Steroid sequestration and tightly bound oestrogen-protein complexes in human breast tumours and a breast cancer cell line

Homogenates of breast tumours taken at surgery were prepared in phosphate-buffered medium in the presence or absence of the protease inhibitors N-alpha-p-tosyl-L-arginine methyl ester (TAME, 10 mM) or soy bean trypsin inhibitor (STI, 1 mg/ml). Aliquots (0.25 ml) were incubated in 5 ml medium, with the addition of excess trypsin (2 mg/ml) to experimental flasks. Oestrogen was measured by means of a radioreceptor assay (RRA) based on rat or human uterine cytosolic oestradiol receptor. In oestrogen receptor positive (ER +ve) tumour homogenates, TAME decreased while STI increased ethyl acetate extractable oestrogen in these preparations. The addition of trypsin enhanced yields of oestrogen in the TAME, but not in the STI or control (no inhibitor) preparations. None of these treatments affected RRA detectable oestrogen in homogenates of ER -ve tumours. Suspensions of ZR-75-1 cells, prepared in Krebs Ringer bicarbonate (KRBG) incubated with trypsin also gave greatly enhanced yields of extractable oestrogen. Fractionation of oestrogens from both tumour homogenates and from the cell line showed coincidence of RRA detectable steroid with oestradiol and oestrone, and, particularly in trypsin flasks, very non-polar components were also found. In the cell-line extracts, HPLC fractionation combined with specific radioimmunoassays confirmed the presence of both oestradiol and oestrone. The major extracted component was oestrone. The data suggest the existence within breast tumour tissue of sequestered pools of steroid requiring proteolytic action for their release. One possibility, consistent with reports in the literature, is that the steroids may themselves be directly conjugated to protein. Their presence in ER +ve but not ER -ve tumours strongly suggests some relationship to the development of hormone-sensitive disease. Alternatively, the phenomenon may be associated with the rigid compartmentalization of the paracrine function of the tissue.     

Epidermal growth factor receptors in breast cancer: association with early relapse and death, poor response to hormones and interactions with neu

Epidermal growth factor receptors (EGFRs) were measured in 221 primary breast cancers by ligand binding with 125I-labelled EGF, and high-affinity sites were quantitated. There was a highly significant inverse relationship between oestrogen receptor (ER) and EGFR (15 EGFR-positive [EGFR+]ER+ and 92 EGFR-negative [EGFR-]ER+: 54 EGFR- ER- and 60 EGFR+ ER-). The relapse-free survival and overall survival were significantly shorter for EGFR+ vs EGFR- tumours (P less than 0.001) by about 2 yr in the case of relapse-free survival. When ER- tumours were substratified by EGFR status, the EGFR- ER- tumours had a prognosis almost as good as the ER+ tumours. In 31 of 184 cases, high expression of neu, correlating with amplification, was found. Expression of neu conferred similar poor prognosis to EGFR expression in all prognostic subgroups. Coexpression of neu and EGFR had an additive adverse effect. Epidermal growth factor receptors (EGFR) and oestrogen receptors (ER) were analysed in 221 patients with primary operable breast cancer by means of radioligand assays. After median follow-up of 24 months (range 3-60 months), there had been recurrences in 99 patients, of whom 72 (median age 56 yr, range 32-77 yr) received tamoxifen alone as first-line treatment for recurrence. 14 patients (19%) showed a response to this therapy and 58 (81%) did not. Of 32 ER+ tumours, 12 (37.5%) showed an objective response to tamoxifen compared with only 2 of 40 (5%) ER- tumours (P less than 0.005). Of 35 EGFR+ tumours, 3 (8.5%) achieved an objective response compared with 11 of 36 (30%) EGFR tumours (P less than 0.05). Only 1 of 28 EGFR+, ER- tumours achieved an objective response. Including patients whose disease remained stable for more than 6 months with the responders, however, EGFR status was a better predictor of response to tamoxifen; 15 of 37 EGFR- patients and 5 of 35 EGFR+ patients responded (P less than 0.01).     

Modulation of oestrone sulphatase activity in breast cancer cell lines by growth factors

Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E₁) and oestradiol. As the formation of E₁ from its sulphate (E₁S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.     

