An essential role of metalloprotease-disintegrin ADAM12 in triple-negative breast cancer

Abstract

In the absence of HER2 overexpression, triple-negative breast cancers (TNBCs) rely on signaling by epidermal growth factor receptor (EGFR/ErbB1/HER1) to convey growth signals and stimulate cell proliferation. Soluble EGF-like ligands are derived from their transmembrane precursors by ADAM proteases, but the identity of the ADAM that is primarily responsible for ligand release and activation of EGFR in TNBCs is not clear. Using publicly available gene expression data for patients with lymph node-negative breast tumors who did not receive systemic treatment, we show that ADAM12L is the only ADAM with an expression level significantly associated with decreased distant metastasis-free survival times. Similar effect was not observed for patients with ER-negative non-TNBCs. There was a positive correlation between ADAM12L and HB-EGF and EGFR in TNBCs, but not in ER-negative non-TNBCs. We further demonstrate that ectopic expression of ADAM12L increased EGFR phosphorylation in a mouse intraductal xenograft model of early breast cancer. Finally, we detect strong correlation between the level of anti-ADAM12L and anti-phospho-EGFR immunostaining in human breast tumors using tissue microarrays. These studies suggest that ADAM12L is the primary protease responsible for the activation of EGFR in early stage, lymph node-negative TNBCs. Thus, our results may provide novel insight into the biology of TNBC.

Fig. 1

Flowchart of analysis according to REMARK criteria [59]

Fig. 2

High expression of ADAM12L is associated with poor prognosis in TNBC patients from the EMC286 cohort. Kaplan-Meier analysis of distant metastasis-free survival fractions for all patients (top), patients with ER-negative or ER-positive tumors (middle), and patients with TNBC or ER-negative non-TNBC (bottom). Patients were rank-ordered according to the expression of ADAM12L (a) or ADAM12S (b) and divided into two groups with mRNA expression levels below or above the median values.

Fig. 3

The prognostic value of ADAM12L in TNBCs from the EMC286 dataset is the highest among other ADAMs encoding membrane-anchored, catalytically-active ADAM proteases. Kaplan-Meier plots of distant metastasis-free survival fractions for TNBC patients from the EMC286 cohort are shown.

Fig. 4

High expression of ADAM12L is associated with poor prognosis in a combined set of node-negative TNBCs without chemotherapy. Gene expression profiles obtained in four independent studies (GEO accession numbers GSE2034, GSE5327, GSE7390, and GSE11121) were merged, as described in Methods. a Log2-transformed, median-centered ADAM12L expression values in TNBC patients without vs with distant metastasis. Mean expression values ± SEM are indicated. b Kaplan-Meier plots of distant metastasis-free survival fractions are shown.

Fig. 5

Increased expression of ADAM12L augments EGFR phosphorylation in a mouse-intraductal (MIND) xenograft model of basal breast cancer. a Detection of a functional EGFR in MCF10DCIS.com cells. Cells were serum-starved for 16 h and then incubated for 30 min with indicated concentrations of EGF. The extent of EGFR phosphorylation (pY1173) was examined by Western blotting. b Stable expression of ADAM12L in MCF10DCIS.com cells. Cells stably transduced with ADAM12L or with empty vector were analyzed by Western blotting using an antibody against the cytoplasmic tail of ADAM12L (left). The nascent form represents the full-length, catalytically inactive, intracellular form of ADAM12L. The mature form represents ADAM12L lacking its N-terminal pro-domain, which is catalytically active and resides predominantly at the cell surface. Cell surface localization of the mature form of ADAM12L was confirmed by flow cytometry after staining of live cells with an antibody recognizing the extracellular domain of ADAM12L. c Immunohistochemical detection of phosphorylated EGFR in mouse intraductal xenografts. MCF10DCIS.com cells stably transduced with ADAM12L or with empty vector were injected into mammary ducts of NSG mice. Six weeks after injection, paraffin-embedded mammary tissue sections were stained with anti-phospho-EGFR (Y1173) antibody, visualized with DAB (brown), and counterstained with hematoxylin (blue). Four representative images are shown in the top and middle rows, respectively. The specificity of staining was confirmed by incubation of duplicate sections with or without primary antibody (bottom row). d Comparison of anti-phospho-EGFR staining intensities in intraductal xenografts of MCF10DCIS.com cells stably transduced with empty vector or ADAM12L. For each cell type, 38 tissue sections from three different mice were analyzed. Quantification was performed as described in Online Resource Methods. All staining intensity values (in arbitrary units) were normalized to the mean value obtained for cells transduced with empty vector.

Fig. 6

Positive correlation between the level of ADAM12L expression and the extent of EGFR phosphorylation in human breast cancer tissue microarrays. a Validation of the specificities of the antibodies used for IHC staining of tissue microarrays. Two cores of the same tumor were stained with anti-ADAM12L antibody or with pre-immune serum (top). Similarly, two cores of the same tumor were stained with anti-pEGFR (Y1173) or with the secondary antibody only (bottom). Positive staining was observed mostly in epithelial cells, with much weaker staining in the stromal compartment. b Two examples of duplicate sections in which epithelial cells stained strongly (top) or weakly (bottom) for both ADAM12L and pEGFR. c Correlation analysis of ADAM12L staining versus pEGFR staining intensities in 142 cores, including 33 cores of TNBC. Staining intensities (in arbitrary units) were quantified as described in Online Resource Methods.