Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr;73(5):476-88.
doi: 10.1002/pros.22589. Epub 2012 Sep 19.

Cathepsin D acts as an essential mediator to promote malignancy of benign prostatic epithelium

Freddie L Pruitt  1 , Yue He, Omar E Franco, Ming Jiang, Justin M Cates, Simon W Hayward
Affiliations

Cathepsin D acts as an essential mediator to promote malignancy of benign prostatic epithelium

Freddie L Pruitt et al. Prostate. 2013 Apr.

Abstract

Background: Stromal-epithelial interactions are important in both development and prostate cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. We investigated whether stromal cathepsin D (CathD) overexpression affects prostate tumorigenesis through a paracrine mechanism.

Methods: Normal prostate fibroblasts (NPF) were retrovirally transduced to overexpress cyclin D1 (CD1) and were designated NPF(CD1) . Cathepsin D expression was knocked down using shRNA in cancer associated fibroblasts (CAF) and NPF(CD1) . We analyzed these stromal cell lines using immunohistochemistry, Western blot, and tissue recombination.

Results: An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue. Overexpression of CD1 in normal prostate fibroblasts (NPF(CD1) ) produced a phenotype similar to, but more moderate than, CAF in a tissue recombination model. Knockdown studies revealed that CathD is required for NPF(CD1) motility and invasive growth in vitro. BPH-1 cell proliferation was found to be induced when cultured with NPF(CD1) conditioned medium, this effect was inhibited when CathD was knocked down in NPF(CD1) cells. Overexpression of CathD in prostate stromal cells induced malignancy in adjacent epithelium, and this transformation was inhibited when stromal CathD expression was knocked down in CAF.

Conclusions: The study presented here demonstrates increased CathD expression is seen in human CAF. The upregulation of CD1 results in concomitant increases in CathD expression. Elevated CathD expression in the stroma contributes to tumor promotion.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Cathepsin D is overexpressed in maligant prostate clinical samples
(A) Representative images from immunohistochemical (top) analysis of CathD expression in normal (left) n = 18 and tumor (right) n = 30 human prostate tissues. Representative images of Masson’s tri-chrome staining (bottom) from normal (left) and tumor (right). Scale bar is equal to 50µm. (B) Quantitation of CathD expression in the prostate comparing normal to malignant tissue. Data are presented as means ±SD.
Figure 2
Figure 2. Evaluation of Cathepsin D as a paracrine mediator of neoplastic epithelial cell growth in tissue recombinants in vivo
(A) Immunohistochemical analysis of CathD expression in recombinations of 1) BPH-1 + NPF, 2) BPH-1 + rUGM, 3) BPH-1 + NPFCD1, 4) BPH-1 + CAF. Scale bar is equal to 50µm (Letters K,S,E, refer to kidney, stroma, and epithelium). (B) Evaluation of densitometric analysis of cell cycle regulators and CathD in BPH-1NPF, BPH-1CAFTD1, BPH-1NPFCD1 cells. Expression of Beta-actin was used for a loading control. Graphical representation of the mean ± SD of band intensities.
Figure 3
Figure 3. Cathepsin D is a critical mediator between BPH-1 cells and NPFCD1 in vitro
(A) Wound healing assay. Confluent monolayers of NPF, NPFCD1, NPFCD1-control, NPFCD1-CathD sh were scratched with a pipette tip. Bar graphs represent the mean ± SD of rate of wound closure over 8 hour period. Significance determined by ANOVA, p-value ≤ 0.005 n=3. (B) 3D outgrowth assays. NPF cell lines were embedded in matrigel and cultured for 14 days, scale bar = 100µm. Images taken at 10x (3B, b). (C) Evaluation of CathD as a paracrine mediator of growth. BPH-1 cells were treated with conditioned media collected from NPF cell lines for 3 days. Cell numbers were quantitated by direct counting, graphical representation of the mean ± SD of the experiment is shown, significance determined by ANOVA p-value ≤ 0.005 n=3. (D) Western blot confirming knockdown of CathD in NPF, NPFCD1, NPFCD1-control, NPFCD1-CathD sh cells (Top). Densitometric analysis of band intensities performed to determine knockdown efficiency (Bottom).
Figure 4
Figure 4. Cyclin D1 and Cathepsin D are required for CAF induced tumorigenicity in vivo
(A) Gross morphology of 2 month grafts of BPH-1 + CAFPSR, BPH-1 + CAFCD1sh, and BPH-1 + CAFCathDsh, scale bar equal to 5cm. (B) H&E staining of BPH-1 + CAFPSR, CAFCD1sh, or CAFCathDsh. BPH-1 + CAFPSR recombinants formed adenosquamous carcinoma as previously described. Scale bar equal to 50µm (C) Western blot confirming knockdown of CD1 and CathD in CAFs (left). Knockdown efficiency determined by performing densitometric analysis of western blot represented by bar graph (middle). Quantiation of tumor volume of 2 month grafts of BPH-1 + CAFPSR, BPH-1 + CAFCD1sh, and BPH-1 + CAFCathDsh (right), graphical representation of the mean ± SD of the grafts is shown in n=6. Significance determined by ANOVA, p-value ≤0.05.
Figure 5
Figure 5. Overexpression of Cathepsin D induces a malignant transformation through activation of TGFβ signaling
(A) Characterization of CathD overexpressing grafts. H&E staining (top) of BPH-1 + BHPrSEV and BPH-1 + BHPrSCathD. Recombinations of BPH-1 + BHPrSCathD, produced malignant transformations. IHC for CathD (middle) strong expression visible in the stroma of recombinations of BPH-1 + BHPrSCathD. Immunoflurescence for CathD (green) and GFP (red) show co-localization for GFP and CathD (yellow) in recombinations of BPH-1 + BHPrSCathD. (B) Masson’s trichrome staining (top) of recombinations of BPH-1 + BHPrSEV and BPH-1 + BHPrSCathD. IHC for p-SMAD2/3 (middle) and Collagen IV±2 (lower) in recombinations of BPH-1 + BHPrSEV and BPH-1 + BHPrSCathD. Scale bar equal to 20µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Weinberg RA. Coevolution in the tumor microenvironment. Nat Genet. 2008;40:494–495. - PubMed
    1. Li H, Fan X. Tumor microenvironment: the role of the tumor stroma in cancer. Journal of cellular biochemistry. 2007 - PubMed
    1. Cunha G. Tissue interactions between epithelium and mesenchyme of urogenital and integumental origin. The Anatomical Record. 1972 - PubMed
    1. Olumi A, Grossfeld G, Hayward S, Carroll P. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999 - PMC - PubMed
    1. Cunha GR, Hayward SW, Wang YZ. Role of stroma in carcinogenesis of the prostate. Differentiation. 2002;70:473–485. - PubMed

Publication types

MeSH terms