Poly(ADP-ribose) Polyremase-1 (PARP-1) Inhibition: A Promising Therapeutic Strategy for ETS-Expressing Tumours.

ETS transcription factors are a highly conserved family of proteins involved in the progression of many cancers, such as breast and prostate carcinomas, Ewing's sarcoma, and leukaemias. This significant involvement can be explained by their roles at all stages of carcinogenesis progression. Generally, their expression in tumours is associated with a poor prognosis and an aggressive phenotype. Until now, no efficient therapeutic strategy had emerged to specifically target ETS-expressing tumours. Nevertheless, there is evidence that pharmacological inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, specifically sensitises ETS-expressing cancer cells to DNA damage and limits tumour progression by leading some of the cancer cells to death. These effects result from a strong interplay between ETS transcription factors and the PARP-1 enzyme. This review summarises the existing knowledge of this molecular interaction and discusses the promising therapeutic applications.

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

Computational characterization of the binding mode between oncoprotein Ets-1 and DNA-repair enzymes.

The Ets-1 oncoprotein is a transcription factor that promotes target gene expression in specific biological processes. Typically, Ets-1 activity is low in healthy cells, but elevated levels of expression have been found in cancerous cells, specifically related to tumor progression. Like the vast majority of the cellular effectors, Ets-1 does not act alone but in association with partners. Given the important role that is attributed to Ets-1 in major human diseases, it is crucial to identify its partners and characterize their interactions. In this context, two DNA-repair enzymes, PARP-1 and DNA-PK, have been identified recently as interaction partners of Ets-1. We here identify their binding mode by means of protein docking. The results identify the interacting surface between Ets-1 and the two DNA-repair enzymes centered on the α-helix H1 of the ETS domain, leaving α-helix H3 available to bind DNA. The models highlight a hydrophobic patch on Ets-1 at the center of the interaction interface that includes three tryptophans (Trp338, Trp356, and Trp361). We rationalize the binding mode using a series of computational analyses, including alanine scanning, molecular dynamics simulation, and residue centrality analysis. Our study constitutes a first but important step in the characterization, at the molecular level, of the interaction between an oncoprotein and DNA-repair enzymes.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis.

Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.

Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function.

Ets-1 is a transcription factor that plays an important role in various physiological and pathological processes, such as development, angiogenesis, apoptosis and tumour invasion. In the present study, we have demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in a caspase-dependent manner in Jurkat T-leukaemia cells undergoing apoptosis, resulting in three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. In vitro cleavage of Ets-1 p51 by caspase 3 produces fragments consistent with those observed in cells undergoing apoptosis. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p51. This region is absent from the Ets-1 p42 isoform, which therefore cannot be cleaved by caspases. In Ets-1 p51, cleavage generates C-terminal fragments containing the DNA-binding domain, but lacking the transactivation domain. The Cp17 fragment, the major cleavage product generated during apoptosis, is devoid of transcriptional activity and inhibits Ets-1 p51-mediated transactivation of target genes by competing with Ets-1 p51 for binding to Ets-binding sites present in the target promoters. In the present study, we have demonstrated that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Furthermore, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative form of the full-length Ets-1 p51 protein.

Ets-1 p51 and p42 isoforms differentially modulate Stromelysin-1 promoter according to induced DNA bend orientation.

The Stromelysin-1 gene promoter contains a palindrome of two Ets-binding sites (EBS) that bind the p51 and p42 isoforms of the human Ets-1-transcription factor. A previous study established that full gene transactivation is associated with a ternary complex consisting of two p51 bound to the two EBS on the promoter. p42, only able to bind one of the two EBS, induces only very weak activity. Here, we investigate the mechanism by which the Stromelysin-1 promoter discriminates between p51 and p42. The differential stoichiometry of the two Ets-1 isoforms arises from the Stromelysin-1 EBS palindrome. The ternary complex requires the presence of two inhibitory domains flanking the DNA-binding domain and the ability to form an intramolecular autoinhibition module. Most importantly, the p51-ternary and the p42-binary complexes induce DNA curvatures with opposite orientations. These results establish that differential DNA bending, via p51 and p42 differential binding, is correlated with the Stromelysin-1 promoter activation process.

Integrin alphavbeta3 upregulates integrin-linked kinase expression in human ovarian cancer cells via enhancement of ILK gene transcription.

