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The epidermal growth factor receptor (EGFR)-src-signal transducers and activators of transcription 3 (STAT3) oncogenic pathway plays a central role in tumorigenesis and is involved not only in cell transformation but also in tumor escape to genotoxic treatments. Despite its importance, the molecular mechanisms by which this signaling pathway induces resistance to DNA damage remain most of the time to be characterized. In this study, we show that the EGFR-src pathway is activated in response to topoisomerase I inhibition. After treatment, this signaling cascade induced the activation of STAT3 and the binding of the transcription factor to the promoter of the Eme1 gene. Eme1 is an endonuclease involved in the processing of DNA damage after topoisomerase I inhibition. These results suggest a model by which the STAT3-mediated activation of Eme1 prevents DNA damage and enhances cell survival in response to topoisomerase inhibition. This survival pathway was inhibited by a combined treatment with a src inhibitor, SKI, and with cetuximab, a monoclonal antibody directed against the EGFR that is widely used in the treatment of colorectal cancers. We therefore propose that the benefit of anti-EGFR therapy relies on an increase of DNA damage generated by topoisomerase I inhibition. [Cancer Res 2008;68(3):815–25]

Supplementary Figure Legend from The EGFR-STAT3 Oncogenic Pathway Up-regulates the Eme1 Endonuclease to Reduce DNA Damage after Topoisomerase I Inhibition

Supplementary Figure 1 from The EGFR-STAT3 Oncogenic Pathway Up-regulates the Eme1 Endonuclease to Reduce DNA Damage after Topoisomerase I Inhibition

Introduction: NSCLC accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor EGFR activating mutations, such as in-frame deletions in exon 19 (Ex19del). Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of anti-EGFR inhibitors such as Osimertinib. The CAM xenograft offers a rich environment in nutrients and embryonic growth factors, favorable for aggressive tumor development, with its highly vascularized membrane and natural immunodeficiency at engraftment (at EDD9). Methods: HCC827 Ex19del cells have been engrafted in ovo, and treated with various doses of osimertinib for seven days (4 treatments). The xenografts were collected and immunohistopathological, transcriptional and genetic analyses were carried out. Based on human transcriptional data, supervised multivariate discrimination between conditions and ontology analysis was performed through GSEA. Results: The xenograft growth was inversely dependent on the exposure dose of osimertinib, with a reduction in tumor weight of 58% for 10 µM (corresponding to 8.3 µg/Kg, T1/2 =4hr). The histological analysis showed epithelial tumor cells composed of solid sheets. The quantity of tumor cells was significantly lower in the treated group, with an area of tumor necrosis. The immunohistochemistry of tumor cells confirmed the human pulmonary epithelial origin, with TTF1 positive in treated and control groups. As expected, the ontology analysis of transcriptomic findings highlighted a downregulation of the EGFR pathway and its downstream effectors. Furthermore, the transcriptomic responses associated with chemotaxis, immune cell recruitment, and angiogenesis were dampened in the presence of osimertinib. This suggests that therapeutic efficacy was not uniquely guided through cell-autonomous mechanisms, but also takes place at the tissue level. We confirmed this in an independent experiment of in ovo HCC827 engraftment, showing a statically significant reduction of 15% in the number of vessels surrounding the xenograft in the osimertinib-treated condition. This demonstrates the advantage of using the in ovo model to decipher the regulations associated with therapeutics in a complex tissue context. Conclusions: For the first time, we have observed that osimertinib modulates angiogenesis and chemotaxis, which apparently reduces immune system recruitment and immune checkpoint responses. The ontologic analysis of transcriptomic findings in the CAM model strongly support the clinical observations of the pharmacological tumoral response to targeted therapy. The fact that immune infiltration was reduced in osimertinib-treated tumors may partially explain the lower response to immunotherapy regimens in osimertinib resistance contexts. Citation Format: David Barthelemy, Arnaud Vigneron, Xavier Rousset, Jerome Guitton, Emmanuel Grolleau, Margaux Raffin, Julie Balandier, Gaelle Lescuyer, Florence Geiguer, Sebastien Couraud, Jean Vaillet, Nazim Benzerdjeb, Lea Payen-Gay. Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4866.

