Phytochemicals Targeting Estrogen Receptors: Beneficial Rather Than Adverse Effects?

In mammals, the effects of estrogen are mainly mediated by two different estrogen receptors, ERα and ERß. These proteins are members of the nuclear receptor family, characterized by distinct structural and functional domains, and participate in the regulation of different biological processes, including cell growth, survival and differentiation. The two estrogen receptor (ER) subtypes are generated from two distinct genes and have partially distinct expression patterns. Their activities are modulated differently by a range of natural and synthetic ligands. Some of these ligands show agonistic or antagonistic effects depending on ER subtype and are described as selective ER modulators (SERMs). Accordingly, a few phytochemicals, called phytoestrogens, which are synthesized from plants and vegetables, show low estrogenic activity or anti-estrogenic activity with potentially anti-proliferative effects that offer nutraceutical or pharmacological advantages. These compounds may be used as hormonal substitutes or as complements in breast cancer treatments. In this review, we discuss and summarize the in vitro and in vivo effects of certain phytoestrogens and their potential roles in the interaction with estrogen receptors.

The actin/MKL1 signalling pathway influences cell growth and gene expression through large-scale chromatin reorganization and histone post-translational modifications.

In addition to soluble factors, mechanical constraints and extracellular matrix stiffness are important regulators of cell fate that are mediated by cytoskeletal modifications. The EMT (epithelial-mesenchymal transition) that occurs during normal development and malignant progression is a typical example of the phenotypic switch associated with profound actin remodelling and changes in gene expression. For instance, actin dynamics control motile cell functions in EMT, in part, through regulating the subcellular localization of the myocardin-related transcription factor MKL1 (megakaryoblastic leukaemia translocation 1), a co-activator of SRF (serum-responsive factor). In the present paper, we show that MKL1 participates also to the control of the cellular switch between growth and quiescence. Experimental disconnection between MKL1 and G-actin (globular actin), by using an MKL1 mutant or enhancing the F (filamentous)-/G-actin ratio, generates a widely open chromatin state and a global increase in biosynthetic activity, classically associated with cell growth. Conversely, G-actin accumulation favours nuclear condensation and cell quiescence. These large-scale chromatin changes rely upon extensive histone modifications, exemplified by that of H3K9 (H3 Lys9) shifting from trimethylation, a heterochromatin mark, to acetylation, a mark of euchromatin. The present study provides the first evidence for a global reversible hetero/euchromatinization phenomenon triggered by the actin/MKL1 signalling pathway.

Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers.

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.

Cyclical DNA methylation of a transcriptionally active promoter.

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

Epigenetic memories: structural marks or active circuits?

A hallmark of living systems is the management and the storage of information through genetic and epigenetic mechanisms. Although the notion of epigenetics was originally given to any regulation beyond DNA sequence, it has often been restricted to chromatin modifications, supposed to behave as cis-markers, specifying the sets of genes to be expressed or repressed. This definition does not take into account the initial view of epigenetics, based on nonlinear interaction networks whose "attractors" can remain stable without need for any chromatin mark. In addition, most chromatin modifications are the steady state resultants of highly dynamic modification and de-modification activities and, as such, seem poorly appropriate to work as long-term memory keepers. Instead, the basic support of epigenetic memory could remain the attractors, to which chromatin modifications belong as do many other components. The influence of chromatin modifications in memory is highly questionable when envisioned as static structural marks, but can be recovered under the dynamic circuitry perspective, thanks to their self-templating properties. Beside their standard repressive or permissive functions, chromatin modifications can also influence transcription in multiple ways such as: (1) by randomizing or inversely stabilizing gene expression, (2) by mediating cooperativity between pioneer and secondary transcription factors, and (3) in the hysteresis and the ultrasensitivity of gene expression switches, allowing the cells to take unambiguous transcriptional decisions.

Deciphering the Molecular Mechanisms Sustaining the Estrogenic Activity of the Two Major Dietary Compounds Zearalenone and Apigenin in ER-Positive Breast Cancer Cell Lines.

The flavone apigenin and the mycotoxin zearalenone are two major compounds found in the human diet which bind estrogen receptors (ERs), and therefore influence ER activity. However, the underlying mechanisms are not well known. To unravel the molecular mechanisms that could explain the differential effect of zearalenone and apigenin on ER-positive breast cancer cell proliferation, gene-reporter assays, chromatin immunoprecipitation (ChIP) experiments, proliferation assays and transcriptomic analysis were performed. We found that zearalenone and apigenin transactivated ERs and promoted the expression of estradiol (E2)-responsive genes. However, zearalenone clearly enhanced cellular proliferation, while apigenin appeared to be antiestrogenic in the presence of E2 in both ER-positive breast cancer cell lines, MCF-7 and T47D. The transcriptomic analysis showed that both compounds regulate gene expression in the same way, but with differences in intensity. Two major sets of genes were identified; one set was linked to the cell cycle and the other set was linked to stress response and growth arrest. Our results show that the transcription dynamics in gene regulation induced by apigenin were somehow different with zearalenone and E2 and may explain the differential effect of these compounds on the phenotype of the breast cancer cell. Together, our results confirmed the potential health benefit effect of apigenin, while zearalenone appeared to be a true endocrine-disrupting compound.

