Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

peer reviewedRationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors

Versatile multicharacterization platform involving tailored superhydrophobic SU-8 micropillars for the investigation of breast cancer estrogen receptor isoforms

International audienceHere, the authors report the fabrication of lotus-leaf-like tailored SU8 micropillars and their application in the context of a multitechnique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, as it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) is also expressed in ERα positive breast cancers and breast cancer cell lines. The superhydrophobic supports were developed by using a two-step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the biological samples resulted in stretched fibers of different diameters which were then characterized by synchrotron x-ray diffraction (XRD), Raman and Fourier-transform infrared spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biomarkers by exploiting a multitechnique in situ characterization approach

The antagonist properties of Bazedoxifene after acute treatment are shifted to stimulatory action after chronic exposure in the liver but not in the uterus

International audienceA promising alternative to conventional hormone therapy for postmenopausal symptoms is treatment combining Bazedoxifene (BZA), a third-generation selective estrogen receptor modulator (SERM), and conjugated equine estrogen (CE). This combination is also known as a tissue-selective estrogen complex (TSEC). Understanding the tissue-specific actions of SERMs and the TSEC remains a major challenge to try to predict their clinical effects. The aim of this study was to compare acute versus chronic treatment with BZA, CE or CE + BZA in two major targets of estrogens, the uterus and the liver. In these two tissues, acute treatment with CE, but not with BZA, induced similar gene expression change than the most important endogenous estrogen, 17-β estradiol (E2). Acute induction of gene expression by E2 or by CE was antagonized by the addition of BZA. Concomitantly, BZA alone or in combination with E2 or CE induced a partial degradation of ERα protein after acute exposure. In uterus, chronic treatment of BZA alone had no impact on tissue weight gain or on epithelial cell proliferation, and also antagonized CE-effect in uterus, thereby mimicking the acute effect. By contrast, in the liver, chronic BZA and CE + BZA elicited agonistic transcriptional effects similar to those of CE alone. In addition, at variance to BZA acute effect, no change in ERα protein abundance was observed after chronic treatment in this tissue. These experimental in vivo data highlight a new aspect of the time-dependent tissue-specific action of BZA or TSEC, i.e. they can act acutely as antagonists but become agonists after chronic treatment. This shift was observed in liver tissue, but not in proliferative sex target such as the uterus

Adaptive β-Cell Neogenesis in the Adult Mouse in Response to Glucocorticoid-Induced Insulin Resistance

International audienceBoth type 1 and type 2 diabetes are characterized by deficient insulin secretion and decreased β-cell mass. Thus, regenerative strategies to increase β-cell mass need to be developed. To characterize mechanisms of β-cell plasticity, we studied a model of severe insulin resistance in the adult mouse and defined how β-cells adapt. Chronic corticosterone (CORT) treatment was given to adult mice and led to rapid insulin resistance and adaptive increased insulin secretion. Adaptive and massive increase of β-cell mass was observed during treatment up to 8 weeks. β-Cell mass increase was partially reversible upon treatment cessation and reinduced upon subsequent treatment. β-Cell neogenesis was suggested by an increased number of islets, mainly close to ducts, and increased Sox9 and Ngn3 mRNA levels in islets, but lineage-tracing experiments revealed that neoformed β-cells did not derive from Sox9- or Ngn3-expressing cells. CORT treatment after β-cell depletion partially restored β-cells. Finally, β-cell neogenesis was shown to be indirectly stimulated by CORT because serum from CORT-treated mice increased β-cell differentiation in in vitro cultures of pancreatic buds. Altogether, the results present a novel model of β-cell neogenesis in the adult mouse and identify the presence of neogenic factors in the serum of CORT-treated mice

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cell.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an Estrogen Receptor (ER) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol (E2) in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear.Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. Methods and Results: Using three complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with E2 and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα sub-functions (membrane/extra-nuclear versus genomic/transcriptional) demonstrated that E2-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. Conclusions: Whereas tamoxifen acts as an anti-estrogen and ERα antagonist in breast cancer, but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα

The activation function-1 of estrogen receptor alpha prevents arterial neointima development through a direct effect on smooth muscle cells

Rationale: 17β-Estradiol (E2) exerts numerous beneficial effects in vascular disease. It regulates gene transcription through nuclear estrogen receptor (ER) via 2 activation functions, AF1 and AF2, and can also activate membrane ER. The role of E2 on the endothelium relies on membrane ER activation, but the molecular mechanisms of its action on vascular smooth muscle cells (VSMCs) are not fully understood. Objective: The aim of this study was to determine which cellular target and which ER subfunction are involved in the preventive action of E2 on neointimal hyperplasia. Methods and Results: To trigger neointimal hyperplasia of VSMC, we used a mouse model of femoral arterial injury. Cre-Lox models were used to distinguish between the endothelial- and the VSMC-specific actions of E2. The molecular mechanisms underlying the role of E2 were further characterized using both selective ER agonists and transgenic mice in which the ER AF1 function had been specifically invalidated. We found that (1) the selective inactivation of ER in VSMC abrogates the neointimal hyperplasia protection induced by E2, whereas inactivation of endothelial and hematopoietic ER has no effect; (2) the selective activation of membrane ER does not prevent neointimal hyperplasia; and (3) ER AF1 is necessary and sufficient to inhibit postinjury VSMC proliferation. Conclusions: Altogether, ER AF1-mediated nuclear action is both necessary and sufficient to inhibit postinjury arterial VSMC proliferation, whereas membrane ER largely regulates the endothelial functions of E2. This highlights the exquisite cell/tissue-specific actions of the ER subfunctions and helps to delineate the spectrum of action of selective ER modulators. © 2015 American Heart Association, Inc

0218: The uterine and vascular actions of estetrol delineate an original distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation

International audienc

The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation.

International audienceEstetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications that should now be considered further