International audience; This work proposes a rapid, simple and accurate analytical method for quantification of the organophosphate flame retardant triphenylphosphate (TPhP) and the biomarker diphenylphosphate (DPhP) in whole blood by a simple and effective sample preparation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification. A liquid–liquid microextraction (µLLE) from 250 µL of mouse whole blood was developed and validated to quantify TPhP and DPhP. Nature of extraction solvent, grinding for cell lysis and purification step were investigated. The use of distilled solvents proved necessary to reduce and control the contamination of the ubiquitous target compounds throughout the analytical process and to maintain a stable and repeatable signal in the analytical controls.The validation was performed by matrix-matched calibration with a correction by labelled internal standards: the limit of quantification was 1 ng/mL for DPhP and 2 ng/mL for TPhP with good linearity over the range up to 48 ng/mL for each compound. Accuracy was between 92.5% and 106.7% with relative standard deviation < 20%. The µLLE-LC-MS/MS method was successfully tested during short exposures of mice to DPhP or TPhP via drinking water or by intravenous injection.

BackgroundAryl phosphate esters (APEs), a main class of organophosphorus ester molecules, are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives in human samples, are poorly characterised.ObjectiveOur aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs and determine its potential deleterious biological effects in chronically exposed mice.MethodsConventional animals (FVB mice) were acutely (intravenous or oral gavage) or chronically (0.1 mg.mL-1, 1 mg.mL-1, 10 mg.mL-1 in drink water) exposed to relevant doses of DPhP or triphenyl phosphate (TPhP), one of its main precursors in the environment. Both molecules were measured in blood and other relevant tissues by liquid chromatography-mass spectrometry (LC-MS). Biological effects of chronic DPhP exposure were addressed through liver multi-omics analysis combining mRNA extraction and sequencing to high resolution LC-MS to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses (immunohistochemistry (IHC) and animal growth measurement).ResultsAcute and chronic exposure to DPhP led to significant levels of this molecule in blood and other tissues, an effect missing with TPhP. Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1 mg.mL-1 in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty-acid catabolic processes centred on acylcarnitine and mitochondrial β-oxidation. Interestingly, mRNA expression confirmed and extended these observations by demonstrating at all tested doses the overall repression of genes involved in lipid catabolic processes and regulated by PPARα, a master regulator of β-oxidation and its associated ketogenesis. IHC analysis confirmed the alteration of these pathways by showing a specific downregulation of Hmgcs2, a kernel target gene of PPARα, at all doses tested, and surprisingly, a strong reduction of the lipid droplet content only at the highest dose. Overall, DPhP absorption led to weight loss, which was significant using the highest dose.ConclusionsOur results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver in particular is essential for controlling fast and feast periods with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular and metabolic disease incidence deserves, in light of our results, further investigations.

Background The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. Results Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. Conclusions Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes. Electronic supplementary material The online version of this article (doi:10.1186/s12900-017-0076-x) contains supplementary material, which is available to authorized users.

National audience

Hydrazine-based liquid propellants are routinely used for space rocket propulsion, in particular monomethylhydrazine (MMH), although such compounds are highly hazardous. For several years, great efforts were devoted to developing a less hazardous molecule. To explore the toxicological effects of an alternative compound, namely (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), we exposed various cellular animal and human models to this compound and to the reference compound MMH. We observed no cytotoxic effects following exposure to TMTZ in animal, as well as human models. However, although the three animal models were unaffected by MMH, exposure of the human hepatic HepaRG cell model revealed that apoptotic cytotoxic effects were only detectable in proliferative human hepatic HepaRG cells and not in differentiated cells, although major biochemical modifications were uncovered in the latter. The present findings indicate that the metabolic mechanisms of MMH toxicity is close to those described for hydrazine with numerous biochemical alterations induced by mitochondrial disruption, production of radical species, and aminotransferase inhibition. The alternative TMTZ molecule had little impact on cellular viability and proliferation of rodent and human dermic and hepatic cell models. TMTZ did not produce any metabolomic effects and appears to be a promising putative industrial alternative to MMH.

