Proliferation in Response to Estrogen

It is well established that the development of the breast is influenced by estrogens (Russo et al, 1999) and that E2 can stimulate the proliferation of breast cancer cell lines and may influence breast cancer growth in vivo (Lippman et al, 1976; Russo et al, 2002). To study this further, non-tumourigenic, and tumourigenic cell line proliferation studies were performed in the presence of E2, ICI 182,780 and a combination of the two. Additionally the role of GPER-1 in mediating cellular proliferation was studied using an agonist and antagonist for this receptor and co-incubation of the GPER-1 antagonist, G-15 with E2.

It was observed that in MCF-10A, MCF-12A and MCF-7 cells, the lack of EGF, resulted in a lack of proliferation. A dependence on EGF has been noted previously in the MCF-10A and MCF-12A cell line (Soule et al, 1990; Paine et al, 1992; Wang et al, 1997), but not in MCF-7 cells. This discrepancy may be due to the very low concentration of serum used and the phenol red-free media, rather than a necessity for EGF itself.

E2 does not appear to greatly influence proliferation of the cell lines studied, although a small increase in cell number was observed in MCF-7 cells in response to E2 treatment. This lack of proliferation in response to the steroid hormone would be expected in the ERα negative MCF-10A and MDA-MB-231 cell lines and has been observed previously (Spink et al, 2006), but it is also common for ER positive non-transformed mammary epithelial cells, such as MCF-12A to be non-proliferative in

104 response to stimulus (Clarke et al, 1997; Russo et al, 1999). Additionally, it is important to remember that MCF-7 cells, although estrogen-responsive, do not always show significant proliferation in response to estrogens, and proliferation in response to these compounds, varies significantly between cell batches (Payne et al, 2000).

The lack of proliferative effect of E2 in MCF-12A cells suggests that E2 does not induce proliferation in non-transformed cell lines, regardless of their ER status, and supports previous work demonstrating that non-transformed mammary epithelial cells do not increase DNA synthesis in response to E2 treatment (Haslam, 1986). Alternatively, it has been hypothesised that in cell cultures, and even in in vivo models, two populations of epithelial cells exist; one which is ER positive and the other which is ER negative. It is the ER negative cells that proliferate in response to paracrine signals from ER positive cells and cells in the surrounding stroma, after stimulation by E2 (Nandi et al, 1995; Clarke et al, 1997; Russo et al, 1999; Mallepell et al, 2006; Gupta et al, 2007). This theory adds an additional level of complexity to the model of hormonal carcinogenesis, whereby the events leading to breast cancer are governed not just by the presence of E2, but also the interactions of the various cell types in the mammary gland and surrounding tissue.

Another possibility is linked to the expression of ERβ. It has been demonstrated that increased expression of this receptor correlates with decreased cell proliferation (Sotoca et al, 2008). Furthermore, in breast tumours, the ratio of ERα to ERβ is higher than though of normal breast tissue (Lazennec et al, 2001). These observations have led to the hypothesis that ERβ suppresses the proliferative effects of ERα, and would mean that reduced ERβ expression correlates with increased proliferation (Covaleda et al, 2008). Indeed, Covaleda et al (2008) demonstrated this effect in breast cancer cell lines. However, the exact mechanism for how ERβ mediates the actions of ERα, to our knowledge, remains elusive. The small stimulatory effect of ICI 182, 780, either alone or in combination with E2, on MCF-12A proliferation could be explained by the confirmed presence of GPER-1 in MCF-12A cells, as both E2 and ICI 182,780 can act as agonists for this receptor (Kamanga-Sollo et al, 2008; Kuhn et al, 2008). Indeed, when MCF-12A cells were treated with the GPER-1 agonist, G-1, increased proliferation was observed. This means that, in MCF-12A cells at least, GPER-1 activation can induce proliferative effects. In addition to this, when the GPER-1 antagonist, G-15 was added to E2 and ICI 182,780 co-incubated MCF-12A cells, the proliferation curve resembled that of solvent controls.

Our observations agree with previously published work showing that E2 has a mitogenic effect on the MCF-7 cell line (Lewis et al, 2005; Spink et al, 2006; Wang et al, 2008b). In fact, out of all four cell

