Patrick Augereau

Chromatin structure of the regulatory regions of pS2 and cathepsin D genes in hormone-dependent and -independent breast cancer cell lines

We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5′-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located −10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor α, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor α, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5′-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions −2.3 Kb and −3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position −4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.

The nuclear receptor transcriptional coregulator RIP140.

The nuclear receptor superfamily comprises ligand-regulated transcription factors that control various developmental and physiological pathways. These receptors share a common modular structure and regulate gene expression through the recruitment of a large set of coregulatory proteins. These transcription cofactors regulate, either positively or negatively, chromatin structure and transcription initiation. One of the first proteins to be identified as a hormone-recruited cofactor was RIP140. Despite its recruitment by agonist-liganded receptors, RIP140 exhibits a strong transcriptional repressive activity which involves several inhibitory domains and different effectors. Interestingly, the RIP140 gene, located on chromosome 21 in humans, is finely regulated at the transcriptional level by various nuclear receptors. In addition, the protein undergoes several post-translational modifications which control its repressive activity. Finally, experiments performed in mice devoid of the RIP140 gene indicate that this transcriptional cofactor is essential for female fertility and energy homeostasis. RIP140 therefore appears to be an important modulator of nuclear receptor activity which could play major roles in physiological processes and hormone-dependent diseases.

Retinoid regulation of human cathepsin D gene expression.

Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration-dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect.

The estrogen-regulated 52K-cathepsin-D in breast cancer: From biology to clinical applications

We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer cell lines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in lambda gt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D.(ABSTRACT TRUNCATED AT 250 WORDS)

The 52K cathepsin-D of breast cancer: structure, regulation, function and clinical value

A major question in cancer research is: why do cancer cells continuously proliferate and invade adjacent tissue. Breast cancer cells are particularly suitable for studying this question since when they are differentiated, they contain estrogen receptors, and estrogen, specifically, triggers their growth. One approach to understanding the estrogen-induced growth stimulation in this model is to identify the estrogen-induced factors involved in the control of cell proliferation [1–6]. Here, we illustrate this approach in a study of growth regulation of MCF7 cells by estrogen, in which we found a secreted 52K protein with mitogenic activity. It was identified as a protease that is also possibly involved in the process of tumor cell metastasis.

Regulation of hormone signaling by nuclear receptor interacting proteins.

Nuclear receptors are ligand-activated transcription factors which regulate the expression of genes critical for the growth of hormone-dependent cancers. Their expression and activity are controlled by various cofactors which are important players in hormone-dependent carcinogenesis. RIP140 is a negative transcriptional regulator which is recruited by agonist-liganded receptors. Its strong repressive activity involves four silencing domains which interact with histone deacetylases (HDACs), carboxyl-terminal binding proteins (CtBPs) and additional partners. RIP140 positively regulates transactivation when nuclear receptors are recruited to target promoters through interaction with the Sp1 transcription factor. In human breast cancer cells, RIP140 expression is upregulated at the transcriptional level by various ligands of nuclear receptors revealing the existence of regulatory loops. The Mdm2 oncogenic ubiquitin-ligase is another protein which directly interacts with nuclear receptors. It is involved in a ternary complex with ERα and p53 and regulates ERα turn-over. In MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) abolished E2-dependent turn-over of ERα without affecting its transactivation potential. Altogether, our results show that RIP140 and Mdm2 are two important regulators of ERα expression and activity and could therefore play major roles in hormone-dependent breast carcinogenesis.

Abstract 4970: Complex regulation of RIP140 gene expression by E2F transcription factors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We recently showed that RIP140 (Receptor Interacting protein of 140 kDa), a regulator of nuclear receptor activity, was involved in the control of cell proliferation and able to repress E2F1 activity. We now demonstrate that RIP140 and E2F1 comprise a novel transcriptional regulatory loop. We first observed that RIP140 mRNA levels increased during the G1/S and G2/M transitions and were reduced in tissues from E2F1 null mice. Furthermore, we identified the RIP140 gene as a transcriptional target of E2F1, showing, by transient transfection, that overexpression of E2F1/DP1 strongly increased transcription of the RIP140 promoter. Bioinformatic analysis identified several binding sites for E2Fs in the proximal promoter region and we evidenced the direct binding of E2F1/DP1 on these sequences by gel shift analysis. However, site directed mutagenesis of these elements only modestly affected transactivation of the RIP140 promoter by E2F1/DP1, thus suggesting an indirect recruitment of E2Fs on the promoter. This was strengthened by the observation that overexpression of E2F1, 2 or 3 alone was very potent in increasing transcription from the RIP140 promoter. Moreover, overexpression of DP1 produced a negative effect on this regulation which was also strongly repressed by RIP140 itself. Altogether, these results revealed a complex control loop between E2Fs and RIP140 and the molecular mechanisms underlying these atypical regulations are currently under investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4970.

Estrogen-induced Pro-cathepsin D and its Relationship to Breast Cancer Invasion and Metastasis

Human breast cancer is characterized by its estrogen dependence, observed in 30 to 50% of the patients, and by its high frequency of metastasis. Moreover, in several estrogen-responsive and -unresponsive human breast cancer cell lines, factors involved in the control of cell proliferation and tumor invasion have been identified and used to develop antibodies allowing direct studies in mammary tumor biopsies. One fruitful approach to identifying such factors has been to detect the proteins, enzymes, and factors secreted in conditioned media by estrogen-receptor-(RE)-positive breast cancer cells following their stimulation by estrogens.