Oestrogenreceptor (ER) positive breast cancer is the most common subtype of breast cancer. Endocrine therapies that target the dependence of this subtype on ER have substantial activity, yet resistance to therapy is inevitable in advanced cancer. Major progress has been made in identifying the drivers of ER positive breast cancer and the mechanisms of resistance to endocrine therapy. This has translated into major advances in the treatment of advanced breast cancer, with a number of targeted therapies that enhance the efficacy of endocrine therapy. Substantial improvements in progression free survival have been demonstrated with mTOR inhibitor and CDK4/6 inhibitors. A new wave of targeted therapies is being developed, including PI3K, AKT, HER inhibitors and new generation of ER degraders. Substantial challenges remain in patient selection, selection of the most appropriate order of therapies, and whether there is cross-resistance between therapies.
Genotype and allele frequencies of the oestrogenreceptor gene (ESR) PvuII restriction site were inves- tigated in populations of the main Czech maternal breeds. 1253 sows and gilts and 396 boars in Large White and 334 sows and gilts and 318 boars in Landrace were genotyped from blood samples by the modified PCR-RFLP procedure as described in Short et al. (1997). In Large White, the frequency of allele B was about 0.51. In Landrace, the frequency of allele B reached from 0.02 for boars to 0.03 for sows. No significant deviations of the observed genotype frequencies from the frequencies expected ac- cording to Hardy-Weinberg equilibrium were found in both breed. Opposite trends in allele frequencies development could be assumed for analysed sows and boars of both breeds. In Large White sows the frequency of allele B raised probably due prefering sows with BB genotype, whereas in Landrace popu- lation the frequency of allele B decreased from yet unknown reasons.
(Corthesy et al 1988, Freedman et al 1989, Klein-Hitpass et al 1990). Steroid receptors have been shown to stimulate transcription from simple promoters containing only an HRE and a TATA box suggesting they are capable of interacting directly with components of the initiation complex (Klein-Hitpass et al 1990, Tora et al 1989b). This Is supported by observations of Tsai and colleagues (1987) that a non-DNA binding protein, later identified as TFIIB, can stabilise the binding of COUP-TF to its response element resulting in the activation of transcription. TFIIB has since been shown to interact with the oestrogen, glucocorticoid and progesterone receptors and it has been suggested that this protein may stabilise the interactions of steroid receptors with their response elements leading to the formation of a more stable receptor/TFIIB/TFIID/TFIIA complex on the DNA and subsequently initiation complex formation (Ing et al 1992). The role of a common intermediary protein in the induction of gene expression by steroid receptors is supported by transcriptional interference ('squelching') studies. Such experiments titrate the ability of two transcription factors to compete with one another for a common component that is required for transcription. In this manner both transcriptional activation functions of the oestrogenreceptor have been observed to squelch the transcriptional activation of the glucocorticoid and progesterone receptors and vice versa indicating all three receptors require a common factor to stimulate transcription (Meyer et al 1989, Tasset et al 1990).
Luciferase oestrogenreceptor-alpha transcriptional assay ERα-positive T47D-KBluc human BCa cells were grown in phenol red–free RPMI 1640 supplemented with 10% CSS for 5 days. The cells were seeded on a 96-well plate (2 × 10 4 cells/well). After 24 hours, the cells were treated with either the test compounds or 4-OHT in the presence of 1 nM E2. The test compounds were screened at two concentrations: 12 μM and 30 μM. 4-OHT was added at a final concentration of 5 μM. For generation of dose–response curves, the compounds were added at a range of 0.1 to 50 μM and 4-OHT was added at a range of 0.000006 to 3 μM. The medium contained 0.1% (v/v) ethanol and 0.1% (v/v) DMSO. Twenty-four hours after treatment, the medium was aspirated and the cells were lysed by adding 50 μl of 1× passive lysis buffer (Promega, Madison, WI, USA). The plates were placed on a shaker at room temperature for 15 minutes and then subjected to two freeze–thaw cycles to help lyse the cells. Next, 20 μl of the lysate from each treatment was transferred onto a white, 96-well, flat-bottomed plate (Corning Life Sciences, Corning, NY, USA), and the luminescent signal was measured after adding 50 μl of the luciferase assay reagent (Promega) on a TECAN M200 PRO microplate reader (Tecan, Männedorf, Switzerland). Differences in growth were normalised against total
The oestrogenreceptor (ER) is a m em ber of the nuclear receptor superfam ily of ligand inducible transcription factors. In the absence of oestrogen the ER exists in the cell as p a rt of an inactive complex w ith heat shock proteins. U pon binding oestrogen the heat shock proteins are displaced, a if the ER is able to bind DNA as a dim er w here it stim ulates transcription from oestrogen responsive elem ents (ERE) in the vicinity of target genes. The ER exists in two forms, the classical ERa and the recently discovered ERp, which are encoded by distinct genes. ERP has a sim ilar b in d in g affinity for 17p-oestradiol as ERa and is capable of activ atin g tran scrip tio n from ERE containing prom oters. W hen co expressed in v itr o or in v iv o ERa and ERP form heterodim ers, w hich bind to an ERE w ith an affinity similar to that of ERa hom odim ers but g reater than that of ERp h o m o d im e r s . The h etero d im er, like the hom odim ers, are capable of binding the steroid receptor coactivator 1 (SRCl) w hen b ound to DNA and stim ulating transcription from ERE containing reporter genes in cell lines. ERa has two well characterised transcriptional activation dom ains, activation function 1 (API) in the N -term in u s and activation function 2 (AF2) in the C -term inus. A com parison of the A Fl and AF2 dom ains from ERa and ERp revealed that ERp does not have an A Fl activity equivalent to that of ERa, while their AF2 activities are similar.
