Dynamic chromatin accessibility during nutritional iron overload reveals a BMP6-independent induction of cell cycle genes.

Iron is essential to organism physiology as it participates in numerous biological processes including oxygen transport, respiration, and erythropoiesis. Although iron is critical to physiology, excess iron is toxic to cells and tissues due to generation of reactive oxygen species. Therefore, well-kept iron homeostasis is a mainstay of proper cell and organ function. Iron overload disorders, caused by nutritional or genetic factors, contribute to many pathologies such as diabetes, non-alcoholic steatohepatitis and hepatocellular carcinoma. The liver is not only vulnerable to the effects of iron overload, it is also the major organ controlling iron homeostasis. During iron overload, Bone Morphogenic Protein (BMP) levels increase and initiate a hepatic response aimed at lowering iron levels. The transcriptional effects of iron overload are not well-characterized and the underlining enhancer regulation is uncharted. Here, we profiled the liver's transcriptome and chromatin accessibility following nutritional iron overload. We found marked changes in gene expression and enhancer accessibility following iron overload. Surprisingly, 16% of genes induced following iron overload participate in propagating the cell cycle. Induction of cell cycle genes was independent of BMP. Genome-wide enhancer landscape profiling revealed hundreds of enhancers with altered activity following iron overload. Characterization of transcription factor motifs and footprints in iron-regulated enhancers showed a role for the Activator Protein 1 (AP-1) transcription factor in promoting cell cycle-related transcription. In summary, we found that the transcriptional program at play during iron overload is bifurcated in which BMP signaling controls iron homeostasis genes while an AP-1-driven program controls cell cycle genes.

The Three Ds of Transcription Activation by Glucagon: Direct, Delayed, and Dynamic.

Upon lowered blood glucose occurring during fasting, glucagon is secreted from pancreatic islets, exerting various metabolic effects to normalize glucose levels. A considerable portion of these effects is mediated by glucagon-activated transcription factors (TFs) in liver. Glucagon directly activates several TFs via immediate cyclic adenosine monophosphate (cAMP)- and calcium-dependent signaling events. Among these TFs, cAMP response element-binding protein (CREB) is a major factor. CREB recruits histone-modifying enzymes and cooperates with other TFs on the chromatin template to increase the rate of gene transcription. In addition to direct signal transduction, the transcriptional effects of glucagon are also influenced by dynamic TF cross talk. Specifically, assisted loading of one TF by a companion TF leads to increased binding and activity. Lastly, transcriptional regulation by glucagon is also exerted by TF cascades by which a primary TF induces the gene expression of secondary TFs that bring about their activity a few hours after the initial glucagon signal. This mechanism of a delayed response may be instrumental in establishing the temporal organization of the fasting response by which distinct metabolic events separate early from prolonged fasting. In this mini-review, we summarize recent advances and critical discoveries in glucagon-dependent gene regulation with a focus on direct TF activation, dynamic TF cross talk, and TF cascades.

Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading.

The cytokines interleukin 1ß and 6 (IL-1ß, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins. Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression. By mapping changes in enhancer landscape and transcription factor occupancy (using ChIP-seq), we show that synergistic gene induction is achieved by assisted loading of STAT3 on chromatin by NF-κB. With IL-6 treatment alone, STAT3 does not efficiently bind 20% of its coordinated binding sites. In the presence of IL-1ß, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac. This facilitates STAT3 binding and synergistic gene expression. Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others. This model reconciles seemingly contradictory reports of NF-κB-STAT3 crosstalk.

TM7SF3, a novel p53-regulated homeostatic factor, attenuates cellular stress and the subsequent induction of the unfolded protein response.

Earlier reported small interfering RNA (siRNA) high-throughput screens, identified seven-transmembrane superfamily member 3 (TM7SF3) as a novel inhibitor of pancreatic ß-cell death. Here we show that TM7SF3 maintains protein homeostasis and promotes cell survival through attenuation of ER stress. Overexpression of TM7SF3 inhibits caspase 3/7 activation. In contrast, siRNA-mediated silencing of TM7SF3 accelerates ER stress and activation of the unfolded protein response (UPR). This involves inhibitory phosphorylation of eukaryotic translation initiation factor 2α activity and increased expression of activating transcription factor-3 (ATF3), ATF4 and C/EBP homologous protein, followed by induction of apoptosis. This process is observed both in human pancreatic islets and in a number of cell lines. Some of the effects of TM7SF3 silencing are evident both under basal conditions, in otherwise untreated cells, as well as under different stress conditions induced by thapsigargin, tunicamycin or a mixture of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta and interferon gamma). Notably, TM7SF3 is a downstream target of p53: activation of p53 by Nutlin increases TM7SF3 expression in a time-dependent manner, although silencing of p53 abrogates this effect. Furthermore, p53 is found in physical association with the TM7SF3 promoter. Interestingly, silencing of TM7SF3 promotes p53 activity, suggesting the existence of a negative-feedback loop, whereby p53 promotes expression of TM7SF3 that acts to restrict p53 activity. Our findings implicate TM7SF3 as a novel p53-regulated pro-survival homeostatic factor that attenuates the development of cellular stress and the subsequent induction of the UPR.

Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

p53 promotes the expression of gluconeogenesis-related genes and enhances hepatic glucose production.

BACKGROUND: The p53 tumor suppressor protein is a transcription factor that initiates transcriptional programs aimed at inhibiting carcinogenesis. p53 represses metabolic pathways that support tumor development (such as glycolysis and the pentose phosphate pathway (PPP)) and enhances metabolic pathways that are considered counter-tumorigenic such as fatty acid oxidation. FINDINGS: In an attempt to comprehensively define metabolic pathways regulated by p53, we performed two consecutive high-throughput analyses in human liver-derived cells with varying p53 statuses. A gene expression microarray screen followed by constraint-based modeling (CBM) predicting metabolic changes imposed by the transcriptomic changes suggested a role for p53 in enhancing gluconeogenesis (de novo synthesis of glucose). Examining glucogenic gene expression revealed a p53-dependent induction of genes involved in both gluconeogenesis (G6PC, PCK2) and in supplying glucogenic precursors (glycerol kinase (GK), aquaporin 3 (AQP3), aquaporin 9 (AQP9) and glutamic-oxaloacetic transaminase 1 (GOT1)). Accordingly, p53 augmented hepatic glucose production (HGP) in both human liver cells and primary mouse hepatocytes. CONCLUSIONS: These findings portray p53 as a novel regulator of glucose production. By facilitating glucose export, p53 may prevent it from being shunted to pro-cancerous pathways such as glycolysis and the PPP. Thus, our findings suggest a metabolic pathway through which p53 may inhibit tumorigenesis.

'Cancer associated fibroblasts'--more than meets the eye.

Cancer associated fibroblasts (CAFs) are a subpopulation of cells that reside within the tumor microenvironment and promotes the transformation process by encouraging tumor growth, angiogenesis, inflammation, and metastasis. CAF-specific proteins serve as both prognostic markers and targets for anticancer drugs. With the growing interest in CAFs, several controversial issues have been raised, including the genomic landscape of these cells, the identity of specific markers, and their cell of origin. Here, we tackle these debated issues and put forward a new definition for 'CAF' as a cell 'state' rather than a cell type. We hope this conceptualization can resolve the ongoing discrepancies revolving around CAF research and aid in designing better anti-cancer treatment strategies.

Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta.

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNß) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNß on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNß treatment.

Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4 by activating p53.

Cytochrome P450 (P450) enzymes are abundantly expressed in the human liver where they hydroxylate organic substrates. In a microarray screen performed in human liver cells, we found a group of eleven P450 genes whose expression was induced by p53 (CYP3A4, CYP3A43, CYP3A5, CYP3A7, CYP4F2, CYP4F3, CYP4F11, CYP4F12, CYP19A1, CYP21A2 and CYP24A1). The mode of regulation of four representative genes (CYP3A4, CYP3A7, CYP4F2 and CYP4F3) was further characterized. The genes were induced in a p53-dependent manner in HepG2 and Huh6 cells (both are cancer-derived human liver cells) and in primary liver cells isolated from human donors. Furthermore, p53 was found to bind to p53-responsive elements in the genes' DNA-regulatory regions and to enhance their transcription in a reporter gene assay. Importantly, when p53 was activated following the administration of either of three different anticancer chemotherapeutic agents (cisplatin, etoposide or doxorubicin), it was able to induce CYP3A genes, which are the main factors in systemic clearance of these agents. Finally, the p53-dependent induction of P450 genes following either Nutlin or chemotherapy treatment led to enhanced P450 enzymatic activity. Thus, in addition to the well-established role of p53 at the tumor site, our data unravels a novel function of hepatic p53 in inducing P450 enzymes and position p53 as a major factor in the hepatic response to xenobiotic and metabolic signals. Importantly, this study reveals a novel pathway for the induction of CYP3As by their substrates through p53, warranting the need for careful consideration when designing systemically administered chemotherapeutic regimens.

