Purpose: Receptor-interacting protein of 140 kDa (RIP140) is a transcriptional cofactor for nuclear receptors involved in reproduction and energy homeostasis. Our aim was to investigate its role in the regulation of E2F1 activity and target genes both in breast cancer cell lines and in tumor biopsies.Experimental Design: Glutathione S-transferase pull-down assays, coimmunoprecipitation experiments, and chromatin immunoprecipitation analysis were used to evidence interaction between RIP140 and E2F1. The effects of RIP140 expression on E2F1 activity were determined using transient transfection and quantification of E2F target mRNAs by quantitative real-time PCR. The effect on cell cycle was assessed by fluorescence-activated cell sorting analysis on cells overexpressing green fluorescent protein-tagged RIP140. A tumor microarray data set was used to investigate the expression of RIP140 and E2F1 target genes in 170 breast cancer patients.Results: We first evidenced the complex interaction between RIP140 and E2F1 and showed that RIP140 represses E2F1 transactivation on various transiently transfected E2F target promoters and inhibits the expression of several E2F1 target genes (such as CCNE1 and CCNB2). In agreement with a role for RIP140 in the control of E2F activity, we show that increasing RIP140 levels results in a reduction in the proportion of cells in S phase in various human cell lines. Finally, analysis of human breast cancers shows that low RIP140 mRNA expression was associated with high E2F1 target gene levels and basal-like tumors.Conclusion: This study shows that RIP140 is a regulator of the E2F pathway, which discriminates luminal-and basal-like tumors, emphasizing the importance of these regulations for a clinical cancer phenotype. Clin Cancer Res; 16(11); 2959-70. ©2010 AACR.Cell cycle control is a fundamental process that governs cell proliferation and is frequently altered during tumorigenesis. E2Fs and their heterodimer partners (DP) are central regulators of cell cycle progression and directly regulate the expression of a broad spectrum of genes involved, for instance, in cell cycle regulation, DNA replication and repair, apoptosis, differentiation, or development (1, 2).E2F1, which was discovered as a protein promoting the transition to S phase, was the founding member of the E2F family, which comprises eight members in mammals. Among this family, some were initially presented as "activator E2Fs" (E2F1, E2F2, and E2F3), whereas the other members were mostly known as transcription repressors, although this classification now seemed too simplistic (reviewed in ref. 2 and references therein). E2F transcriptional activity was shown to be regulated by a large number of coactivators or corepressors, including the so-called pocket proteins, which form the retinoblastoma (RB) tumor suppressor family (pRB, together with the related proteins p107 and p130; ref. 3). RB attenuates E2F action by recruiting transcriptional corepressors such as histone deacetylases to E2F-regulated promoters, thus med...

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. HistoryIn the early 90's, one of the main goals for several laboratories working on nuclear receptor (NR) signaling was to identify associated proteins which could act as transcriptional coregulators. The efforts were initially focused on partners of the ligand binding domain (LBD) encompassing the ligand-dependent activating function (AF-2) because it was the most convenient (due to the existence of inactivating mutations and to the use of antagonist ligands). RIP140 (Receptor Interacting Protein of 140 kDa) was one of the first NR transcriptional cofactors to be isolated. It was first identified by far-western blotting in human cancer cell extracts using a chimeric radiolabeled probe containing the ligand binding domain (LBD) of the mouse ERα fused to the glutathione-S-transferase [Cavailles et al., 1995]. In the presence of estradiol, this probe detected several bands corresponding to RIP140 and to the p160 family of coactivators. Using the same strategy, the RIP140 cDNA was then isolated from a cDNA expression library established from ZR75-1 breast cancer cells [Cavailles et al., 1995].The mouse RIP140 cDNA was isolated 3 years later from a mouse embryonic library using a yeast two hybrid strategy using the LBD of the orphan TR2 receptor as a bait [Lee et al., 1998]. Currently, the cDNA sequences from several species including rat, dog, chicken, xenopus and zebra fish have been deposited in databases. The RIP140 gene is also known as NRIP1 (Nuclear Receptor-Interacting Protein 1) which is the official symbol provided by the HUGO gene nomenclature committee. Protein domain structureThe human RIP140 protein comprises 1158 amino acids with an overall important identity between species (83% of amino acid identity between human and mouse sequences). When the...