Yvan de Launoit

Differential expression patterns of the PEA3 group transcription factors through murine embryonic development

ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.

Synergistic Activation of Human Immunodeficiency Virus Type 1 Promoter Activity by NF-κB and Inhibitors of Deacetylases: Potential Perspectives for the Development of Therapeutic Strategies

ABSTRACT The transcription factor NF-κB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-κB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of κB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IκBα. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-κB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.

Expression of the PEA3 group of ETS-related transcription factors in human breast-cancer cells

The PEA3 group of transcription factors belongs to the ets family and is composed of 3 known members, PEA3, ERM and ER81, which are more than 95% identical within the DNA‐binding ETS domain and exhibit 50% aa identity overall. Recently, transgenic mice bearing the c‐erbB‐2/neu oncogene have been shown to over‐express PEA3 mRNA in mammary adenocarcinomas, suggesting a role for this gene family in mammary tumorigenesis. In the present work we characterized the mRNA expression levels of PEA3‐group genes in a series of human epithelial breast cell lines. Each of the 3 genes was highly expressed in normal human HMEC 1001‐7 and HMEC 219‐4 cells. In breast‐cancer cell lines, the 3 genes were highly expressed in the ER−MDA‐MB‐436, MDA‐MB‐330, MDA‐MB‐231 and BT‐20 cell lines, but not in the ER+MDA‐MB‐134‐VI and ZR‐75‐1 cells. In an attempt to characterize the PEA3‐group proteins in breast‐cancer cells, we first produced and characterized specific antibodies against each of these 3 proteins. The anti‐ERM and anti‐ER81 antibodies recognized specific strong bands at approximately 72 kDa and 62 kDa, corresponding to ERM and ER81, respectively, in MDA‐MB‐231 and Hs‐578T cells expressing significant levels of the 3 mRNAs. No protein was detected in MCF‐7 cells expressing low levels of mRNA for PEA3‐group‐family genes, or in ZR‐75‐1 cells, where mRNA was undetectable by Northern blot. Although in vitro‐translated PEA3 is specifically immunoprecipitated by anti‐PEA3 anti‐serum, we were unable to immunoprecipitate PEA3 protein from MDA‐MB‐231 and Hs‐578T cells. In order to study the transcription factor activity of ERM, PEA3 and ER81 proteins in mammary‐cancer cells, we tested their ability to transactivate a reporter plasmid containing 3 Ets‐binding sites, and were able to show that, in all the breast‐cancer cells tested, transfected ERM, PEA3 and ER81 are able to transactivate. Although the target genes of the PEA3 group of transcription factors in breast‐cancer cells have yet to be determined, these genes have a potential role in the regulation of growth and the progression of human breast cancer.Int. J. Cancer 70:590–597.

The cardenolide UNBS1450 is able to deactivate nuclear factor κB–mediated cytoprotective effects in human non–small cell lung cancer cells

Non–small cell lung cancers (NSCLC) are associated with very dismal prognoses, and adjuvant chemotherapy, including irinotecan, taxanes, platin, and Vinca alkaloid derivatives, offers patients only slight clinical benefits. Part of the chemoresistance of NSCLCs results from the constitutive or anticancer drug-induced activation of the nuclear factor-κB (NF-κB) signaling pathways. The present study shows that human A549 NSCLC cells display highly activated cytoprotective NF-κB signaling pathways. UNBS1450, which is a cardenolide belonging to the same class of chemicals as ouabain and digitoxin, affected the expression and activation status of different constituents of the NF-κB pathways in these A549 tumor cells. The modifications brought about by UNBS1450 led to a decrease in both the DNA-binding capacity of the p65 subunit and the NF-κB transcriptional activity. Using the 3-(4,5-dimethylthiazol-2yl)-dephenyltetrazolium bromide colorimetric assay, we observed in vitro that UNBS1450 was as potent as taxol and SN38 (the active metabolite of irinotecan) in reducing the overall growth levels of the human A549 NSCLC cell line, and was more efficient than platin derivatives, including cisplatin, carboplatin, and oxaliplatin. The chronic in vivo i.p. and p.o. UNBS1450 treatments of human A549 orthotopic xenografts metastasizing to the brains and the livers of immunodeficient mice had a number of significant therapeutic effects on this very aggressive model. [Mol Cancer Ther 2006;5(2):391–9]

The M-type receptor PLA2R regulates senescence through the p53 pathway

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.

The PEA3 Group of ETS-related Transcription Factors

The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.

Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells.

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.

Senescent Keratinocytes Die by Autophagic Programmed Cell Death

Normal cells reach senescence after a specific time and number of divisions, leading ultimately to cell death. Although escape from this fate may be a requisite step in neoplastic transformation, the mechanisms governing senescent cell death have not been well investigated. We show here, using normal human epidermal keratinocytes, that no apoptotic markers appear with senescence. In contrast, the expression of several proteins involved in the regulation of macroautophagy, notably Beclin-1 and Bcl-2, was found to change with senescence. The corpses occurring at the senescence growth plateau displayed a large central area delimited by the cytokeratin network that contained a huge quantity of autophagic vacuoles, the damaged nucleus, and most mitochondria. 3-methyladenine, an inhibitor of autophagosome formation, but not the caspase inhibitor zVAD, prevented senescent cell death. We conclude that senescent cells do not die by apoptosis, but as a result of high macroautophagic activity that targets the primary vital cell components.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.