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. 2011 Nov;179(5):2625-36.
doi: 10.1016/j.ajpath.2011.07.016. Epub 2011 Sep 9.

Kallikrein-related peptidase 14 acts on proteinase-activated receptor 2 to induce signaling pathway in colon cancer cells

Valérie Gratio  1 , Céline Loriot, G Duke Virca, Katerina Oikonomopoulou, Francine Walker, Eleftherios P Diamandis, Morley D Hollenberg, Dalila Darmoul
Affiliations

Kallikrein-related peptidase 14 acts on proteinase-activated receptor 2 to induce signaling pathway in colon cancer cells

Valérie Gratio et al. Am J Pathol. 2011 Nov.

Abstract

Serine proteinases participate in tumor growth and invasion by cleaving and activating proteinase-activated receptors (PARs). Recent studies have implicated PAR-1 and PAR-4 (activated by thrombin) and PAR-2 (activated by trypsin but not by thrombin) in human colon cancer growth. The endogenous activators of PARs in colon tumors, however, are still unknown. We hypothesize that the kallikrein-related peptidase (KLK) family member KLK14, a known tumor biomarker, is produced by colonic tumors and signals to human colon cancer cells by activating PARs. We found that i) KLK14 mRNA was present in 16 human colon cancer cell lines, ii) KLK14 protein was expressed and secreted in colon cancer cell lines, and iii) KLK14 (0.1 μmol/L) induced increases in intracellular calcium in HT29, a human colon cancer-derived cell line. KLK14-induced calcium flux was associated with internalization of KLK14-mediated activation of PAR-2. Furthermore, KLK14 induced significant extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and HT29 cell proliferation, presumably by activating PAR-2. A PAR-2 cleavage and activation-blocking antibody dramatically reduced KLK14-induced ERK1/2 signaling. Finally, ectopic expression of KLK14 in human colon adenocarcinomas and its absence in normal epithelia was demonstrated by IHC analysis. These results demonstrate, for the first time, the aberrant expression of KLK14 in colon cancer and its involvement in PAR-2 receptor signaling. Thus, KLK14 and its receptor, PAR-2, may represent therapeutic targets for colon tumorigenesis.