Background: Ubiquitin modification of histones regulates gene expression. Results: USP42 targets histone H2B at promoters, leading to decreased ubiquitylation. This correlates with the regulation of transcription driven by a number of transcription factors. Conclusion: USP42 contributes to the modulation of transcription. Significance: The identification of histone H2B as a target for USP42 extends our understanding of the factors that can regulate gene expression., Ubiquitin-specific peptidase 42 (USP42) is a deubiquitylating enzyme that can target p53 and contribute to the stabilization of p53 in response to stress. We now show that USP42 can also regulate transcription independently of p53. USP42 co-localized with RNA polymerase II (RNA Pol II) in nuclear foci, bound to histone H2B, and deubiquitylated H2B. Depletion of USP42 increased H2B ubiquitylation at a model promoter and decreased both basal and induced transcription from a number of promoters. These results are consistent with a role for USP42 in regulating transcription by deubiquitylating histones.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.

In addition to acting as a transcriptional cofactor for p53, ASPP1 has been shown to function in the cytoplasm to regulate the nuclear localization and activity of YAP/TAZ. We show here that the ability of ASPP1 to activate YAP results in the decreased expression of LATS2, which lowers the ability of p53 to induce p21, cell-cycle arrest and senescence. ASPP1 expression peaks in S-phase, and down-regulation of ASPP1 leads to a reduction in DNA synthesis and enhanced senescence in response to drugs that impede DNA replication. These activities of cytoplasmic ASPP1 in opposing p53-mediated p21 expression are in contrast to the role of nuclear ASPP1 in cooperating with p53 to induce the expression of apoptotic target genes, and may help to dampen p53 activity in normal cells.

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.

The ASPP (apoptosis-stimulating protein of p53) family of proteins can function in the nucleus to modulate the transcriptional activity of p53, with ASPP1 and ASPP2 contributing to the expression of apoptotic target genes. In this study, we describe a new function for cytoplasmic ASPP1 in controlling YAP (Yes-associated protein)/TAZ. ASPP1 can inhibit the interaction of YAP with LATS1 (large tumor suppressor 1), a kinase that phosphorylates YAP/TAZ and promotes cytoplasmic sequestration and protein degradation. This function of ASPP1 therefore enhances nuclear accumulation of YAP/TAZ and YAP/TAZ-dependent transcriptional regulation. The consequence of YAP/TAZ activation by ASPP1 is to inhibit apoptosis, in part through the down-regulation of Bim expression, leading to resistance to anoikis and enhanced cell migration. These results reveal a potential oncogenic role for cytoplasmic ASPP1, in contrast to the tumor-suppressive activity described previously for nuclear ASPP1.

Comment on: Lane DP, et al. Cell Cycle 2009; 9:540-7 and Lane DP, et al. Cell Cycle 2009; 9:748-54.

Constitutive activation of STAT3 (signal transducer and activator of transcription) has been reported in several primary cancers and tumor cell lines where it induces cell transformation through a combined inhibition of apoptosis and cell-cycle activation. Several studies have suggested that STAT3 prevents cell-cycle arrest and cell death through upregulation of survival proteins and downregulation of tumor suppressors. As a consequence of anti-apoptotic and proliferative lesions, we propose that this oncogenic pathway is also involved in intrinsic drug resistance and that STAT3-expressing tumors are resistant to chemotherapeutic agents. If this hypothesis is correct, the detection of the activated form of this protein should help to define subsets of tumors that fail to respond to chemotherapy. Furthermore, interfering with the STAT3 oncogenic pathway might restore the sensitivity to anticancer drugs.

