ADAM12 induces estrogen-independence in breast cancer cells

Abstract

Antiestrogen therapy has been used successfully to prolong disease-free and overall survival of ER positive breast cancer patients. However, 50% of patients with ER+ tumors fail to respond to such therapy or eventually acquire resistance to endocrine therapy, resulting in tumor progression and mortality. It is imperative, therefore, to understand the mechanisms that lead to hormone refractory breast cancer in order to develop therapeutics that can modulate the resistance to antiestrogen therapy. The protease, ADAM12, can be detected in the urine of breast cancer patients and its levels correlate with disease status, stage, and cancer risk. Within the context of this study, the authors have investigated the role of the two distinct isoforms of ADAM12 in breast tumor cell proliferation and as potential mediators of endocrine resistance. Using stable clones of ADAM12-overexpressing MCF-7 cells, the authors analyzed proliferation rates of these ER+ breast tumor cells both in estrogen-depleted medium and in the presence of the antiestrogens, tamoxifen, and ICI 182,780. Acquired estrogen resistance in these cells was analyzed using phospho-RTK analysis. Upregulation and phosphorylation of proteins were detected via immunoprecipitation and immunoblotting. EGFR and MAPK inhibitors were used to explore the mechanism of acquired estrogen resistance in breast tumor cells. It was observed that overexpression of the two isoforms, transmembrane ADAM12-L, and secreted ADAM12-S, in breast tumor cells promoted estrogen-independent proliferation. In ADAM12-L-expressing cells, estrogen-independence was a direct result of increased EGFR expression and MAPK activation, whereas, the mechanism in ADAM12-S-expressing cells may be enhanced IGF-1R signaling. The importance of the EGFR signaling pathway in the estrogen-independent growth of ADAM12-L expressing cells was highlighted by the effect of EGFR inhibitors AG1478 and PD15035 or MAPK inhibitor U0126, each of which abolished the antiestrogen resistance in these cells. Taken together, these results demonstrate that ADAM12 isoforms confer a proliferative advantage to MCF-7 cells in the absence of estrogen stimulation, and suggest that downregulation of ADAM12 in combination with endocrine therapy may represent a useful pharmacological approach to breast cancer therapy.

Fig. 1. ADAM12 expression promotes estrogen-independent growth

Proliferation rates of WT MCF-7 (black) and representative ADAM12 clones (ADAM12-S; red, ADAM12-L; blue) grown in the presence of estrogen are comparable (a, left panel). In the absence of exogenous estrogen, ADAM12 clones proliferated significantly faster (a, right panel). ADAM12-expressing cells retain ERα mRNA (left panel) and protein expression (right panel) (b). MDA-MB-231 lysate was used as a negative control for ERα protein expression. Individual ADAM12-L and ADAM12-S clones are indicated as C1, C2, C3 respectively.

Fig. 2. Effect of antiestrogen treatment on ADAM12-expressing cells

WT MCF-7 and ADAM12 clones were treated with tamoxifen (3μM) or ICI 182,780 (100nM). In the presence of tamoxifen (a, left panel) and ICI 182,780 (a, right panel) ADAM12-L (blue circle; A12-LC1) and ADAM12-S-expressing (red square; A12-SC4) cells proliferated significantly faster than WT MCF-7 (black diamond) cells. Tamoxifen and ICI 182,780 treatment resulted in significantly greater growth inhibition (versus vehicle control) in WT MCF-7 cells as compared to ADAM12-S-expressing (ADAM12-S clones 1 and 4) and ADAM12-L-expressing (ADAM12-L clones 1 and 4) respectively. These results are expressed as the mean (SD) of three independent experiments. ERα protein expression remained unchanged in vehicle control and tamoxifen treated cells, however, there was complete loss of ERα expression in ICI 182,780 treated cells (c).

Fig. 3. ADAM12-L expression confers estrogen independent growth capability to breast tumor cells via upregulation of EGFR expression

Receptor tyrosine kinase (RTK) profile of WT MCF-7 and ADAM12 clones (a, upper panel). Densitometric analysis indicated increased levels of pEGFR and pIGF-1R in ADAM12-L (~2-fold) and ADAM12-S clones (~1.3-fold) (a, lower panel). Individual ADAM12-L and ADAM12-S clones are indicated as C1, C2, C3 respectively. Effect of IGF-1 or EGF treatment of ADAM12-expressing clones. Increased pIGF-1R levels were detected in both ADAM12-S and ADAM12-L expressing cells as compared to WT MCF-7 cells (b), whereas increased pEGFR levels were only detected in the ADAM12-L-expressing cells (c). pMAPK levels were elevated in IGF-1 treated ADAM12-S clones as compared to WT MCF-7 (b). Comparative expression of EGF receptor family members in ADAM12-L clones (d). ADAM12-L-expressing clones had increased protein expression of EGFR and HER3 (ErbB3), whereas HER2 (ErbB2) and HER4 (ErbB4) levels remained unchanged (d). EGFR transcript levels were also upregulated in ADAM12-L clones as compared to WT MCF-7 (e). pEGFR levels were higher in ADAM12-L-expressing clones as compared to WT MCF-7, GAPDH is used as a loading control (f).

Fig. 4. ADAM12 expression stimulates amphiregulin shedding in breast tumor cells

ADAM12 expression stimulated shedding of amphiregulin (AR) but not for HB-EGF, EGF, TGFα or epiregulin. Immunoblot of conditioned medium (a) or amphiregulin elisa (b,c) indicated increased shedding of AR in response to ADAM12 expression in MCF-7 and T47-D cells respectively. The results are representative of three independent experiments.

Fig. 5. EGFR and MAPK inhibition results in loss of estrogen independent growth in ADAM12-expressing MCF-7 cells

MCF-7 cells displayed ≥ 75% growth inhibition (vs vehicle control) when treated with ICI 182,780, whereas ADAM12-L and ADAM12-S-expressing cells were inhibited significantly less (~43%; p≤ 0.01 and ~31%; p ≤ 0.001) compared to WT MCF-7 cells. Treatment of ADAM12-L-expressing cells with the EGFR inhibitor AG1478 (10μM) or the selective EGFR inhibitor, PD15035 (1μM) or the MAPK inhibitor U0126 (10μM) in combination with the ER inhibitor ICI 182,780, resulted in a complete loss of resistance to the estrogen inhibitor ICI 182,780. Similarly, AG1478 and U0126 treatment of ADAM12-S-expressing clones led to 61-82% growth inhibition (a). These results are expressed as the mean (SD) of three independent experiments conducted with WT MCF-7 and two individual clones of ADAM12-L- and ADAM12-S-expressing cells respectively. Treatment with the MAPK inhibitor U0126, resulted in significant downregulation of pMAPK levels while total MAPK remained unchanged (b). Similarly, treatment of ADAM12-L-expressing clone LC1 or WT MCF-7 with EGFR inhibitors PD15035 or AG1478 also resulted in downregulation of pMAPK levels (c).

Fig. 6. ADAM12 expression is upregulated in tamoxifen-resistant breast tumor cells

ADAM12 expression remains unchanged for tamoxifen-sensitive (MCF-N2) and tamoxifen-resistant (MCF-T17, MCF-T52) cells under basal conditions (vehicle control; white bars). When tamoxifen (1μM) was added to the medium ADAM12-S expression was significantly higher in tamoxifen-resistant clones MCF-T17 (15-fold; *p≤0.05) and MCF-T52 (7.5-fold; *p≤0.05) (a). Similarly, ADAM12-L expression was also upregulated in tamoxifen-resistant clones MCF-T17 (2-fold increase) and MCF-T52 (9-fold; p≤0.05) (b).