Mutant p53R273H attenuates the expression of phase 2 detoxifying enzymes and promotes the survival of cells with high levels of reactive oxygen species.

Uncontrolled accumulation of reactive oxygen species (ROS) causes oxidative stress and induces harmful effects. Both high ROS levels and p53 mutations are frequent in human cancer. Mutant p53 forms are known to actively promote malignant growth. However, no mechanistic details are known about the contribution of mutant p53 to excessive ROS accumulation in cancer cells. Herein, we examine the effect of p53(R273H), a commonly occurring mutated p53 form, on the expression of phase 2 ROS-detoxifying enzymes and on the ability of cells to readopt a reducing environment after exposure to oxidative stress. Our data suggest that p53(R273H) mutant interferes with the normal response of human cells to oxidative stress. We show here that, upon oxidative stress, mutant p53(R273H) attenuates the activation and function of NF-E2-related factor 2 (NRF2), a transcription factor that induces the antioxidant response. This effect of mutant p53 is manifested by decreased expression of phase 2 detoxifying enzymes NQO1 and HO-1 and high ROS levels. These findings were observed in several human cancer cell lines, highlighting the general nature of this phenomenon. The failure of p53(R273H) mutant-expressing cells to restore a reducing oxidative environment was accompanied by increased survival, a known consequence of mutant p53 expression. These activities are attributable to mutant p53(R273H) gain of function and might underlie its well-documented oncogenic nature in human cancer.

Regulation of lipid metabolism by p53 - fighting two villains with one sword.

Both cellular and systemic metabolism of lipids are paramount for homeostasis, and their malfunction leads to devastating pathologies. Recently, exciting findings have linked the p53 tumor suppressor to the regulation of lipid metabolism. Here, we summarize these findings showing a clear role for p53 in enhancing lipid catabolism while inhibiting its anabolism. We also describe the multitude of genes regulated by p53 that participate in or regulate systemic lipid transport. From the compilation of available data a scenario is emerging in which p53 regulates genes involved in lipid metabolism - both in a cancer-preventive effort and, intriguingly, as a means to prevent atherosclerosis. Thus, by regulating lipid metabolism, p53 fights the two major causes of death worldwide - atherosclerosis and cancer.

Cancer research, a field on the verge of a paradigm shift?

The theoretical framework for the field of cancer research is based on two main principles. The first is that cancer advances in a stepwise manner, with each alteration driving cells further toward a malignant state. Second, to cure cancer we must target only cancer-specific properties. Here, we analyze the birth and propagation of the cancer research paradigm. We believe the current paradigm is immersed in crisis and that the field would benefit from integrating theories within and outside the normal modes of research to compile a new framework, with the hope of faster progress and significantly fewer cancer-related deaths.

Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature.

Concomitant expression of mutant p53 and oncogenic Ras, leading to cellular transformation, is well documented. However, the mechanisms by which the various mutant p53 categories cooperate with Ras remain largely obscure. From this study we suggest that different mutant p53 categories cooperate with H-Ras in different ways to induce a unique expression pattern of a cancer-related gene signature (CGS). The DNA-contact p53 mutants (p53(R248Q) and p53(R273H)) exhibited the highest level of CGS expression by cooperating with NFκB. Furthermore, the Zn(+2) region conformational p53 mutants (p53(R175H) and p53(H179R)) induced the CGS by elevating H-Ras activity. This elevation in H-Ras activity stemmed from a perturbed function of the p53 transcription target gene, BTG2. By contrast, the L3 loop region conformational mutant (p53(G245S)) did not affect CGS expression. Our findings were further corroborated in human tumor-derived cell lines expressing Ras and the aforementioned mutated p53 proteins. These data might assist in future tailor-made therapy targeting the mutant p53-Ras axis in cancer.

p53, a novel regulator of lipid metabolism pathways.

BACKGROUND & AIMS: In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor. METHODS: Analysis of gene expression following alteration of p53 status in several human- and mouse-derived cells using microarray analysis, quantitative real-time PCR, chromatin immunoprecipitation, and reporter gene assays. A functional assay was performed to determine lipid transfer activity. RESULTS: We identified a novel role for the p53 protein in regulating lipid and lipoprotein metabolism, a process not yet conceived as related to p53, which is known mainly for its tumor suppressive functions. We revealed a group of 341 genes whose expression was induced by p53 in the liver-derived cell line HepG2. Twenty of these genes encode proteins involved in many aspects of lipid homeostasis. The mode of regulation of three representative genes (Pltp, Abca12, and Cel) was further characterized. In addition to HepG2, the genes were induced following activation of p53 in human primary hepatic cells isolated from liver donors. p53-dependent regulation of these genes was evident in other cell types namely Hep3B cells, mouse hepatocytes, and fibroblasts. Furthermore, p53 was found to bind to the genes' promoters in designated p53 responsive elements and thereby increase transcription. Importantly, p53 augmented the activity of secreted PLTP, which plays a major role in lipoprotein biology and atherosclerosis pathology. CONCLUSIONS: These findings expose another facet of p53 functions unrelated to tumor suppression and render it a novel regulator of hepatic lipid metabolism and consequently of systemic lipid homeostasis and atherosclerosis development.