In addition to its function as a cyclin-dependent kinase (cdk) inhibitor, p21waf1 fulfills additional roles involved in DNA replication and transcriptional regulation that could also contribute to cell cycle arrest. In this study, we have shown that p21waf1 functions as a transcriptional repressor of the myc and cdc25A genes. Ectopic expression of the cell cycle inhibitor down-modulates myc and cdc25A transcription but has no effect on cdk4 levels. Using chromatin immunoprecipitation, we found that p21waf1 is recruited to the promoters of these two genes together with the STAT3 and E2F1 transcription factors. Its presence on DNA is associated with an inhibition of the recruitment of the p300 histone acetylase and with a down-regulation of histone H4 acetylation. The same effect was also observed following DNA damage because topoisomerase inhibitors such as sn38 or doxorubicin also induce the association of p21waf1 with DNA. Following transcriptional repression of the myc and cdc25A genes, cells were arrested in the fraction with 4 N DNA content. By contrast, the expression of these two genes remains elevated in the absence of the cell cycle inhibitor, and p21waf1-/- cells re-replicate their DNA and become polyploid. In light of these results, we propose that p21waf1 simultaneously targets cdk and transcriptional regulators to prevent the expression of oncogenic pathways upon DNA damage.

Following several key discoveries on hypoxia-inducible factors, we have observed an explosion of studies investigating how the hypoxic microenvironment provokes bioenergetic alterations. This is particularly relevant for cancer cells, as they are often exposed to hypoxic conditions in the course of tumor progression. Thus, interest in the measurement of oxygen consumption at the tissue, cell, or mitochondrion level has been revived. Here, we describe the basic principles of cellular respiration and survey some of the conventional methods for measuring O2 consumption in intact or permeabilized cells.

Following several key discoveries on hypoxia-inducible factors, we have observed an explosion of studies investigating how the hypoxic microenvironment provokes bioenergetic alterations. This is particularly relevant for cancer cells, as they are often exposed to hypoxic conditions in the course of tumor progression. Thus, interest in the measurement of oxygen consumption at the tissue, cell, or mitochondrion level has been revived. Here, we describe the basic principles of cellular respiration and survey some of the conventional methods for measuring O2 consumption in intact or permeabilized cells.

In addition to its function as a tumour suppressor, p53 is also involved in an increasing number of pathology associated with aging. Several activities of p53 appear contribute to its role in aging; one function that might be particularly relevant in this context is the regulation of senescence. The control of ROS and senescence by p53 may help to explain how p53 can function to both restrain and promote aging.

International audience; The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.

The epidermal growth factor receptor (EGFR)-src-signal transducers and activators of transcription 3 (STAT3) oncogenic pathway plays a central role in tumorigenesis and is involved not only in cell transformation but also in tumor escape to genotoxic treatments. Despite its importance, the molecular mechanisms by which this signaling pathway induces resistance to DNA damage remain most of the time to be characterized. In this study, we show that the EGFR-src pathway is activated in response to topoisomerase I inhibition. After treatment, this signaling cascade induced the activation of STAT3 and the binding of the transcription factor to the promoter of the Eme1 gene. Eme1 is an endonuclease involved in the processing of DNA damage after topoisomerase I inhibition. These results suggest a model by which the STAT3-mediated activation of Eme1 prevents DNA damage and enhances cell survival in response to topoisomerase inhibition. This survival pathway was inhibited by a combined treatment with a src inhibitor, SKI, and with cetuximab, a monoclonal antibody directed against the EGFR that is widely used in the treatment of colorectal cancers. We therefore propose that the benefit of anti-EGFR therapy relies on an increase of DNA damage generated by topoisomerase I inhibition. [Cancer Res 2008;68(3):815–25]

