Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis
Graphical abstract
Introduction
Breast cancer is the most common malignant tumors, ranking second in mortality among American women (Smith et al., 2003). Despite advances in the diagnosis and treatment of breast cancer, metastasis remains responsible for approximately 90% of patient deaths. Metastasis comprises multiple biological processes involving adhesive interactions and invasion (Brooks et al., 2010). Adhesion of tumor cells to matrix components is a hallmark of solid tumor cells and an essential step for tumor metastasis (Schmidmaier and Baumann, 2008). Deregulated cell adhesion is frequently observed in a number of pathologic conditions including cancer progression, while the regulation of cell–cell and cell–extracellular matrix (ECM) adhesion is strictly controlled in normal cells (Bourboulia and Stetler-Stevenson, 2010).
Estrogen has long been known to enhance progression of hormone-sensitive breast cancer. Based on this knowledge, hormonal therapies that interfere with estrogen receptor (ER) functions are currently applied in patients with ER-positive breast cancer and beneficial effects have been achieved, yet initial or acquired resistance to endocrine therapies frequently arises and, particularly cancer recurs as metastasis (Saha Roy and Vadlamudi, 2012), indicating a more complex receptor mechanism involved in cancer progression. Several reports showed that G protein coupled estrogen receptor (GPR30) mediates a wide range of estrogenic responses, including rapid non-genomic actions such as activation of Src tyrosine kinase, mitogen-activated kinases and phosphoinositide 3-kinases, as well as genomic effects (Acconcia and Kumar, 2006, Maggiolini and Picard, 2010). Importantly, several studies revealed that GPR30 is over-expressed in invasive breast cancer and associated with metastatic phenotype (Filardo et al., 2006, Liu et al., 2009). Despite its clinical importance, little is known regarding how GPR30 is implicated in breast cancer metastasis.
Calpain, a calcium-dependent cysteine protease ubiquitously expressed as mu-calpain (calpain 1) and m-calpain (calpain 2), is involved in a variety of biological activities, and has been shown to be associated with cancer (Storr et al., 2011), including breast cancer (Libertini et al., 2005, Smith et al., 2003, Storr et al., 2012), renal cell carcinoma (Braun et al., 1999), and osteosarcoma (Fan et al., 2009). Calpain has also been found to be implicated in the process of cell adhesion. For instance, calpain induces cleavage of the talin head from the rod domain, which may facilitate talin banding to integrin and make integrin activation so as to anchor cells to ECM (Moser et al., 2009). We previously reported that 17-beta-estradiol (E2) induces activation of calpain, leading to the generation of processed cyclin E in breast cancer cells (Hou et al., 2012), yet the receptor mechanism(s) that underlies calpain activation remains unclear.
Pure antiestrogen fulvetrant (ICI 182 780, ICI) has been found to stimulate, through activation of GPR30, proliferation and migration in breast cancer cells (Pandey et al., 2009) and enhance proliferation in both endometrial and ovarian cancer cells (Albanito et al., 2007, Vivacqua et al., 2006), which indicates an estrogen-like action of ICI. In the present study, by using MCF-7 breast cancer cells as a model system, we addressed whether E2 and ICI act on cell–matrigel adhesion that may facilitate metastasis and whether this action occurs via a novel GPR30 to calpain pathway.
Section snippets
Reagents and antibodies
17β-Estradiol (E2), fulvestrant (ICI 180 780) and G1 were obtained from Sigma–Aldrich (St. Louis, MO), and dissolved in dimethyl sulfoxide (DMSO) as stock solutions at 10− 5 M, 0.02 M, and 0.1 M, respectively, and stored at 4 °C. G15 (Sigma–Aldrich), MEK1/2 inhibitor U0126, calpeptin (N-benzyloxycarbonyl- L-leucylnorleucinal) and calpain inhibitor I (ALLN) (Calbiochem, San Diego, CA) were dissolved in DMSO at 0.1 M, 0.01 M, 0.1 M and 0.1 M, respectively, and stored at − 20 °C as stock solutions. Matrigel
E2 and ICI promote cell–matrigel adhesion with increased calpain activity
Initially, to assess the impacts of E2 and ICI on cell–matrigel adhesion, MCF-7 breast cancer cells (ERα +/GPR30 +) were treated with E2 or ICI for 24 h. We observed that both ICI and E2 tremendously promote cell adhesion to matrigel (Fig. 1Aa), a reconstituted basement membrane. As calpain can be activated by E2 (Hou et al., 2012), we further asked whether calpain is activated coincident with enhanced cell adhesion by both E2 and ICI. To do this, calpain 1 (large subunit) autolysis or proteolysis
Discussion
It is well documented that estrogens exert actions on breast normal and cancer tissues and cells via classical ERs (ER-α/ER-β), regulating a variety of physiological and pathological processes. There is a notion that ERα expression correlates with a lower metastasis risk of breast cancer, and this could be explained by the estrogen modulation of cell behaviors including cell–cell adhesion. For instance, a recent study showed that estrogens enhance cell–cell adhesion by inducing the formation of
Conflict of interest statement
All the authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grants 31360252, 30860093 and 81302804), and the Startup Foundation for Doctors of Guiyang Medical University ((2013)09).
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These authors contributed equally to this work.