Cathepsin K regulates the tumor growth and metastasis by IL-17/CTSK/EMT axis and mediates M2 macrophage polarization in castration-resistant prostate cancer

Abstract

A common stage of advanced prostate cancer is castration-resistant prostate cancer (CRPC), greater understanding of which is required in order to address and solve the clinically difficult challenge. Cathepsin K (CTSK) is a cysteine protease that usually has a strong activity of degrading extracellular matrix and is related to osteoclast-mediated bone destruction. However, the mechanism of CTSK-regulation in CRPC is still unclear to us. The current study aimed to analyze the expression of differentially expressed genes (DEGs) in patient samples (from localized PC and CRPC). Interestingly, we found that CTSK to be significantly up-regulated in CRPC. Through further signal pathway enrichment analysis, we found that the IL-17 signaling pathway to be highly correlated with CTSK. The oncogenic functions of CTSK and IL-17 in CRPC were proven by a series of in vivo and in vitro experiments. Possible downstream molecules of CTSK were investigated, which could serve as control elements to regulate the expression of EMT, thereby facilitating the metastasis and excessive proliferation of PC cells. Expression of CTSK was related to high concentration of M2 tumor-associated macrophages (TAMs) M2 in CRPC. A CTSK-mediated feedback circuit between TAMs and CRPC tissues was indicated in the process of transfer, proving the possibility of CTSK could be use as an available therapeutic target for CRPC.

Fig. 1. Over-expression of CTSK-associated IL-17A in castration-resistant prostate cancer.

A Principle description of the work procedure used to study the “IL-17A/CTSK” axis in CRPC. The expression profiles data of various types of prostate cancer (#GSE70770, GSE32982 and GSE32269). B Heatmap of 23 up-regulated genes and 85 down-regulated genes mined from GSE70770, GSE32982 and GSE32269. The lower bar shows different groups, the pink one represents the group of CRPC and the orange red one represents the group of localized PC. C The bar chart shows KEGG pathway enrichment data for DEGs. Pink represents up-regulated pathways and blue represents down-regulated pathways. D Circle diagram was adopted for further exploration to clarify the expression genes involved in the three significantly up-regulated pathways. E Estimate the therapeutic values of CTSK by analyzing the ROC curve (p < 0.001).

Fig. 2. Higher CTSK expression in metastatic prostate cancer/castration-resistant prostate cancer and the associated poor prognosis.

A WB assay to examine the CTSK protein expression in CRPC tissues (T) and normal prostate tissues (N), GAPDH protein served as control. B The scatter diagram represents the relative CTSK expression in CRPC and normal tissues. C WB assay to examine the CTSK and AR-V7 protein expression in non-metastatic (nmPC) and metastatic (mPC) cancer tissues, β-tubulin protein served as control. D The scatter diagram represents the relative CTSK expression in nmPC and mPC tissues. E Comparing CTSK and IL-17A expression in BPH, ADPC, and CRPC groups by IHC staining. F The histogram represents comparison of the expression of CTSK and IL-17A in three kinds of groups. G Univariate analyses (log-rank) and Kaplan–Meier survival curves for PC patients with distinct CTSK expression levels. H Univariate analyses (log-rank) and Kaplan–Meier survival curves for PC patients with distinct IL-17A expression levels. Data are shown as mean ± s.e.m. *p < 0.05.

Fig. 3. Effect of IL-17A on the growth of prostate cancer cells via its influence on the expression of CTSK both in vivo and in vitro.

A WB assay to examine the CTSK protein expression in PC cells, β-tubulin protein served as control. B Treated with 15 ng/ml restructuring human IL-17A in DU145 cells for 0, 6, 12, 18, 24, 30, and 36 h. WB assay to examine the CTSK protein expression, GAPDH protein served as control. C WB assay to examine the CTSK/IκBα protein expression, GAPDH protein served as control. Treated with 15 ng/ml restructuring human IL-17A in DU145 cells for 0–36 h and one group is added 10 µg/ml CHX at 24 h and collected at 36 h. D DU145 cells are treated with control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A, and conducted colony formation assay. E Quantization of D. F Wound healing assay in DU145 cells transfected with negative control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A. G Quantization of F. H Transwell assay in DU145 cells transfected with negative control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A. I Quantization of H. J, K DU145 cells treated with negative control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A are injected subcutaneously into the abdomen of mice. The effect of negative control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A on the growth of PCa. L Measuring tumor sizes of mice daily for 2 weeks when the tumor is 1.5–2 mm in diameter. M Quantification of tumors in different groups of mice in L. N, O Comparing CTSK expression in negative control siRNA, IL-17A, CTSK siRNA, CTSK siRNA + IL-17A groups by IHC staining.

Fig. 4. Enhancement of EMT by IL-17A via CTSK induction.

A Subcutaneous tumor tissue of mice is stained for EMT markers (β-catenin, vimentin, E-cadherin). The mice tumor tissues express excessive CTSK. B Subcutaneous tumor tissue of mice is stained for EMT markers (β-catenin, vimentin, E-cadherin). The mice tumor tissues express a small amount of CTSK. C Phase-contrast photomicrographs of human prostate cancer cells in monolayer culture. D–F Western blot analysis of EMT markers in human prostate cancer cells.

Fig. 5. Immune infiltration in the combined sample dateset.

A The bar chart shows the composition of the 21 immune-infiltrating cells at various sample, with each column representing a sample. B The histogram shows the composition of immune-infiltrating cells in all samples. C Comparison of the content of various immune-infiltrating cells in CRPC samples and localized PC samples. D The heatmap shows the amount of various immune-infiltrating cells in each sample, and each column represents a sample. The grouping of samples is shown in the annotation above. E The concentrations of various immune-infiltrating cells in the dataset for GSE32269 with each column representing one sample.

Fig. 6. Correlation between the color modules and immune-infiltrating cells.

A Determine soft-thresholding power in WGCNA. B Tree diagram of head 5000 genes gathered based on different metrics (1-TOM). C Correlation heat map between modules and immune-infiltrating cells. D The corresponding DEGs in each module.

Fig. 7. High expression of CTSK is related to high enrichment of M2 macrophages of tumor-associated in CRPC.

A–D IF assay analysis of the correlation between CD206 and CTSK in prostate cancer tissues. E Quantification of immunofluorescence by confocal microscopy. Histograms show quantification of 10–25 different images per condition. F Correlation curve of CTSK and CD206 expression in localized PC tissues by quantification of immunofluorescence, eight sites are selected for each tissue. G Correlation curve of CTSK and CD206 expression in CRPC tissues by quantification of immunofluorescence, eight sites are selected for each tissue. H The profile mechanism of the regulation and mechanism of IL-17A/CTSK-mediated CRPC metastasis.