STAT3 transcription factors are implicated in cellular transformation and chemotherapy resistance. During the treatments, STAT3 interacts with the transcriptionnal repressor Rb and the cell cycle inhibitor p21. These two proteins inhibit its transcriptionnal activity and induce senescence following mitotic catastrophe. However, the constitutive activity of STAT3 prevents Rb and p21 activation, increses cellular resistance to topoisomerase II inhibitors, and generates genomic instability. Finally, two STAT3 inhibitors sensitize colorectal cells to topoisomerase I inhibitors. We also show that STAT3 upregulates the expression of several DNA repair genes such as Emel, thereby enhancing resistance to genotoxic treatment. As a consequence, we propose that STAT3 should be considered as a predictive marker of drug resistance and its inhibition, in tumor where it is active, could sensitize the tumors to conventional treatment., Les facteurs de transcription STAT3 sont activés dans de nombreuses tumeurs et leurs effets sur la prolifération et la survie suggéraient fortement que ces protéines puissent être impliquées à la fois dans la transformation cellulaire et dans l'échappement aux traitements classiques de chimiothérapie.Nous nous sommes donc intéressés aux différents aspects impliquant STAT3 dans la réponse de lignées cellulaires aux agents génotoxiques de chimiothérapie, et plus particulièrement aux inhibiteurs de topoisomérase. Durant ces traitements, STAT3 interagit avec le répresseur transcriptionnel Rb et l'inhibiteur du cycle cellulaire p21. Ces deux protéines inhibent son activité transcriptionnelle, notamment sur les gènes c-myc et cdc25A, et permettent la mise en place de la sénescence induite par les dommages de l'ADN et la catastrophe mitotique. Cependant, en présence d'une activité constitutive de STAT3 induite par l'oncogène v-src, STAT3 empêche l'activation de Rb et de p21, favorise la résistance des cellules aux inhibiteurs de topoisomérase II, et génère de l'instabilité génomique. Finalement,deux inhibiteurs de STAT3, le cetuximab, un anticorps monoclonal dirigé contre l'EGFR, et un inhibiteur de la tyrosine kinase c-src, sensibilisent des cellules colorectales aux inhibiteurs de topoisomérase I. L'inhibition de STAT3 empêche l'activation du gène Eme1 qui induit l'expression d'une protéine de réparation de l'ADN.STAT3 est donc un facteur de résistance aux inhibiteurs de topoisomérase. Sa détection pourrait ainsi permettre de mieux prédire la réponse des patients à ces inhibiteurs, et son inhibition, dans les tumeurs où il est actif, pourrait permettre de les sensibiliser aux inhibiteurs de topoisomérase.

In addition to its function as a cyclin-dependent kinase (cdk) inhibitor, p21waf1 fulfills additional roles involved in DNA replication and transcriptional regulation that could also contribute to cell cycle arrest. In this study, we have shown that p21waf1 functions as a transcriptional repressor of the myc and cdc25A genes. Ectopic expression of the cell cycle inhibitor down-modulates myc and cdc25A transcription but has no effect on cdk4 levels. Using chromatin immunoprecipitation, we found that p21waf1 is recruited to the promoters of these two genes together with the STAT3 and E2F1 transcription factors. Its presence on DNA is associated with an inhibition of the recruitment of the p300 histone acetylase and with a down-regulation of histone H4 acetylation. The same effect was also observed following DNA damage because topoisomerase inhibitors such as sn38 or doxorubicin also induce the association of p21waf1 with DNA. Following transcriptional repression of the myc and cdc25A genes, cells were arrested in the fraction with 4 N DNA content. By contrast, the expression of these two genes remains elevated in the absence of the cell cycle inhibitor, and p21waf1-/- cells re-replicate their DNA and become polyploid. In light of these results, we propose that p21waf1 simultaneously targets cdk and transcriptional regulators to prevent the expression of oncogenic pathways upon DNA damage.

