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 du rôle modulateur de STAT1 sur les réponses cellulaires induites par deux stratégies anticancéreuses (-Chimiothérapie par les agents alkylants )

Les membres de la famille des facteurs STAT ont des rôles modulateurs très différents. Dans cette famille, STAT1 et STAT3 sont très similaires, peuvent réguler les mêmes gènes cibles mais sont caractérisés par des rôles opposés. STAT1 est considéré comme un anti-oncogène tandis que STAT3 serait plutôt un oncogène. Le facteur de transcription STAT1 est impliqué dans plusieurs voies de signalisations visant à protéger la cellule. STAT1 est un facteur antiprolifératif et proapoptotique. Le rôle modulateur de STAT1 de l action de certains agents génotoxiques a été démontré et semble dépendre à la fois de l agent et du type cellulaire étudiés. STAT3 est souvent activé dans les tumeurs et est localisé dans le noyau. Ses fonctions pro-prolifératives et anti-apoptotiques en font une cible de choix pour des traitements antitumoraux. Dans un premier temps, nous avons montré que STAT1 régulait l expression de MLH1, protéine essentielle du système Mismatch Repair (MMR), et de c-Abl. L absence de STAT1 était associée à la persistance des lésions de l ADN, un arrêt du cycle cellulaire en phase G 2 /M et à une sensibilité accrue à l agent alkylant MNNG. En outre, STAT1 entrait dans la composition d un complexe actif STAT1/MLH1/c-Abl/p53 qui pourrait moduler le devenir cellulaire après exposition au MNNG. Dans un deuxième temps, nous avons montré que l inhibition de STAT3 par un oligonucléotide leurre (ODN) contenant une séquence GAS piégeait les dimères de STAT3 activés dans le cytoplasme en empêchant la liaison aux aryophérines et induisait la mort cellulaire via STAT1. Cependant, notre ODN peut aussi lier STAT1 et nécessite donc une optimisation pour accroître sa spécificité. L association d une stratégie ODN leurre à une chimiothérapie classique pourrait permettre de potentialiser l action des anticancéreux et de diminuer leurs effets secondaires.STAT family members have various modulating roles. Within this family, STAT1 and STAT3 are very similar and can regulate the same target genes but do have opposite effects. Whereas STAT1 is considered as an anti-oncogene, STAT3 is rather an oncongene. The transcription factor STAT1 modulates several pathways involved in cellular protection. STAT1 has antiproliferative and proapoptotic effects. A role for STAT1 as a modulator of the effect of genotoxic agents has been demonstrated and seems to depend on both the type of genotoxic agent and of the cell line studied. STAT3 is often activated in several types of cancers and is located in the nucleus. Anticancer strategies targeting the pro-proliferative and antiapoptotic STAT3 are promising. We have first shown that STAT1 regulated the expression of MLH1, an essential factor of the mismatch repair (MMR) system, and of c-Abl. STAT1 deficiency was associated with DNA lesions persistency, sustained G 2 /M cell cycle arrest and increased sensibility to the alkylating agent MNNG. Also, STAT1 entered into an active complex including STAT1/MLH1/c-Abl/p53 which could modulates the cell fate following MNNG exposure. Second, we have shown that inhibition of STAT3 with a decoy oligonucleotide (ODN) containing a GAS sequence could trap activated STAT3 dimers in the cytoplasm. This ODN impaired binding of STAT3 to karyopherin and induced a STAT1-dependent cell death. However, this ODN can also bind to STAT1 and hence it needs to be optimized in order to improve STAT3-specificity. The combination of a decoy-ODN strategy with a conventional chemotherapy should allow to synergize the action of anticancer drugs and to decrease their side effects.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Syndrome de Chanarin-Dorfman

