Nongenomic mechanisms

In addition to its classic genomic actions, recent evidence suggests that estrogen can also mediate rapid, nongenomic signaling events through binding to ER localized at the cell membrane.50 A small pool of ER has been shown to be tethered to the plasma membrane through either binding to the lipid raft proteins caveolin-1 and flotillin or complexing with a range of membrane-associated signal transduction proteins such as growth factor receptors and G proteins.51 In breast cancer cells, signaling via membrane ER, or membrane-initiated steroid signaling (MISS), has been shown to involve the coupling of ER with EGFR, HER2, IGF-1R and SRC, providing important mitogenic signals to epithelial cells through the subsequent recruitment and activation of downstream p38 and ERK1/2 MAP kinase pathways (Figure 10.2, 2b).5051 Interestingly, MISS has also been shown to subsequently impact on ER transcriptional activity through MAP kinase-mediated phosphoryla-tion of nuclear ER and its associated coactiva-tor AIB-1 in human epidermal growth factor receptor 2 (HER2)-overexpressing cells, providing an integrated genomic/nongenomic signaling network which can mediate both acute and long-term actions of estrogen.51,52

Estrogens inhibit negative elements of growth factor signaling pathways

As well as the positive influences exerted by estrogens on growth factor signaling pathways detailed above, it is notable that in parallel they diminish (while antiestrogens induce) the expression of the growth inhibitory factor TGFP in several estrogen-responsive human breast cancer cell lines, possibly via activation of the p38 pathway (Figure 10.2, 3).53 Estrogens thus serve to inhibit the expression of a factor, which can act through the p38/JNK pathways, to induce programed cell death.54,55 Additionally, however, it is of particular significance that estrogens have been reported to inhibit expression of tyrosine phosphatases in ER-positive breast cancer cells to increase growth factor mitogenic activity, while both steroidal and nonsteroidal antiestrogens increase phosphatase activity.56,57 Tamoxifen, for example, inhibits the mitogenic activity of EGF by promoting significant dephosphoryla-tion of EGFR, an effect that reduces MAP

kinase signaling and is believed to be ER mediated.56

The estrogen receptor interacts with growth factor-induced nuclear transcription factors, coactivators/ corepressors, and additional proteins to target a diversity of response elements

An important feature of growth factor signaling is its potential to activate several profiles of nuclear transcription factors, which subsequently serve to promote the expression of genes participating in a diversity of endpoints, including cell cycle progression (Figure 10.2, 4). For example, as stated previously, in addition to its phosphorylation of the ER protein, growth factor-induced MAP kinase (ERK1/2) directly activates Elk-1/p62TCF.58 This latter transcription factor subsequently forms a ternary complex with p67SRF (serum response factor) and primes Fos expression via the c-fos serum response element.58 Similarly, JNK phosphory-lates the c-Jun protein which subsequently het-erodimerises with Fos.9,17 The resultant complex, AP-1, is of central importance since it directly targets the 12-O-tetradecanoyl-phor-bol-13 acetate-responsive element (TPA-RE), a sequence found in the promoters of many genes involved in a plethora of cellular endpoints, including proliferation and survival.59

In light of this, it has been reported that estrogens can significantly enhance growth factor induced AP-1 activity in hormonesensitive breast cancer cells.60 This feature is believed to be a consequence of productive protein/protein interactions between the estrogen receptor and the AP-1 complex,22 a phenomenon also demonstrated to occur between ER and other transcription factors such as SP-1.61 Thus, ER appears able to activate genes containing AP-1 sites in their promoters,62 providing a mechanism whereby ER signaling may be markedly diversified. Initial studies suggested that antiestrogens antagonized growth factor-induced AP-1 activity, with maximal inhibition by pure antiestrogens.60 In contrast to the above, ER may repress the activity of the transcription factor NFkB,63 which regulates expression of many cytokines and growth factors.64

Finally, it should be remembered that ER/ERE-mediated gene transcription is also significantly enhanced by the recruitment of several coactivators and/or by overcoming the effects of corepressor proteins. Indeed, ERK1/2 and p38 MAP kinase-mediated serine phosphorylation of coactivators such as SRC-1, GRIP1 and AIB1, and the corepressor SMRT, has been shown to regulate their ability to associate with ERa and influence its transcriptional activity.65-69 Additional proteins also under growth factor/MAP kinase regulation have been show to interact with the ER, including the cell cycle protein cyclin D1 and the orphan member of the nuclear receptor superfamily, the estrogen-related receptor alpha (ERRalpha).70,71 Cyclin D1 can activate ER by direct binding, as well as by recruiting coactivators of the SRC-1 family to the ER,70 whilst ERRalpha has been shown to compete directly with ER, and consequently repress transcriptional activity via this receptor.71

Steroid hormone and growth factor signaling pathways influence common growth regulatory genes

In order for cells to proliferate, they initially need to be recruited into the cell cycle and then be induced to progress through it. These outcomes are orchestrated by at least two series of events, which can be jointly influenced by steroid hormone and growth factor-directed MAP kinase signaling pathways:72 firstly, the induction of intermediate early response genes, such as c-fos,73 c-jun,73,74 and c-myc;72,75 and secondly, the regulation of G1 cyclins (e.g. cyclin D1), and their partner kinases and inhibitors which are involved in restriction point control.72,76 Joint activation of these pathways by estrogens and growth factor-induced MAP kinase would, at a minimum, reinforce mitogenic signals to responsive cells, and might even result in syn-ergistic interactions between overlapping elements (Figure 10.2, 5).