Most breast cancers overexpress oestrogenreceptor-α (ER-α), and this molecule is the major target of the anti- estrogens, such as tamoxifen, used to treat the disease. Not all patients respond to anti-oestrogen treatment and many who do respond later relapse. The reasons for treat- ment failure are still unclear. The suggestion that mutation of ER-α might have a role in the formation of breast cancer and subsequent response to treatment was raised by the detection of a somatic A908G (Lys303→Arg; K303R) mutation in the gene encoding ER-α. This mutation was reported in a significant proportion of breast hyperplasia  and also in the majority of invasive cancers and all metas- tases tested [2,3]. The K303R ER-α variant apparently exhibits a hypersensitivity to oestradiol , a characteristic that might allow breast cancers to respond to much lower levels of oestrogenic stimulation with a subsequent impact on malignant progression and the effectiveness of anti-oes-
inversely correlated to oestrogenreceptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1β, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP- 1β were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1β were expressed to a greater degree in HER2-positive than in HER2-negative patients. The expression of most of the studied cytokines was correlated to levels of activator protein-1, which is known to regulate numerous cytokines. Overexpression of MCP-1 and MIP-1β were linked to B lymphocyte, T lymphocyte and macrophage infiltration, whereas high levels of IL-8 were correlated with high macrophage content in tumour. Moreover, IL-8 positive tumours exhibited increased vascularization.
Flax contains large amounts of hormone-like compounds, especially lignans. These so-called phy- toestrogens are thought to inhibit the cell growth of hormone-sensitive cancers. Hence, we ana- lysed the influence of flax root extracts at various stages of maturity on the proliferation and cy- totoxicity in oestrogen-receptor-positive breast cancer cells (MCF7) in vitro. Flax root extracts were prepared by using lignan extraction. The extracted compounds were analysed by Pyroly- sis-Field Ionisation Mass Spectrometry. Various extract concentrations were applied to the cells to test for proliferation (BrdU test) and cytotoxicity (LDH test). A significantly higher inhibition of cell proliferation was observed with an extract made from 9-week-old flax roots in comparison with that of 3- and 6-week-old roots. Older roots contained more lignans and other phenolic sub- stances than younger roots. The maturity grade of plants or their various parts is thus important for the production and concentration of secondary metabolites and leads to different biological effects on breast cancer cell growth.
The mutations in the conserved region therefore may weaken the contacts between TAF-2 and its target(s) or possibly affect an earlier step required for transcriptional activation that has not been examined. The conserved amino acids identified as being important for transcriptional activation may contact the target(s) of TAF-2 directly or be important for the structure of the interacting surface either directly or by allowing posttranslational modifications. In the wild-type receptor the removal of the N-terminus (in the mutant MOR 121-599) had no significant effect on the ability of the oestrogenreceptor to stimulate transcription from pERE BLCAT whilst it reduced the level of transcriptional activation, approximately 5-fold, when pERE MLTCAT was used. This may be due to the presence of different endogenous factors binding to the promoter, differences in the TATA box sequence or differences in the positions of factor binding sites. With regards to the model it is possible that a factor binding to the promoter of pERE BLCAT is able to substitute for TAF-1 in cooperating with TAF-2 when TAF-1 is removed. pERE MLTCAT being a minimal promoter may lack a binding site for a suitable factor. Mutations in TAF-2, however, affect the ability to cooperate with the endogenous transcription factors bound to pERE BLCAT resulting in a decrease in the level of transcription.
