**1. Introduction**

450 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

Woods, A & Couchman, J R (1994). Syndecan 4 heparan sulfate proteoglycan is a selectively

Woods, A, Longley, R L, Tumova, S & Couchman, J R (2000). Syndecan-4 binding to the high

Yang, J, Price, M A, Li, G Y, Bar-Eli, M, Salgia, R, Jagedeeswaran, R, Carlson, J H, Ferrone, S,

fibroblasts. *Arch Biochem Biophys,* Vol. 374, No. 1, pp, 66-72.

transition. *Cancer Res,* Vol. 69, No. 19, pp, 7538-7547.

*S A,* Vol. 96, No. 6, pp, 3071-3076.

183-192.

protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1. *Proc Natl Acad Sci U* 

enriched and widespread focal adhesion component. *Mol Biol Cell,* Vol. 5, No. 2, pp,

affinity heparin-binding domain of fibronectin drives focal adhesion formation in

Turley, E A & McCarthy, J B (2009). Melanoma proteoglycan modifies gene expression to stimulate tumor cell motility, growth, and epithelial-to-mesenchymal

> Estrogen plays a major part in the regulation of cell proliferation and survival, controlling female physiology, reproduction and behaviour (Musgrove and Sutherland, 2009). It however assumes a more malevolent role in its association with breast cancer pathogenesis. Consequently, therapies have been designed to block the actions of estrogen mediated through its receptors (ERα and ERβ), or to simply reduce its levels in the body (Zilli et al., 2009). Since Beatson (1896) first introduced ovariectomy over a century ago as the first therapeutic modality to reduce the adverse effects of estrogen, endocrine therapy has developed into the cornerstone of breast cancer treatment for those 60-70% of patients whose tumours over-express ER and/or progesterone receptor (PR) (Massarweh and Schiff, 2007; Zilli et al., 2009). For three decades, selective estrogen receptor modulators (SERMS), predominantly tamoxifen, have proved to be effective agents for the suppression of breast cancer growth in both early and advanced disease (Normanno et al., 2005). Tamoxifen has significantly improved the quality of life and survival of many patients with metastatic disease, as well as displaying prophylactic benefit, particularly in women with ductal carcinoma-*in situ* (Fisher et al., 1999).

> However, about half of ER+ patients with advanced disease and nearly all patients with metastatic disease fail to respond to first-line tamoxifen therapy. About 40% of patients receiving tamoxifen as adjuvant therapy experience tumour relapse and die from their disease, and a third of women treated with tamoxifen for 5 years develop recurrent disease within 15 years (Normanno et al., 2005). The introduction of pure estrogen antagonists such as fulvestrant, to overcome the apparent disadvantage of tamoxifen with its partial agonist properties, did not resolve the resistance problem (Osborne and Schiff, 2011). Second line therapy with other endocrine agents designed to inhibit peripheral extra-gonadal synthesis of estrogen in postmenopausal women produces some beneficial effects but for the most part serves merely to delay onset of endocrine resistance (Massarweh and Schiff, 2007). This refractiveness to continued administration of anti-estrogens and aromatase inhibitors poses a significant therapeutic problem that has been addressed by a large number of studies. Several theories have been proposed to explain this phenomenon, based on observations made with a variety of *in vitro* cellular models (Normanno et al., 2005). The consensus opinion seems to be that whereas *de novo* resistance is most likely due to low levels of ER expression, *acquired* resistance is predominantly the consequence of an attenuated response to other peptide growth factors that normally play a subsidiary role in cell proliferation.

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 453

a manageable number, and given the designation of 'gene signature' by virtue of selectively

In a separate scenario, new insights have been gained into our understanding of cell differentiation from studies that have demonstrated that epithelial cells have the potential to trans-differentiate into mesenchymal cells (epithelial to mesenchymal transition: EMT) and vice versa (mesenchymal to epithelial transition: MET). Many recent reports have indicated that this process, which was previously observed during transition between developmental stages, is synonymous with the process of tumour metastasis. Both processes share similar pathways of activation. Our recent data (Luqmani et al., 2009; Al Saleh, 2010; Al Saleh et al., 2011a) suggests that there may also be causal links between the development of endocrine resistance and the onset of EMT. In this report we summarise the molecular pathways of ER activity, the mechanisms proposed to account for resistance and finally review the evidence

ERα and ERβ are transcribed from distinct genes located on separate chromosomes (6 and 14, respectively) (Green et al., 1986; Kuiper et al., 1996). These receptors differ in their tissue distribution, with ERα being highly expressed in the pituitary gland, ovaries (thecal and interstitial cells), uterus, liver, kidneys, adrenals and the mammary glands while ERβ is found mainly in the prostate, bone, ovaries (granulosa cells), lungs and in various parts of the central and peripheral nervous system (Emmen et al., 2005; Kuiper et al., 1997). Nevertheless, ERα and ERβ do overlap in their expression in some tissues (Zilli et al., 2009). More importantly, the two receptors have different roles in breast development. Only ERα appears to be essential for ductal growth although both receptors are present in the breast. ERα-knockout mice show very little growth of mammary ducts, while ERβ-knockout mice develop a normal mammary gland with regular ductal branching (Förster et al., 2002; Lubahn et al., 1993). This suggests that ERβ might be exerting pro-differentiative and antiproliferative functions. In addition, increased ERα/ERβ ratio in breast cancer as compared with benign tumours and normal tissues suggest that ERα is most closely associated with breast cancer pathogenesis, while ERβ can protect against the mitogenic activity of estrogens in pre-malignant lesions (Roger et al., 2001; Shaw et al., 2002). It has even been suggested that the estrogen-induced proliferation of ER+ breast cancer cells can be inhibited by ERβ over-expression (Ström et al., 2004; Williams et al., 2008). Thus ERα remains the main focus of attention in studies on breast cancer. Unless otherwise specified, 'ER' in this review will