Importantly, in the archetypal endocrine responsive breast cancer cell line MCF-7, growth factor signaling leads to increased MAP kinase activity, which appears critical for their growth, since substantial growth inhibition is achieved with the MEK1 inhibitor PD098059. Significantly, however, the increases in growth factor-induced MAP kinase activity, which facilitate the productive crosstalk with ER signaling described above, are only short-lived due to a highly efficient negative feedback of phos-phorylation of these enzymes.7 Such a negativefeedback system stems not only from phosphatases effectively targeting MAP kinases, but additionally from distinct nonaberrant expression patterns of growth factor receptors and intracellular signaling elements comprising the network upstream of MAP kinase acti-vation.77 This serves to tailor input signals to the precise growth requirements of the cells and maintain the modest levels of proliferation which are characteristic of endocrine responsive disease.

In contrast to the above, in several instances elevated activation of ERK1/2 MAP kinase and upstream regulators of this pathway have been associated with the more aggressive growth of de novo and acquired endocrine resistant cells.40,44,45,52,78-83 Significantly, within our in-house breast cancer cell models of acquired resistance to tamoxifen and faslodex,78,79 not only was PD098059 shown to be a highly effective inhibitor of the growth of the anti-hormone-resistant cells, but arrest of cell proliferation was also achieved with ZD 1839, an EGFR selective tyrosine kinase inhibitor, and herceptin, an inhibitor of c-erbB2.78,79 Such data indicate that these erbB receptors are direct upstream regulators of MAP kinase-induced growth regulation in these resistant cell lines. erbB receptors do not appear to be the only regulators of MAP kinase activity in tamoxifen resistance, as Cui et al84 have reported that reduced activity of MAP kinase phosphatase 3 (MKP3), a negative regulator of ERK1/2 MAP kinase, may also play a role in the generation of the acquired tamoxifen-resistant phenotype. More recently, activation of the p38 signaling pathway has also been implicated in endocrine resistance, with enhanced levels of phosphorylated p38 being observed in a xenograft model of acquired tamoxifen-resistant MCF-7 cells.85

In addition to directly driving cell growth, we have also demonstrated that the increased EGFR/HER2/ERK1/2 MAP kinase signaling observed in our tamoxifen-resistant variant can efficiently phosphorylate serine 118 within the AF-1 domain of ER.86 It is possible that such ligand-independent activation of ER may play a role in tamoxifen resistance, as cell lines resistant to this antiestrogen, in common with their clinical counterparts, continue to express ER at an equivalent level to that observed in the parental cell line.87-91 Indeed, increased ER phosphorylation has been reported in breast cancer cell lines resistant to tamoxifen and long-term estrogen deprivation,92-96 and more recently in ovariectomized mice bearing tumor xenografts from aromatase-transfected MCF-7 cells.97 This MAP kinase-dependent phosphorylation of ER allows recruitment of several AF-1 coactivators, such as p68 RNA helicase, and subsequent reactivation of ER as a nuclear transcription factor, resulting in expression of detectable levels of the estrogen regulated genes, in particular amphiregulin, in our tamoxifen-resistant cell line.86 Although the temporal sequence of these events remains to be established during the development of tamoxifen resistance, we have postulated that EGFR/MAP kinase/ER-driven increases in expression of amphiregulin may serve to establish a self-propagating autocrine signaling loop, allowing the emergence and maintenance of efficient EGFR/MAP kinase-promoted resistant growth.86 It should also be noted that, as mentioned previously, MAP kinase signaling can directly promote phos-phorylation of ER coactivators, which can result in their increased nuclear localization and enhance their impact on ER function.65-69

Indeed, overexpression of the coactivator AIB1 has been shown to correlate with resistance to tamoxifen in breast cancer patients, and EGFR/HER2/ERK1/2 MAP kinase-dependent phosphorylation of this coactivator has also been proposed to mediate tamoxifen resistance in HER2 overexpressing MCF-7 cells.52,94

Targeting the ER with the pure antiestro-gen faslodex, which acts by promoting ERa degradation to deplete ERa protein expression,1,98,99 can effectively interrupt the autocrine signaling loop established in our tamoxifen-resistant cell line, reducing activation of EGFR, c-erbB2 and MAP kinase, and potently inhibiting cell growth.87 However, exposure of these cells to exogenous EGF lig-ands not only activates EGFR, c-erbB2 and ERK1/2 MAP kinase, but also supports substantial tumor cell growth in the presence of faslodex. Thus, strengthening the EGFR pathway it appears able to entirely circumvent the catastrophic effects of this antiestrogen on the ER protein in such cells.87 EGFR ligand-treated cells are thus refractory to the growth inhibitory effects of both tamoxifen and faslodex (i.e. complete endocrine insensitiv-ity), data which certainly implies that the primary growth regulatory role for ER in the tamoxifen-resistant cells is to maintain the efficiency of EGFR signaling. In agreement with these findings, it has recently been reported that exogenous treatment of breast cancer cells with either fibroblast growth fac-tor-1, heregulin beta-1 or vascular endothelial growth factor, and subsequent activation of ERK1/2 MAP kinase signaling, can similarly overcome the growth inhibitory actions of faslodex.100,101 In this context, it is also of considerable interest that Oh et al39 have demonstrated that high levels of constitutive Raf kinase activity, leading to hyperactivation of MAP kinase, imparts MCF-7 cells with an ability to grow in the absence of estrogen. Such cells have lost their ERs and showed no activation of transfected EREs reporter gene constructs. Importantly, this effect was abrogated by inhibiting MAP kinase, thus restoring ER expression.

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