Ab-induced ectodomain shedding mediates hepatocyte growth factor receptor down-regulation and hampers biological activity

Abstract

Targeting tyrosine kinase receptors (RTKs) with specific Abs is a promising therapeutic approach for cancer treatment, although the molecular mechanism(s) responsible for the Abs' biological activity are not completely known. We targeted the transmembrane RTK for hepatocyte growth factor (HGF) with a monoclonal Ab (DN30). In vitro, chronic treatment of carcinoma cell lines resulted in impairment of HGF-induced signal transduction, anchorage-independent growth, and invasiveness. In vivo, administration of DN30 inhibited growth and metastatic spread to the lung of neoplastic cells s.c. transplanted into immunodeficient nu/nu mice. This Ab efficiently down-regulates HGF receptor through a molecular mechanism involving a double proteolytic cleavage: (i) cleavage of the extracellular portion, resulting in "shedding" of the ectodomain, and (ii) cleavage of the intracellular domain, which is rapidly degraded by the proteasome. Interestingly, the "decoy effect" generated by the shed ectodomain, acting as a dominant negative molecule, enhanced the inhibitory effect of the Ab.

Fig. 1.

DN30 impairs HGFR activation and signal transduction. (A) Evaluation of HGFR activation. GTL16 cells were exposed to DN30 for 4 h. HGFR was immunoprecipitated from cell lysates, and Western blots were probed with the indicated Abs. The upper band corresponds to intracellular HGFR precursor (p170); the lower band (p145) is the mature form. DN30 treatment resulted in a decrease of receptor activation more pronounced than receptor down-regulation, as indicated by band density quantification. (B) Analysis of HGFR signaling. Cells were pretreated with either VSV-G or DN30 and stimulated with HGF for the indicated times. Akt phosphorylation was evaluated in total cell lysates. As shown, DN30 reduced both basal and HGF-induced Akt activation.

Fig. 2.

DN30 inhibits the transformed phenotype of cancer cells in vitro. (A) Anchorage-independent growth of GTL16 cells. Cells pretreated with either DN30 or VSV-G for 48 h were seeded in 0.5% agar and maintained in the presence of the indicated amounts of Abs with or without HGF (20 ng/ml). Anchorage-independent growth was drastically inhibited in the presence of DN30, even at low doses. (B) Invasion assay. MDA-MB-435 β4 cells were pretreated with the indicated antibodies for 24 h before seeding on a Matrigel-coated Transwell chamber. The lower chamber was filled with DMEM/2% FBS plus 100 ng/ml HGF. After 24 h, migrated cells were stained and counted. Invasive capacity in response to HGF is expressed as fold increase compared with nonstimulated cells. As shown, DN30 treatment significantly impaired cell invasion.

Fig. 3.

DN30 inhibits tumor growth in vivo. (A and B) Tumorigenesis assay. Nude mice were injected s.c. with 1.5 × 10⁶ GTL16 cells. After tumor appearance, mice displaying tumors of the same size were selected and then injected in situ in the tumor twice a week with 2 μg/g of either VSV-G or DN30. (A) Tumor volume was measured at different time points. Mice were killed after 4 weeks of treatment, and tumor weight was evaluated (B). In mice treated with DN30, tumors were significantly smaller than in control mice (P < 0.05). (C) Evaluation of HGFR activation. Tumor sections from mice treated with VSV-G (a), or DN30 (b) were stained with anti-human phospho-HGFR. HGFR activation was strongly decreased in mice treated with DN30. Magnification ×40.

Fig. 4.

DN30 treatment interferes with tumor progression in vivo. (A) Nude mice were inoculated s.c. with 2.5 × 10⁶ MDA-MB-435 β4 cells and treated with the indicated doses of VSV-G or DN30, administered i.p. (IP), or in situ (IS). As shown, DN30 inhibited tumor growth. (B) Analysis of lung metastases. Metastases were counted by microscopic observation of the lung sections after hematoxylin/eosin staining. A dose-dependent reduction of metastases number was evident in DN30-treated mice. (C and D) Evaluation of tumor vascularization. Blood vessels staining on tumor histological sections was performed with an anti-mouse CD31 Ab. Number and area of vessels were evaluated by fluorescence microscopy. As shown, both the number and the area of vessels were reduced in response to DN30 treatment.

Fig. 5.

DN30 induces HGFR down-regulation. GTL16 cells (A) and MDA-MB-435 β4 (B) were treated with DN30 for the indicated times. Equal amounts of total cell lysates were processed for Western blotting and probed with anti-HGFR or, as a loading control, with anti-Hsp70 antibodies. As shown, DN30 induced HGFR down-regulation in both overexpressing cells (GTL16) and in cells expressing normal levels of HGFR (MDA-MB-435 β4).

Fig. 6.

Ab-induced and ligand-dependent down-regulation exploit different pathways. HeLa (Upper) and GTL16 (Lower) cells were pretreated with lactacystine (lact), concanamycin (conc), or both for 2 h before treatment with HGF or DN30. HGFR down-regulation was evaluated on total cell lysates. In the presence of the proteasome inhibitor (lact), ligand-induced HGFR down-regulation was impaired, whereas Ab-induced was not. In this condition, a 60-kDa fragment [intracellular domain (ICD)], detectable by an Ab directed against the intracellular portion, accumulated in cells.

Fig. 7.

DN30 induces proteolytic cleavage of HGFR and shedding of the extracellular domain (ECD). (A) Supernatants obtained from metabolically labeled GTL16 cells were collected and immunoprecipitated with an anti-HGFR Ab directed against the extracellular domain. As shown, DN30, but not HGF, induced shedding of HGFR ectodomain. (B and C) HGFR shedding is dose- and time-dependent. Cells were stimulated either with increasing amounts of DN30 or for different times.

Fig. 8.

Activation of signal transduction is not required for HGFR shedding. COS-7 cells were transfected with the indicated HGFR mutants and, 48 h later, were treated with DN30 for 4 h. Equal amounts of total cell lysates and conditioned media were processed for Western blotting. As shown, DN30 was able to induce down-regulation and ectodomain (ECD) shedding of all HGFR mutants. HGFR mutants: Met KD, HGFR kinase dead; Met Double, HGFR mutant lacking the docking tyrosines 1349/1356; Met-GFP, HGFR mutant where the whole intracellular portion was replaced by the GFP sequence.

Fig. 9.

The HGFR shed ectodomain behaves as a dominant negative molecule. (A) Cells pretreated for 72 h with DN30 were stimulated with HGF in either the presence (lanes 1–3) or absence (lanes 4–6) of the shed HGFR ectodomain in the culture medium. As shown, shed HGFR ectodomain impaired Akt activation. (B and C) GTL16 (B) and HeLa (C) cells were stimulated with HGF in the presence of control medium (lanes 1–3), medium containing HGFR ectodomain (lanes 4–6), or the same medium depleted of the shed ectodomain (lanes 7–9). As shown, the depleted medium was no longer able to prevent Akt activation.