Deoxyribonucleic acid methyl transferases 3a and 3b associate with the nuclear orphan receptor COUP-TFI during gene activation.

Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.

Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen-responsive breast cancer cell lines.

Estrogen receptor alpha (ERα) is generally considered to be a good prognostic marker because almost 70% of ERα-positive tumors respond to anti-hormone therapies. Unfortunately, during cancer progression, mammary tumors can escape from estrogen control, resulting in resistance to treatment. In this study, we demonstrate that activation of the actin/megakaryoblastic leukemia 1 (MKL1) signaling pathway promotes the hormonal escape of estrogen-sensitive breast cancer cell lines. The actin/MKL1 signaling pathway is silenced in differentiated ERα-positive breast cancer MCF-7 and T47D cell lines and active in ERα-negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells, which have undergone epithelial-mesenchymal transition. We showed that MKL1 activation in MCF-7 cells, either by modulating actin dynamics or using MKL1 mutants, down-regulates ERα expression and abolishes E2-dependent cell growth. Interestingly, the constitutively active form of MKL1 represses PR and HER2 expression in these cells and increases the expression of HB-EGF, TGFß, and amphiregulin growth factors in an E2-independent manner. The resulting expression profile (ER-, PR-, HER2-) typically corresponds to the triple-negative breast cancer expression profile.

Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring.

Inhibition of histone deacetylases (HDACs) leads to growth arrest, differentiation, or apoptosis of tumor cell lines, suggesting HDACs as promising targets for cancer therapy. At present, only one HDAC inhibitor (HDACi) is used in therapy: suberoylanilide hydroxamic acid (SAHA). Here, we describe the synthesis and biological evaluation of a new series of compounds derived from SAHA by substituting short alkyl chains at various positions of the phenyl ring. Such modifications induced variable effects ranging from partial loss of activity to increased potency. Through molecular modeling, we describe a possible interaction between HDAC7 proline 809, a residue that is strictly conserved within class 2 enzymes only, and the amide group of HDACi, while nuclear magnetic resonance experiments indicated that dimethyl m-substitution may stabilize the inhibitor in the active site. Our data provide novel information on the structure-activity relationship of HDACi and suggest new ways for developing second generation SAHA-like molecules.

T-box factors: targeting to chromatin and interaction with the histone H3 N-terminal tail.

T-box transcription factors play a crucial role in development where they are implicated in patterning and cell fate decisions. Tbx2 and Tbx3 have also been implicated in several cancers including melanoma, and can act as antisenescence factors through their ability to repress p19(ARF) and p21(CIP1) expression. Although several target genes for T-box factors have been identified, it is unknown whether this family of proteins can bind chromatin, a property that would facilitate the epigenetic reprogramming that occurs in both development and cancer progression. Here, we show that Tbx2 has the potential to recognize mitotic chromatin in a DNA-dependent fashion, can interact specifically with the histone H3 N-terminal tail, a property shared with Tbx4, Tbx5 and Tbx6, and can also recognize nucleosomal DNA, with binding to nucleosomes being antagonized by the presence of the histone tails. Strikingly, in vivo Tbx2 co-localization with pericentric heterochromatin appears to be regulated and ectopic expression of Tbx2 leads to severe mitotic defects. Taken together our results suggest that Tbx2, and most likely other members of the T-box family, are able to target chromatin and may indicate a role for the T-box factors in epigenetic reprogramming events.

A neural-specific splicing event generates an active form of the Wiskott-Aldrich syndrome protein.

Actin polymerization is required for cellular events such as podosome, lamellipode or filopode formation in migrating cells, and members of the Wiskott-Aldrich syndrome protein (WASP) family have essential roles in regulating actin dynamics at the cell leading edge. However, WASP proteins need first to be activated in order to be able to target actin polymerization. Here, we show the occurrence of a neural-specific splicing event, which is favoured by the nuclear orphan receptor chicken ovalbumin upstream promoter-transcription factor I, and generates a truncated WASP protein deleted of exon 2-encoded amino acids. This deletion relocates the protein to the plasma membrane and induces the formation of actin-rich podosome-like structures that also contain paxillin and vinculin. Furthermore, expression of the truncated protein in PC12 cells, as well as in primary neurons, stimulates neuritogenesis. These data underscore the importance of the neural-specific splicing of WASP RNA during development.