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Figures

Figure 1
Figure 1
Expression of KLK14 in human colon cancer cell lines (A) and in HT29 cells versus human normal colonic epithelial cells (B). Total RNA (4 μg) was reverse transcribed and PCR amplified with KLK14 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. B: A single PCR-amplified product of the predicted size (485 bp) for KLK14 was visualized after electrophoresis on 2% agarose gel. The breast cancer cell line (MDA-MB-468) and the prostate cancer cell line (PC-3) were used as positive controls.
Figure 2
Figure 2
Immunodetection of KLK14 in human colon cancer cells. A: Immunodetection of KLK14 in paraffin sections from HT29 (left upper panel) and MDA-MB-468 cells (left lower panel). No immunoreactivity was detected when the primary antibody was omitted (right panels). B: Immunofluorescence detection of KLK14. HT29 cells were fixed using 2% paraformaldehyde. Left: KLK14 protein was evident in the cytoplasm of HT29 cells. Right: No immunofluorescence was observed when the anti-KLK14 antibody was omitted. Original magnification, ×630. The inset shows confocal microscopic immunocytochemical localization of KLK14 in HT29 cells. Original magnification, ×630; zoom, ×4. Arrows show perinuclear staining of KLK14.
Figure 3
Figure 3
KLK14 induces calcium mobilization in HT29 cells via PARs. HT29 cells were loaded for 60 minutes at 37°C using Fura-2/AM. A: Cells were challenged with the indicated concentrations of KLK14. B: Cells were challenged by the addition of the TRAP that coactivates PAR-1 and PAR-2, followed by sequential challenges with AP1, AP2, and KLK14 (0.1 μmol/L). Addition of the agonists is indicated by arrows. These results are representative of three independent experiments.
Figure 4
Figure 4
KLK14 initiates changes in intracellular Ca2+ mobilization via PAR-2 but not PAR-1 in HT29 cells. HT29 cells were loaded for 60 minutes at 37°C using Fura-2/AM. A: HT29 cells were challenged first with thrombin (0.01 μmol/L) followed by a second challenge with KLK14 (0.1 μmol/L). B: Cells were first challenged with KLK14 followed by a second challenge with AP1 (TFFLLR-NH2, 100 μmol/L). Note that cells are still responsive to AP1. C: First, cells were challenged twice with the PAR-2–specific agonist peptide, 2-furoyl-LIGRLO-NH2 (2-fLIGRLO-NH2, 10 μmol/L), and then with KLK14 (0.1 μmol/L). A subsequent challenge with AP1 (TFFLLR-NH2, 100 μmol/L) showed that cells were still responsive. Administration of the compounds is indicated by arrows. These results are representative of three independent experiments.
Figure 5
Figure 5
KLK14 induces loss of PAR-2 from the surface of HT29 cells. A: Immunofluorescence detection of PAR-2 in HT29 cells treated with KLK14 (0.1 μmol/L), thrombin (0.01 μmol/L), trypsin (0.01 μmol/L), or vehicle (control) for 15 minutes at 37°C. Cells were fixed using 2% paraformaldehyde and were immunostained with a PAR-2 mAb that recognizes an epitope in the N-terminal extracellular domain of PAR-2 that overlaps the activating-cleavage site. Original magnification, ×630. B: Confocal photomicrographs of unstimulated (control) and KLK14-, thrombin-, and trypsin-stimulated HT29 cells. Cells were fixed and permeabilized with acetone and immunostained with PAR-2 mAb. Original magnification, ×630; zoom, ×4. Results are representative of two independent experiments.
Figure 6
Figure 6
KLK14 activates p42/p44 MAPK in HT29 cells. A: Immunoblot with phospho-specific p42/p44 MAPK antibodies on quiescent HT29 cell lysates treated with or without KLK14 (0.1 μmol/L) for the indicated periods. Results are representative of two separate experiments. B: Dose-dependent activation of p42/p44 MAPK phosphorylation by KLK14. Quiescent HT29 cells were stimulated with the indicated concentrations of KLK14 for 10 minutes. To confirm equal protein loading, the membranes were stripped and incubated with p42/p44 MAPK antibody. Results are representative of three separate experiments. C: Cells grown in serum-free medium were preincubated with anti–PAR-2 (mAb 13-8) (200 nmol/L) for 2 hours and then were challenged for 5 minutes with trypsin (0.01 μmol/L) or with KLK14 (2 nmol/L). Cell lysates were then directly analyzed for ERK1/2 phosphorylation with anti-phospho-ERK1/2. The blot was subsequently stripped and reprobed with anti-ERK1/2 to verify equal protein loading lanes. The figure shows a representative immunoblot from two separate experiments. Densitometric analysis of the phospo-p42/p44 MAPK divided by p42/p44 MAPK is represented in the left panels. Data are the mean ± SD of two separate experiments.
Figure 7
Figure 7
The ability of KLK14 to induce cell proliferation in HT29 cells. Cells were seeded in medium containing 10% FCS. After 3 days, cells were washed and covered with serum-free medium for 48 hours. Quiescent cells were grown for 96 hours in serum-free medium without control, with 100 μmol/L AP2, 1 nmol/L trypsin, or 1 nmol/L KLK14. After 72 hours, cells from triplicate wells were counted for each condition. Data are the mean ± SEM of three different experiments. *P < 0.001, KLK14-, AP2-, or trypsin-treated cells versus control cells.
Figure 8
Figure 8
KLK14 secretion in colon cancer cell lines. Supernatants were collected from colon cancer cells in culture, and KLK14 expression was estimated by sandwich-type ELISA (see Materials and Methods). Protein values represent the mean ± SEM concentration of KLK14 expressed by 1 × 106 cells.
Figure 9
Figure 9
Representative immunostaining for KLK14 in paraffin sections of normal colonic mucosa and colonic tissues from patients with adenocarcinomas. KLK14 immunoreactivity is absent in normal sigmoid glandular mucosa from control subjects (A) and in an adjacent section from a colonic mucosa distant from an adenocarcinoma (B). Asterisks show stromal cell staining. C–E: High and variable immunoreactivity (white arrows) is seen in the epithelial cells of adenocarcinomas of three different patients. The black arrow in the enlarged area in D points to positive immunoreactivity in the cytoplasmic compartment of epithelial cells. Scale bars = 100 μm. Original magnification, ×200.
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