DNA-damaging drugs stop tumor cell proliferation by inducing apoptosis, necrosis, or senescence. Cyclin-dependent kinase inhibitor p21waf1 is an important regulator of these responses, promoting senescence and preventing aberrant mitosis that leads to cell death. Because tumors expressing oncogenic tyrosine kinases are relatively resistant to DNA-damaging agents, the effects of Src on cellular responses to anticancer drug Adriamycin were investigated. Src expression increased drug survival in HT1080 fibrosarcoma cells, as measured by the colony formation assay, and strongly inhibited Adriamycin-induced senescence. Src also decreased the number of apoptotic cells while increasing the fraction of cells dying through necrosis. In addition, Src inhibited the G2 and G1 tetraploidy checkpoints of Adriamycin-treated cells, permitting these cells to proceed into mitosis and subsequently double their DNA content. Inhibition of senescence and G2-G1 checkpoints in Src-expressing cells was associated with the failure of these cells to up-regulate p21waf1 in response to Adriamycin. The failure of p21waf1 induction, despite increased expression of p53 and its binding to p21waf1 promoter, was mediated by the up-regulation of c-Myc, a negative regulator of p21waf1 transcription. Conversely, ectopic expression of p21waf1 inhibited Myc transcription in Src-expressing cells, an effect that was associated with the interaction of p21waf1 with the STAT3 transcription factor at the Myc promoter. These results reveal a complex effect of Src on cellular drug responses and provide an explanation for the effect of this oncogene on cellular drug resistance.

The STAT3 (signal transducer and activator of transcription) transcription factor functions as down-stream effector of growth factor signaling. Whereas STAT3 activation is transient in normal cells, constitutively activated forms of the transcription factor have been detected in several cancer cell lines and primary tumors. Through the up-regulation of cell cycle and survival genes, STAT3 plays important roles in cell growth, anti-apoptosis, and cell transformation yet the molecular basis for this behavior is poorly understood. In this study, we show that STAT3 and its transcriptional cofactors are recruited to the promoter of the Cdc25A gene to activate its expression. Using chromatin immunoprecipitations, we observed that Myc is recruited to this promoter following STAT3 DNA binding. Moreover, small interfering RNA-mediated knockdown of Myc specifically inhibits the STAT3-mediated activation of Cdc25A. Reduction in Myc protein level results in defective recruitment of the CREB-binding protein, Cdk9, and RNA polymerase complexes, indicating that Myc is necessary for STAT3 transcription. Surprisingly, the association of STAT3 with the Cdc25A promoter does not necessarily lead to transcriptional induction because this protein also functions as a transcriptional repressor of the Cdc25A gene. Following hydrogen peroxide stimulation, STAT3 forms a repressor complex with the retinoblastoma (Rb) tumor suppressor to occupy the Cdc25A promoter and block its induction. In coimmunoprecipitations and ChIP experiments, Rb was found to associate with STAT3 on DNA and we provide evidence that Rb binds directly to the transcription factor. Thus, we propose that Myc and STAT3 cooperate to induce the expression of Cdc25A and that their transcriptional activity is normally regulated by the Rb tumor suppressor gene.

During the G2 phase of the cell cycle, the Aurora-A kinase plays an important role in centrosome maturation and progression to mitosis. In this study, we show in colorectal cell lines that Aurora-A expression is downregulated in response to topoisomerase I inhibition. Using chromatin immunoprecipitation assays, we have observed that the Myc transcription factor and its Max binding partner are associated with the Aurora-A promoter during the G2 phase of the cell cycle. RNA interference experiments indicated that Myc is involved in the regulation of the Aurora-A gene. Following topoisomerase I inhibition, the expression of Myc decreased whereas Mad was upregulated, and the association of Myc and Max with the promoter of the kinase was inhibited. In parallel, an increased association of Mad and Miz-1 was detected on DNA, associated with an inhibition of the recruitment of transcriptional coactivators. Interestingly, a gain of H3K9 trimethylation and HP1γ recruitment was observed on the Aurora-A promoter following sn38 treatment, suggesting that this promoter is located within SAHF foci following genotoxic treatment. Since Aurora-A is involved in centrosome maturation, we observed as expected that topoisomerase I inhibition prevented centrosome separation but did not affect their duplication. As a consequence, this led to G2 arrest and senescence induction. These results suggest a model by which the Aurora-A gene is inactivated by the G2 checkpoint following topoisomerase I inhibition. We therefore propose the hypothesis that the coordinated overexpression of Myc and Aurora-A, together with a downregulation of Mad and Miz-1 should be tested as a prognosis signature of poor responses to topoisomerase I inhibitors.

