Claude Beaudoin

The PEA3 Group of ETS-related Transcription Factors

The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.The ets genes encode eukaryotic transcription factors that are involved in tumorigenesis and developmental processes. The signature of the Ets family is the ETS-domain, which binds to sites containing a central 5-GGAA/T-3 motif. They can be sub-classified primarily because of the high amino acid conservation in their ETS-domains and, in addition, in the conservation of other domains generally characterized as transactivating. This is the case for the PEA3 group, which is currently made up of three members, PEA3/E1AF, ER81/ETV1 and ERM, which are more than 95% identical in the ETS-domain and more than 85% in the transactivation acidic domain. The members of the PEA3 group are activated through both the Ras-dependent and other kinase pathways, a function which emphasizes their involvement in several oncogenic mechanisms. The expression pattern of the three PEA3 group genes during mouse embryogenesis suggests that they are differentially regulated, probably to serve important functions such as tissue interaction. Although the target genes of these transcription factors are multiple, their most frequently studied role concerns their involvement in the metastatic process. In fact, PEA3 group members are over-expressed in metastatic human breast cancer cells and mouse mammary tumors, a feature which suggests a function of these transcription factors in mammary oncogenesis. Moreover, when they are ectopically over-expressed in non-metastatic breast cancer cells, these latter become metastatic with the activation of transcription of matrix metalloproteinases or adhesion molecules, such as ICAM-1.

Crosstalk between androgen receptor and epidermal growth factor receptor-signalling pathways: a molecular switch for epithelial cell differentiation.

In the male, androgens promote growth and differentiation of sex reproductive organs through ligand activation of the androgen receptor (AR). Here, we show that androgens are not major actors of the cell cycle arrest associated with the differentiation process, and that the epidermal growth factor (EGF)-mediated signalling interferes with AR activities to regulate androgen response when epithelial cells are differentiated. Higher AR expression and enhanced androgen responsiveness correlate with reduction of phosphorylated ERK1/2 over differentiation. These modifications are associated with recruitment of cells in phase G(0)/G(1), up-regulation of p27(kip1), down-regulation of p21(Cip1) and p53 proteins, and accumulation of hypo-phosphorylated Rb. Exposure to EGF reduces AR expression levels and blocks androgen-dependent transcription in differentiated cells. It also restores p53 and p21(Cip1) levels, Rb hyper-phosphorylation, ERK1/2 activation and promotes cell cycle re-entry as p27(kip1) protein levels are decreased. Treatment with a MEK inhibitor reverses the EGF-mediated AR down-regulation in differentiated cells, thus suggesting the existence of an inverse correlation between EGF and androgen signalling in non-tumoural epithelia. Interestingly, when androgen signalling is set in differentiated cells, dihydrotestosterone exerts an inhibitory effect on ERK activity but paradoxically does not modify EGFR (ErbB1) phosphorylation, indicating that androgens are able to disrupt the EGFR-ERK cascade. Overall, our data demonstrate the existence of a balance between AR and mitogen-activated protein kinase activities that favours either the maintenance of differentiated conditions or the enhancement of cell proliferation capacities.

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

ERM is a member of the ETS transcription factor family. High levels of the corresponding mRNA are detected in a variety of human breast cancer cell lines, as well as in aggressive human breast tumors. As ERM protein is almost undetectable in these cells, high degradation of this transcription factor has been postulated. Here we have investigated whether ERM degradation might depend on the proteasome pathway. We show that endogenous and ectopically expressed ERM protein is short-lived protein and undergoes proteasome-dependent degradation. Deletion mutagenesis studies indicate that the 61 C-terminal amino acids of ERM are critical for its proteolysis and serve as a degradation signal. Although ERM conjugates with ubiquitin, this post-translational modification does not depend on the C-terminal domain. We have used an Ets-responsive ICAM-1 reporter plasmid to show that the ubiquitin–proteasome pathway can affect transcriptional function of ERM. Thus, ERM is subject to degradation via the 26S proteasome pathway, and this pathway probably plays an important role in regulating ERM transcriptional activity.

