Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma

Abstract

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors.

Keywords: NF-κB; inflammation; kallikrein; microRNA (miRNA); oral cancer; protease; protease-activated receptor; serine protease.

FIGURE 1.

Immunohistochemical analysis of PAR-2 and KLK5 expression in pre-malignant oral lesions and OSCC. A and B, PAR-2 expression in pre-malignant oral lesions. A, 24% of lesions exhibited negative or low PAR-2 expression; B, 76% demonstrated moderate to high PAR-2 expression. PAR-2 antibody was used at a 1:50 dilution. Staining was detected using an avidin-biotin horseradish peroxidase system. C and D, PAR-2 expression in oral cancer. C, 24% of lesions exhibited negative or low PAR-2 expression; D, 76% demonstrated moderate to high PAR-2 expression. Antibody dilutions as in A. E and F, KLK5 expression in pre-malignant lesions. E, 37% of lesions exhibited negative or low KLK5 expression; F, 63% demonstrated moderate to high KLK5 expression. KLK5 antibody was used at a 1:50 dilution. G and H, KLK5 expression in oral cancer. Note that KLK5 expression in OSCC was previously published in Pettus et al. (17). In that study, 22% of lesions exhibited negative or low KLK5 expression, with 78% staining positive or strong positive. Magnification, ×200.

FIGURE 2.

PAR-2 expression and activation in OSCC cell lines. A, Western blot showing elevated PAR-2 protein in SCC1 and SCC25 cell lines relative to immortalized oral keratinocyte line OKF6/T. Cell lysates were electrophoresed on 9% SDS-polyacrylamide gels and electroblotted to Immobilon. Upper panel, blots were probed with murine anti-PAR-2 antibody (1:100 dilution) followed by an HRP-conjugated secondary antibody (1:4000 dilution). Loading controls (lower panel) were probed with mouse anti-GAPDH (1:500) and an HRP-conjugated secondary antibody (1:10,000). The experiment was repeated in triplicate, and a representative blot is shown. B–E, flow cytometry analysis of cell surface PAR-2. B and D, OKF6/T; C and E, SCC1 cell lines were incubated on ice with vehicle (B and C) or anti-PAR-2 antibody (1:100) (D and E) followed by FITC-conjugated secondary antibody (1:500) and evaluated using a Beckman/Coulter FC500 cell sorter. F–I, analysis of PAR-2 activation-induced calcium signaling. Cell lines were loaded with Fura2AM and incubated with the PAR-2 agonist peptide SLIGRL-NH₂ (50 μm) or KLK5 (3.23 μm), as indicated. Shown in each trace is a representative single cell Ca²⁺ response measured as 340/380 fluorescence intensity (F.I.) ratio. The calcium response is the difference in the peak value and baseline value. F, peptide (SLIGRL-NH₂, 50 μm) activation of PAR-2 in SCC1 cell line. G, peptide (SLIGRL-NH₂, 50 μm) activation of PAR-2 in OKF6/T cell line. H, KLK5 (3.23 μm) activation of PAR-2 in SCC1 cell line. I, de-sensitization experiment showing lack of additional calcium response in SCC1 cells treated with SLIGRL-NH₂ (50 μm) followed by KLK5 (3.23 μm).

FIGURE 3.

PAR2 induces RelA nuclear translocation. Cells grown on coverslips in serum-free media were untreated (A, C, and E) or treated with TNFα (100 ng/ml, 1.9 nm, 30 min) (B), SLIGRL-NH₂ (20 μm, 85 min) (D), or KLK5 (3.23 μm, 60 min) (F). After washing and fixing/permeabilization, cells were treated with antibody to RelA, a FITC secondary, and mounted. Nuclear translocation of RelA was assessed on an inverted AMG EVOS All-In-One digital microscope.

FIGURE 4.

PAR-2 activation regulates expression of mRNAs and microRNAs in OSCC cell lines. A, PAR-2 activation increases pro-inflammatory mRNAs. PAR-2 was activated in SCC1 or SCC25 cell lines, as indicated, by incubation with SLIGRL-NH₂ (20 μm, 2 h) followed by immediate RNA isolation and processing for RT-qPCR as described under “Materials and Methods.” Data shown are fold-change in expression of IL8 (black bar), IL1A (dark gray bar), and MMP9 (light gray bar) relative to untreated cells. Experiments were repeated in triplicate; error bars show S.E. B, PAR-2 activation decreases anti-inflammatory microRNAs. PAR-2 was activated in SCC1 or SCC25 cell lines, as indicated, by incubation with SLIGRL-NH₂ (20 μm, 2 h) followed by immediate RNA isolation and processing for RT-qPCR as described under “Materials and Methods.” Data shown are fold-change in expression of let-7d (black bar), miR-23b (dark gray bar), and miR-200c (light gray bar) relative to untreated cells. Experiments were repeated in triplicate; error bars show S.E. C, PAR-2-mediated microRNA suppression is reversed by Nf-κB inhibitors. Cells were incubated with vehicle (control, black bar) or pretreated overnight with inhibitors as indicated: sc-514 (100 μm, dark gray bar), SB747651A (20 μm, light gray bar), or pertussis toxin (PTX) (1 μg/ml, 8.5 nm white bar) prior to activation of PAR-2 with SLIGRL-NH₂ (20 μm, 2 h) in the continued presence of inhibitor. RNA was isolated immediately and used as template for RT-qPCR. Data shown are fold-change in expression of let-7d, miR-23b, and miR-200c relative to SLIGRL-NH₂-activated cells without the addition of an inhibitor. Experiments were performed in duplicate.

FIGURE 5.

KLK5 silencing inhibits tumor growth and progression in an orthotopic murine OSCC xenograft model. Cells (0.8 × 10⁶ in 30 μl of PBS) with diminished KLK5 expression (designated SCC25-KLK5-KD) or vector controls (SCC25-Vec) were injected into the lateral border of the base of the tongue and allowed to grow for 9 weeks. Following sacrifice, tumors were harvested, processed for histology, and stained with H&E. A and B, representative tumors formed from SCC25-Vec control cells are poorly circumscribed with invasive cords of tumor cells, characteristic of moderately differentiated OSCC. Magnification, ×200. C, section of SCC25-Vec tumor immunostained with anti-KLK 5 antibody (1:200) and peroxidase-conjugated secondary antibody. Magnification, ×400. D and E, representative tumors formed from SCC25-KLK5-KD cells with silenced KLK5 expression are well circumscribed with pushing tumor margins and keratin pearls (*), characteristic of well differentiated OSCC. Magnification, ×200. F, representative section of SCC25-KLK5-KD tumor immunostained with anti-KLK5 antibody. Magnification, ×400. G, quantitation of tumor area. Tumors formed from SCC25-Vec cells were ∼8-fold larger than SCC25-KLK5-KD tumors (p = 0.002).

FIGURE 6.

KLK5 silencing reduces tumor inflammatory infiltrate. A–C, inflammatory cells were enumerated from H&E-stained sections for each high power field containing or immediately adjacent to tumor nests. Results were presented as counts per high power field. Magnification A and B, ×200. *, p = 0.001. D–F, tumor sections were evaluated for mast cell infiltration by staining for mast cell tryptase using murine anti-tryptase (1:100) and the Vector M.O.M. (mouse-on-mouse) kit according to the manufacturer's specifications. Tryptase-positive mast cells were quantified as described under “Materials and Methods.” Magnification C and D, ×200. *, p = 0.004.