Tamoxifen behaves as an estrogen agonist in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance. Gefitinib's ability to eliminate this cross-talk and to restore tamoxifen's antitumor effects should be tested in the clinic.

Estrogen receptors (ERs) mediate most of the biological effects of estrogen in mammary and uterine epithelial cells by binding to estrogen response elements in the promoter region of target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ER␣ in the absence of estrogen. Although PI 3-kinase increased the activity of both estrogen-independent activation function 1 (AF-1) and estrogendependent activation function 2 (AF-2) of ER␣, AKT increased the activity of only AF-1. PTEN and a catalytically inactive AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI 3-kinase utilizes AKTdependent and AKT-independent pathways in activating ER␣. The consensus AKT phosphorylation site Ser-167 of ER␣ is required for phosphorylation and activation by AKT. In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT pathway, reduced phosphorylation of ER␣ in vivo. Moreover, AKT overexpression led to up-regulation of estrogen-regulated pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate that AKT protects breast cancer cells from tamoxifen-induced apoptosis. Taken together, these results define a molecular link between activation of the PI 3-kinase/AKT survival pathways, hormone-independent activation of ER␣, and inhibition of tamoxifen-induced apoptotic regression.

Deaths from breast cancer have fallen markedly over the past decade due, in part, to the use of endocrine agents that reduce the levels of circulating oestrogens or compete with oestrogen for binding to its receptor. However, many breast tumours either fail to respond or become resistant to endocrine therapies. By understanding the mechanisms that underlie this resistance, we might be able to develop strategies for overcoming or bypassing it.

The mechanisms involved in resistance to estrogen deprivation are of major importance for optimal patient therapy and the development of new drugs. Long term culture of MCF-7 cells in estrogen (E2)-depleted medium (long term estrogen deprivation; LTED) results in hypersensitivity to E2 coinciding with elevated levels of estrogen receptor (ER) ␣ phosphorylated on Ser 118 and MAPK, together with several of its downstream targets associated previously with ER␣ phosphorylation. Our data suggest elevated MAPK activity results from enhanced ERBB2 expression in the LTED cells versus the wild-type (wt), and treatment with the tyrosine kinase inhibitor ZD1839 revealed increased sensitivity in both transcription and proliferation assays. Similarly the MEK inhibitor U0126 decreased transcription and proliferation in the LTED cells and reduced their sensitivity to the proliferative effects of E2, while having no effect on the wt. However, the complete suppression of MAPK activity in the LTED cells did not inhibit ER␣ Ser 118 phosphorylation suggesting that ER activity remained ligand-dependant. The LTED cells also expressed elevated levels of insulin-like growth factor-1R, and inhibition of phosphatidylinositol 3-kinase activity with LY294002 reduced basal ER␣ transactivation by 70% in the LTED cells compared with the wt. However, LY294002 had no effect on ER␣ Ser 118 phosphorylation. These data suggest that although elevated levels of MAPK occur during LTED and influence the phenotype, this is unlikely to be the sole pathway operating to achieve adaptation.The knowledge that steroids play a pivotal role in the development of breast cancer has been exploited clinically by the development of endocrine treatments (1). These have sought to perturb the steroid hormone environment of the tumor cells, predominately by withdrawal or antagonism of estrogen. Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumors to circumvent the need for steroid hormones, while in most cases retaining the nuclear steroid receptors (2). The identification of the factors and pathways responsible for the development of these resistant conditions is therefore paramount for the design of new diagnostics and therapeutic regimes (reviewed by Ali and Coombes (3)).In an attempt to elucidate these mechanisms, our laboratory and others (4 -6) have developed in vitro models to study the molecular changes associated with long term estrogen deprivation (LTED).1 Our previously published studies demonstrated that MCF-7 cells deprived of E2 for over 80 weeks passed through three distinct phases: quiescent (LTED-Q) followed by a hypersensitive phase (LTED-H), where basal cell growth was stimulated by the addition of E2 at concentrations below 10 Ϫ13

Purpose: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer.Experimental Design: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n ¼ 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X).Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling.Conclusions: This study demonstrates that patientspecific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention. Personalized profiling detects rising ctDNA ahead of clinical relapse. A-E, Plasma levels of ctDNA across serial plasma time points for five patients with breast cancer (one per panel). Mean VAFs are denoted by a dark blue circle, and solid lines represent the average VAF profile over time. The lead time is calculated as the time interval between clinical relapse (red triangle) and molecular relapse (blue triangle). CA 15-3 levels are graphed over time (teal circle), and the baseline levels (32 U/mL) are marked in light blue. F, Summary of percent VAF and number of targets detected at molecular and clinical relapse for all ctDNA-positive samples. Data are from 13 relapsed patients, excluding three patients with only one plasma time point. Coombes et al.

