LH and FSH promote migration and invasion properties of a breast cancer cell line through regulatory actions on the actin cytoskeleton

Abstract

Reproductive hormones influence breast cancer development and progression. While the actions of sex steroids in this setting are established, tentative evidence suggests that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) may also play a role, yet this remains elusive. We here identify that T-47D breast cancer cells express functional receptors for FSH and LH, and that these hormones regulate breast cancer cell motility and invasion through the control of the actin cytoskeleton and the formation of cortical actin aggregates and focal adhesion complexes. Such actions are mediated by the cytoskeletal controllers Moesin and focal adhesion kinase (FAK). Moesin is recruited rapidly by FSH and LH through a signaling cascade requiring the G protein Gα₁₃ and the Rho-associated kinase, ROCK-2. FSH and LH activate FAK via a Gα_i/β and c-Src-dependent signaling cascade. Both cascades involve signaling to phosphatidylinositol-3 kinase and Akt. FSH and LH receptors and the related signaling intermediates are necessary for the actions of gonadotrophins on breast cancer cell cytoskeletal rearrangement, migration and invasion. These findings provide original information on the actions of gonadotrophins on breast cancer cells and may have clinical implications for the use of drugs that modulate gonadotrophins in breast cancer patients.

Introduction

Breast cancer (BC) is the most frequent malignancy in Western countries, affecting approximately one out of eight women, and one-fifth die for the disease (Janssens et al., 2007). Most breast cancers are hormone-dependent, express receptors for estrogen and are affected by therapies that decrease the levels of these hormones or interfere with their receptors, such as aromatase inhibitors or tamoxifen. Although the available evidence is scanty and contradictory, there are indications that gonadotrophins may play a regulatory role in the breast as much as in breast cancer (Huhtaniemi, 2010).

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are glycoprotein hormones synthesized and secreted by pituitary gonadotrophs in response to gonadotrophin-releasing hormone (GnRH) (Wildt et al., 1981, Knobil, 1981). GnRH agonists, that suppress the release of LH and FSH (and therefore block estradiol production in the ovaries), are effective in the treatment of breast cancer in fertile women (Robertson and Blamey, 2003). In parallel, a few studies suggest that women exposed to gonadotrophins for ovulation induction (Pappo et al., 2008) or to drugs that raise gonadotrophins, such as clomiphene citrate (Lerner-Geva et al., 2006, Orgeas et al., 2009), may incur in mildly elevated increases in breast cancer risk. It is difficult to discriminate from this clinical data any possible effects of gonadotrophins from those of estrogens, that are likely prevalent. However, the hypothetical concept that gonadotrophins might regulate breast cancer would be particularly relevant in postmenopausal women, when elevated FSH and LH along with the highest breast cancer incidence rate are found (Bray et al., 2004).

Actions of gonadotrophins outside the gonads have been identified in a variety of tissues (Rao and Lei, 2007), including the breast (Jiang et al., 2002a, Jiang et al., 2002b). Breast cancers (Meduri et al., 1997, Meduri et al., 2003) and breast cancer cell lines (Bodek et al., 2003) express the HCG/LH receptor, and gonadotrophins have been proposed to enhance breast cancer tumorigenesis, however this is supported by weak evidence and hence debated (Huhtaniemi, 2010). Recent reports suggest that gonadotrophin receptors may be important in tumor-associated endothelial cells in a wide variety of cancers, including breast cancer (Radu et al., 2010).

Local progression and distant metastasis are the main reason for morbidity and mortality in women affected by breast cancer (Janssens et al., 2007). While different therapeutic interventions against cancer cell proliferation are established, strategies to counteract cancer cell invasion or dissemination are not yet available. Recent evidence highlights that hormone-sensitive cancers, including breast and endometrial cancer, can be driven to invade the surrounding environment by sex steroids through the recruitment of signaling pathways that regulate cell movement and interaction with the extracellular environment (Fu et al., 2008a, Giretti et al., 2008, Flamini et al., 2009). Activation of dynamic remodeling of the actin cytoskeleton and of cell membrane adhesive properties is central for these actions and selected intermediates have been identified as targets of sex steroids. For instance, estrogen-induced threonine⁵⁵⁸ phosphorylation of the actin-binding protein Moesin enhances breast cancer cell movement and invasion by promoting the formation of molecular bridges between actin, integrins, and focal adhesion complexes at specialized cell membrane sites such as ruffles and pseudopodia (Giretti et al., 2008). Moreover, in the presence of estrogens, the focal adhesion complex regulator, focal adhesion kinase (FAK), is phosphorylated at tyrosine³⁹⁷ and localizes to the plasma membrane where it endows cells with higher motility through increased adhesion to the extracellular matrix (Fu et al., 2010, Sanchez et al., 2010).

In the present paper we investigated whether FSH and LH affect the motility or invasion of breast cancer cells. We here describe that FSH and LH act through their receptors in T-47D breast cancer cells by modulating Moesin and FAK, turning into cell membrane remodeling and enhanced horizontal motility and invasion of three-dimensional matrices, and we characterize the relevant signaling cascades.