Le syndrome de Chanarin-Dorfman (CDS) est une entité rare entraînant une ichtyose congénitale et une accumulation de gouttelettes lipidiques dans différents tissus (peau, foie, muscle…). Une anomalie dans le gène ABHD5 (appelé aussi CGI-58), en perturbant l’activité de la triglycéride-lipase et des facteurs lipolytiques, est à l’origine de cette pathologie. Nous rapportons l’observation d’une fillette de 7 ans présentant depuis la naissance une ichtyose érythrodermique. Elle présente un déficit pelvien stable depuis la petite enfance, peu de plaintes fonctionnelles, avec des CPK élevées autour de 1 000 UI/l. La biopsie musculaire met en évidence une accumulation de gouttelettes lipidiques dans les myocytes, conduisant, avec le tableau clinique, au diagnostic de syndrome de Chanarin-Dorfman

Performance Characteristics of Oncomine Focus Assay for Theranostic Analysis of Solid Tumors, A (21-Months) Real-Life Study

International audienceNext generation sequencing analysis is crucial for therapeutic decision in various solid tumor contexts. The sequencing method must remain accurate and robust throughout the instrument lifespan allowing the biological validation of patients’ results. This study aims to evaluate the long-term sequencing performances of the Oncomine Focus assay kit allowing theranostic DNA and RNA variants detection on the Ion S5XL instrument. We evaluated the sequencing performances of 73 consecutive chips over a 21-month period and detailed the sequencing data obtained from both quality controls and clinical samples. The metrics describing sequencing quality remained stable throughout the study. We showed that an average of 11 × 106 (±0.3 × 106) reads were obtained using a 520 chip leading to an average of 6.0 × 105 (±2.6 × 105) mapped reads per sample. Of 400 consecutive samples, 95.8 ± 16% of amplicons reached the depth threshold of 500X. Slight modifications of the bioinformatics workflow improved DNA analytical sensitivity and allowed the systematic detection of expected SNV, indel, CNV, and RNA alterations in quality controls samples. The low inter-run variability of DNA and RNA—even at low variant allelic fraction, amplification factor, or reads counts—indicated that our method was adapted to clinical practice. The analysis of 429 clinical DNA samples indicated that the modified bioinformatics workflow allowed detection of 353 DNA variants and 88 gene amplifications. RNA analysis of 55 clinical samples revealed 7 alterations. This is the first study showing the long-term robustness of the Oncomine Focus assay in clinical routine practice

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

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells

International audienceThe aim of our study was to investigate whether myofibroblasts and the chemokine monocyte chemoattractant protein‐1 (MCP‐1)/CCL2 may play a role in hepatocellular carcinoma progression. We observed that hepatic myofibroblast LI90 cells express MCP‐1/CCL2 mRNA and secrete this chemokine. Moreover, myofibroblast LI90 cell‐conditioned medium (LI90‐CM) induces human hepatoma Huh7 cell migration and invasion. These effects are strongly reduced when a MCP‐1/CCL2‐depleted LI90‐CM was used. We showed that MCP‐1/CCL2 induces Huh7 cell migration and invasion through its G‐protein–coupled receptor CCR2 and, to a lesser extent, through CCR1 only at high MCP‐1/CCL2 concentrations. MCP‐1/CCL2's chemotactic activities rely on tyrosine phosphorylation of focal adhesion components and depend on matrix metalloproteinase (MMP)‐2 and MMP‐9. Furthermore, we observed that Huh7 cell migration and invasion induced by the chemokine are strongly inhibited by heparin, by β‐D‐xyloside treatment of cells and by anti‐syndecan‐1 and ‐4 antibodies. Finally, we developed a 3‐dimensional coculture model of myofibroblast LI90 and Huh7 cells and demonstrated that MCP‐1/CCL2 and its membrane partners, CCR1 and CCR2, may be involved in the formation of mixed hepatoma‐myofibroblast spheroids. In conclusion, our data show that human liver myofibroblasts act on hepatoma cells in a paracrine manner to increase their invasiveness and suggest that myofibroblast‐derived MCP‐1/CCL2 could be involved in the pathogenesis of hepatocellular carcinoma