A disintegrin and metalloproteinase-10 (ADAM-10) mediates DN30 antibody-induced shedding of the met surface receptor

Abstract

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown. Here, we report that A Disintegrin And Metalloproteinase (ADAM)-10, but not ADAM-17, is required for DN30-induced Met shedding. Knockdown of ADAM-10 in different tumor cell lines or abrogation of its proteolytic activity by natural or synthetic inhibitors abolished Met down-regulation on the cell surface as well as reduction of Met activation. Moreover, hepatocyte growth factor-induced tumor cell migration and invasion were impaired upon ADAM-10 knockdown. Thus, the therapeutic effect of DN30 involves ADAM-10-dependent Met shedding, linking for the first time a specific metalloprotease to target therapy against a receptor tyrosine kinase.

FIGURE 1.

A, shedding of pre-Met upon administration of DN30 appeared to be time-dependent. Representative Western blots for pre-Met and Met in A549 cells, incubated with 80 μg/ml DN30 for 1.5 h and 3 h, are shown. α-Tubulin (α-Tub) was used to normalize protein levels. Densitometric analysis (n = 3): without DN30, 100.00%; 1.5 h DN30, 79.4 ± 3.7; 3 h DN30, 25.9 ± 4.7). B, DN30-induced shedding of pre-Met was inhibited by addition of recTIMP-1. Representative Western blots for pre-Met and Met in A549 cells cultured with 1 μg/ml recTIMP-1 for 1 h prior to addition of 80 μg/ml DN30 and further incubation for 5 h with or without 1 μg/ml recTIMP-1 are shown. α-Tubulin was used to normalize protein levels. Densitometric analysis (n = 3): without DN30 without TIMP-1, 100.00%; with DN30 without TIMP-1, 56.8 ± 8.4; without DN30 with TIMP-1, 155.2 ± 36.5; with DN30 with TIMP-1, 152.4 ± 27.5). C, DN30-induced shedding of pre-Met was inhibited by addition of recTIMP-3. Representative Western blots for pre-Met and Met in A549 cells cultured with 1 μg/ml recTIMP-3 for 1 h prior to addition of 80 μg/ml DN30 and further incubation for 5 h with or without 1 μg/ml recTIMP-3 are shown. α-Tubulin was used to normalize protein levels. Densitometric analysis (n = 3): without DN30 without TIMP-3, 100.00%; with DN30 without TIMP-3, 22.5 ± 0.7; without DN30 with TIMP-3, 82.1 ± 2.4; with DN30 with TIMP-3, 61.8 ± 6.6). D, DN30-induced shedding of pre-Met in GTL-16 cells was stimulated by PMA. Cells were incubated with or without 80 μg/ml DN30 and 100 nm PMA for 4 h. Supernatants were analyzed afterward with Western blotting. A representative Western blot of three experiments is shown. n.s., not significant.

FIGURE 2.

A–C, ADAM-10, but not ADAM-17, knockdown inhibited DN30-induced shedding of pre-Met. Representative Western blots (of three independent experiments) for pre-Met and Met, ADAM-10, and ADAM-17 in A549 (A), SKOV3ip (B), and GTL-16 (C) cells, cultured 5 h with or without 80 μg/ml DN30 (A–C) are shown. α-Tubulin was used to normalize protein levels. Knockdown cell lines (shADAM-10 or shADAM-17, respectively) and control cell lines (shNT) were generated using shRNAi. Densitometric analysis: A: A549shNT without DN30, 100%; with DN30, 48.0% ± 8.2%; n = 3; shADAM17 without DN30, 100%; with DN30, 56.3% ± 8.1%; n = 3; shADAM10 without DN30, 100%; with DN30, 103.7% ± 2.6%; n = 3. B, SKOV3ipshNT without DN30, 100%; with DN30, 38.1% ± 12.7%; n = 3; shADAM17 without DN30, 100%; with DN30, 51.6% ± 14.3%; n = 3; shADAM10 without DN30, 100%; with DN30, 105.3% ± 4.6%; n = 3. C, GTL16shNT without DN30, 100%; with DN30, 63.4% ± 4.2%; n = 3; shADAM17 without DN30, 100%; with DN30, 55.2% ± 5.6%, n = 3; shADAM10 without DN30, 100%; with DN30, 90.8% ± 9.2%, n = 3. Furthermore, in SKOV3ip (B) and GTL-16 cells (C), elevated levels of ADAM-17 after DN30 administration and increased ADAM-10 protein upon knockdown of ADAM-17 were detected. D, representative Western blot of supernatants and immunoprecipitated protein from supernatants of GTL-16shNT or GTL-16shADAM-10 cells are shown, cultured for 5 h with 80 μg/ml DN30. Knockdown of ADAM-10 reduced Met shedding. E, representative Western blot of A549 cells of three independent experiments cultured with or without 80 μg/ml DN30 and the indicated amounts of GI254023X, showed that ADAM-10-specific inhibition by GI254023X abolished Met shedding. Densitometric analysis: without DN30 without GI254023X, 100%; with DN30 without GI254023X, 42.7% ± 5.6%; with DN30 1 μm GI254023X, 46.6% ± 12.8%; with DN30 3 μm GI254023X, 81.8% ± 33.2%; with DN30 5 μm GI254023X, 125.1% ± 28.2%. n.s., not significant.