In what is now referred to as the nuclear or genomic action of ER, binding of estrogen induces activation of the receptor by initiating its dissociation from cognate heat shock proteins, and leads to conformational changes, dimerisation and autophosphorylation (Osborne & Schiff, 2005). The activated ER binds to estrogen response elements (EREs) located in the promoter regions upstream of estrogen-regulated genes. Frasor et al., (2003) observed from microarray analysis of gene expression in MCF-7 cells that about 70% of such estrogen-regulated genes were actually down-regulated following treatment with estradiol. Many of these genes are transcriptional repressors, or genes with anti-proliferative or proapoptotic function. On the other hand, there is increased expression of genes inducing cell proliferation and survival. Up-regulation of gene expression is mediated through two domains; activating function-1 (AF-1) and activating function-2 (AF-2). AF-1 is a hormone

**2. Mechanisms of estrogen receptor induced cell proliferation** 

circumscribing a particular sub-group of patients.

for the above hypothesis.

refer to ERα.

These molecules exert their action through a variety of trans-membrane receptors that possess intrinsic tyrosine kinase activity. Fig 1 depicts the various potential influences that govern the behaviour of breast cancer cells.

Fig. 1. Factors affecting growth and proliferation of breast cancer cells. Breast epithelial cells are subject to various influences that can either promote or inhibit cellular activity. (A) Endocrine stimulation by a variety of hormones, most significantly estrogen, promotes long term effects. (B) Autocrine stimulation involves, under various conditions, the production and secretion of a number of peptides that act back on the producer cell to modify its activity through membrane bound receptors that frequently possess intrinsic tyrosine kinase activity which initiates a signalling cascade that terminates in the action of transcriptional regulators to modify gene expression. (C) Paracrine stimulation is effected by the action of mediators which include the listed peptide growth factors as well as others originating from myoepithelia (in the normal breast) and stromal elements that include fibroblasts and macrophages in tumours. All of these pathways have been found to operate both *in vitro* (in tumour-derived cell lines) and *in vivo*, but their relative contributions vary considerably in both cases and may be influenced not only by biological heterogeneity but also by therapeutic interventions.

It is also a general experience that endocrine resistance is associated with increased aggressiveness and frequent metastasis (Hiscox et al., 2007), characteristics that more often typify ER-ve tumours. Identification of ligands, receptors and downstream signaling molecules with increased activity in the resistant phenotype, both in cell culture and in tumour biopsies, has highlighted a bewildering collection of molecules that may play a direct causative role, be a consequence or simply innocent bystanders in the progressive cellular change towards endocrine independence. For the purposes of therapeutic discrimination, attempts have been made to reduce this plethora, generated principally by microarray analyses (eg Charafe-Jauffret et al., 2006 ; Luqmani et al., 2009; Al Saleh, 2010) to

These molecules exert their action through a variety of trans-membrane receptors that possess intrinsic tyrosine kinase activity. Fig 1 depicts the various potential influences that

Fig. 1. Factors affecting growth and proliferation of breast cancer cells. Breast epithelial cells are subject to various influences that can either promote or inhibit cellular activity. (A) Endocrine stimulation by a variety of hormones, most significantly estrogen, promotes long term effects. (B) Autocrine stimulation involves, under various conditions, the production and secretion of a number of peptides that act back on the producer cell to modify its activity through membrane bound receptors that frequently possess intrinsic tyrosine kinase activity which initiates a signalling cascade that terminates in the action of transcriptional regulators to modify gene expression. (C) Paracrine stimulation is effected by the action of mediators which include the listed peptide growth factors as well as others originating from myoepithelia (in the normal breast) and stromal elements that include fibroblasts and macrophages in tumours. All of these pathways have been found to operate both *in vitro* (in tumour-derived cell lines) and *in vivo*, but their relative contributions vary considerably in

both cases and may be influenced not only by biological heterogeneity but also by

It is also a general experience that endocrine resistance is associated with increased aggressiveness and frequent metastasis (Hiscox et al., 2007), characteristics that more often typify ER-ve tumours. Identification of ligands, receptors and downstream signaling molecules with increased activity in the resistant phenotype, both in cell culture and in tumour biopsies, has highlighted a bewildering collection of molecules that may play a direct causative role, be a consequence or simply innocent bystanders in the progressive cellular change towards endocrine independence. For the purposes of therapeutic discrimination, attempts have been made to reduce this plethora, generated principally by microarray analyses (eg Charafe-Jauffret et al., 2006 ; Luqmani et al., 2009; Al Saleh, 2010) to

govern the behaviour of breast cancer cells.

therapeutic interventions.

a manageable number, and given the designation of 'gene signature' by virtue of selectively circumscribing a particular sub-group of patients.

In a separate scenario, new insights have been gained into our understanding of cell differentiation from studies that have demonstrated that epithelial cells have the potential to trans-differentiate into mesenchymal cells (epithelial to mesenchymal transition: EMT) and vice versa (mesenchymal to epithelial transition: MET). Many recent reports have indicated that this process, which was previously observed during transition between developmental stages, is synonymous with the process of tumour metastasis. Both processes share similar pathways of activation. Our recent data (Luqmani et al., 2009; Al Saleh, 2010; Al Saleh et al., 2011a) suggests that there may also be causal links between the development of endocrine resistance and the onset of EMT. In this report we summarise the molecular pathways of ER activity, the mechanisms proposed to account for resistance and finally review the evidence for the above hypothesis.
