Mechanisms Of Resistance In The Estrogen Receptor Biology Essay

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The estrogen receptor is over expressed in approximately 60-70% of breast cancer patients forming the basis for endocrine therapy. Different classes of endocrine agents act to suppress stimulation of breast cancer cells by estrogen either by interfering with the activity of the estrogen receptor (ER) or reducing estrogen biosynthesis. (Musgrov & Sutherland, 2009) The final decision on the type of agent to be used depends on the patient and tumor characteristics.

Despite the fact that the use of these agents has improved survival and reduced mortality, many patients fail to benefit from endocrine therapy due to the development of intrinsic or acquired resistance. It has been documented that up to 25% of breast cancer cases are intrinsically resistant. In fact, almost all patients develop resistance with time. This phenomenon has affected a clinically significant proportion of women leading to disease progression, metastasis and eventually, mortality (Dawood & Cristofanilli, 2007). With the development of cellular models, researchers have been able to investigate and describe the mechanisms by which breast cancer cells acquire this behavior.

Before interpreting those models, it is important to understand the mechanism of action of the ER, which is a typical nuclear located receptor. By far, two ERs have been described; ER-α and ER-β. Estrogen freely diffuses through the membranes of cells and it migrates into the nucleus where it interacts with its receptor. When estrogen binds, the ER dissociates from heat shock proteins and undergoes conformational changes, dimerization and phosphorylation ultimately leading to activation of the receptor. The activated receptor then binds to specific DNA sequences, such as estrogen response elements (EREs), which are located upstream of estrogen-dependent genes and they alter transcription. However, EREs are not the main transcriptional promotors. This is referred to as the classical mode of action of estrogen. A study conducted on the cellular effects of estradiol on the proliferative function of cells concluded that 70% of estrogen-regulated genes are downregulated upon its exposure and many of those genes are transcriptional repressors or genes with antiproliferative function. It has also been demonstrated that genes with pro-proliferative function are upregulated (Normanno et al. 2005, Ring & Dowsett, 2004). This clarifies the role of estrogen in the pathogenesis of breast cancer and explains why its continuous and prolonged exposure, whether endogenous or exogenous may produce or sustain the growth of cancer cells.

The upregulation of pro-proliferative genes is mediated by two domains; Activating function-1 (AF-1), which is at the N-terminus of the receptor and AF-2, which is in the ligand binding domain. AF-1 is regulated by phosphorylation and is hormone independent whereas AF-2 is hormone dependent. Although those two domains act synergistically, they may independently activate certain gene promoters. Additionally, the transcriptional activity of the ER may be modulated by co-regulatory proteins which may either be co-activators and thus stimulate ER activity via interacting with AF-2, or co-repressor proteins which reduce the rate of initiation of transcription via recruiting histone deacetylase complexes ultimately leading to chromatin condensation (Normanno et al. 2005, Shou et al. 2004). The relevance of those coregulators will be explained in further detail later on.

The estrogen receptor may also interact with other transcriptional factors such as the Fos/Jun activating protein-1 (AP-1) complex to regulate gene expression. This is the non-classical mechanism of action of estrogen in mediating its genomic effects (refer to figure 1). Additionally, ER-α may interact with kinases such as IGF-1R, Src, PI3K, MAPK, EGFR and ErbB-2 producing its non-genomic effects. The levels of the mentioned kinases regulate the membrane functions of ER via phosphorylating co-activators which activate ER-α function. The genomic and non-genomic effects of the ER may overlap and interactions between them do exist (Normanno et al. 2005).

(Figure 1) --- ??

Types of agents

Several strategies have been developed to inhibit the ER pathway in the management of breast cancer. Tamoxifen is a selective estrogen receptor modulator (SERM) that competitively inhibits the ER. While its effects are not purely inhibitory, its overall effect on cellular function is tissue-dependent and is influenced by the presence of coregulatory proteins. Selective ER downregulators (SERDs), such as fulvestrant, have also been developed. Those agents potently target the ER by inducing its destabilization and degradation. Another approach relies on estrogen withdrawal and may be achieved by aromatase inhibitors such as anastrozole or letrozole which reduce the peripheral production of estrogen. Recent clinical trials have concluded that these agents have a superior effect compared to tamoxifen and are likely to be effective in patients who are resistant to tamoxifen though longer follow up studies are required to confirm this. These agents are also being used as a first line therapy for postmenopausal women. (Schiff et al, 2005 and Normanno et al, 2005).

Mechanisms of Resistance

Loss of ER-α expression or function

As mentioned earlier, breast cancer patients may either be classified as ER positive (ER+) or negative (ER-). Patients with the ER+ phenotype are thought to have a better prognosis as this indicates that they are candidates for and will respond to endocrine therapy. Breast cancer tumor cells which are ER- are classified as intrinsically resistant as the expression of this receptor is the main predictor of response to endocrine therapy. Histone deacetylation and DNA demethylation are thought to be responsible for the silencing of ER-α (Parl 2003). --- Get another source!