Transcriptional activity of ATF3 in the stromal compartment of tumors promotes cancer progression.

Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.

TMPRSS2/ERG promotes epithelial to mesenchymal transition through the ZEB1/ZEB2 axis in a prostate cancer model.

Prostate cancer is the most common non-dermatologic malignancy in men in the Western world. Recently, a frequent chromosomal aberration fusing androgen regulated TMPRSS2 promoter and the ERG gene (TMPRSS2/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between TMPRSS2/ERG and other defective pathways in cancer progression. However, the unveiling of more specific pathways in which TMPRSS2/ERG takes part, requires further investigation. Using immortalized prostate epithelial cells we were able to show that TMPRSS2/ERG over-expressing cells undergo an Epithelial to Mesenchymal Transition (EMT), manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. These findings were corroborated in vivo, where the control cells gave rise to discrete nodules while the TMPRSS2/ERG-expressing cells formed malignant tumors, which expressed EMT markers. To further investigate the general transcription scheme induced by TMPRSS2/ERG, cells were subjected to a microarray analysis that revealed a distinct EMT expression program, including up-regulation of the EMT facilitators, ZEB1 and ZEB2, and down-regulation of the epithelial marker CDH1(E-Cadherin). A chromatin immunoprecipitation assay revealed direct binding of TMPRSS2/ERG to the promoter of ZEB1 but not ZEB2. However, TMPRSS2/ERG was able to bind the promoters of the ZEB2 modulators, IL1R2 and SPINT1. This set of experiments further illuminates the mechanism by which the TMPRSS2/ERG fusion affects prostate cancer progression and might assist in targeting TMPRSS2/ERG and its downstream targets in future drug design efforts.

SPATA18, a spermatogenesis-associated gene, is a novel transcriptional target of p53 and p63.

The transcription factor p53 functions not only to suppress tumorigenesis but also to maintain normal development and homeostasis. Although p53 was implicated in different aspects of fertility, including spermatogenesis and implantation, the mechanism underlying p53 involvement in spermatogenesis is poorly resolved. In this study we describe the identification of a spermatogenesis-associated gene, SPATA18, as a novel p53 transcriptional target and show that SPATA18 transcription is induced by p53 in a variety of cell types of both human and mouse origin. p53 binds a consensus DNA motif that resides within the first intron of SPATA18. We describe the spatiotemporal expression patterns of SPATA18 in mouse seminiferous tubules and suggest that SPATA18 transcription is regulated in vivo by p53. We also demonstrate the induction of SPATA18 by p63 and suggest that p63 can compensate for the loss of p53 activity in vivo. Our data not only enrich the known collection of p53 targets but may also provide insights on spermatogenesis defects that are associated with p53 deficiency.

p53 Regulates the Ras circuit to inhibit the expression of a cancer-related gene signature by various molecular pathways.

In this study, we focus on the analysis of a previously identified cancer-related gene signature (CGS) that underlies the cross talk between the p53 tumor suppressor and Ras oncogene. CGS consists of a large number of known Ras downstream target genes that were synergistically upregulated by wild-type p53 loss and oncogenic H-Ras(G12V) expression. Here we show that CGS expression strongly correlates with malignancy. In an attempt to elucidate the molecular mechanisms underling the cooperation between p53 loss and oncogenic H-Ras(G12V), we identified distinguished pathways that may account for the regulation of the expression of the CGS. By knocking-down p53 or by expressing mutant p53, we revealed that p53 exerts its negative effect by at least two mechanisms mediated by its targets B-cell translocation gene 2 (BTG2) and activating transcription factor 3 (ATF3). Whereas BTG2 binds H-Ras(G12V) and represses its activity by reducing its GTP loading state, which in turn causes a reduction in CGS expression, ATF3 binds directly to the CGS promoters following p53 stabilization and represses their expression. This study further elucidates the molecular loop between p53 and Ras in the transformation process.

A novel translocation breakpoint within the BPTF gene is associated with a pre-malignant phenotype.

Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma--one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis.