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Activation of rapid oestrogen signalling in aggressive human breast cancers.

ABSTRACT: Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ER? is required for the formation of the ER?/Src/PI3K complex and that ER? is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.

SUBMITTER: Poulard C

PROVIDER: S-EPMC3494876 | biostudies-other | 2012 Nov

REPOSITORIES: biostudies-other

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Activation of rapid oestrogen signalling in aggressive human breast cancers.

Poulard Coralie C Treilleux Isabelle I Lavergne Emilie E Bouchekioua-Bouzaghou Katia K Goddard-Léon Sophie S Chabaud Sylvie S Trédan Olivier O Corbo Laura L Le Romancer Muriel M

EMBO molecular medicine 20121015 11

Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well ...[more]

PMID: 23065768

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Project description:IntroductionAn emerging hypothesis suggests that cytokines could play an important role in cancer as potential modulators of angiogenesis and leucocyte infiltration.MethodsA novel multiplexed flow cytometry technology was used to measure the expression of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 [p70], IL-13, IL-17, granulocyte colony-stimulating factor [CSF], granulocyte-macrophage CSF, IFN-gamma, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1beta, tumour necrosis factor [TNF]-alpha) at the protein level in 105 breast carcinoma. B lymphocyte, T lymphocyte and macrophage levels were determined by immunohistochemistry.ResultsFourteen of the 17 cytokines were expressed in breast carcinoma, whereas only nine cytokines could be detected in normal breast. Most cytokines were more abundant in breast carcinoma than in normal breast, with IL-6, IL-8, granulocyte CSF, IFN-gamma, MCP-1 and MIP-1beta being very abundant. IL-2, IL-6, IL-8, IL-10, IFN-gamma, MCP-1, MIP-1beta and TNF-alpha, and to a lesser extent IL-1beta and IL-13 exhibited levels of expression that were inversely correlated to oestrogen receptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1beta, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP-1beta were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1beta 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-1beta 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.ConclusionWe found that multiple cytokines were overexpressed in oestrogen receptor negative breast carcinoma, and that the three major cytokines--MCP-1, MIP-1beta and IL-8--were correlated with inflammatory cell component, which could account for the aggressiveness of these tumours.

Project description:PurposeOestrogen receptor β is believed to exert a cardioprotective effect against ischaemic injury. Nonetheless, the mechanism underlying its protective action remains to be fully elucidated. Recently, increased attention has been focused on Notch1 signalling for ameliorating cardiac ischaemic injury. Here, we hypothesised that oestrogen receptor β activation attenuates myocardial infarction (MI)-induced cardiac damage by modulating the Notch1 signalling pathway.MethodsMale C57BL/6 mice were used to establish an MI model through the ligation of the anterior descending branch of the left coronary artery. Two chemical drugs, 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN) and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester (DAPT), a specific inhibitor of Notch1 signalling) were administered via intraperitoneal injection to change oestrogen receptor β and Notch1 activities. Immunohistochemistry, western blot analysis, enzyme-linked immunosorbent assay (Elisa) assessment and echocardiography were used in this study to analyse cardiac oxidative stress, apoptosis, infraction volume, fibrosis and cardiac function.ResultsDPN-mediated oestrogen receptor β activation effectively protected cardiomyocytes from MI-induced oxidative damage and apoptosis. Furthermore, oestrogen receptor β activation reduced the infarct size and lowered the levels of myocardial enzymes in the serum, thereby leading to greater overall cardiac function improvement. Ischaemic injury-induced myocardial fibrosis was attenuated by oestrogen receptor β activation. Nevertheless, all of these cardioprotective effects of oestrogen receptor β activation were almost abrogated by DAPT administration, i.e. DAPT attenuated the anti-oxidative and anti-apoptotic effects and the decrease in infarct and fibrotic areas and reversed cardiac functional recovery. The levels of phospho-phosphatidylinositol-3-kinase (PI3K) and phospho-protein kinase B (Akt) were increased after DPN administration, and this change was reversed after DAPT was administered.ConclusionsAll of these new findings indicate that oestrogen receptor β activation is effective in ameliorating MI-induced cardiac dysfunction by enhancing Notch1 signalling and that PI3K/Akt signalling is the downstream mediator.

Project description:IntroductionOestrogens can mediate some of their cell survival properties through a nongenomic mechanism that involves the mitogen-activated protein kinase (MAPK) pathway. The mechanism of this rapid signalling and its dependence on a membrane bound oestrogen receptor (ER), however, remains controversial. The role of G-protein-coupled receptor and epidermal growth factor (EGF) receptor in an ER-independent signalling pathway modulated by oestrogen was investigated.MethodsER-positive and ER-negative breast cancer cell lines (MCF-7 and SKBR3) and primary breast cancer cell cultures were used in this study. Cell proliferation was assessed using standard MTT assays. Protein and cAMP levels were detected by Western blotting and ELISA, respectively. Antigen localization was performed by immunocytochemistry, immunohistochemistry and immunofluorescence. Protein knockdown was achieved using small interfering RNA technologies.ResultsEGF and oestrogen, alone and in combination, induced cell proliferation and phosphorylation of MAPK proteins Raf and ERK (extracellular signal regulated kinase)1/2 in both ER-negative SKBR3 and ER-positive MCF-7 human breast cancer cell lines. Increased Raf phosphorylation was also observed in primary human breast cultures derived from ER-positive and ER-negative breast tumours. Oestrogen induced an increase in intracellular cAMP in ER-negative SKBR3 human breast cancer cells. Oestrogen-mediated cell growth and phosphorylation of MAPK was modified by the EGF receptor antagonist AG1478, the G-protein antagonist pertussis toxin, and the angiotensin II receptor antagonist saralasin. Knockdown of angiotensin II type 1 receptor (AT1) protein expression with small interfering RNA attenuated oestrogen-induced Raf phosphorylation in ER-negative cells. AT1 receptor was found to be expressed in the cell membrane of breast tumour epithelial cells.ConclusionThese findings provide evidence that, in breast cancer cells, oestrogen can signal through AT1 to activate early cell survival mechanisms in an ER-independent manner.

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Activation of rapid oestrogen signalling in aggressive human breast cancers.

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Activation of rapid oestrogen signalling in aggressive human breast cancers.

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