Estrogen receptor ß exon 3-deleted mouse: The importance of non-ERE pathways in ERß signaling.

In 1998, an estrogen receptor ß (ERß) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERß mutant mouse was generated by deleting ERß exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ERß. In the present study, we created an ERß exon 3-deleted mouse (ERß-Δex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ERß protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ERß antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ERß-Δex3 protein. Moreover, with nuclear extracts from ERß-Δex3 prostates, there was an ERß-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ERß-Δex3 mouse. We conclude that very little of ERß transcriptional activity depends on binding to classical estrogen response elements (EREs).

Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients.

BACKGROUND: ESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations. METHODS: We screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations (E380Q, Y537S, Y537N, Y537C, and D538G) using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated. RESULTS: We detected ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment. CONCLUSIONS: We have shown that there are distinct populations of ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.

Differential expression of estrogen receptor α, ß1, and ß2 in lobular and ductal breast cancer.

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERß1 and ERß2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERß. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERß-negative disease, lobular cancer expresses both ERα and ERß but with very few Ki67-positive cells. ERß2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERß2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERß2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERß was expressed, but in the high-grade lobular cancer ERß was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERß agonists in preventing in situ ductal cancers from becoming invasive.

Clinical significance of estrogen receptor ß in breast and prostate cancer from biological aspects.

Breast and prostate cancers are among the most common of all cancers. They are referred to as hormone-dependent cancers, because estrogen and androgen are involved in their development and growth. The effects of these hormones are mediated by their respective receptors, estrogen receptor (ER) α and androgen receptor. Around 18 years ago, a second ER, ERß, which has a very similar structure to ERα, was discovered. Its function has been investigated using a variety of methods and biological systems, leading to our present understanding that ERß can interact with or inhibit ERα and androgen receptor function directly and/or indirectly, suppress cell growth, and influence responsiveness to endocrine therapy. In order to apply the "inhibition of cell growth" function to cancer treatment, several specific ERß agonists have been synthesized and are being tested for effectiveness in cancer treatment. We need to keep our eyes on ERß.

Prognostic role of PIK3CA mutations of cell-free DNA in early-stage triple negative breast cancer.

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3-kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor-positive breast cancer patients and, during the past few years, PIK3CA mutations of cell-free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early-stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse-free survival (RFS; P = 0.0072) and breast cancer-specific survival (BCSS; P = 0.016), according to the log-lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early-stage TNBC patients and it was associated with PI3K pathway-dependent AR phosphorylation.

Estrogen receptor ß represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity.

INTRODUCTION: The inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERß in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERß can modulate pathways implicated in endocrine resistance development. METHODS: T47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERß were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit. RESULTS: Expression of ERß in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERß. Furthermore, ERß increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERß expression increased the sensitivity of these breast cancer cells to tamoxifen. CONCLUSIONS: Our results suggest a link between expression of ERß and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERß presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERß as a target in selected groups of breast cancer.

Correction: Estrogen Signalling and the Metabolic Syndrome: Targeting the Hepatic Estrogen Receptor Alpha Action.

[This corrects the article DOI: 10.1371/journal.pone.0057458.].

Estrogen receptor-alpha signaling in growth of the ventral prostate: comparison of neonatal growth and postcastration regrowth.

A role for estrogen receptor (ER)-alpha in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ERalpha(-/-) mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ERalpha(-/-) mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ERalpha and FGF10. ERalpha was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ERalpha or ERbeta with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ERalpha agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3-5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ERalpha is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ERalpha can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.

Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients.

BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). RESULTS: Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients.

BACKGROUND: The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. METHODS: The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. RESULTS: cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. CONCLUSIONS: We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Sex differences and effects of oestrogen in rat gastric mucosal defence.

