Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

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 KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-ß1 (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.

CFL1 expression levels as a prognostic and drug resistance marker in nonsmall cell lung cancer.

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=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.

Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin.

Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H(2)O(2)) 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.

p53 and ATF-2 partly mediate the overexpression of COX-2 in H(2)O (2)-induced premature senescence of human fibroblasts.

Cyclooxygenase-2 and release of prostaglandin E2 are up-regulated in replicative senescence of dermal and prostate fibroblasts and in H(2)O(2)-induced premature senescence of IMR-90 lung fibroblasts expressing the catalytic subunit of telomerase. Inhibition of cyclooxygenase-2 activity by specific chemical inhibitor or siRNA attenuates the H(2)O(2)-induced increase of senescence associated beta-galactosidase positive cells and attenuates growth arrest. In this work, p38(MAPK) activation and increased DNA binding activities of ATF-2 and p53 are shown to mediate cyclooxygenase-2 overexpression in premature senescence.

Normal or stress-induced fibroblast senescence involves COX-2 activity.

Cyclooxygenase-2 (COX-2) is an inducible enzyme of the prostaglandin biosynthesis pathway. It is involved in many stress responses, and its activity can produce oxidative damage, suggesting it could participate in senescence. In this study, COX-2 expression is shown to increase during senescence of normal human dermal or prostatic fibroblasts, and the ensuing prostaglandin E(2) (PGE(2)) production to increase about 10-fold. Enhancing this COX-2 activity by supplying exogenous arachidonic acid accelerates the occurrence of the major markers of senescence, cell-size increase, spreading, senescence-associated-beta-galactosidase (SA-beta-Gal) activity and growth plateau. Conversely, blocking this COX-2 activity with the specific inhibitor NS398 partially inhibited the occurrence of these markers. COX-2 expression and PGE(2) production are also increased about 10-fold during both NF-kappaB- or H(2)O(2)-induced senescence. Using NS398 or small interferent RNA specifically targeting COX-2 attenuated the appearance of the SA-beta-Gal activity and growth arrest in both stress situations. Taken together, these findings indicate that COX-2 is highly up-regulated during both normal and stress-induced fibroblast senescence and contributes to the establishment of the senescent characteristics.

Knocking down p53 with siRNA does not affect the overexpression of p21WAF-1 after exposure of IMR-90 hTERT fibroblasts to a sublethal concentration of H2O2 leading to premature senescence.

Premature senescence of IMR-90 human diploid fibroblasts (HDFs) expressing telomerase was induced by exposure to sublethal concentration of H(2)O(2), with appearance of several biomarkers of cellular senescence like enlarged cell shape, senescence-associated beta-galactosidase (SA ss-gal) activity, and cell cycle arrest. The induction of stress-induced premature senescence (SIPS) was associated with a transient increase in DNA-binding activity of p53 and an increased expression of p21(WAF-1). p53 small interferent RNA (siRNA) affected the basal level of p21(WAF-1) mRNA but did not affect the overexpression of p21(WAF-1) after stress. This siRNA approach confirms previous results obtained with other methods.

Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts.

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.

Establishment of H2O2-induced premature senescence in human fibroblasts concomitant with increased cellular production of H2O2.

Premature senescence of human fibroblasts is established after exposure to an acute sublethal concentration of H(2)O(2). Overexpression of transforming growth factor-beta1 (TGF-beta1) was shown to be responsible for the appearance of the biomarkers of senescence in these conditions. Other studies have shown that incubation of human fibroblasts with TGF-beta1 leads to overexpression of H(2)O(2). In this work, we show an increased production of H(2)O(2) by human fibroblasts as premature senescence is established after an initial exposure to H(2)O(2).

From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing.

The Hayflick limit-senescence of proliferative cell types-is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of 'feeder cells' can trigger either SIPS or prolonged reversible G(0) phase of the cell cycle. In vivo, 'proliferative' cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.

Stress-induced premature senescence: from biomarkers to likeliness of in vivo occurrence.

The similarities between the biomarkers of stress-induced premature senescence and replicative senescence are reviewed. The possibility of existence of 'molecular scars', i.e. long-term changes observed after subcytotoxic stress and not observed in replicative senescence, is considered. Lastly, the likeliness of existence of stress-induced premature senescence in vivo is discussed. The possible effects on normal and pathological tissue ageing are predicted.

Stress-induced premature senescence or stress-induced senescence-like phenotype: one in vivo reality, two possible definitions?

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.