Cystatin C is downregulated in prostate cancer and modulates invasion of prostate cancer cells via MAPK/Erk and androgen receptor pathways

Abstract

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (r(s) (2) = -0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.

Figure 1. Immunohistochemical analysis of cystatin C expression in TMA with malignant and benign samples from patients with localized prostate cancer.

A). Immunohistochemical analysis of cystatin C expression in benign and PCa specimens. Sections representing benign, tissue and tumors of Gleason grade 3 and grade 5 are shown. Representative pictures were obtained using a 40x objective. B). The graph of quantitative analysis of immunohistochemical staining of Cystatin C (score 0-negative, 1- moderate, 2- strong, 3- very strong) shows the comparison between 448 benign and cancer specimens (average staining of duplicates of each specimen). The paired Wilcoxon's rank sum test analyses were used to assess the comparison between the groups. The mean values of intensities of staining (horizontal lines) with error bars representing 95% confidence intervals for the mean are shown. The boxes represent the distribution of the expression of cystatin C in the groups. C) Overall survival in patients with high or low expression of cystatin C in prostate cancer samples. Kaplan-Meier survival analysis was performed. D) Survival curves of time to relapse as evaluated as a biochemical recurrence measured as by raise of PSA for the low (intensity score 0–1.5) and high (intensity score 2–3) cystatin C expression.

Figure 2. Kaplan-Meier survival analysis in 99 patients with advanced prostate cancer.

Overall survival in a group of 99 patients with the most advanced prostate cancer (Gleason grade 4–5) which were characterized by high expression of AR and were separated to different groups based on cystatin C levels (low- intensity score 0–1.5 and high- intensity score 2–3).

Figure 3. Cystatin C is expressed in the media of prostatic cells lines.

ELISA assay of supernatants from three different prostate cancer cells lines, which were plated 24 h or 48 h before experiment was carried out. Data are shown as average of triplicates±SD for 3 experiments.

Figure 4. The effect of knockdown of cystatin C on the invasion of PC3 cells.

A). Immunoblotting of cystatin C in PC3 cells after treatment with control (siCtr) and cystatin C (siCys) siRNA. B–C) Invasion assay in matrigel- coated Boyden chambers of PC3 cells with knockdown of cystatin C. Representative image of invading cells is shown in (B) and quantitative analysis of invasion by measuring absorbance after staining of invading cells with Cell Stain Solution containing crystal violet supplied in the Transwell Invasion assay (Chemicon, Millipore, CA) (C) Data±SD are representative for at least 3 experiments;* p<0.01 (Student T-test).

Figure 5. Cystatin C decreases invasion of prostate cancer cells.

A) Immunoblot of cystatin C in PC3 cells after stable transfection with pcDNA3.1 and cystatin C-pcDNA3.1 plasmids. Stable clones were established after 2 weeks of selection on neomycin. B–C) Invasion assay of PC3 cells with overexpression of control or cystatin C plasmids. Representative images of cells are shown in B and quantification (absorbance) of data +SD from 3 independent experiments is shown in C. *p<0.05 (Student T-test).

Figure 6. The role of Erk1/2 and Smad2 in cystatin C regulated invasion of PC-3 cells.

A). Immunoblot analysis of P-Smad2 in PC-3 cells after silencing of Smad2. B–C). Invasion assay in PC-3 cells after silencing of Smad2 simultaneously with knockdown of cystatin C. The representative pictures are shown in B and quantitative results of 3 independent experiments are presented in C. *p<0.05 (Student T-test). D). Immunoblot with antibody against phosphorylated (Ser383)-Elk1 and cystatin C in the lysates from PC3 cells transfected transiently with siRNA cystatin C and control siRNA and co-treated with Erk2 inhibitor (25 µM). Note that Erk2 inhibitor blocks downstream phosphorylation of Elk1 a target of Erk2 in cells with knockdown of cystatin C. Data are representative for 2 experiments. E–F). Invasion assay in PC-3 cells after concomitant silencing of cystatin C and inhibition of Erk2 with selective inhibitor. The representative pictures are shown in D and quantitative results of 2 independent experiments are presented in E. *p<0.05, #p<0.01 (Student T-test).

Figure 7. AR regulates the invasion in the absence of cystatin C.

A–B). Invasion assay of PC3 cells transfected with control siRNA or against cystatin C and coexpressing AR. The pictures from representative experiment are shown in A and the quantitative absorbances of invading cells (*p<0.05, cystatin C siRNA versus siRNA control, #p<0.05 AR versus CMV, Student T-test) after stating with Cell Stain Solution supplied in the Transwell Invasion assay (Chemicon, Millipore, CA) are shown in B. The data are representative for 3 independent experiments.