AIM: To evaluate sex differences and the effects of oestrogen administration in rat gastric mucosal defence. METHODS: Sex differences in gastric mucus thickness and accumulation rate, absolute gastric mucosal blood flow using microspheres, the integrity of the gastric mucosal epithelium in response to a chemical irritant and the effects of oestrogen administration on relative gastric mucosal blood flow in an acute setting was assessed in an in vivo rat experimental model. Subsequently, sex differences in the distribution of oestrogen receptors and calcitonin gene related peptide in the gastric mucosa of animals exposed to oestrogen in the above experiments was evaluated using immunohistochemistry. RESULTS: The absolute blood flow in the GI-tract was generally higher in males, but only significantly different in the corpus part of the stomach (1.12 ± 0.12 mL/min•g in males and 0.51 ± 0.03 mL/min•g in females) (P = 0.002). After removal of the loosely adherent mucus layer the thickness of the firmly adherent mucus layer in males and females was 79 ± 1 µm and 80 ± 3 µm respectively. After 60 min the mucus thickness increased to 113 ± 3 µm in males and 121 ± 3 µm in females with no statistically significant difference seen between the sexes. Following oestrogen administration (0.1 followed by 1 µg/kg•min), mean blood flow in the gastric mucosa decreased by 31% [68 ± 13 perfusion units (PFU)] in males which was significantly different compared to baseline (P = 0.02). In females however, mean blood flow remained largely unchanged with a 4% (5 ± 33 PFU) reduction. The permeability of the gastric mucosa increased to a higher level in females than in males (P = 0.01) after taurocholate challenge. However, the calculated mean clearance increase did not significantly differ between the sexes [0.1 ± 0.04 to 1.1 ± 0.1 mL/min•100 g in males and 0.4 ± 0.3 to 2.1 ± 0.3 mL/min•100 g in females (P = 0.065)]. There were no significant differences between 17ß-Estradiol treated males (mean ratio of positive staining ± SEM) (0.06 ± 0.07) and females (0.11 ± 0.11) in the staining of ERα (P = 0.24). Also, there were no significant differences between 17ß-Estradiol treated males (0.18 ± 0.21) and females (0.06 ± 0.12) in the staining of ERß (P = 0.11). Finally, there were no significant differences between 17ß-Estradiol treated males (0.04 ± 0.05) and females (0.11 ± 0.10) in the staining of CGRP (P = 0.14). CONCLUSION: Gastric mucosal blood flow is higher in male than in female rats and is reduced in male rats by oestrogen administration.

Expression of estrogen receptor (ER) (beta)cx protein in ER(alpha)-positive breast cancer: specific correlation with progesterone receptor.

Estrogen receptor (ER) (beta)cx, a splice variant of ERbeta, is a dominant repressor of ER(alpha) function. In this study we investigated the possibility that because the progesterone receptor (PR) gene is a downstream target of activated ER(alpha), in ER(alpha)-positive breast cancers, expression of ER(beta)cx would result in repression of PR. In ER(alpha)-positive MCF-7 cells, stable transfection of an ER(beta)cx expression vector resulted in reduced expression of PR without affecting ER(alpha) expression. In breast cancers, immunohistochemical evaluation of ER(alpha)-positive foci for the expression of PR and ER(beta)cx revealed a significant correlation between a PR-negative phenotype and the presence of ER(beta)cx within the foci. However, when entire lesions were evaluated by Allred scoring in 115 ER(alpha)-positive breast cancer specimens, the presence of two distinct groups of patients could be discerned. One group expressed ER(beta)cx and had very reduced levels of PR expression, as expected. The second group showed both ER(beta)cx and high levels of PR. To evaluate the role of ER(beta)cx in sensitivity to tamoxifen, 18 core needle biopsies, obtained before preoperative treatment with tamoxifen, were investigated. The results show that expression of ER(beta)cx in primary lesions correlated with a poor response to tamoxifen, especially in cancers with a low PR expression in Allred score. This is the first evidence that evaluation of ER(beta)cx along with PR may contribute to a better characterization of ER(alpha)-positive breast cancers.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients.

BACKGROUND: The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. RESULTS: ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). METHODS: A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). CONCLUSIONS: We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Estrogen receptor (ER) beta1 and ERbetacx/beta2 inhibit ERalpha function differently in breast cancer cell line MCF7.