Oestrogen Receptor Status of Breast Tumour Biopsies in Malaysian Patients Med J Malaysia VoI 38 No 2 June 1983 STATUS OF BREAST MALAYSIAN PATIENTS OESTROGEN RECEPTOR TUMOUR BIOPSIES IN SHAHARUDDIN AZI[.]
Human dental pulp cells (hDPCs) ex press oestrogenreceptor (ER) isoforms ER , ER 1 and ER 2, as well as a 7-transmembrane G protein-coupled receptor, GPR30, that mediates rapid oestrogen signalling . Following osteogenic differentiation of these cells ER 1 and ER 2 were up regulated approximately 50-fold while ER and GPR30 were down regulated, but to a much lesser degree (approximately 2- fold). ER was characterised as a 59 kDa protein following SDS-PAGE. ER was detected in both nuclear and cytoplasmic cell compartments following immunofluorescence of cultured cells. Furthermore isoform specific antibodies detected both ER 1 and ER 2 in DPC cultures and in situ analysis of ER expression in decalcified tooth/pulp sections identified the odontoblast layer of pulp cells juxtaposed to the tooth enamel as strongly reactive for both ER isoforms. Finally the use of isoform specific agonists identified ER as the main receptor responsible for the pro-
confers selective binding to E2 since it would not accommodate the hydrogen bond accepting 3-keto group at the A -ring o f other steorid horm ones, such as progesterone. Although the ER antagonists, Tamoxifen and Raloxifene, are held in place by sim ilar electrostatic interactions as observed for E2 binding, these antagonists induce a distinct conformational change in the ER LBD as a result of their bulky side-chains (Brzozowski et ah, 1997; Shiau et ah, 1998). The side- chains of these antagonists project from the ligand binding cavity and are stabilised by a salt-bridge with D351. As a result, helix 12 adopts an alternative position and resides in a hydrophobic cleft formed betw een helices 3, 4 and 5. The com plem entarity of the hydrophobic cleft with the inner surface o f helix 12 indicates that the alternative position is not an artifact produced by the crystal lattice. In fact, the antagonist induced intram olecular interactions provided im portant clues for the delineation of the ER coactivator docking surface (see Results and Discussion). It is worth noting the critical role o f D351 in the action of antagonists. This is supported by the identification of a point mutation, D351Y in E R a , from tum ours which are stim ulated by T am oxifen in athym ic mice (Catherine et al., 1995). Breast cancer cells stably transfected with this mutant receptor respond to both Tamoxifen and Raloxifene as agonist suggesting that this may be one mechanism for the development o f Tam oxifen resistance in breast tumours (Levenson and Jordan, 1998). From a structural point o f view, it can be envisaged that the side-chains of antagonists may adopt an alternative position or are simply untethered in the absence of D351 which allow helix 12 to assume its native position.
activation of the EGFR  and downstream phosphory- lation of ERK . We, therefore, measured activation of ERK signalling by ER and assessed the effect of the specific EGFR inhibitor gefitinib. Activation of ER sig- nalling with 17b-estradiol caused increased phosphoryla- tion of ERK which could be abrogated by treatment with tamoxifen or gefitinib, suggesting that ligand depen- dent ER signalling transactivates EGFR to phosphorylate ERK (Figure 3E). In mammosphere culture, gefitinib sig- nificantly reduced the effect of oestrogen in ER positive primary cells and lines (Figure 3F). However, with the exception of primary 3, the MFC remained significantly higher compared to control cultures which were not exposed to oestrogen. These findings support the hypoth- esis that EGFR mediated paracrine signalling plays a role in the response to oestrogen but suggests other paracrine signals are also important.