Role of inflammation and oxidative stress in post‐menopausal oestrogen‐dependent breast cancer

Weight gain and obesity are among the most important risk factors for post-menopausal oestrogen-dependent breast cancer (EDBC). Weight gain is associated with oxidative stress, which in turn promotes breast cancer progression. We carried out a prospective study in 216 consecutive post-menopausal breast cancer patients aiming to examine the correlations between traditional prognostic factors (tumour size, T, nodal, N, grading, G, and metastasis status, M), and body mass index (BMI), leptin, pro-inflammatory cytokines (Interleukin, IL,-6 and tumour necrosis factor-alpha, TNF-α), and oxidative stress (reactive oxygen species, ROS, glutathione peroxidase, GPx, superoxide dismutase, SOD) among patients with oestrogen receptor (ER)+ and ER− breast cancers. Distribution of T, N and M categories did not differ between ER+ and ER− breast cancer patients. ER− patients showed a higher incidence of G3 tumours. Weight, BMI, leptin, IL-6 and ROS were higher in ER+ compared with ER− patients. Among ER+ patients, BMI, leptin, IL-6 and ROS correlated with T and M. Leptin, IL-6 and ROS were positively correlated also with N. Among ER− patients, BMI and leptin did not correlate with any of prognostic parameters, whereas a positive correlation between IL-6, ROS and M was found. Multivariate regression analysis showed that BMI, leptin, IL-6 and ROS were predictive for T, N and M in ER+ patients. Weight gain, inflammation and oxidative stress are involved in EDBC prognosis. Their modulation through antidiabetic, anti-inflammatory and antioxidants drugs combined with endocrine therapy may constitute a targeted approach in post-menopausal EDBC.     

Association between breast cancer subtypes and response to neoadjuvant anastrozole

Considerable heterogeneity exists amongst oestrogen receptor positive (ER+ve) breast cancer in both its molecular profile and response to therapy. Attempts to better define variation amongst breast tumours have led to the definition of four main “intrinsic” subtypes of breast cancer with two of these classes, Luminal A and B, composed almost entirely of ER+ve cancers. In this study we set out to investigate the significance of intrinsic subtypes within a group of ER+ve breast cancers treated with neoadjuvant anastrozole. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with anastrozole was analyzed on Illumina 48K microarrays. Gene-expression based subtypes and risk of relapse (ROR) scores for tumours pre- and post-treatment were determined using the PAM50 method. Amongst pre-treatment samples, all intrinsic subtypes were found to be present, although luminal groups were represented most highly. Luminal A and B tumours obtained similar benefit from treatment, as measured by the proportional fall in the proliferation marker Ki67 upon treatment (mean suppression = 75.5% vs 75.7%). Tumours classified as basal and Her2-like showed poor reductions in Ki67 upon treatment. Residual Ki67 staining after two weeks remained higher in the Luminal B group. ROR score was significantly associated with anti-proliferative response to AI and with clinical response. These results suggest that in the short-term, Luminal A and B tumours may gain similar benefit from an AI but that the higher residual Ki67 level seen in Luminal B is indicative of poorer long term outcome.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.     

TWIST represses estrogen receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.     

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.     

Minimising immunohistochemical false negative ER classification using a complementary 23 gene expression signature of ER status

Background

Expression of the oestrogen receptor (ER) in breast cancer predicts benefit from endocrine therapy. Minimising the frequency of false negative ER status classification is essential to identify all patients with ER positive breast cancers who should be offered endocrine therapies in order to improve clinical outcome. In routine oncological practice ER status is determined by semi-quantitative methods such as immunohistochemistry (IHC) or other immunoassays in which the ER expression level is compared to an empirical threshold[1], [2]. The clinical relevance of gene expression-based ER subtypes as compared to IHC-based determination has not been systematically evaluated. Here we attempt to reduce the frequency of false negative ER status classification using two gene expression approaches and compare these methods to IHC based ER status in terms of predictive and prognostic concordance with clinical outcome.