Estrogen receptor (ER) alpha plays an important role in the proliferation and progression of breast cancer. In order to explore the function of wild-type ERbeta (ERbeta1) and its variant form, ERbetacx/beta2, stable transformants of ERalpha-positive breast cancer MCF7 cells with ERbeta1 or ERbetacx/beta2 expression vector were established. Constitutive expression of ERbeta1 or ERbetacx/beta2 reduced the S phase population of the cell cycle in dish culture and the number of colonies in an anchorage-independent assay. DNA-protein complexes of ERE with nuclear extracts from ERbeta1 transformants were observed in the electrophoretic mobility shift assay, while no complex was observed for ERbetacx/beta2 transformants. Reporter gene assay using estrogen-responsive element (ERE)-luciferase showed less responsiveness to estrogen in these transformants compared with parental cells. Endogenous mRNA expression of two known estrogen-responsive genes, cathepsin D and IGFBP4, was weakly induced by estrogen in ERbeta1 and ERbetacx/beta2 transformants compared with parental cells. A comprehensive gene expression analysis using our custom-made cDNA microarray showed that MCF7 and ERbeta1 transformants had a similar gene expression profile, whereas ERbetacx/beta2 showed a distinct profile from others. These results indicate that ERbeta1 and ERbetacx/beta2 inhibit ERalpha function differently in MCF7 cells.

Differential regulation of estrogen receptor (ER)alpha and ERbeta in primate mammary gland.

Estrogen, mainly estradiol (E2), and progesterone (P) are essential for the growth and differentiation of the breast, but their roles in breast cancer are highly debated. To understand how E2 and P influence cell proliferation and differentiation, it is essential to know how their receptors are regulated. Because of limited tissue availability, little is known about regulation of the two estrogen receptors (ERalpha and ERbeta) and the two progesterone receptor isoforms (PR-A and PR-B) in the normal human breast. What we know comes from rodent studies, which are not always pertinent for the human breast. We report now on regulation of gonadal hormone receptors during the menstrual cycle, pregnancy, and lactation in rhesus monkey mammary gland and on the relationship of these receptors to proliferation. We found that ERalpha but not ERbeta is down-regulated when E2 levels increase and when cells enter the cell cycle. PR-B but not PR-A is expressed in proliferating cells. Thus under normal conditions, the ratio of ERalpha to ERbeta in the breast depends on plasma concentrations of E2. Elevated expression of ERalpha (as occurs in postmenopausal women) is a normal response to loss of E2 and indicates nonproliferating cells. As selective receptor ligands become available, they will be helpful in delineation of the functions of these receptors.

HDAC6 expression is correlated with better survival in breast cancer.

PURPOSE: The structure and function of chromatin can be altered by modifications to histone. Histone acetylation in vivo is a dynamic reversible process governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC6 is a unique isoform among the HDACs, and a gene expression pattern study, with cDNA microarray in MCF-7 cells, showed the HDAC6 gene to be late responsive, estrogen induced, and up-regulated. This led us to hypothesize that there was a link between levels of HDAC6 expression and the metastatic potential of breast cancer and also, therefore, the prognosis of these patients. EXPERIMENTAL DESIGN: In the present study, the level of HDAC6 mRNA expression was analyzed with quantitative real-time reverse transcription-PCR, in 135 female patients with invasive breast cancer. HDAC6 protein expression was also determined by immunohistochemistry. An association was sought between HDAC6 expression and various clinicopathologic factors. RESULTS: HDAC6 mRNA was expressed at significantly higher levels in breast cancer patients with small tumors measuring less than 2 cm, with low histologic grade, and in estrogen receptor alpha- and progesterone receptor-positive tumors. By contrast, no relationship was found between HDAC6 mRNA expression and any of the other clinicopathologic factors, namely, age, menopausal status, and axillary lymph node involvement. Patients expressing high levels of HDAC6 mRNA and protein had a better prognosis than those expressing low levels, in terms of disease-free survival. However, multivariate analysis failed to show that HDAC6 mRNA and protein are an independent prognostic factors for disease-free survival and overall survival. Furthermore, the patients with high levels of HDAC6 mRNA tended to be more responsive to endocrine treatment than those with low levels. Specific HDAC6 staining was found in the nucleus of some normal epithelial cells and in the cytoplasm of the majority of cancer cells. Although postmenopausal patients showed higher HDAC6 protein expression, there were no relationship between protein expression and any other clinicopathologic factors. CONCLUSIONS: We conclude that the levels of HDAC6 mRNA expression may have potential both as a marker of endocrine responsiveness and also as a prognostic indicator in breast cancer. Additional investigations are warranted concerning the relationship between HDAC6 expression and response to endocrine therapy.

The expression of ER beta cx in human breast cancer and the relationship to endocrine therapy and survival.