During treatment with tamoxifen, loss of ER-α expression may arise as an acquired mechanism of resistance in ER+ tumors. It has been hypothesized that the overactivation of MAPK leads to the loss of ER-α expression. This has been studied with the aid of ER-α+ MCF-7 cells. The ER-α downregulation that occurred in these cell lines supports claims that certain MAPK genes are inversely regulated compared with estrogen related genes. This cell line was compared with profiles generated from Er-α+ versus Erα- breast tumors and it very well predicted the ER-α status of clinical breast tumors in 4 different clinical studies. This suggests that these cell lines closely mimic the situation in ER- tumors indicating that this pathway is one of the major ones operating in ER- breast cancer (Creighton et. al, 2006). --- MAPK role. However loss of ER-α is uncommon (Sarvilinna et. al, 2006) and almost 20% of tamoxifen resistant patients eventually respond to second-line treatment with fulvestrant or aromatase inhibitors. (Normanno et. Al, 2005)

Apart from the loss of Er-α expression, an alteration in the function of the receptor may also result in resistance. This is thought to occur due to a mutation which involves a single amino acid substitution converting the receptor into a hypersensitive state where an enhanced binding to co-activators is observed even in the presence of low estrogen levels. However, this mutation is rarely found in primary breast carcinomas (Sarvilinna et. al, 2006) and is mainly detected in ER- tumors. --- (Fuqua et al, 2000 and normanno)

The lack or loss of progesterone receptor (PgR) expression is also thought to affect the response to endocrine agents. It has been reported that ER+/PgR+ patients respond more significantly to adjuvant therapy with tamoxifen. They have a much reduced risk of recurrence and mortality compared to those with ER+/PgR- patients. This suggests that the loss of the PgR may lead to a sustained activation of growth signaling pathways. Cui et al. 2003 demonstrated that the activation of PI3K/AKT pathway is associated with the downregulation of PgR. --- do I need to reference cui et al?


The role of ER-β in endocrine resistance is not clearly understood as multiple studies have produced conflicting results. There is evidence that ERα and ERβ can be coexpressed in some cell types and independently in others though the most frequently occurring status is the ERα+/ERβ+ phenotype (Murphy et al, 2005).

A study on aimed at investigating the effect of ERβ upregulation on estrogen and tamoxifen responsiveness in MCF-7 cells did not support the hypothesis that ERβ1 upregulation in the presence of ERα leads to resistance. This however does not exclude its role as other factors weren't taken into consideration such as altered cofactor expression or activity. Their results showed that the overexpression of ERβ negatively modulates ERα mediated growth in a dose dependent fashion in ERα+ cells. This may be a mechanism for differential estrogen sensitivity. However, it did not inhibit all ERα-regulated acitivities as endogenous PR expression, which is considered a specific downstream marker of ERα activity, was not affected but was actually upregulated. It is thought that this ERβ mediated PR regulation is due to increased transcription as steady state PR MRNA levels were also increased though this needs further investigation (Murphy et al, 2005). Studies on preinvasive mammary tumors showed that the expression of ERβ is downregulated during tumprigenesis suggesting that its ability to modulate ERα is altered at this stage (and during progression) (Roger et. al. 2001).

Another study (Wu et al. 2011) was conducted to examine the effects of endoxifen (a major active metabolite of tamoxifen) on breast cancer cell lines expressing ERβ. This was achieved by transfecting MCF7, Hs578T and U2OS cells with full length ERβ. It was found that endoxifen stabilizes ER-β protein in contrast to its downregulating action on ERα. They also found that its inhibitory effect on estrogen target genes seemed to be more potent when ER-β is expressed. This suggests that ER-β may be responsible for the variations in response to endoxifen. Additionally, an enhanced sensitivity to the antiestrogenic effects of endoxifen was observed in the presence of ER-β.


Owing to their influence on the transcriptional activity of the ER, altered expression of certain co-regulators may also be involved in the response to endocrine therapy. The current view of transcription regulation by estrogen suggests that coactivator overexpression and/or corepressor downregulation would favour breast tumor growth (Girault et al, 2006).

AIB1 is an ER coactivaor that is sometimes amplified in breast cancer and is thought to assist in the regulation of estrogen-dependent cell proliferation. Su et al, 2008 transfected BT474 breast cancer cells with an RNA interference expression vector specially targeting AIB1 mRNA and assessed its influence on cell proliferation and cell cycle distribution. They found that in ER+ breast cancer cells that over-express AIB1, tamoxifen behaves like an estrogen agonist as its inhibitory effects on cell proliferation were restored upon downregulation of AIB1 (Su et al, 2008).

