Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H 2 O 2 , with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-␤1 (TGF-␤1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated ␤-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-␤1 or its receptor (TGF-␤ RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated ␤-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-␤1 in stressful conditions, leading to SIPS and overexpression of these four genes.Normal human diploid fibroblasts (HDFs) 1 exposed to various types of noncytotoxic oxidative stress display a senescentlike phenotype coined "stress-induced premature senescence" or SIPS (1, 2). Such stressful conditions include exposure to hydrogen peroxide (3, 4), tert-butylhydroperoxide (t-BHP) (5), hyperoxia (6), UV light (7), and radioactivity (8). Many biomarkers of replicative senescence appear in SIPS: typical cell morphology (5), irreversible growth arrest, lack of response to mitogenic stimuli (4), sharp decrease of the DNA synthesis, and an increase in cells positive for the senescent-associated ␤-galactosidase activity (SA ␤-gal) (9). A long term overexpression of the cyclin-dependent kinase inhibitor p21waf-1 was observed in SIPS induced by H 2 O 2 (10) or t-BHP (9). p21waf-1 inhibits the cyclin D/cyclin-dependent kinase 4 and 6 complexes, leading to hypophosphorylation of the retinoblastoma protein (Rb). A long term hypophosphorylation of Rb over several weeks was indeed observed in SIPS induced by H 2 O 2 or t-BHP, explaining the block of the cell cycle, through Rb-mediated inhibition of the E 2 F transcription factor (9, 10). Last, several genes overexpressed in senescent HDFs, such as fibronectin, osteonectin, SM22, and apolipoprotein J (clusterin), displayed a similar increase in mRNA level in SIPS induced by t-BHP or H 2 O 2 (9).In different experimental models, an overexpression of either SM22 (11), apolipoprotein J (12), osteonectin (13), or fibronectin (14) is induced by extracellular addition of transforming growth factor-␤1 (TGF-␤1). Moreover, incubation of HDFs with TGF-␤1 triggers the appearance of a senescent-like morphology (15, 16) and growth arrest (17).Two main arguments favor the hypothesis that oxidative stress-induced premature senescence could be triggered by a pRb-mediated TGF-␤1 overexpression. First, it has been shown that AT...

Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation1,2. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H2O2) can damage proteins and induce apoptosis, but the role of specific protein oxidation in this process has not been defined. We found that the actin-binding protein cofilin is a key target of oxidation. When oxidation of this single regulatory protein is prevented, oxidant-induced apoptosis is inhibited. Oxidation of cofilin causes it to lose its affinity for actin and to translocate to the mitochondria, where it induces swelling and cytochrome c release by mediating opening of the permeability transition pore (PTP). This occurs independently of Bax activation and requires both oxidation of cofilin Cys residues and dephosphorylation at Ser 3. Knockdown of endogenous cofilin using targeted siRNA inhibits oxidant-induced apoptosis, which is restored by re-expression of wild-type cofilin but not by cofilin containing Cys to Ala mutations. Exposure of cofilin to TnCl results in intramolecular disulphide bonding and oxidation of Met residues to Met sulphoxide, but only Cys oxidation causes cofilin to induce mitochondrial damage.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic, mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRASG12V gene in wild-type KRAS tumor cells led to Treg induction. We also demonstrate that mutant KRAS induces the secretion of interleukin-10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

BACKGROUND: Nonsmall cell lung cancer (NSCLC) is the major determinant of overall cancer mortality worldwide. Despite progress in molecular research, current treatments offer limited benefits. Because NSCLC generates early metastasis, and this behavior requires great cell motility, herein the authors assessed the potential value of CFL1 gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker. METHODS: Metadata analysis of tumor tissue microarray was applied to examine expression of CFL1 in archival lung cancer samples from 111 patients, and its clinicopathologic significance was investigated. The robustness of the finding was validated using another independent data set. Finally, the authors assayed in vitro the role of CFL1 levels in tumor invasiveness and drug resistance using 6 human NSCLC cell lines with different basal degrees of CFL1 gene expression. RESULTS: CFL1 levels in biopsies discriminate between good and bad prognosis at early tumor stages (IA, IB, and IIA/B), where high CFL1 levels are correlated with lower overall survival rate (P < .0001). Biomarker performance was further analyzed by immunohistochemistry, hazard ratio (P < .001), and receiver-operating characteristic curve (area ¼ 0.787; P < .001). High CFL1 mRNA levels and protein content are positively correlated with cellular invasiveness (determined by Matrigel Invasion Chamber System) and resistance (2-fold increase in drug 50% growth inhibition dose) against a list of 22 alkylating agents. Hierarchical clustering analysis of the CFL1 gene network had the same robustness for stratified NSCLC patients. CONCLUSIONS: This study indicates that the CFL1 gene and its functional gene network can be used as prognostic biomarkers for NSCLC and could also guide chemotherapeutic interventions. Cancer 2010;116;3645-55.

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.