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. 2008 Jun 27;283(26):18393-401.
doi: 10.1074/jbc.M801329200. Epub 2008 Apr 22.

The ectodomain shedding of E-cadherin by ADAM15 supports ErbB receptor activation

Abdo J Najy  1 , Kathleen C Day, Mark L Day
Affiliations

The ectodomain shedding of E-cadherin by ADAM15 supports ErbB receptor activation

Abdo J Najy et al. J Biol Chem. .

Abstract

The zinc-dependent disintegrin metalloproteinases (a disintegrin and metalloproteinases (ADAMs) have been implicated in several disease processes, including human cancer. Previously, we demonstrated that the expression of a catalytically active member of the ADAM family, ADAM15, is associated with the progression of prostate and breast cancer. The accumulation of the soluble ectodomain of E-cadherin in human serum has also been associated with the progression of prostate and breast cancer and is thought to be mediated by metalloproteinase shedding. Utilizing two complementary models, overexpression and stable short hairpin RNA-mediated knockdown of ADAM15 in breast cancer cells, we demonstrated that ADAM15 cleaves E-cadherin in response to growth factor deprivation. We also demonstrated that the extracellular shedding of E-cadherin was abrogated by a metalloproteinase inhibitor and through the introduction of a catalytically inactive mutation in ADAM15. We have made the novel observation that this soluble E-cadherin fragment was found in complex with the HER2 and HER3 receptors in breast cancer cells. These interactions appeared to stabilize HER2 heterodimerization with HER3 and induced receptor activation and signaling through the Erk pathway, supporting both cell migration and proliferation. In this study, we provide evidence that ADAM15 catalyzes the cleavage of E-cadherin to generate a soluble fragment that in turn binds to and stimulates ErbB receptor signaling.

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Figures

FIGURE 1.
FIGURE 1.
ADAM15 and HER2 expression in breast cancer. A, ADAM15 and HER2 are simultaneously overexpressed in seven different breast cancer cDNA microarrays. B, a graphical representation of ADAM15 and HER2 expression in normal and tumor breast tissues using the published Richardson_Breast_2 array available on the World Wide Web.
FIGURE 2.
FIGURE 2.
ADAM15 cleaves E-cadherin in breast cancer cells. A, GFP-tagged and endogenous ADAM15 in MCF-7 cells are indicated (Lysate). Due to the intense banding pattern observed in ADAM15-overexpressing (ADAM15-GFP) cells, a lower exposure of the GFP fusion protein was cropped into the ADAM15 panel. Shown is an analysis of sE-cad in the conditioned media (CM) of ADAM15-overexpressing cells. B, ADAM15 expression was down-regulated in MCF-7 cells using a stable shRNA against ADAM15 (Lysate), and sE-cad was assessed in the conditioned media. Full-length E-cadherin (FL/E-cad) remained unchanged in these experiments.
FIGURE 3.
FIGURE 3.
E-cadherin is a substrate for ADAM15. A, immunocytochemistry analysis of ADAM15 (green) and E-cadherin (red) in MCF-7 cells. Shown is confocal microscopy at ×400 concentration. Isolated ADAM15 and E-cadherin were co-incubated. ADAM15 cleaves full-length E-cadherin (FL/E-cad) into its soluble fragment (sE-cad) in a time-dependent (B) and concentration-dependent (C) manner. D, E-cadherin and ADAM15 were co-incubated with vehicle (veh) or 1,10-phenanthroline (1,10P). E, wild-type (WT) or catalytically dead (CD) ADAM15 were co-incubated with isolated E-cadherin. Isolated E-cadherin and ADAM15 alone were loaded as controls on the end lanes. Whole cell lysate (WCL) was used to demarcate the E-cadherin banding pattern.
FIGURE 4.
FIGURE 4.
Soluble E-cadherin mediates HER2-HER3 heterodimerization through ErbB receptor binding. ADAM15 knockdown MCF-7 whole cell lysates were immunoprecipitated (IP) with HER2 (A), HER3 (B), or isotype IgG. Control shScrm MCF-7 cells were treated with either vehicle (veh) or trastuzumab (TZM) for 24 h prior to immunoprecipitation either with HER2 (C), HER3 (D), or isotype IgG under growth factor-depleted conditions. Immunoblotting with E-cadherin (E-cad), HER2, or HER3 antibodies were used to assess E-cadherin binding to ErbB receptors and receptor dimerization. The input lane (2% of the amount of protein used for the immunoprecipitation) was used for the E-cadherin banding pattern.
FIGURE 5.
FIGURE 5.
Soluble E-cadherin mediates HER2-HER3 phosphorylation and induces ErbB-mediated cell signaling. Phosphotyrosine (pTyr) status was assessed in control shScrm or shADAM15 (shA15) MCF-7 cells in response to serum starvation. Whole cell lysates were collected and immunoprecipitated (IP) with HER2 (A), HER3 (B), or isotype IgG prior to immunoblotting with antibodies specific to Tyr(P) (pTyr), HER2, or HER3 (C). An increase in Erk activation (pErk) in MCF-7 cells was observed at 24 and 26 h after growth factor withdrawal.
FIGURE 6.
FIGURE 6.
ADAM15 supports cell migration and proliferation. A, control shScrm or shADAM15 MCF-7 cells were abraded with a 10-μl pipette tip, and wound closure was monitored over time. The columns represent the mean of three separate experiments quantitated in four different samples. Bars, S.D. *, p < 0.04. B, control shScrm or shADAM15 MCF-7 cells were grown under serum-depleted conditions, and cell proliferation was analyzed as a function of time. The columns represent the mean of three separate experiments quantitated in eight different samples. Bars, S.D. *, p < 4.6E-05; **, p < 5.5E-04.
FIGURE 7.
FIGURE 7.
HER2 stimulation by exogenous soluble E-cadherin. A, E-cadherin-negative SKBr3 cells were treated with either vehicle (veh) or the extracellular Fc-Ecad, and lysates were then immunoprecipitated (IP) with HER2 or isotype IgG. Immunoblotting with E-cadherin (E-cad) or HER2 was performed to detect Fc/E-cadherin binding. B-D, Fc/E-cadherin fusion protein mediated HER2-HER3 heterodimerization and HER3 phosphorylation as well as increased Erk activation (pErk) and cell proliferation in SKBr3 cells. Columns represent the mean of three separate experiments quantitated in eight different samples. Bars, S.D. *, p < 5E-04; **, p < 0.001.
FIGURE 8.
FIGURE 8.
Model for ADAM15-dependent activation of HER2 by soluble E-cadherin. Serum depletion stimulates ADAM15 activation, which in turn cleaves E-cadherin. The liberated E-cadherin fragment (sE-cad) mediates ErbB activation.
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