Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer

Abstract

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.

Figure 1

Membrane-tethered PAR1-i3 and i1 pepducins block Ca²⁺ flux and migration. A: Model of PAR1 based on the X-ray structure of rhodopsin from Swift et al. Protease cleaved receptor results in generation of a new tethered ligand. The topologic arrangement of the i1 to i4 loops and G proteins are illustrated. Schematic diagram of palmitoylated PAR1 i3 and i1 pepducins and their amino acid sequence composition are shown. B: Effect of PAR1 pepducins on human platelets Ca²⁺ flux. Platelets were preincubated with indicated concentrations of pepducins and stimulated with SFLLRN. Effect of blockade is expressed as the percentage of full Ca²⁺ signal generated with SFLLRN in the absence of inhibitors. C: Migration of A549 cells toward 0.3 nmol/L thrombin or 10 μmol/L PRSFLLRN in the presence or absence of 3 μmol/L P1pal-7, P1pal-10S, or P1pal-i1-11 after 18 hours. Basal migration (-), migration of A549 cells toward PAR1 ligand (Veh). Cell migration is expressed as fold migration of ligand-mediated migration divided by basal migration.

Figure 2

Differential inhibition of PAR1-ERK activation by i3- and i1-derived PAR1 pepducins. A–C: Western blots of lysates from A549 cells untreated, or treated with thrombin, SLIGKV, or AYPGKF. Indicated samples were pretreated with various concentrations (0.1 to 3 μmol/L) of P1pal-7, P1pal-10S, or P1pal-i1-11 before stimulation and immunoblotted with anti-phospho-ERK. D–F: Quantification of the Western blots in A–C were conducted by densitometry and results are expressed relative to maximum (fold max) phosphorylation of pERK.

Figure 3

PAR expression profile in lung cancer. Lung cancer cell lines (n = 7) from the NCI-60 panel were characterized for surface expression of PAR1, PAR2, PAR3, and PAR4 (A) as previously described.B:PAR mRNA expression profile in patient samples. Quantitative real-time PCR analysis of PAR1 to PAR4 (corrected for actin expression as internal control) and expressed as fold increase over nonmalignant lung tissue. Migration of lung carcinoma cell lines toward fibroblast conditioned media is expressed as cell number ± SD. C: Silencing of PAR1 expression in A549 and HOP62 cells with shPAR1 or vector control. Surface expression of PAR1 was determined by flow cytometry using SFLLR-Ab. D: Migration of A549 and HOP62 lung cancer cells toward NIH-3T3 conditioned media supplemented with RWJ-56110 (10 μmol/L) and SCH7979 (50 μmol/L) or vehicle (0.2% dimethyl sulfoxide). Migration of lung cancer cells treated with shPAR1 treatment or appropriate controls toward NIH-3T3 conditioned media. Basal migration toward conditioned media (vehicle treated or vector control) was normalized to 100%. *P < 0.05, **P < 0.01.

Figure 4

PAR1 is a promigratory factor in lung cancer cells. A–F: Inhibition of lung cancer migration in adenocarcinomas A549, HOP62, H1299 and H522, H226 squamous, and HOP92 large cell using PAR1 pepducins. Pepducins at 3 μmol/L (P1pal-7, P1pal-10S, P1pal-i1-11, or negative control P1pal-19EE) were added to the lower well. G: Cell from primary lung tumors or nonmalignant control were pretreated with PAR1 pepducins and migration was determined (*P < 0.05, **P < 0.01).

Figure 5

Monotherapy with PAR1-i3 pepducins inhibits tumor progression of A549 xenografts. A and B: A549 mice treated with vehicle, P1pal-7, P1pal-i1-11, P1pal-10S, X1/2-i1, X1/2-i3, or Avastin. Each time point represents the mean ± SE. C: Final tumor volume at the completion of the experiment was calculated as a percentage of untreated vehicle control. D: Serum-starved A549 adenocarcinoma cells were pretreated with P1pal-7, P1pal-10S, P1pal-i1-11, vehicle, or PD98059 and stimulated with thrombin. VEGF production was assessed by enzyme-linked immunosorbent assay. Data are expressed as mean ± SD (*P < 0.05, **P < 0.01).

Figure 6

Pharmacokinetics of PAR1 pepducins. A: CF-1 mice were injected subcutaneously with P1pal-7 (3 mg/kg, open circles), P1pal-7 (10 mg/kg, closed circles), P1pal-10S (10 mg/kg, gray circles), or P1pal-i1-11 (10 mg/kg, open squares) in 20% dimethyl sulfoxide and blood was collected at indicated time points (n = 3). Pepducin levels in plasma were quantified with LC/MS/MS. B: Daily (D1 to D6) plasma levels of P1pal-7 after subcutaneous injection of 10 mg/kg P1pal-7 were measured at 1 hour (open circles) and 24 hours (open squares).