Altered TGF-β signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis

Abstract

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-β receptor II (TGFβR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFβR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-β) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFβR2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFβR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-β1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFβR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-β1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-β1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-β signaling to tumor promotion by tumor stromal cells.

Figure 1. Prostate stroma regulates the fate of prostate epithelial cells

Heterotypic tissue recombinants of rat urogenital mesenchyme (rUGM) and carcinoma associated fibroblasts (CAF) in different ratios with BPH1 cells were sub-renal capsule grafted to male hosts mice for 8 weeks before the tissues were harvested. A.(a-e). H&E shows differentiation of BPH1 cells to benign cords when combined with 100%UGM. As the ratio of CAF cells increased, malignant transformation of BPH1 cells was observed and the epithelium became progressively less organized and more invasive. (b-e). Immunofluorescence labeling of αSMA/Vimentin (f-j), γActin/SV40 (k-o) and desmin/cytokeratin (p-t) showed a more muscular stroma surrounding areas with high percentage of rUGM (>75%). Markers of normal stromal differentiation were dramatically decreased when the ratio of CAF/rUGM increased to more than 50%. Hoescht 33258 staining of the nucleus (blue) confirmed the human nature of the stromal cells. B. Gross picture of BPH1 cells recombined with rUGM or CAF cells (a & b) showing epithelial invasion into the kidney parenchyma (*). rUGM induced benign glandular differentiation, which was separated by a thick layer of differentiated γSMA (+) fibroblasts (green) from the underlying kidney. C. Tumor volumes were quantitated, graphical representation of the mean ±SD of the grafts is shown n=6. The volume of the tumors increased with the percentage of CAF.

Figure 2. The prostate tumor stroma shows heterogeneous P-Smad2 expression, a surrogate marker of response to TGF-ϐ

A. Immunohistochemical staining of tissue recombinants for phospho-Smad2. BPH1+CAF recombinants showed the highest proportion of phospho-Smad2 positive stromal cells (~50%) compared to either BPH1+BHPrS1 (<10%) or BPH1+rUGM (<25%). B. Phospho-Smad2 expression in human prostate cancer tissue array. Normal prostate epithelial cells were surrounded by cells expressing low levels of phospho-Smad2. Stromal cells in the tumors as well as those in normal looking areas in the peripheral zone of cancer patients had a phospho-Smad2 SCORE of more than 2.5 representing more than 50% of positive cells (see text for details) C. Addition of TGF-ϐ1 ligand to cultured fibroblasts induced the expression αSMA and phospho- Smad2 in a subpopulation of CAF while the BHPrS1 showed a more homogeneous pattern of expression. Note morphologic changes to CAF with serum compared to BHPrS. This phenomena may be the result of the ability of CAF to remodel collagen matrices.

Figure 3. Prostate epithelial cell responses to a heterogeneous stroma

A. MTT assay of BPH1 cells exposed to CAF, BHPrS1^−EV, BHPrS1^−DN, or BHPrS1^−EV/BHPrS1^−DN conditioned medium (CM). Epithelial cells proliferated faster in the presence of CAF and BHPrS1^−EV/BHPrS1^−DN CM as compared to medium conditioned by the other fibroblasts. B. BPH1+BHPrS1^−EV/BHPrS1^−DN, BHPrS1^−EV, or BHPrS1^−DN tissue recombinants were grafted into male SCID mice for 8 weeks before harvesting the tissues. BPH1+BHPrS1^−EV/BHPrS1^−DN recombinants produced the largest tumors as shown by the gross appearance (arrowhead) and quantitation of tumor volume. C. Histological examination revealed malignant transformation of BPH1 only with mixed BHPrS1^−EV/BHPrS1^−DN stromal cells (a-c). The adeno-squamous phenotype with invasion into the kidney resembled BPH1+CAF recombinants. The arrow indicate invasion into the host kidney. Immunofluorescence staining of SV40 confirmed the invasion of BPH1 cells into the kidney parenchyma (*) of BPH1+BHPrS1^−EV/BHPrS1^−DN grafts, no invasion was seen in the other recombinants (d-f). The presence of BHPrS1^−DN increased the proportion of Ki67 positive cells in the BPH1+BHPrS1^−EV/BHPrS1^−DN and BPH1+BHPrS1^−EV recombinants (g-i). Malignant transformation of BPH1 cells was accompanied by changes in the stromal compartment towards a myofibroblast phenotype, as noted by the increased expression of vimentin and αSMA (j-l).

Figure 4. Molecular consequences of heterogeneous stromal cell mixes

BHPrS1^−EV, BHPrS1^−DN, and BHPrS1^−EV/BHPrS1^−DN mixtures were cultured for 48 hr before RNA and protein isolation. A. RT-PCR analysis showed increased SDF1α expression and down- regulation of TGFϐR3 in BHPrS1^−EV/BHPrS1^−DN compared to the other groups. B. PCR Array analysis validated the increased expression of TGF-ϐ1 in BHPrS1^−EV/BHPrS1^−DN and BHPrS1^−DN cells (arrow) and showed dysregulation of several genes involved in tumor progression. C. Evaluation by densitometric analysis of the protein expression of downstream signaling and potential paracrine mediators of tumor progression showed increased TGF-ϐ signaling (exemplified by phospho-Akt, αSMA, and p27) and CD90 in BHPrS1^−EV/BHPrS1^−DN mixtures.

Figure 5. Induction of CAF phenotype by over-expression of TGF-ϐ1 and SDF1α in normal prostate fibroblasts

A. ELISA was used to test and corroborate the expression of the transgenes in fibroblasts. Fibroblasts expressing constitutively active TGF-ϐ1 ligand showed an increased SDF1α expression. However, over-expressing SDF1α in fibroblasts had no effect on TGF-ϐ1 levels. B. BHPrS1^−TGFϐ1 and BHPrS1^−SDF1α were recombined with BPH1 cells and xenografted under the kidney capsule for about 8 weeks. The volume of the tumors composed of BHPrS1^−TGFϐ1+BPH1 and BHPrS1^−SDF1α+BPH1 were significantly larger than the controls. Note the invasive characteristic of the tumors (arrows) C. Histological examination revealed malignant trasnformation of the epithelial cells (a-c). TGFϐ1-expressing fibroblasts had a greater impact on the proliferation of BPH1 cells as shown by Ki67 staining as compared to BHPrS1^−SDF1α cells (d-f). BHPrS1^−EV fibroblasts exhibited light blue stromal Trichrome staining indicative of some collagen in the stroma. In contrast recombinants composed of BHPrS1^−SDF1α stained predominantly red suggesting a more muscular phenotype. Intense blue Trichrome staining in the stroma of BHPrS1^−TGFϐ1+BPH1 recombinants revealed the extensive collagen deposition in these tumors. (g-i). Intense SDF1α staining corroborated the secretion of the chemokine in grafts composed of SDF1α-expressing compared to TGFϐ1-expressing fibroblasts (j-l).