PURPOSE: Estrogen receptor (ER) alpha-positive breast cancer is often treated with endocrine therapy using either antiestrogens or aromatase inhibitors. However, 30% of patients who receive endocrine therapy will derive no benefit from such treatments and may indeed suffer adverse effects. Currently, there are no ways to predict response to such treatments. ER beta cx, a variant of ER beta, has a dominant-negative effect over ER alpha, and its expression thought to modulate response to endocrine treatment may represent a predictor of response to endocrine therapy. EXPERIMENTAL DESIGN: We investigated the expression of the ER beta cx in 82 frozen breast samples (8 benign, 1 ductal carcinoma in situ, and 73 malignant) by Western blot analysis. The relationship between the expression of ER beta cx variants with prognosis and outcome of endocrine therapy was examined. RESULTS: There was a statistically significant association between the presence of ER beta cx and the response to endocrine therapy (Fisher's exact test, P = 0.04). We also examined the influence of the ER beta cx status of a tumor on time to progression and death. There was a relationship between the presence of ER beta cx and survival, with patients whose tumors express ER beta cx having a longer survival rate (P = 0.05). Cell-type specificity of expression was assessed by immunohistochemistry on a selection of histological samples. CONCLUSIONS: On the basis of this small group of patients, we conclude that the expression of ER beta cx correlated with favorable response to endocrine therapy. A larger study involving the staining of archival material is currently underway to confirm these preliminary results.

Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens.

Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/µL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.

Differential role of MACC1 expression and its regulation of the HGF/c­Met pathway between breast and colorectal cancer.

The newly identified gene, metastasis­associated in colon cancer 1 (MACC1), is suggested to be a transcriptional regulator of c­Met, leading to cancer progression in colorectal cancer. To date however, little is known of the role of MACC1 in breast cancer. In a series of 300 breast cancer patients, we analyzed the association of MACC1 mRNA and protein expression with breast cancer survival using Cox proportional hazard models. In an in vitro study, we evaluated activities of c­Met protein after transfection with a MACC1­harboring plasmid as well as the binding ability of MACC1 to the c­Met promoter using a chromatin immunoprecipitation (ChIP) assay. In survival analyses, reduced MACC1 expression was associated with patient mortality. MACC1 expression was an independent prognostic factor in multivariate analysis. In the cell lines tested, MACC1 expression was much higher in colorectal than in breast cancer cells. After cells were transfected with MACC1, c­Met expression was not induced in MCF7 cells, whereas corresponding c­Met expression was upregulated in SW480 cells. Further, SW480 cells transfected with MACC1 showed enhanced migratory ability, whereas in MDA­MB­231 cells, transfection of MACC1 had no impact on this ability. In ChIP assay, the binding of MACC1 to the c­Met promoter was suggested in SW480 cells, but not in MCF7 cells. In conclusion, our findings provide some novel insights into the role of MACC1 in breast cancer, indicating that it plays different roles in breast and several other cancers. There is a possibility that MACC1 does not modulate the transcriptional role of c­Met signaling in breast cancer.

Immunohistochemical analysis in ethinylestradiol-treated breast cancers after prior long-term estrogen-deprivation therapy.

BACKGROUND: Estrogen receptor (ER) positive breast cancer can often be treated by hormone therapy; however a certain population of ER-positive patients become resistant to hormone therapy after long-term hormone treatment. Ethinylestradiol (EE2) is a derivative of estrogen, which has shown promising effects in these patients. METHODS: We successfully obtained tissue samples from 6 patients undergoing EE2 treatment and examined 13 well-known breast cancer-related factors by immunohistochemistry. Of the 6 patients, 5 responded but one patient did not. RESULTS: Before EE2 treatment, staining for both ER and androgen receptor (AR) was strong in the nucleus, and the progesterone receptor (PgR) was almost no staining. EE2 treatment significantly down-regulated ER and up-regulated PgR while nuclear and cytosolic AR were oppositely down- and up-regulated, respectively. Cytosolic staining of BRCA1 was significantly up-regulated by EE2 whereas nuclear staining tended to decrease. Individual comparisons suggested less induction of PgR and decreasing AKT but increasing pAKT in the non-responder following EE2 treatment. CONCLUSIONS: Our observations revealed that EE2 activated ER downstream genes; however it did not stimulate cell growth. This suggests that hormone resistant cells might receive growth signals from a non-genomic pathway and this may be reflected in their sensitivity to EE2 treatment.