Fig 1: KLK4 regulates protein expression through PAR1 in prostate‐derived stromal cells. (A–B) FGF1 protein in 30 μg of total cellular proteins was determined using FGF1‐ELISA in (A) WPMY1 cells treated for 6, 12 or 18 h with mKLK4, KLK4 (20 nm) or AP1 (100 μm), or (B) WPMY1 cells transfected with PAR1‐siRNA or control‐siRNA and treated with KLK4 or mKLK4 (20 nm) for 24 h. Results are expressed as mean ± SD from three biological replicates. (C) TAGLN expression was determined by immunofluorescent detection in WPMY1 cells treated for 48 h with mKLK4, KLK4 (20 nm) or AP1 (100 μm). Nuclei were stained using DAPI. Representative images are shown, scale bar: 20 μm. (D) TAGLN expression was determined by western blot in WPMY1 cells transfected with PAR1‐siRNA or control‐siRNA and treated for 24 and 48 h with mKLK4 or KLK4 (20 nm). Densitometry analysis was performed using imagej software on three independent experiments. (E–G) WPMY1 cells transfected with FGF1‐siRNA or control‐siRNA were treated for 24 h with mKLK4 or KLK4 (20 nm). (E). Gene expression was obtained by RTqPCR with expression observed for WPMY1 cells control‐siRNA treated with mKLK4 as reference. Results are presented as mean ± SD of three biological replicates. (F) The amount of FGF1 protein in 30 μg of total cellular proteins was determined using FGF1‐ELISA. Results are expressed as mean ± SD calculated on three biological replicates. (G) TAGLN protein expression was determined by western blot as in D. Densitometry analysis was performed using imagej software on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to reference.
Fig 2: Schematic for the possible involvement of KLK4 in early stages of prostate cancer. KLK4 is produced by premalignant cells in benign prostatic hyperplasia and PIN lesions and acts on prostate fibroblast stromal cells through activation of PAR1 as well as other undetermined pathways conducting to gene regulation of IL8, FGF1, LOX, αSMA, TAGLN and VEGFA (PAR1 dependent) and FGF5, ESR1 and SFRP1 (PAR1 independent). In response to KLK4 stimulation, prostate fibroblast stromal cells present a higher proliferation rate and a modification of their secretome (increase in Dkk‐1, GDF15, HGF/SF and decrease in IGFBP3, MCP1 and PDGF‐AA) in favour of a proangiogenic response (increase in IL8 and VEGF), which could ultimately lead to the development of a proangiogenic microenvironment necessary for prostate cancer progression. We could also hypothesize that modification of prostate fibroblast secretome after KLK4 stimulation could directly influence proliferation/migration/survival of prostate cancer cells.
Fig 3: KLK4 regulates gene expression in prostate‐derived NPF/CAF. (A) NPFs isolated from two patients were treated for 24 h with KLK4 and mKLK4 (20 nm) and gene expression analysed (RTqPCR). Gene expression observed for NPF cells treated with mKLK4 was used as reference for each patient. Results are presented as mean ± SD of two biological replicates. (B) The amount of FGF1 protein in 30 μg of total cellular protein in NPF/CAFs from 2 different patients was determined using FGF1‐ELISA. Results are expressed as mean ± SD calculated on three biological replicates. (C) TAGLN protein expression was determined by western blot (as in Fig. 4D). Mann–Whitney test with *P < 0.05, **P < 0.01, ***P < 0.001. (D) Gene expression was analysed in matched NPF/CAF isolated from five patients. Gene expression observed for NPF cells was used as reference for each patient. Results are presented as mean ± SD of three technical replicates. Statistical analysis was performed using one‐way ANOVA test and Bonferroni's multiple comparison, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: KLK4 induces CAF‐related features in prostate stromal cells through PAR1. (A) Gene expression of αSMA, ESR1 and SFRP1 was determined by RTqPCR in WPMY1 cells treated with mKLK4, KLK4 or AP1 for 24 h. Results are expressed as mean ± SD calculated on three biological replicates, *P < 0.05, **P < 0.01, ***P < 0.001. (B–C) WPMY1 cells were treated every 48 h with mKLK4, KLK4 (20 nm) or AP1 (100 μm) for 6 days. (B) αSMA and TAGLN expression was determined by western blot (left panel). Densitometry analysis was performed using imageJ on three independent experiments. αSMA expression was also determined by immunofluorescent staining (right panel) and the fluorescence quantified (Fig. S2B). (C). Proliferation was measured by direct cell counting using InCell analyzer and cellprofiler software based on nuclei staining (DAPI). Results are presented as mean ± SD of three biological replicates, *P < 0.05, **P < 0.01, ***P < 0.001. (D) Conditioned media (CM) of WPMY1 cells treated for 48 h with KLK4 and mKLK4 (20 nm) were analysed using a protein array (Human XL Cytokine array). For each factor analysed, results are expressed as mean ± SD of relative intensity of duplicate spots compared to mean intensity of six positive control spots present on each array (%). Full analysis of the cytokine array can be found in Table S4. (E) IL8 and VEGF concentrations were determined by ELISA in conditioned media (CM) from wild‐type WPMY1 cells or WPMY1 cells transfected with PAR1‐siRNA or control‐siRNA treated for 24 h with mKLK4 or KLK4 (20 nm). Results are expressed as mean ± SD calculated on three biological replicates. Statistical analysis was performed using t‐test, Mann–Whitney with *P < 0.05, **P < 0.01, ***P < 0.001 compared to reference. (F) Effect of CM prepared in E on proliferation of HUVEC cells was analysed. Left panel: HUVEC growth in the presence of WPMY1‐derived CM was followed by live cell imaging (Incucyte) for 48 h. Relative confluence was calculated using confluence at 24 h of HUVEC cells treated with mKLK4‐treated WPMY1's CM as reference. Right panel: HUVEC growth in the presence of WPMY1‐derived CM or endothelial cells growth medium (EGM) was analysed by DNA assay after 48 h of treatment in the presence of an IgG isotype control or a VEGF‐neutralizing IgG. Relative fluorescence intensity was calculated using mKLK4‐treated WPMY1's CM containing IgG isotype control as reference. Results are presented as mean ± SD of three biological replicates, *P < 0.05, **P < 0.01, ***P < 0.001 compared to reference.
Supplier Page from MilliporeSigma for Anti-TAGLN antibody produced in rabbit