Cancer cells with stemness properties - generally designated cancer stem cells (CSCs) - are at the apex of the hierarchical organisation of tumours and are believed to drive tumour recurrence and metastasis formation. However, how they perform these neoplastic activities in a nutritive environment that is distinct from the one in an established tumour is unknown. Here, we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy necessary for the tumorigenic potential of CSCs. By regulating MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this regulation, glucocorticoids activate the hexosamine biosynthetic pathway and rewire the metabolism of CSCs. The anabolic efficiency of these cells increases then, fostering tumour and metastasis development. Together, our findings suggest that inhibition of glucocorticoid metabolic activity could be an original strategy for CSC eradication and tumour treatment; Les cellules cancéreuses ayant des propriétés souches, appelées cellules souches cancéreuses (CSCs), sont au sommet de l'organisation hiérarchique des tumeurs et sont soupçonnées d'être responsable des rechutes et de la formation de métastases. Cependant, nous nous sommes demandé comment ces cellules sont-elles capables de supporter le stress métabolique lié à l'oncogène et de croitre dans un environnement nutritif défavorable distinct de celui d'une tumeur établie ? Nous avons mis en évidence dans ce travail le rôle de la réponse aux glucocorticoïdes dans le contrôle de la plasticité mammaire et l'induction d'une plasticité métabolique nécessaire au maintien du potentiel tumorigène des CSCs. Nous avons montré qu'en régulant la reprogrammation cellulaire via l'induction du gène MAFB, les glucocorticoïdes contrôlent le caractère souche des cellules épithéliales malignes en activant la voie de biosynthèse des hexosamines. L'efficacité anabolique de ces cellules est alors augmentée, favorisant le développement de la tumeur et des métastases. Ensemble, nos résultats suggèrent que l'inhibition de l'activité métabolique des glucocorticoïdes pourrait être une stratégie pour éliminer les CSCs et traiter les tumeurs mammaires

Cancer cells with stemness properties - generally designated cancer stem cells (CSCs) - are at the apex of the hierarchical organisation of tumours and are believed to drive tumour recurrence and metastasis formation. However, how they perform these neoplastic activities in a nutritive environment that is distinct from the one in an established tumour is unknown. Here, we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy necessary for the tumorigenic potential of CSCs. By regulating MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this regulation, glucocorticoids activate the hexosamine biosynthetic pathway and rewire the metabolism of CSCs. The anabolic efficiency of these cells increases then, fostering tumour and metastasis development. Together, our findings suggest that inhibition of glucocorticoid metabolic activity could be an original strategy for CSC eradication and tumour treatment, Les cellules cancéreuses ayant des propriétés souches, appelées cellules souches cancéreuses (CSCs), sont au sommet de l'organisation hiérarchique des tumeurs et sont soupçonnées d'être responsable des rechutes et de la formation de métastases. Cependant, nous nous sommes demandé comment ces cellules sont-elles capables de supporter le stress métabolique lié à l'oncogène et de croitre dans un environnement nutritif défavorable distinct de celui d'une tumeur établie ? Nous avons mis en évidence dans ce travail le rôle de la réponse aux glucocorticoïdes dans le contrôle de la plasticité mammaire et l'induction d'une plasticité métabolique nécessaire au maintien du potentiel tumorigène des CSCs. Nous avons montré qu'en régulant la reprogrammation cellulaire via l'induction du gène MAFB, les glucocorticoïdes contrôlent le caractère souche des cellules épithéliales malignes en activant la voie de biosynthèse des hexosamines. L'efficacité anabolique de ces cellules est alors augmentée, favorisant le développement de la tumeur et des métastases. Ensemble, nos résultats suggèrent que l'inhibition de l'activité métabolique des glucocorticoïdes pourrait être une stratégie pour éliminer les CSCs et traiter les tumeurs mammaires