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. 2007 Feb;117(2):337-45.
doi: 10.1172/JCI29518. Epub 2007 Jan 11.

Targeting TACE-dependent EGFR ligand shedding in breast cancer

Paraic A Kenny  1 , Mina J Bissell
Affiliations

Targeting TACE-dependent EGFR ligand shedding in breast cancer

Paraic A Kenny et al. J Clin Invest. 2007 Feb.

Abstract

The ability to proliferate independently of signals from other cell types is a fundamental characteristic of tumor cells. Using a 3D culture model of human breast cancer progression, we have delineated a protease-dependent autocrine loop that provides an oncogenic stimulus in the absence of proto-oncogene mutation. Targeting this protease, TNF-alpha-converting enzyme (TACE; also referred to as a disintegrin and metalloproteinase 17 [ADAM17]), with small molecular inhibitors or siRNAs reverted the malignant phenotype in a breast cancer cell line by preventing mobilization of 2 crucial growth factors, TGF-alpha and amphiregulin. We show that TACE-dependent ligand shedding was prevalent in a series of additional breast cancer cell lines and, in all cases examined, was amenable to inhibition. Using existing patient outcome data, we demonstrated a strong correlation between TACE and TGFA expression in human breast cancers that was predictive of poor prognosis. Tumors resulting from inappropriate activation of the EGFR were common in multiple tissues and were, for the most part, refractory to current targeted therapies. The data presented here delineate the molecular mechanism by which constitutive EGFR activity may be achieved in tumor progression without mutation of the EGFR itself or downstream pathway components and suggest that this important oncogenic pathway might usefully be targeted upstream of the receptor.

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Figures

Figure 1
Figure 1. Upregulation of an autocrine growth factor loop during a model of breast cancer progression.
(A) T4-2 (malignant) cells, which grow independently of exogenous EGF, had significantly higher activity of EGFR than their phenotypically normal counterpart, S1 (nonmalignant) cells. The level of EGFR phosphorylation is consistent with activation by a soluble factor produced in these cells. Ponceau S staining was used as a loading control. (B) S1 cells were starved of EGF for 12 hours and then stimulated for 10 minutes with either T4-2 conditioned medium (CM) or 5 ng/ml EGF. A 5 minute pretreatment with Iressa (0.3 nM) abolished MAPK activation induced by the conditioned medium and by EGF. (C) RT-PCR analysis shows that AREG and TGFA were transcriptionally upregulated in T4-2 relative to S1 cells. GAPDH was used as a loading control. (D) ELISA of conditioned medium shows that T4-2 cells secreted significantly more AREG and TGF-α than did S1 cells. (E) S1 cell proliferation in the presence of all EGFR ligands (860 pM) was significantly different from control (Ctrl).
Figure 2
Figure 2. Inhibition of sheddase activity reverts the malignant phenotype of T4-2 cells by suppressing mobilization of growth factors and downregulating EGFR pathway activity.
(A) T4-2 cells grown in 3D lrECM cultures formed continuously proliferating, disorganized, apolar colonies. (B) T4-2 cells treated with EGFR inhibitor (80 nM AG1478) underwent morphological reversion, forming small, smooth, spherical, growth-arrested colonies. (C) T4-2 cells treated with a broad-spectrum MMP/ADAM inhibitor (20 μM TAPI-2) underwent morphological reversion similar to that of EGFR inhibitor–treated cells. (D) Absence of tissue polarity as demonstrated by α6 integrin staining of vehicle-treated T4-2 cells. (E) Restoration of tissue polarity as demonstrated by α6 integrin staining of TAPI-2–treated T4-2 cells. Scale bars: 100 μm (A–C); 10 μm (D and E). (F) Analysis of cross-sectional area of T4-2 cells treated with either vehicle, AG1478, or TAPI-2 for 4 days. ***P < 0.001 versus control. (G) TAPI-2 treatment (24 hours) reduced the basal activity of kinases downstream of EGFR, but cells remained competent to respond to exogenous EGF (860 pM, 5-minute stimulation). (H) TAPI-2 treatment resulted in a dose-dependent reduction in T4-2 cell proliferation that was completely overcome by addition of soluble EGF. **P < 0.01; ***P < 0.001 compared with 0 μM TAPI-2. (I) ELISA of conditioned medium from TAPI-2–treated T4-2 cells, indicating that it suppressed the shedding of both AREG and TGF-α.
Figure 3
Figure 3. TACE cleaves AREG and TGF-α and is necessary for T4-2 cell proliferation.
(A) RT-PCR analysis showing TACE expression in S1 and T4-2 cells. GAPDH was used as a loading control. (B) ELISA analysis of EGFR ligand shedding in T4-2 cells transfected with 3 siRNA oligos, either individually or as a pool. Ligand shedding was proportional to the level of TACE expression. (C) Reversion of the malignant phenotype of T4-2 cells in 3D lrECM culture following transfection of siRNA against TACE. Left insets: Phase-contrast micrographs of transfected cells grown on plastic. Right insets: α6 integrin immunostaining of representative colonies. Original magnification, ×100; ×600 (right insets). (D) ELISA analysis of the conditioned medium from the experiment shown in C.
Figure 4
Figure 4. TAPI-2–induced reversion of T4-2 cells is a direct result of inhibition of growth factor ectodomain shedding.
(A) Schematic representation of full-length (pro-) and deletion mutants (ΔTM) of AREG and TGF-α. Deletion mutants lack both the transmembrane and the cytoplasmic domains and can thus be secreted without requiring TACE activity. (B) T4-2 cells overexpressing full-length or deletion growth factor constructs were susceptible to reversion by the EGFR inhibitors, but cells expressing either soluble AREG or soluble TGF-α escaped the TAPI-2–induced reversion. Scale bar: 100 μm. (C) Analysis of cross-sectional area of T4-2 cells and derivatives in response to pharmacological inhibition of EGFR and TACE. Horizontal bars represent median values. (D) Higher-magnification (×600) analysis of representative colonies from B. Colonies expressing the soluble mutants of AREG and TGF-α remain disorganized and apolar in the presence of TAPI-2.
Figure 5
Figure 5. Suppression of growth factor shedding by TAPI-2 in a panel of breast cancer cell lines.
(A) Three AREG-expressing breast cancer lines were treated with 20 μM TAPI-2 or vehicle for 90 minutes, and AREG shedding was quantified by ELISA. (B) Two breast cancer cell lines expressing TGF-α were identified and treated as in A, and TGF-α shedding was quantified by ELISA. TAPI-2 suppressed TGF-α shedding. (C) Each cell line was treated with TAPI-2 for either 1 or 5 hours. Downregulation of MAPK activity was detected in those cell lines expressing EGFR.
Figure 6
Figure 6. Kaplan-Meier survival analysis of 295 human breast tumors stratified by marker expression level.
High levels of (A) TACE and (B) TGFA predict poor survival. High levels of (C) AREG or (D) ERα are correlated with good outcome (AREG and ERα are related; see Discussion). P values represent the log-rank comparison between the upper and lower quartiles of marker expression evaluated at 5 and 10 years after surgery.
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