The glucocorticoid receptor interferes with progesterone receptor-dependent genomic regulation in breast cancer cells.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

Inhibitor of growth 1 (ING1) acts at early steps of multiple DNA repair pathways.

ING proteins are tumor suppressors involved in the regulation of gene transcription, cell cycle arrest, apoptosis, and senescence. Here, we show that ING1b expression is upregulated by several DNA-damaging agents, in a p53-independent manner. ING1b stimulates DNA repair of a variety of DNA lesions requiring activation of multiple DNA repair pathways. Moreover, Ing1(-/-) cells showed impaired genomic DNA repair after H2O2 and neocarzinostatin treatment and this defect was reverted by overexpression of ING1b. Two tumor-derived ING1 mutants failed to promote DNA repair highlighting the physiological importance of the integrity of the PHD domain for ING1b DNA repair activity and suggesting a role in the prevention of tumor progression. Ing(-/-) cells showed higher basal levels of γ-H2AX and, upon DNA damage, γ-H2AX increase was greater and with faster kinetics compared to wild-type cells. Chromatin relaxation by Trichostatin A led to an exacerbated damage signal in both types of cells, but this effect was dependent on Ing1 status, and more pronounced in wild-type cells. Our results suggest that ING1 acts at early stages of the DNA damage response activating a variety of repair mechanisms and that this function of ING1 is targeted in tumors.

Insights into estrogen receptor alpha modulation by cholestenoic acids.

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.

Cholestenoic acid analogues as inverse agonists of the liver X receptors.

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.

E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

SOX-11 regulates LINE-1 retrotransposon activity during neuronal differentiation.

Activity of the human long interspersed nuclear elements-1 (LINE-1) retrotransposon occurs mainly in early embryonic development and during hippocampal neurogenesis. SOX-11, a transcription factor relevant to neuronal development, has unknown functions in the control of LINE-1 retrotransposon activity during neuronal differentiation. To study the dependence of LINE-1 activity on SOX-11 during neuronal differentiation, we induced differentiation of human SH-SY5Y neuroblastoma cells and adult adipose mesenchymal stem cells (hASCs) to a neuronal fate and found increased LINE-1 activity. We also show that SOX-11 protein binding to the LINE-1 promoter is higher in differentiating neuroblastoma cells, while knock-down of SOX-11 inhibits the induction of LINE-1 transcription in differentiating conditions. These results suggest that activation of LINE-1 retrotransposition during neuronal differentiation is mediated by SOX-11.

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.