanced human prostate cancer specimens. Jagged1 expressed by tumor cells may activate Notch signaling in both adjacent tumor cells and cells in tumor microenvironment. However, it remains undetermined whether increased Jagged1 expression reflects a cause for or a consequence of tumor progression in vivo. To address this question, we generated a novel R26-LSL-JAG1 mouse model that enables spatiotemporal Jagged1 expression. Prostate specific upregulation of Jagged1 neither interferes with prostate epithelial homeostasis nor significantly accelerates tumor initiation or progression in the prostate-specific Pten deletion mouse model for prostate cancer. However, Jagged1 upregulation results in increased inflammatory foci in tumors and incidence of intracystic adenocarcinoma. In addition, Jagged1 overexpression upregulates Tgfβ signaling in prostate stromal cells and promotes progression of a reactive stromal microenvironment in the Pten null prostate cancer model. Collectively, Jagged1 overexpression does not significantly accelerate prostate cancer initiation and progression in the context of loss-of-function of Pten, but alters tumor histopathology and microenvironment. Our study also highlights an understudied role of Notch signaling in regulating prostatic stromal homeostasis.

Figure 1. Generation of the R26-LSL-JAG1 model

(A) Schematic illustration of the targeting vector. (B) Southern blot analysis identifies ES clone with correct homologous recombination. Wild type and recombined ES clones show bands of 10 and 4 kb, respectively.

Figure 2. Successful prostate specific JAG1 expression in the PB-JAG1 model

(A) Bioluminescence imaging of a PB-JAG1 and a control mouse. (B) qRT-PCR analysis of Jagged1 in 52-wk-old PB-JAG1 and control mice. (C) Coimmunostaining of JAG1 and K14 in prostate tissues of PB-JAG1 and control mice. Bars=25μm. (D–E) Western blot analyses of JAG1 (D) and Notch1 and Notch2 intracellular domains (N1ICD and N2ICD) (E) in prostate tissues of PB-JAG1 and control mice. Individual lanes represent results from different mice. PrEC: primary human prostate basal epithelial cells. NRAEV: normalized relative arbitrary expression value. (F) qRT-PCR analysis of Hey1 and Ccnd1 in PB-JAG1 and control mice. *: p<0.05, ***: p<0.001.

Figure 3. JAG1 upregulation does not alter prostate homeostasis

(A) Representative images of prostate tissues from 1-yr old control and PB-JAG1 mice. Dot plot shows quantification of prostate weights. Bars=5mm. (B) H&E staining of different prostate lobes of 1-yr old control and PB-JAG1 mice. Bars=50μm. (C) Immunostaining of K5 and K8 in prostate tissues of 1-yr old control and PB-JAG1 mice. Bars=25μm. (D) Immunostaining of BrdU and cleaved caspase 3 (CC3) in prostate tissues of 1-yr old control and PB-JAG1 mice. Arrows point to cells with positive staining. Bars=25μm. Dot plot shows means ± s.d.

Figure 4. JAG1 upregulation does not affect tumor progression in the Pten null model

(A) Representative images of urogenital organs and prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Dot plot shows quantification of prostate weight. Bars=1cm. (B–D) H&E staining of prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice shows formation of high grade PIN lesions (B) and intracystic adenocarcinoma (arrow, C) with reactive stroma (D). Bars=50μm. (E) H&E staining of seminal vesicles of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Bars=300μm. (F–G) Immunostaining of Pten, pAKT, K5, K8, P63 and AR in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Black bars=50μm, white bars=25μm.

Figure 5. JAG1 upregulation augments inflammatory foci and blood vessel density

(A) H&E staining of prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Black arrows denote inflammatory foci. Bars=50μm. (B) FACS plots of CD45 staining in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Dot plot shows means ± s.d. of percentage of CD45⁺ cells. (C) Immunostaining of CD31 in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Red arrows point to CD31-expressing vessels. Bars=50μm. Dot plot shows means ± s.d. of blood vessel number per field. (D) FACS plots of myeloid derived suppressor cells (MDSCs) in blood and tumor tissues in 1-year-old PB-Pten-JAG1 and PB-Pten mice. Bar graphs show quantification. *: p<0.05, **: p<0.01.

Figure 6. Jagged1 upregulation promotes formation of reactive stroma

(A) Trichrome staining of prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Dot plot shows means ± s.d. of percentage of blue collagen staining per field. Bars=50μm. (B) Immunostaining of Procollagen1 in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Bars=50μm. (C) Coimmunostaining of Smooth muscle actin (SMA) and Vimentin (Vim) in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Bars=100μm. (D) Immunostaining of Tenascin C (TNC) in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Asterisks denote background staining in epithelial cells while arrows point to stronger staining in stromal cells. Bars=50μm. (E) Immunostaining of pSmad3 in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice. Dot plot shows means ± s.d. of percentage of pSmad3⁺ stromal cells. Bars=50μm. (F) Dot graphs show means ± s.d. of expression of Tgfβ1 and Tgfβ3 in prostate tissues of 1-yr old PB-Pten and PB-Pten-JAG1 mice by qRT-PCR. *: p<0.05.

Figure 7. Notch signaling regulates prostate stromal cell biology

(A) qRT-PCR analysis of expression of Notch receptors and target genes in FACS-sorted prostate stromal cells of 10-wk-old wild type mice. Bar graphs show means ± s.d. from 3 independent experiments. (B) Schematic illustration of coculture experimental approach. (C) qRT-PCR analysis of gene expression. Bar graphs show means ± s.d. of 1 out of 2 independent representative experiments. **: p<0.01, ***: p<0.05.