Methodology/Principal Findings

Firstly, ER status was discriminated by fitting the bimodal expression of ESR1 to a mixed Gaussian model. The discriminative power of ESR1 suggested bimodal expression as an efficient way to stratify breast cancer; therefore we identified a set of genes whose expression was both strongly bimodal, mimicking ESR expression status, and highly expressed in breast epithelial cell lines, to derive a 23-gene ER expression signature-based classifier. We assessed our classifiers in seven published breast cancer cohorts by comparing the gene expression-based ER status to IHC-based ER status as a predictor of clinical outcome in both untreated and tamoxifen treated cohorts. In untreated breast cancer cohorts, the 23 gene signature-based ER status provided significantly improved prognostic power compared to IHC-based ER status (P = 0.006). In tamoxifen-treated cohorts, the 23 gene ER expression signature predicted clinical outcome (HR = 2.20, P = 0.00035). These complementary ER signature-based strategies estimated that between 15.1% and 21.8% patients of IHC-based negative ER status would be classified with ER positive breast cancer.

Conclusion/Significance

Expression-based ER status classification may complement IHC to minimise false negative ER status classification and optimise patient stratification for endocrine therapies.     

Characterization and biological relevance of a 29-kDa, oestrogen receptor-related protein

The properties of a monoclonal antibody (D5) that can immunoprecipitate human oestradiol receptor (ER) under some but not all conditions are described. The antibody recognises a 29-kDa serine phosphoprotein that is qualitatively and quantitatively related to ER but not other steroid receptors or binding proteins. p29 will not complex with untreated cytosol ER but, after ammonium sulphate, KCl, heat or phosphatase treatments, interaction occurs that can be detected by immunoprecipitation with D5; molybdate and GTP inhibit complex formation. In human endometrium, p29 is increased by oestrogen and decreased by progestins. IRMA and histochemical assays for p29 have been developed and applied to a large series of human breast tumours. Most, but not all ER+ tumours are p29+, whilst ER-tumours are rarely p29+ unless they are also PR+. p29 predicts for clinical response to hormone therapy. ER+ p29+ tumours have a higher response rate than the ER+ p29-tumours. We do not know if p29 is a previously undetected component of the oestradiol receptor machinery or whether it is a product of oestrogen action.     

Discrepancies between antibody (EIA) and saturation analysis of oestrogen receptor content in breast tumour samples

The use of different techniques for assay of oestrogen receptors (ER) in breast cancer raises the question of their relative effectiveness in measuring concentrations of functional receptors. Data were obtained on soluble receptors from supernatants from 58 primary breast tumour homogenates, using the ligand ([3H]oestradiol) binding assay with dextran-coated charcoal (DCC) separation, either at a single saturating ligand dose, or by Scatchard analysis, and by using the Abbott enzyme immunoassay (EIA) kit. As previous reports have shown, the two methods gave reasonably good correlation (r = 0.8), but EIA values were systematically higher than DCC (slope = 3.0). Similar values were obtained when the ER + ve/progesterone receptor (PR) + ve subgroup were examined separately (n = 34, r = 0.86, slope = 3.0). However the two sets of data were in much better agreement in the ER + ve/PR - ve subgroup (n = 10, r = 0.98, slope = 1.24). When analysed by isoelectric focusing on polyacrylamide gels (IEF), two major specific binding components were identified, at pI 6.1 and at pI 6.6. Both isoforms were present in 50/66 ER + ve PR + ve breast tumour samples, but only the pI 6.6 (4S) was present in most ER + ve/PR - ve samples (13/20). It appears that, compared with DCC, the EIA method gives much higher values for the 8S isoform, whereas the two methods detect the 4S isoform with similar sensitivity. In assays on the tumour cell lines, T47D and MCF-7, still greater discrepancies, at least 10-fold, were found between EIA and DCC data.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

Aberrant oestrogen receptor species in human meningioma tissue.

Meningiomas are very rich in progestin receptors (PR) whereas oestrogen receptors (ER) are seldomly found and only at low concentrations. These tumours might possess an ER which is defective in oestrogen binding but still functional in stimulating oestrogen-responsive genes such as PR. In human meningiomas a polymerase chain reaction fragment including the DNA binding domain, the hinge region and the ligand binding domain of ER was amplified. The size of the fragment obtained was as expected from wild type mRNA sequences. Moreover, a variant, which was overexpressed in meningiomas, with a major deletion in exons 2-6 was detected.     

Wnt Pathway Activity in Breast Cancer Sub-Types and Stem-Like Cells

Introduction

Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.

Methods

We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.

Results

Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.

Conclusions

Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.     

Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.