ERβ1 downregulation occurs in many breast cancers but the responsible molecular mechanisms remain unclear. Here we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumours confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-UTR of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer.

BACKGROUND:

Cyclooxygenase-2 (COX-2) is selectively over-expressed in colorectal tumours. The mechanism of COX-2 induction in these tumours is not fully understood, although evidence suggests a possible link between nuclear factor (NF)-κB and COX-2. We hypothesised an association between COX-2 expression and NF-κB-p65, NF-κB-p50 and IκB-kinase-α (IKKα) in both epithelial and stromal cells in human colorectal cancer.

Methods:

Using immunohistochemistry, we measured COX-2, NF-κB-p65, NF-κB-p65 nuclear localisation sequence (NLS), NF-κB-p50, NF-κB-p50 NLS and IKKα protein expression in matched colorectal biopsy samples comprising both non-tumour and adjacent tumour tissue from 32 patients with colorectal cancer.

Results:

We have shown that stromal cells of malignant and surrounding normal colorectal tissue express COX-2. In all cell types of malignant tissue, and in vascular endothelial cells (VECs) of neighbouring normal tissue, COX-2 expression was strongly associated with NF-κB-p65 expression (Pearson's correlation, P=0.019 for macrophages, P=0.001 for VECs, P=0.002 for fibroblasts (malignant tissue), and P=0.011 for VECs (non-malignant tissue)) but not NF-κB-p50 or IKKα.

Conclusions:

These data suggest that in these cells COX-2 induction may be mediated through activation of the canonical NF-κB pathway. Finally, the lack of association between COX-2, NF-κB-p65 or IKKα in stromal cells with the clinical severity of colorectal cancer as determined by Duke's stage, suggests that COX-2, NF-κB-p65 and IKKα expression are possibly early post-initiation events, which could be involved in tumour progression.

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

Male breast cancer is uncommon, accounting for less than 1% of all breast cancers. Carcinoma metastatic to the male breast is also unusual, with metastatic prostatic carcinoma being among the most common primary sites from which such tumours derive. Metastatic prostatic cancer and primary breast cancer may be histologically indistinguishable without immunohistochemistry because both often infiltrate with a cribriform architecture. Distinguishing between primary and metastatic disease within the breast is important because the treatment options for each are radically different. Following a case in which metastatic prostatic disease was initially wrongly diagnosed as primary breast cancer, a small series of male breast cancers was examined for expression of prostate specific antigen (PSA) and prostatic acid phosphatase to assess the usefulness of these markers in making this distinction. Focal expression of PSA was found in one of 11 cases of male breast cancer. These results indicate that PSA should be used with caution in this context.

Aim: Two oestrogen receptors (ERs) have been identified to date—the “classic” ERα and the more recently described ERβ. Although much is known about ERα at the mRNA and protein levels, our knowledge of the expression and distribution of ERβ protein is much more limited. The aim of this study was to compare the cellular distribution of ERα and ERβ in normal human mammary gland.

Methods: Formalin fixed, paraffin wax embedded material was obtained from reduction mammoplasty specimens, normal tissue adjacent to breast tumour, or fibroadenoma. Sections were immunohistochemically stained for ERα, ERβ, and the progesterone receptor. The staining pattern for each antibody was evaluated and compared.

Results: ERα was restricted to the cell nuclei of epithelial cells lining ducts and lobules. Although ERβ was also seen in these cells, additional strong staining was detected specifically in the cell nuclei of myoepithelial cells. Occasional staining was seen in surrounding stromal and endothelial cell nuclei and in lymphocytes.

Conclusions: ER subtypes have distinct distribution patterns in the normal mammary gland. The widespread distribution of ERβ suggests that it may be the dominant ER in the mammary gland where it may be acting as a natural suppressor.

British Journal of Cancer (2002) 87, 687–687. doi:10.1038/sj.bjc.6600534 www.bjcancer.com

© 2002 Cancer Research UK

Oestrogen receptor (ER) is used routinely to predict endocrine responsiveness in patients with breast cancer. A second ER, ERβ has been described but its significance remains undefined; most studies have described mRNA levels rather than protein expression. Here, we demonstrate for the first time, immunohistochemical detection of ERβ in archival breast tumours. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17β-oestradiol (E2), leading to increases in reductive 17β-hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception α (ERα). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL-6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose-dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 p M. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERα+cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERα by IL-6. © 2000 Cancer Research Campaign

Enzymes modulating local steroid availability play an important role in the progression of human breast cancer. These include isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD), aromatase and steroid sulphatase (STS). The aim of this study was to investigate the expression, by reverse transcription polymerase chain reaction, of 17-HSD types I–IV, aromatase and steroid STS in a series of 51 human breast tumour biopsies and 22 primary cultures of epithelial and stromal cells derived from these tumours, giving a profile of the steroid-regulating network for individual tumours. Correlations between enzyme expression profiles and expression of the interleukin (IL)-6 gene were also sought. All except one tumour expressed at least one isoform of 17-HSD, either alone or in combination with aromatase and STS. Expression of 17-HSD isoforms I–IV were observed in nine tumours. Of the 15 tumours which expressed three isoforms, a combination of 17-HSD II, III and IV was most common (6/15 samples). The majority of tumours (n = 17) expressed two isoforms of 17-HSD with combinations of 17-HSD II and IV predominant (7/17 samples). Eight tumours expressed a single isoform and of these, 17-HSD I was in the majority (5/8 samples). In primary epithelial cultures, enzyme expression was ranked: HSD I (86%) > STS (77%) > HSD II (59%) > HSD IV (50%) = aromatase (50%) > HSD III (32%). Incidence of enzyme expression was generally reduced in stromal cultures which were ranked: HSD I (68%) > STS (67%) > aromatase (48%) > HSD II (43%) > HSD IV (28%) > HSD III (19%). Expression of IL-6 was associated with tumours that expressed ≥ 3 steroid-converting enzymes. These tumours were of higher grade and tended to come from patients with family history of breast cancer. In conclusion, we propose that these enzymes work in tandem with cytokines thereby providing sufficient quantities of bioactive oestrogen from less active precursors which stimulates tumour growth. © 1999 Cancer Research Campaign

As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.

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Timing of surgery in premenopausal patients with breast cancer remains controversial. Angiogenesis is essential for tumour growth and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic cytokines. We aimed to determine whether the study of VEGF in relation to the menstrual cycle could help further the understanding of this issue of surgical intervention. Fourteen premenopausal women were recruited, along with three post-menopausal women, a woman on an oral contraceptive pill and a single male subject. Between eight and 11 samples were taken per person, over one menstrual cycle (over 1 month in the five controls) and analysed for sex hormones and VEGF165. Serum VEGF was significantly lower in the luteal phase and showed a significant negative correlation with progesterone in all 14 premenopausal women. No inter-sample variations of VEGF were noted in the controls. Serum from both phases of the cycle from one subject was added to MCF-7 breast cancer cells; VEGF expression in the supernatant was lower in the cells to which the luteal phase serum was added. The lowering of a potent angiogenic cytokine in the luteal phase suggests a possible decreased potential for micrometastasis establishment in that phase. This fall in VEGF may be an effect of progesterone and should be the focus of future studies.

The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified.

Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

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