Cross talk between the Estrogen Receptor (ER) and ErbB2/HER-2 pathways have long been implicated in breast cancer aetiology and drug response 1 , yet no direct connection at a transcriptional level has been shown. We now show that estrogen-ER and tamoxifen-ER complexes directly repress ErbB2 transcription via a cis-regulatory element within the ERBB2 gene. We implicate the Paired Box 2 gene product (Pax2), in a novel role, as a crucial mediator of ER repression of ErbB2 by the anti-cancer drug tamoxifen. We show that Pax2 and the ER coactivator AIB-1/SRC-3 compete for binding and regulation of ErbB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ErbB2 by ER-Pax2 links these two important breast cancer subtypes and suggests that aggressive ErbB2 positive tumours can originate from ER positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.The genomic mapping of Estrogen Receptor binding sites has revealed insight into how ER functions in breast cancer cells, including the finding that ER rarely binds to promoter regions and that ER loading on the chromatin requires the presence of pioneer factors, such as FoxA1 2-4 . We have replicated genome-wide ER Chromatin Immunoprecipitation (ChIP)-on-chip analyses in ER positive MCF-7 cells. Identification of the ER binding sites using a false discovery rate of 5% revealed 8,525 ER sites, with excellent representation (86%) of the published ER binding profile 2 (Supplementary data 2). Included within the new, more extensive list, was an ER binding site within the intron of the ERBB2/HER-2 genomic region ( Figure 1a). Sequence analysis of all 8,525 ER binding sites revealed a statistical enrichment (p-value < 0.0001) for the Paired Box (Pax) transcription factor motif (GTCANGN(A/G)T) ( Figure 1b). Little is known about the role that Pax proteins play in hormone signalling, however, Pax2 was shown to be expressed in a subset of breast cancers and was recently identified as a tamoxifen-regulated effector in endometrial cancer cells 5,6 .5 To whom correspondence should be addressed (jason.carroll@cancer.org.uk). NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 August 11. Tamoxifen is one of the most successful and effective therapies in the treatment of breast cancer, but tamoxifen resistance is inevitable 7 . Tamoxifen resistant breast tumours are characterised by elevated ErbB2 levels 8 and ER positive cell line models over expressing ErbB2 acquire resistance to tamoxifen 9 . We assessed Pax2 binding to a select number of ER binding sites adjacent to important estrogen regulated genes, including the newly identified binding site within the ERBB2 gene. Pax2 was generally recruited only after tamoxifen treatment, with the exception of the ER binding site within ERBB2 (Figure 1c), where Pax2 was recruited to the ER binding site after both estrogen and tamoxifen...

Using a transient co‐transfection system, we show that the human oestrogen receptor (hER) becomes phosphorylated in the presence of oestradiol (E2) as well as in the presence of the anti‐oestrogens 4‐hydroxy‐tamoxifen (OHT) and ICI 164, 384 (ICI), although at lower efficiencies than with E2. There are multiple sites of phosphorylation in hER; using deletion and point mutants one of these sites has been mapped in the N‐terminal A/B region at serine 118. Mutation of this serine to alanine caused, in a number of cell types, a significant reduction in transcriptional activation by hER from reporter genes containing an oestrogen response element (ERE), but did not affect the DNA binding properties or nuclear localization of hER. Thus phosphorylation of serine 118 is important for the action of the transcription activation function 1 (AF‐1) located in the A/B region of the oestrogen receptor.

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localise to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequiste for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knockin of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, CDK7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knockin mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine resistant breast cancer mediated by ER mutations.