Chantal Vercamer

Involvement of Rel/Nuclear Factor-κB Transcription Factors in Keratinocyte Senescence

After a finite doubling number, normal cells become senescent, i.e., nonproliferating and apoptosis resistant. Because Rel/nuclear factor (NF)-κB transcription factors regulate both proliferation and apoptosis, we have investigated their involvement in senescence. cRel overexpression in young normal keratinocytes results in premature senescence, as defined by proliferation blockage, apoptosis resistance, enlargement, and appearance of senescence-associated β-galactosidase (SA-β-Gal) activity. Normal senescent keratinocytes display a greater endogenous Rel/NF-κB DNA binding activity than young cells; inhibiting this activity in presenescent cells decreases the number of cells expressing the SA-β-Gal marker. Normal senescent keratinocytes and cRel-induced premature senescent keratinocytes overexpressed manganese superoxide dismutase (MnSOD), a redox enzyme encoded by a Rel/NF-κB target gene. MnSOD transforms the toxic O 2 into H2O2, whereas catalase and glutathione peroxidase convert H2O2 into H2O. Neither catalase nor glutathione peroxidase is up-regulated during cRel-induced premature senescence or during normal senescence, suggesting that H2O2 accumulates. Quenching H2O2 by catalase delays the occurrence of both normal and premature cRel-induced senescence. Conversely, adding a nontoxic dose of H2O2 to the culture medium of young normal keratinocytes induces a premature senescence-like state. All these results indicate that Rel/NF-κB factors could take part in the occurrence of senescence by generating an oxidative stress via the induction of MnSOD.

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.

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age.

Expression of an Ets-1 dominant-negative mutant perturbs normal and tumor angiogenesis in a mouse ear model

We and others have shown that members of the Ets family of transcription factors are involved in morphogenic properties of endothelial cells in vitro. To investigate the role of these factors in the transcriptional regulation of angiogenesis in vivo, we set up a nontraumatic model that allows daily macroscopic examination of both growth factor- and tumor-induced angiogenesis in mouse ears. In the same animal, we were thus able to record variations in the patterns of neovessels induced and cell populations recruited by the angiogenic factors FGF-2 and VEGF. In this model, inhibition of FGF-2-induced angiogenesis by the pharmacological compound TNP-470 was readily observed, demonstrating that the mouse ear model is also useful in the evaluation of antiangiogenic strategies. Our functional analysis of Ets transcription factors activity utilized a competitor protein, Ets1-DB, a dominant negative Ets1 mutant lacking the transactivation domain. Retrovirus-mediated expression of Ets1-DB inhibited FGF-2-induced angiogenesis, while the expression of Ets1-DB in cancerous and stromal cells disturbed tumor-induced angiogenesis. These results illustrate the value of the ear model and highlight the role of Ets family members in the transcriptional regulation of tumor angiogenesis.

MnSOD Upregulation Induces Autophagic Programmed Cell Death in Senescent Keratinocytes

Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H2O2 overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H2O2 level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H2O2. This H2O2-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

Constitutive expression of the DNA-binding domain of Ets1 increases endothelial cell adhesion and stimulates their organization into capillary-like structures.

We previously reported that the Ets1 transcription factor is expressed in endothelial cells during angiogenesis both in normal and pathological development. We analyse here the effects of the stable expression of an Ets transdominant negative mutant (Ets1-DB), consisting in an Ets1 protein lacking its transactivation domain. A retrovirus containing the Ets1-DB sequence fused to an IRES-Neo sequence was designed and used to infect brain capillary (IBE) and aorta (MAE) mouse endothelial cell lines. Cells expressing this Ets1 mutant were examined for proliferation, migration and adhesion. Consistent changes were observed on cell morphology, with increased spreading and modifications in the organization of the cytoskeleton, and increased cell adhesion. We investigated the ability of endothelial cells to organise into capillary-like structures using three-dimensional gels. On Matrigel, all endothelial cell lines formed a cord-like network within 24 h, with an increased ability of Ets1-DB cells to spread on this substrate. In long term cultures, IBE cells expressing Ets1-DB showed a higher capacity to form branched structures; this effect was potentiated by FGF2. These results demonstrate a role of the Ets transcription factors in the regulation of the adhesive and morphogenetic properties of endothelial cells.

Ets-1 controls breast cancer cell balance between invasion and growth

Ets‐1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets‐1 or a dominant negative mutant in MMT breast cancer cells, we previously highlighted the key role of Ets‐1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets‐1 decreased the density of breast cancer cells cultured in three‐dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two‐dimensional plastic or matrix‐coated dishes. Ets‐1 overexpression was deleterious to anchorage‐independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets‐1 was associated with epithelial‐to‐mesenchymal transition features and modulated the ratio of Ki67‐positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA‐mediated knockdown of Ets‐1 in human breast cancer cell lines also decreased colony growth, both in anchorage‐independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets‐1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets‐1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models.

ATF6α regulates morphological changes associated with senescence in human fibroblasts

Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated β-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-β-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts.

Identification of a gene signature of a pre-transformation process by senescence evasion in normal human epidermal keratinocytes

BackgroundEpidemiological data show that the incidence of carcinomas in humans is highly dependent on age. However, the initial steps of the age-related molecular oncogenic processes by which the switch towards the neoplastic state occurs remain poorly understood, mostly due to the absence of powerful models. In a previous study, we showed that normal human epidermal keratinocytes (NHEKs) spontaneously and systematically escape from senescence to give rise to pre-neoplastic emerging cells.MethodsHere, this model was used to analyze the gene expression profile associated with the early steps of age-related cell transformation. We compared the gene expression profiles of growing or senescent NHEKs to post-senescent emerging cells. Data analyses were performed by using the linear modeling features of the limma package, resulting in a two-sided t test or F-test based on moderated statistics. The p-values were adjusted for multiple testing by controlling the false discovery rate according to Benjamini Hochberg method.The common gene set resulting of differential gene expression profiles from these two comparisons revealed a post-senescence neoplastic emergence (PSNE) gene signature of 286 genes.ResultsAbout half of these genes were already reported as involved in cancer or premalignant skin diseases. However, bioinformatics analyses did not highlight inside this signature canonical cancer pathways but metabolic pathways, including in first line the metabolism of xenobiotics by cytochrome P450. In order to validate the relevance of this signature as a signature of pretransformation by senescence evasion, we invalidated two components of the metabolism of xenobiotics by cytochrome P450, AKR1C2 and AKR1C3. When performed at the beginning of the senescence plateau, this invalidation did not alter the senescent state itself but significantly decreased the frequency of PSNE. Conversely, overexpression of AKR1C2 but not AKR1C3 increased the frequency of PSNE.ConclusionsTo our knowledge, this study is the first to identify reprogrammation of metabolic pathways in normal keratinocytes as a potential determinant of the switch from senescence to pre-transformation.