105 lines analysed, MCF-7 cells were the only cells to show increased proliferation in response to E2 treatment. The impact of estrogens on MCF-7 proliferation has been widely studied. Several factors are believed to contribute to the mitogenic effect of E2 in this cell line. Firstly, E2 is able to induce the transcription of the proto-oncogenic transcription factor, c-fos via genomic and non-genomic mechanisms (Prall et al, 1997; Sabbah et al, 1999; Duan et al, 2002). C-fos proteins form dimers with members of the Jun transcription factor family to form the AP-1 transcription factor that consequently mediates the transcription of various genes involved in differentiation and proliferation. Consequently, the expression of cyclin D1, a protein involved in cell cycle progression, is up-regulated in response to E2 in the MCF-7 cell line and this correlates with increased proliferation (Prall et al, 1997; Planas-Silva et al, 2001). Estrogens also down-regulate the expression of the cyclin dependent kinase (CDK) inhibitor p27kip1 thus preventing the interactions of cyclins and CDKs which is necessary for cell cycle progression (Foster et al, 2003). Finally, in terms of the cell cycle, E2 treatment induces the hyperphosphorylation of the Rb protein. In its hyperphosphorylated state Rb becomes liberated from elongation actor-2 (EF2), allowing the transcription of genes required for passage through the G1/S phase checkpoint and consequently, increased proliferation (Foster & Wimalasena, 1996; Hurd et al, 1997; Dinda et al, 2002). These E2-induced alterations in gene expression or phosphorylation status present plausible mechanisms by which estrogens induce their proliferative effects. As stated above, MCF-7 cells were the only cells to have a proliferative response to E2. Co-incubation of E2 with ICI 182,780 or G-15 in this cell line, seemed to significantly reduce this effect. It, therefore, would appear that both the ER and GPER-1 are capable of mediating the proliferative effects of estrogens in MCF-7 cells. Strangely however, G-1, a GPER-1 agonist, appeared to have an anti-proliferative effect. The reason for this remains unclear and warrants further investigation, although is not in the scope of this thesis.

As described in the results section, ICI 182,780 treatment of MCF-7 cells, either alone or in combination with E2, prevented cell proliferation, which agrees with previous findings that ICI 182,780 and other ER antagonists induce growth arrest in this cell line (Lippman et al, 1976; Carroll et al, 2000; Lewis et al, 2005; Martin et al, 2005; Wang et al, 2008b).

The lack of response of ERα negative MDA-MB-231 to E2 and ICI 182,780 is not surprising, considering the absence of ERα in this cell line. These cells do, however, express ERβ, and given the lack of response to E2, this may suggest that ERβ does not mediate the proliferative effects of estrogens. Similar observations with E2, ICI 182,780 and even tamoxifen have been demonstrated previously (Lippman et al, 1976; Rai et al, 2005; Wang et al, 2008b). These cells were the only cell line that

106 proliferated in the absence of growth factors, which suggests that they have become self-sufficient in terms of growth signals, a common hallmark of cancer (Hanahan & Weinberg, 2000). Indeed, Ras mutations, as displayed in MDA-MB-231 cells, results in increased production of TGFα (Ciardiello et al, 1988; Cancer Genome Project). This means that MDA-MB-231 are capable of producing growth factors, and may, therefore, mean that they are capable of surviving in an environment where growth factors are limited.

Finally, the role of GPER-1 in proliferation was investigated. All of the cell lines tested are competent for this receptor. In MCF-7 cells, the only cell line in which E2 increased proliferation, GPER- 1 antagonism did not reverse this effect. This suggests that, at least in the case of MCF-7 cells, GPER-1 does not mediate the mitogenic influences of estrogens. In MCF-12A cells however, ICI 182,780 and G- 1, both GPER-1 agonists, did increase proliferation. This may implicate that GPER-1 in this cell line can mediate changes in proliferation, although E2 itself does not increase induce this effect. It remains to be seen whether GPER-1 is implicated in the apoptotic process in the cell lines studied or indeed in three- dimensional (3D) cultures of MCF-12A cells, which will consist of work in later chapters. Nevertheless, it would appear that GPER-1 in the non-tumourigenic ER-competent MCF-12A cell line may be implicated in cellular proliferation.

The data presented here suggests that estrogens may not impact greatly on cellular proliferation in these cell lines, and, therefore, may be exerting their effects via other mechanisms. However, as discussed above, these studies were performed using individual cell lines. In the human mammary gland, multiple cell types are present, and these signal to one another to induce alterations in cell growth and behaviour. Therefore, it is plausible that the lack of significant proliferation in response to E2 is due to the absence of paracrine signalling mechanisms with other cell types. Indeed, as mentioned earlier, there is evidence that suggests that responses of both normal and malignant mammary epithelium to estrogens is not as a result of estrogens directly, and that estrogens instead induce the release of growth factors, such as EGF and TGFα, which, in turn proceed to induce proliferation (Vonderhaar, 1987; Salomon et al, 1992). Further experiments, such as the co-culture of ER positive and ER negative cells, or of epithelial cells with cells of the stroma, would shed further light on this, however for the purpose of the work to be described in later chapters this is not necessary at this stage.

The work presented here has not conclusively shown that E2 acts as a mitogen in ER-competent cell lines or that the primary function of GPER-1 is associated with proliferation. It has, however, established an understanding of how the cells lines tested respond to estrogens and will be important,

107 particularly in the case of the MCF-12A data, for future work. The observations here will allow us to compare the differences between monolayer cultures of MCF-12A cells, and those grown in 3D. This is important to investigate as we are aiming to utilise a 3D model of mammary epithelial MCF-12A cells, to more accurately characterise the impact of estrogens in a more physiologically relevant setting, and highlight the failings of only conducting experiments in traditional monolayer cultures.

In the proceeding chapters we will investigate the impact of estrogenic compounds on 3D cultures of MCF-12A cells and try to elude the role of the ER and GPER-1 in mammary carcinogenesis and the mechanisms involved using a 3D model.

108

CHAPTER III: Establishing Three Dimensional Cultures of Non-