The overexpression of other coactivators such as SRC-1 are thought to enhance the transcriptional role of estrogen and augment the agonist activity of 4-hydroxytamoxifen but there was no evidence of its overexpression in clinical samples from tamoxifen-resistant tumors (Ring and Dowsett, 2004). Conversely, low MRNA levels of SRC-1 and the corepressor N-CoR before initiating tamoxifen therapy is associated with the acquisition of tamoxifen resistnance (Sarvilinna et al, 2006).

Co-repressors + Adaptation to estrogen withdrawal --- incomplete

Growth factor signalling --- incomplete

As mentioned earlier, coregulatory proteins may modulate the transcriptional effects of the ER. Those effects are augmented when the receptor is estrogen bound due to the recruitment of coactivators. In contrast, when tamoxifen is bound, the recruitment of corepressors is favoured thus inhibiting the transcriptional activity. Tamoxifen exhibits both agonist and antagonist effects and the dominance of either effect depends on the level of those coregulators which when altered, leads to resistance. By phosphorylating the ER and accessory proteins, signaling from multiple kinase pathways may modify the activity of those corgeulators and so, augment the genomic function of ER even in the presence of tamoxifen. (Massarweh & Schiff, 2006)

A model system with high AIB1 and HER2 expression was developed in order to investigate the mechanism by which cells which overexpress those complexes develop resistance. Tamoxifen resistant MCF-7 cells engineered to overexpress HER2 (MCF-7/HER2-18) were treated with estrogen, tamoxifen and EGF in absence of geftinib (EGFR inhibitor). Estrogen deprivation completely inhibited the growth of those cells whereas tamoxifen stimulated their growth. Upon exposure to both estrogen and tamoxifen, molecular cross talk between ER and HER2 pathways was increased in MCf-7/HER2-18 compared to MCF-7 cells with cross phosphorylation and activation of both the ER and EGFR/HER2 receptor and other signaling molecules. Coactivator complexes such as ER and AIB1 were recruited by tamoxifen to pS2 (an ER-regulated gene) in MCf-7/HER2-18 cells whereas the corepressors NCoR and histone deacetylase 3 were recruited in MCF-7 cells. These findings suggest that tamoxifen behaves as an estrogen agonist in cells that overexpress AIB1 and HER2. Gefitinib was able to block this cross talk and reestablished corepressor complexes with tamoxifen bound ER thus restoring the antitumor effects of tamoxifen. (Shou et al. 2004)

The above effects are associated with the genomic actions of nuclear located ER. It has been found that a small portion of ER may be membrane located and can initiate rapid cell response by directly interacting with a variety of signaling pathways. For instance, an interaction between ER and IGFR which eventually activates IGFR downstream signaling has been described and is stimulated by tamoxifen. This however is blocked by fulvestrant and MAPK inhibitors. Also, ER may interact with HER2 in the membrane thus protecting HER2-overexpressing cells from the antitumor effects of tamoxifen. EGFR may also be phosphorylated and activated by ER. Many of such interactions exist leading to the activation of secondary messengers and other downstream kinase pathways eventually producing cell proliferation, growth and survival. Nuclear ER and its transcriptional machinery may also be influenced by these kinase signals promoting the genomic activity of the receptor. In such conditions, it is insufficient to utilize a single target for therapy but rather, a combination of agents which target both ER and other pathways are necessary to overcome the growth of the cancer cells (Massarweh & Schiff, 2006).

Clearly, the actions of the ER, both genomic and non-genomic, are influenced by growth factor signaling as they may be augmented by the overexpression of growth factor receptors such as EGFFR/HER2. When HER2 is overexpressed, tamoxifen may stimulate the growth of MCF-7 cells, probably due to its activation of the non-genomic pathway of ER. Apparently, those de novo resistant HER2 over-expressing cells are sensitive to estrogen deprivation and the pure ER antagonist fulvestrant indicating that the receptor remains functional and is still regulating the growth of those cells. Therefore, a reduction of the crosstalk between ER and EGFR/HER2 may be achieved by removing the ligand or downregulating the ER and so, the probability for de novo resistance is reduced.

Acquired resistance may also occur as a consequence of such growth factor receptor pathways. Long term tamoxifen-treated MCF-7 cells eventually become resistant and they show an increased expression of EGFR. The same has been observed with fulvestrant suggesting a role for this growth factor in acquired resistance to both drugs. However, it has been observed that continued growth in tamoxifen-treated cells is actually stimulated by the agonist effects of tamoxifen. Those cells continue to express ER and respond to subsequent treatment with fulvestrant indicating that this form of resistance cannot be fully explained by growth factor pathways but is also mediated by ER signaling. This implies that endocrine therapy alone is not adequate in such cases and that growth factor receptor inhibitors are also required.

--- See Schiff et al 2005