ZR-75-1 breast cancer cells are completely dependent on oestrogen for growth. In standard medium without added oestrogen, growth is strongly reduced. Addition of anti-oes- trogen completely abolishes the growth of these cells . Oestrogen-induced cell proliferation is mediated by the transcription activation function of the ERα. To identify the early effects (that is, the transcription targets) of oestrogen stimulation, the expression profile was analysed after a 6- hour high-dose pulse of 100 nM oestrogen. From Fig. 1 and Additional file 1 it is clear that limited changes in gene expression (115 genes with |DE| ≥ 1.60) have occurred during this short treatment compared with mock-stimulated cultures. Over 75% of these genes seemed to be induced. Among these genes are well-known oestrogen targets such as TFF1 (PS2), CSTD (cathepsin D), CCND1 (cyclin D1) and PGR (progesterone receptor). These and several novel genes were rapidly induced by oestrogen both in the parental ZR-75-1 cells and in the BCAR3-transfected cells (Fig. 1 and Additional files 123). An extended picture emerges after continuous exposure to the regular dose of 1 nM oestrogen. About 400 genes exhibit consistent changes (at least 1.6-fold) in expression, of which about 60% of the sequences exhibit a significant decrease of gene expression (up to sevenfold) and 40% are increased (up to more than 10-fold). The genes specifically modu- lated by oestrogen comprise members of all functional compartments and processes in the cell. One-quarter of the early-induced genes remain expressed (DE > 1.60) during continuous exposure to oestrogen (see Fig. 1), whereas the expression of others is turned off (for example AMD1, BCL2, CCND1, GJA1, MEIS3, RIP140, RUNX1 and STC1) or even downregulated (for example HIF1A, IL6ST, MYB, PC4 and UGT2B7). Definitions of these and
More than one GPCR may participate in rapid oestrogen sig- nalling, and it is likely that further complexity in oestrogen- mediated GPCR signalling may occur as a result of coupling of different G protein heterodimers with the same receptor. Angiotensin II receptor is of particular interest as a candidate, oestrogen-interacting GPCR. Inwang and colleagues  demonstrated expression of angiotensin II type 1 (AT1) recep- tors in both normal and diseased human breast tissues. Other studies showed that activation of AT1 receptor stimulates growth factor pathways such as tyrosine kinase phosphoryla- tion and induces an increase in phospholipase C, leading to activation of downstream proteins such as MAPK , Janus kinases and STAT (signal transducers and activators of tran- scription) proteins . More recently, a study by Greco and colleagues  conducted in MCF-7 cells and primary breast cancer cells revealed that AT1 receptor regulates mitogenic signalling pathways by two simultaneous mechanisms, one
This suggests that oestrogen may mediate proliferation of hPASMCs and may contribute to PAH pathology. We investigated this further by examining ERa-mediated proliferation in female hPASMCs. We demon- strate for the first time that oestrogen can induce proliferation of hPASMCs via ERa activation. In addition, we show an ERb agonist, DPN, and a GPER agonist G1, have no effect on proliferation of PASMCs suggesting that ERa is the receptor that mediates estrogen- induced proliferation in hPASMCs. Moreover, we determine the pro-proliferative effect of oestrogen is dependent on activation of downstream PI3K/Akt and ERK/MAPK signalling. ERK and Akt signalling pathways are closely involved in cardiac hypertrophy and pulmonary vascular remodelling and oestrogen has been shown to regulate activa- tion of Akt signalling in right heart failure. 32,41 Additionally, selective activation of ERa in endothelial cells in aorta increases ERK expression
In this paper, we herein report on the predictive and prognostic factors in female patients with EBC including: axillary nodal status, tumour size, tumour histological type/grade, age at presentation and oestrogenreceptor (ER) status. These features were correlated with actual outcome, with the primary objective of determining which of these clinico-pathological features was most influential within our setting, and which offered the best prognostication for patients involved. Secondary objectives of this paper were also to document incidence, menopausal profiles, and histological features of patients with EBC within our region.
Other combinations involve therapies that target the HER family of growth factor receptors using either antigrowth factor-receptor antibodies (for example, tras- tu zumab) or small molecule tyrosine-kinase inhibitors (such as geﬁ tinib and lapatinib). A randomized trial of ﬁ rst-line geﬁ tinib plus anastrozole versus anastrozole alone in women with oestrogenreceptor (ER)-positive advanced breast cancer reported that patients who received the combination therapy experienced signiﬁ - cantly longer progression-free survival (PFS) and an improvement in clinical beneﬁ t rate, but a lower response rate [30,31]. In neither of the mentioned trials using the combination of geﬁ tinib plus anastrozole were patients selected on the basis of overexpression of growth factor receptors. How ever, two studies have included HER-2 status in selection criteria. Th us, in patients with known ER-positive/HER2-positive tumours, the addition of lapatinib to letrozole signiﬁ cantly reduced the risk of progression and im proved median PFS; clinical beneﬁ t rate was also signiﬁ cantly greater for the combination ; a preplanned analysis was also able to show an impact of combination therapy on PFS in the HER2- negative population. Finally, a randomized phase III trial in patients with known hormone receptor-positive/ HER2-positive metastatic breast cancer recently reported a doubling of PFS with the addition of trastuzumab to anastrozole compared with anastrozole alone . In these combination studies, it is possible that the additional beneﬁ t of targeted therapy is separate from the endocrine eﬀ ects of AIs. However, preclinical studies and measurements of biological markers suggest synergy or cross-talk between signalling systems. Th e hypothesis is therefore that acquired resistance to AIs in patients with ER-positive/HER2-negative tumours may be caused by adaptive epidermal growth factor receptor or HER2 upregulation and this might be prevented or delayed by agents directed against these targets.