ERRα induces H3K9 demethylation by LSD1 to promote cell invasion

Significance Dynamic demethylation of histone residues plays a crucial role in the regulation of gene expression. Lysine Specific Demethylase 1 (LSD1) can remove both transcriptionally permissive and repressive histone marks. How these activities are controlled is not clearly understood. Here, we show that the estrogen-related receptor α (ERRα) induces LSD1 to erase repressive marks in vitro. Through such a mechanism, LSD1 and ERRα commonly activate a set of transcriptional targets that include genes involved in the cellular capacity to invade the extracellular matrix. This process is a hallmark of cancer progression, to which high expression of both LSD1 and ERRα are strongly correlated. Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.

NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells

The chaperone nucleophosmin (NPM1) is over-expressed in the epithelial compartment of prostate tumours compared to adjacent healthy epithelium and may represent one of the key actors that support the neoplastic phenotype of prostate adenocarcinoma cells. Yet, the mechanisms that underlie NPM1 mediated phenotype remain elusive in the prostate. To better understand NPM1 functions in prostate cancer cells, we sought to characterize its impact on prostate cancer cells behaviour and decipher the mechanisms by which it may act. Here we show that NPM1 favors prostate tumour cell migration, invasion and colony forming. Furthermore, knockdown of NPM1 leads to a decrease in the growth of LNCaP-derived tumours grafted in Nude mice in vivo. Such oncogenic-like properties are found in conjunction with a positive regulation of NPM1 on the ERK1/2 (Extracellular signal-Regulated Kinases 1/2) kinase phosphorylation in response to EGF (Epidermal Growth Factor) stimulus, which is critical for prostate cancer progression following the setting of an autonomous production of the growth factor. NPM1 could then be a target to switch off specifically ERK1/2 pathway activation in order to decrease or inhibit cancer cell growth and migration.

Bile acid homeostasis controls CAR signaling pathways in mouse testis through FXRalpha

Bile acids (BAs) are molecules with endocrine activities controlling several physiological functions such as immunity, glucose homeostasis, testicular physiology and male fertility. The role of the nuclear BA receptor FXRα in the control of BA homeostasis has been well characterized. The present study shows that testis synthetize BAs. We demonstrate that mice invalidated for the gene encoding FXRα have altered BA homeostasis in both liver and testis. In the absence of FXRα, BA exposure differently alters hepatic and testicular expression of genes involved in BA synthesis. Interestingly, Fxrα-/- males fed a diet supplemented with BAs show alterations of testicular physiology and sperm production. This phenotype was correlated with the altered testicular BA homeostasis and the production of intermediate metabolites of BAs which led to the modulation of CAR signaling pathways within the testis. The role of the CAR signaling pathways within testis was validated using specific CAR agonist (TCPOBOP) and inverse agonist (androstanol) that respectively inhibited or reproduced the phenotype observed in Fxrα-/- males fed BA-diet. These data open interesting perspectives to better define how BA homeostasis contributes to physiological or pathophysiological conditions via the modulation of CAR activity.

Cholesterol: A Gatekeeper of Male Fertility?

Cholesterol is essential for mammalian cell functions and integrity. It is an important structural component maintaining the permeability and fluidity of the cell membrane. The balance between synthesis and catabolism of cholesterol should be tightly regulated to ensure normal cellular processes. Male reproductive function has been demonstrated to be dependent on cholesterol homeostasis. Here we review data highlighting the impacts of cholesterol homeostasis on male fertility and the molecular mechanisms implicated through the signaling pathways of some nuclear receptors.

Lipid Homeostasis and Ligands for Liver X Receptors: Identification and Characterization

Screening of bona fide ligands for nuclear receptors is a real tour de force as the identified molecules are supposed to be able to activate the targeted proteins in cell culture as well as in vivo. Indeed orphan nuclear receptors are putative pharmacologically targets for various diseases. It is thus necessary to have quick and reproductive systems that help in identifying new ligands, agonist or antagonist, before using them in vivo in animal models to check for secondary effects. Here, we describe the transient transfections (homologous and heterologous) used for the screening of ligands for liver X receptor α (LXRα, NR1H3) in HeLa cells.