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

DNA-dependent protein kinase is a novel interaction partner for Ets-1 isoforms.

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction partner proteins. We used our previously developed, recombinant biotinylated Ets-1 that conserves native Ets-1 properties to identify new interaction partners. Here, based on results from streptavidin pull-down assays, mass spectrometry and co-immunoprecipitation, we report a novel interaction partner for Ets-1 isoforms: a heterotrimeric complex of DNA-dependent protein kinase (DNA-PK), made up of Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs). Kinase assays performed in vitro showed that DNA-PK phosphorylates the Ets-1 protein. Furthermore, we demonstrated that Ku86, but not Ku70 or DNA-PKcs, down-regulated the transcriptional activity of Ets-1 when analysed using a reporter gene assay. These results illustrate how detecting novel molecular interactions may provide new clues for understanding the diverse functions of Ets-1.

Ets-1 binds cooperatively to the palindromic Ets-binding sites in the p53 promoter.

Due to its autoinhibition for DNA binding, the Ets-1 transcription factor must interact with partners to enhance its affinity for DNA. In a study on the stromelysin-1 promoter, we showed that Ets-1 binds cooperatively to two Ets-binding sites (EBS) organized in palindrome, thereby circumventing the need for a binding partner to counteract autoinhibition. This leads to the formation of an Ets-1-DNA-Ets-1 ternary complex necessary for promoter activation. Here we show that Ets-1 also binds cooperatively to the EBS palindrome of the human p53 promoter, despite the presence of a degenerate EBS to which Ets-1 cannot otherwise bind. Transcriptional transactivation through this palindrome fully correlates to Ets-1 binding. Thus, the cooperative binding model that we initially proposed for the stromelysin-1 promoter may be a general mechanism of Ets-1 binding to palindromic EBS separated by 4bp and a way to counteract binding site degeneracy.

Identification of Novel Interaction Partners of Ets-1: Focus on DNA Repair.

The transcription factor Ets-1 (ETS proto-oncogene 1) shows low expression levels except in specific biological processes like haematopoiesis or angiogenesis. Elevated levels of expression are observed in tumor progression, resulting in Ets-1 being named an oncoprotein. It has recently been shown that Ets-1 interacts with two DNA repair enzymes, PARP-1 (poly(ADP-ribose) polymerase 1) and DNA-PK (DNA-dependent protein kinase), through two different domains and that these interactions play a role in cancer. Considering that Ets-1 can bind to distinctly different domains of two DNA repair enzymes, we hypothesized that the interaction can be transposed onto homologs of the respective domains. We have searched for sequence and structure homologs of the interacting ETS(Ets-1), BRCT(PARP-1) and SAP(DNA-PK) domains, and have identified several candidate binding pairs that are currently not annotated as such. Many of the Ets-1 partners are associated to DNA repair mechanisms. We have applied protein-protein docking to establish putative interaction poses and investigated these using centrality analyses at the protein residue level. Most of the identified poses are virtually similar to our recently established interaction model for Ets-1/PARP-1 and Ets-1/DNA-PK. Our work illustrates the potentially high number of interactors of Ets-1, in particular involved in DNA repair mechanisms, which shows the oncoprotein as a potential important regulator of the mechanism.

Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: a useful tool for studying interactions between Ets-1 and its partners.

Identification of Ets-1 interaction partners is critical for understanding its properties. Ets-1 DNA-binding is governed by an intramolecular mechanism called autoinhibition. Ets-1 increases its DNA-binding affinity by counteracting autoinhibition through binding either to a particular organization of Ets binding sites (EBS) in palindrome, as in the Stromelysin-1 promoter, or to EBS adjacent to DNA-binding sites of its partners by combinatorial interactions, as in the Collagenase-1 promoter. Identification of new Ets-1 interaction partners should allow the identification of new functions for this transcription factor. To this end, we fused a biotin tag to Ets-1 protein in order to copurify it and its partners by affinity. For the first time, we cloned, produced in Escherichia coli and purified a biotinylated recombinant Ets-1 protein using the T7-Impact system (New England Biolabs), adapted to induce biotinylation. Nearly 100% biotinylation was attained without altering Ets-1 properties. Biotinylated Ets-1 bound to and transactivated the Stromelysin-1 promoter the same way as native Ets-1 did. It also conserved interactions with known Ets-1 partners such as c-Jun, Erk-2 and Runx-1. In addition, streptavidin pull-down and surface plasmon resonance assays demonstrated that biotinylated Ets-1 is a useful tool for qualitative and quantitative studies of Ets-1 interaction with its partners.

Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.

MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-µM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD.

The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage.

Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.

Synthetic EthR inhibitors boost antituberculous activity of ethionamide.

The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-containing drugs, including ethionamide, are activated by the mycobacterial monooxygenase EthA, the production of which is controlled by the transcriptional repressor EthR. Here we identify drug-like inhibitors of EthR that boost the bioactivation of ethionamide. Compounds designed and screened for their capacity to inhibit EthR-DNA interaction were co-crystallized with EthR. We exploited the three-dimensional structures of the complexes for the synthesis of improved analogs that boosted the ethionamide potency in culture more than tenfold. In Mycobacterium tuberculosis-infected mice, one of these analogs, BDM31343, enabled a substantially reduced dose of ethionamide to lessen the mycobacterial load as efficiently as the conventional higher-dose treatment. This provides proof of concept that inhibiting EthR improves the therapeutic index of thiocarbamide derivatives, which should prompt reconsideration of their use as first-line drugs.

ETS-1 transcription factor binds cooperatively to the palindromic head to head ETS-binding sites of the stromelysin-1 promoter by counteracting autoinhibition.

Stromelysin-1 (matrix metalloproteinase-3) is a member of the matrix metalloproteinase family. Regulation of its gene expression is critical for tissue homeostasis. Patterns of increased co-expression of stromelysin-1 and ETS-1 genes have been observed in pathological processes. Stromelysin-1 promoter is transactivated by ETS proteins through two palindromic head to head ETS-binding sites, an unusual configuration among metalloproteinase promoters. By using surface plasmon resonance, electrophoretic mobility shift assay, and photo-cross-linking, we showed that full-length human ETS-1 (p51) binds cooperatively to the ETS-binding site palindrome of the human stromelysin-1 promoter, with facilitated binding of the second ETS-1 molecule to form an ETS-1.DNA.ETS-1 ternary complex. The study of N-terminal deletion mutants allowed us to conclude that cooperative binding implied autoinhibition counteraction, requiring the 245-330-residue region of the protein that is encoded by exon VII of the gene. This region was deleted in the natural p42 isoform of ETS-1, which was unable to bind cooperatively to the palindrome. Transient transfection experiments showed a good correlation between DNA binding and promoter transactivation for p51. In contrast, p42 showed a poorer transactivation, reinforcing the significance of cooperative binding for full transactivation. It is the first time that ETS-1 was shown to be able to counteract its own autoinhibition.

EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator.

Ethionamide (ETH) is an important second-line antitubercular drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross-resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro-drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin-containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR-binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer-extension of the ethA transcriptional start site indicated that it is located within the EthR-binding region. Taken together, bacterial two-hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member.

Association of a new c-Cbl related protein with the very first stages of apoptosis induction.

This study investigates the involvement of the c-cbl proto-oncogene during the first stages of the apoptotic process. We have already shown that a c-Cbl aptotosis-related protein of 90 kDa (CARP 90) is detected very rapidly in the cytoplasm as well as in the nucleus of murine thymocytes after hydrocortisone (HC) treatment. We report here that this protein appeared as well after in vivo treatment of mice by gamma-irradiation or injection of anti-CD3 monoclonal antibody, two potent thymic apoptosis inductors, providing a close relationship between the occurrence of apoptosis and the appearance of CARP 90. We showed that CARP 90 and p120(cbl) share numerous epitopes strikingly suggesting that CARP 90 is coded by c-cbl. In addition, KO mice do not sustain CARP 90 appearance. We finally showed that CARP 90 contains N- and C-terminal end epitopes of p120(cbl), which suggests that CARP 90 is an alternative spliced form of c-cbl. This protein was also observed under gamma-irradiation in tissues of different origin, which enlarges the physiological significance of this phenomenon. The very rapid CARP 90 appearance under apoptotic conditions in the nucleus of cells originating in different tissues makes this protein if not a possible new actor of the apoptotic process, at least an interesting marker of this process.