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

doi:10.1016/j.taap.2014.01.005

Toxicology and Applied Pharmacology

Volume 275, Issue 2, 1 March 2014, Pages 176-181

https://doi.org/10.1016/j.taap.2014.01.005 Get rights and content

Highlights

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Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells.
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GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2.
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Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

Abstract

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer.

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).

References (37)

F. Acconcia et al.
Signaling regulation of genomic and nongenomic functions of estrogen receptors
Cancer Lett.
(2006)
L. Albanito et al.
G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells
Cancer Res.
(2007)
E.A. Ariazi et al.
The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells
Cancer Res.
(2010)
A. Baki et al.
Autolysis parallels activation of mu-calpain
Biochem. J.
(1996)
I. Bieche et al.
Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer
Oncogene
(2001)
D. Bourboulia et al.
Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion
Semin. Cancer Biol.
(2010)
C. Braun et al.
Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type
Int. J. Cancer
(1999)
S.A. Brooks et al.
Molecular interactions in cancer cell metastasis
Acta Histochem.
(2010)
G.Q. Du et al.
The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells
Biochem. Biophys. Res. Commun.
(2012)
D.G. Fan et al.
Silencing of calpain expression reduces the metastatic potential of human osteosarcoma cells
Cell Biol. Int.
(2009)

E. Ferreira et al.

Selection of suitable reference genes for quantitative real-time PCR in apoptosis-induced MCF-7 breast cancer cells

Mol. Biotechnol.

(2012)

E.J. Filardo et al.

Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF

Mol. Endocrinol.

(2000)

E.J. Filardo et al.

Distribution of GPR30, a seven membrane-spanning estrogen receptor, in primary breast cancer and its association with clinicopathologic determinants of tumor progression

Clin. Cancer Res.

(2006)

A. Glading et al.

Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation

Mol. Cell. Biol.

(2004)

N. Goto et al.

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-beta signaling pathway

Cancer Sci.

(2011)

J. Hou et al.

17beta-estradiol induces both up-regulation and processing of cyclin E in a calpain-dependent manner in MCF-7 breast cancer cells

FEBS Lett.

(2012)

S. Johnston

Fulvestrant and the sequential endocrine cascade for advanced breast cancer

Br. J. Cancer

(2004)

K. Keyomarsi et al.

Cyclin E and survival in patients with breast cancer

N. Engl. J. Med.

(2002)

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¹: These authors contributed equally to this work.

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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

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents and antibodies

E2 and ICI promote cell–matrigel adhesion with increased calpain activity

Discussion

Conflict of interest statement

Acknowledgments

Signaling regulation of genomic and nongenomic functions of estrogen receptors

Cancer Lett.

G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells

Cancer Res.

The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells

Cancer Res.

Autolysis parallels activation of mu-calpain

Biochem. J.

Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer

Oncogene

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion

Semin. Cancer Biol.

Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type

Int. J. Cancer

Molecular interactions in cancer cell metastasis

Acta Histochem.

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

Biochem. Biophys. Res. Commun.

Silencing of calpain expression reduces the metastatic potential of human osteosarcoma cells

Cell Biol. Int.