A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes.</p> <p>Results</p> <p>New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1.</p> <p>Conclusions</p> <p>Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.</p

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

<p>Abstract</p> <p>Background</p> <p>The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear.</p> <p>Results</p> <p>The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death.</p> <p>Conclusions</p> <p>The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.</p

Etude de l efficacité d oligonucléotides leurres de STAT3 dans des cellules tumorales (Analyse de la spécificité et élaboration de séquences discriminantes)

STAT3 est un facteur de transcription de la famille des STATs Signal Transducer and Activator of Transcription . Son activation par des cytokines comme l'IL-6 entraine sa localisation nucléaire, l'activation de gènes cibles et la prolifération cellulaire. Des oligonucléotides leurres en épingle à cheveux (hpdODNs) et porteurs de la séquence consensus de STAT3 induisent la mort de cellules de lignée de cancer du colon dans lesquelles STAT3 est activé. Cependant, le hpdODN est également reconnu par STAT1 activé et protège les cellules contre l'apoptose induite par l'IFNy. Nous montrons que le hpdODN interagit avec STAT3 dans le cytoplasme et bloque sa pénétration nucléaire en empêchant son interaction avec l'importine, essentielle au transport nucléaire de STAT3 activé. Dans le but d'obtenir un hpdODN spécifique de STAT3, l'analyse comparative du DNA Binding Domain (DBD) de STAT3 et STATI complexé à l'ADN cible a été réalisée avec un programme d'analyse 3D. Les substitutions de nucléotides ont été testées dans les cellules de lignée de cancer du colon traitées ou non à l'IFNy. Un hpdODN spécifique a été conçu : il inhibe STAT3 et a une action minimale sur STAT1. Des hpdODNs spécifiques de STATS ont également été analysés et testés. Ils induisent la mort de cellules tumorales sanguines dans lesquelles STATS est activé. Par ailleurs, la pénétration des hpdODNs a pu être améliorée par utilisation de nanoparticules magnétiques. Ces résultats avec les hpdODNs suggèrent que le DBD de STAT3 est une bonne cible ; ce hpdODN pourrait être utilisé comme base pour la conception de molécules interagissant avec le DBD mais capables de traverser la membrane par elles mêmes.STAT3 is a transcription factor of the STAT Signal Transducer and Activator of Transcription family. Triggering of cytokine receptors, such as IL-6, activates kinases of the JAK family and results in phosphorylation and dimerization of STAT3. The dimerized STAT3 penetrates the nucleus, binds its targets and activates their transcription. STAT3 is frequently activated in tumor cells; its inhibition generally leads to the death of these cells. STAT1 is another STAT family member; its activation is most of the time linked with cell death or resistance to pathogens. Intriguingly, STAT1 and STAT3, through their DNA Binding Domain (DBD) interact with very similar DNA sequences. In order to achieve specific inhibition of STAT3, hairpin decoy oligodeoxynucleotide sequences (hpdODN) were used. Transfection of the hpdODNs resulted in cell death in the colon carcinoma cell line SW480, demonstrating the efficiency of targeting STAT3's DBD. However, STAT1-dependent Interferon y-induced cell death was blocked by the hpdODN in these cells, suggesting a lack of specificity. Analysis of the mechanism of action of the hpdODN showed that its interaction with activated dimeric STAT3 takes place in the cytoplasm. We further demonstrated that binding of the hpdODN to dimeric STAT3 prevented the binding of importin thereby impairing importin-mediated transport through the nuclear pore. Finally, STAT3 hpdODN complexed with magnetic nanoparticles was tested and found to improve penetration of the hpdODN. An hpdODN inhibiting STATS was tested and found to induce death of hematologic tumor cells with activated STAT5.To further investigate and improve the hpdODN's specificity, comparative 3D analysis of the STAT3 and STAT1 DBDs was conducted. Modifications in the hpdODN sequence were subsequently tested in the SW480 cell line. The new hpdODN induced cell death without preventing interferon y-induced cell death, indicating that the DBD can be used as a basis for specific inhibitory reagents.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Electrostatic assembly of a DNA superparamagnetic nano-tool for simultaneous intracellular delivery and in situ monitoring.

International audienceA superparamagnetic γFe(2)O(3) nanocarrier was developed, characterized by spectroscopic methods and evaluated for the delivery of a decoy oligonucleotide (dODN) in human colon carcinoma SW 480 cells. This nanoparticle-dODN bioconjugate (γFe(2)O(3)@dODN) was designed to target the signal transducer and activator of transcription 3, STAT3, a key regulator of cell survival and proliferation. We exploited a simple precipitation-redispersion mechanism for the direct and one-step complexation of a labeled decoy oligonucleotide with iron oxide nanoparticles (NPs). The cell internalization of the decoy γFe(2)O(3)@dODN nanoparticles is demonstrated and suggests the potential for DNA delivery in biological applications. Despite the increasing use of NPs in biology and medicine, convenient methods to quantify them within cells are still lacking. In this work, taking advantage of the nonlinear magnetic behavior of our superparamagnetic NPs, we have developed a new method to quantify in situ their internalization by cells. FROM THE CLINICAL EDITOR: In this study, the authors demonstrate methods to quantify superparamagnetic nanocarriers within cells, taking advantage of the nonlinear magnetic behavior of the studied NPs

Easily controlled grafting of oligonucleotides on γFe2O3 nanoparticles: physicochemical characterization of DNA organization and biological activity studies.

International audienceWe report a one-step process to functionalize superparamagnetic iron oxide nanoparticle (SPIO-NP) surfaces with a controlled number of oligonucleotides. For this study, we use a specific oligonucleotide targeting the signal transducer and activator of transcription 3 (STAT3), a key regulator of cell survival and proliferation. This oligonucleotide is self-complementary and can adopt a hairpin structure. It is labeled with the fluorescein amidite group at the 3'-end. The polyanionic DNA is electrostatically attracted onto the positively charged surface of the bare SPIO-NPs. During synthesis, the molar ratio between the oligonucleotides and nanoparticles was varied from 17.5 to 175. For particles with a mean diameter of 10 nm, a nanoparticle surface saturation is observed corresponding to 70 DNA strands per particle. The increase of DNA density per nanoparticle is correlated to a transition from the hairpin structure adsorbed horizontally on the nanoparticle surface to a vertically ordered surface packing assembly. An in vitro study on human colon carcinoma cell line SW480 shows that the kinetics of internalization and biological activity of the NPs seem to be dependent on the oligonucleotide density. Cell death and the kinetics of internalization are favored by a low density of oligonucleotides

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells

International audienceThe SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy

Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4.

International audienceThe male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SSCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events