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

Reprogrammation des cellules MCF7 issues de cancer du sein par hyperhydroxymethylation de l’ADN

Reprogrammation des cellules MCF7 issues de cancer du sein par hyperhydroxymethylation de l’AND. EpiPhase 201

Identification of a new isoform of the human estrogen receptor-alpha (hER-α) that is encoded by distinct transcripts and that is able to repress hER-α activation function 1

A new isoform of the human estrogen receptor-alpha (hER-α) has been identified and characterized. This 46 kDa isoform (hERα46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERα66). hERα46 is encoded by a new class of hER-α transcript that lacks the first coding exon (exon 1A) of the ER-α gene. We demonstrated that these Δ1A hER-α transcripts originate from the E and F hER-α promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERα46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERα46 is a powerful inhibitor of hERα66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominant-negative action is exerted may involve heterodimeri zation of the two receptor isoforms and/or direct competition for the ER-α DNA-binding site. hERα66/hERα46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERα46 in cellular proliferation

Transient cyclical methylation of promoter DNA.

Sara Kangaspeska, Brenda Stride : These authors equally contributed to this work.International audienceMethylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor alpha (ERalpha) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERalpha and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERalpha and of pS2 transcripts

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance

International audienceEstrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy

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

Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications

International audienceEstrogen receptor alpha (ERα) has been recognized now for several decades as playing a key role in reproduction and exerting functions in numerous nonreproductive tissues. In this review, we attempt to summarize the in vitro studies that are the basis of our current understanding of the mechanisms of action of ERα as a nuclear receptor and the key roles played by its two activation functions (AFs) in its transcriptional activities. We then depict the consequences of the selective inactivation of these AFs in mouse models, focusing on the prominent roles played by ERα in the reproductive tract and in the vascular system. Evidence has accumulated over the two last decades that ERα is also associated with the plasma membrane and activates non-nuclear signaling from this site. These rapid/nongenomic/membrane-initiated steroid signals (MISS) have been characterized in a variety of cell lines, and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS and the generation of mice expressing an ERα protein impeded for membrane localization have begun to unravel the physiological role of MISS in vivo. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators based on the integration of the physiological and pathophysiological roles of MISS actions of estrogens

Selective Liver Estrogen Receptor Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice

International audienceHepatocyte estrogen receptor α (ERα) was recently recognized as a relevant molecular target for nonalcoholic fatty liver disease (NAFLD) prevention. The present study defined to what extent hepatocyte ERα could be involved in preserving metabolic homeostasis in response to a full (17β-estradiol [E2]) or selective (selective estrogen receptor modulator [SERM]) activation. Ovariectomized mice harboring a hepatocyte-specific deletion ( mice) and their wild-type (WT) littermates were fed a high-fat diet (HFD) and concomitantly treated with E2, tamoxifen (TAM; the most used SERM), or vehicle. As expected, both E2 and TAM prevented all HFD-induced metabolic disorders in WT mice, and their protective effects against steatosis were abolished in mice. However, while E2 still prevented obesity and glucose intolerance in mice, hepatocyte deletion also abrogated TAM-mediated control of food intake as well as its beneficial actions on adiposity, insulin sensitivity, and glucose homeostasis, suggesting a whole-body protective role for liver-derived circulating factors. Moreover, unlike E2, TAM induced a rise in plasma concentration of the anorectic hepatokine growth differentiation factor 15 (Gdf15) through a transcriptional mechanism dependent on hepatocyte ERα activation. Accordingly, ERα was associated with specific binding sites in the regulatory region in hepatocytes from TAM-treated mice but not under E2 treatment due to specific epigenetic modifications. Finally, all the protective effects of TAM were abolished in HFD-fed knockout mice. We identified the selective modulation of hepatocyte ERα as a pharmacologic strategy to induce sufficient anorectic hepatokine Gdf15 to prevent experimental obesity, type 2 diabetes, and NAFLD