The ovarian-derived steroid hormone estrogen is one of the main regulators of mammary epithelial cell proliferation and differentiation. While estrogen is required for normal mammary gland development, cumulative exposure to estrogen during a woman's lifetime is a high risk factor for breast malignancy. The action of estrogen is exerted by binding and activating the estrogen receptors, ERα and ERβ. Studies on the expression patterns of estrogen receptors and gene knock-out mouse models indicate that ERα is the primary estrogen receptor for mammary epithelial cell proliferation and differentiation. While ERα is expressed exclusively in the epithelial cells, ERβ is expressed in both epithelial and stromal cells but the expression of ERβ in the epithelial cells is very weak [1–4]. In ERβ knock-out mice, the mammary glands can still form the bronchoalveolar structure and lactate normally [5, 6]. In ERα knock-out mice, however, the mammary gland remains at rudimentary duct stage without further development [7–9]. While the role of ERβ in breast cancer is less understood, ERα is one of the most known oncogenes in breast cancer [10–12]. Abnormal expression of ERα is found in about 70–80% of human breast cancers, and about 50% of ERα-positive breast cancer patients respond to anti-estrogen therapy [10, 13]. When overexpressed in the mammary gland epithelial cells of transgenic mice, ERα leads to mammary malignancy [14, 15].
Despite the significant role of ERα in mediating estrogen-induced cell proliferation in normal mammary development and breast cancer, the mechanism is still not fully understood. In the normal mammary gland, ERα expression is found only in a subpopulation of the mammary epithelial cells and the percentage of ERα-positive cells is affected by the physiologic conditions. Unlike most other cellular receptors, ERα expression is not found in proliferating mammary epithelial cells [1–3, 16–18]. This observation leads to the prevailing concept that ERα mediates cell proliferation in a paracrine manner [1, 19]. In this paracrine model, ERα-positive cells don't proliferate; the ERα-positive cells, however, when stimulated by estrogen, will produce and release paracrine growth factors, which in turn stimulate the neighboring cells to proliferate [1, 9]. The EGF family member amphiregulin could be one of those important paracrine growth factors involved in ERα-mediated mammary gland epithelial cell proliferation . It is not clear why ERα-positive cells cannot proliferate, or why ERα does not stimulate cell proliferation in an autocrine mode or in both autocrine and paracrine modes. A recent study by Cheng et al. challenged the paradigm that ERα cannot mediate ERα-positive cell proliferation . The authors proposed that ERα is degraded early in the cell cycle, which accounts for the dissociation of the ERα-staining and cell proliferation marker.
In contrast to the mechanism in normal mammary tissues, ERα might mediate cancer cell proliferation via paracrine and/or autocrine modes in primary breast cancers. In ERα-positive primary breast cancers, the percentage of ERα-staining cells varies from 10% to 50% or higher [1, 3, 21, 22]. Most of the ERα-staining cells are not stained with Ki-67 in these ERα-positive primary breast tumors [1, 3, 23, 24]. In some primary breast tumors, however, ERα does colocalize with the cell proliferation marker Ki-67 in a sub-population of tumor cells; the percentage of ERα and Ki-67 duel-staining cells varies among different patients, from 0% to ~5% of total cells [1, 3]. The co-staining of ERα and Ki-67 suggests that ERα-positive cells in these primary breast tumors might be capable of proliferating, i.e., ERα might be able to mediate cell proliferation via autocrine fashion in these primary breast tumor cells.
Using breast cancer cell line models, ERα has been shown to have non-genomic effect in addition to the traditional genomic action . In its genomic action as a transcriptional factor, ERα activation in MCF-7 cells can induce the expression of cyclin D and myc to promote cell cycle progression [26–29]. When stimulated by estrogen, the effect on cell proliferation in MCF-7 cells is usually not obvious until 5–7 days after the stimulation [30–32]. It is not clear why the impact is delayed for such long time and that raises the question whether the activation of ERα alone is sufficient to drive cell cycle progression through all the phases for mitosis. For the non-genomic action of ERα, cell signaling initiated from the cell surface can activate multiple pathways such as the ERK and AKT pathways [33–35]. Using the EGFR inhibitor, Levine and colleagues demonstrated that EGFR is required for the cell surface ERα-activated signaling transduction . Marks and colleagues demonstrated that inhibition of the MAPK and PI3K-AKT pathways can prevent estrogen-induced mitogenesis in MCF-7 cells . Considering all of the information derived from different perspectives, it is very likely that MCF-7 cells and some other ERα-positive breast cancer cell lines might be regulated by ERα via the autocrine as well as the paracrine modes. In this study, we demonstrated that ERα is colocalized with Ki-67 in MCF-7, T47D, and ZR75-1 cells, the ERα-positive breast cancer cell lines used in our study. Using MCF-7 cell line, we demonstrated that ERα is present in all the phases of cell cycle and activation of ERα in G1 phase promotes cell cycle progression through mitogenesis, supporting the autocrine mode of regulation. Finally, we demonstrated that EGFR activation is not required for the autocrine regulation of cell proliferation by ERα.