FIGURE 3.

A, representative Western blot for phosphorylated Met in GTL-16 cells, cultured with or without 80 μg/ml DN30 is shown. α-Tubulin was used to normalize protein levels. Knockdown cell lines (shADAM-10 or shADAM-17, respectively) and a control cell line (shNT) were generated using shRNAi. ADAM-10 knockout impaired DN30-induced inhibition of Met phosphorylation, whereas ADAM-17 deficiency had no effect on this inhibition. Densitometric analysis: GTL16shNT without DN30, 100%; with DN30, 42.9% ± 1.3%; n = 3; shADAM17 without DN30, 100%; with DN30, 21.9% ± 0.9%, n = 3; shADAM10 without DN30, 100%; with DN30, 97.1% ± 4.4%; n = 3. B and C, representative images of immunohistochemical staining for phosphorylated Met in GTL-16shNT and GTL-16shADAM-10 cells, respectively, cultured either for 5 h with or without 80 μg/ml DN30 (B) or additionally with or without 1 μg/ml recTIMP-1 (C) are shown. Blue signal, DAPI. Green signal, phosphorylated Met. B, DN30-induced shedding reduced Met phosphorylation only when ADAM-10 was not knocked down. Scale bars, 50 μm. Densitometric analysis: GTL16 shNT without DN30, 100.0% ± 5.8%; with DN30, 42.5% ± 2.0%; n = 15; shADAM10 without DN30, 100.0% ± 6.7%; with DN30, 98.2% ± 6.6%, n = 15. C, incubation with the natural ADAM-10 inhibitor TIMP-1 also impaired DN30-induced reduction of Met phosphorylation. Scale bars, 50 μm. Densitometric analysis: GTL16 without TIMP-1 without DN30, 100.0% ± 6.9%; without TIMP-1 with DN30, 42.6% ± 2.6%; n = 15; with TIMP-1 without DN30, 100.0% ± 4.4%; with TIMP-1 with DN30, 85.6% ± 4.3%, n = 15. Besides, a weak Met phosphorylation upon incubation with recTIMP-1 was detected even after administration of DN30. D, incubation with the natural ADAM-10 and ADAM-17 inhibitor TIMP-3 also impaired DN30-induced reduction of Met phosphorylation. Scale bars, 50 μm. Densitometric analysis: GTL16 without TIMP-3 without DN30, 100.0% ± 6.3%; without TIMP-3 with DN30, 36.3% ± 1.5%; n = 15; with TIMP-3 without DN30, 100.0% ± 5.3%; with TIMP-3 with DN30, 88.2% ± 3.4%; n = 15. n.s., not significant.

FIGURE 4.

A, representative microscopic view of A549shNT or A549shADAM-10 cells in culture. Cells were seeded at low density and incubated with or without 80 μg/ml DN30 for 5 h. After colony formation, cells were stimulated with 20 ng/ml recHGF. ADAM-10 appeared to be essential for DN30-induced inhibition of tumor cell scattering. Scale bars, 50 μm. B, analysis of the invasiveness of A549shNT and A549shADAM-10 cells, incubated with or without 80 μg/ml DN30 for 5 h, was analyzed in a Transwell® invasion assay. ADAM-10 was essential for DN30-induced inhibition of tumor cell invasion. A549shNT, 100.00 ± 20.24; A549shNT + DN30, 28.13 ± 11.13; A549shADAM-10, 100.00 ± 26.96; A549shADAM-10 + DN30, 77.29 ± 7.25. Error bars, S.E., n = 3 each. n.s., not significant.

FIGURE 5.

Involvement of ADAM-10 in the DN30-mediated therapeutic effect. A, binding of the ligand HGF to the extracellular portion of Met induces receptor dimerization and phosphorylation of the tyrosine residues 1234 and 1235 thereby leading to the activation of the invasive growth program. B, binding of the monoclonal antibody to the extracellular portion of Met induces ADAM-10-mediated shedding of the receptor thereby reducing the number of available receptors on the cell surface. Activation of the invasive growth program is impaired as HGF can no longer bind to Met. C, under ADAM-10-deficient conditions, DN30 can still bind to Met but cannot induce shedding of the Met receptor. Consequently, receptor levels on the cell surface are not reduced. Met signaling and the invasive growth program can still be activated by HGF.