Role of Oncogene-Induced Senescence in Thyroid Carcinogenesis

Oncogene-induced senescence (OIS) is an irreversible G1 cell cycle arrest triggered by aberrant expression of oncogenes. The arrest observed during senescence is implemented mainly through activation of p53 and the upregulation of the cyclin-dependent kinase (CDK) inhibitors, p16INK4a and p21CIP1. In vivo OIS may act as a barrier to neoplastic transformation, and bypass of OIS is a prerequisite for tumorigenesis. In this work the role of OIS in thyroid carcinogenesis was investigated by in combination of in vitroand in vivo approaches. We found that the expression of different thyroid tumour-associated oncogenes (BRAF, RAS, RET and TRK) in human primary thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks. For instance, cells appeared flatted and enlarged; they displayed senescence-associated B-galactosidase activity (SA-β-Gal), senescence associated heterochromatic foci (SAHF), and high expression levels of the CDK inhibitors p16INK4a and p21CIP1 and p53. Additionally, using RNA-interference strategy we directly assessed the individual contribution of p16INK4a in BRAFV600E-induced senescence. In our experimental setting the inactivation of p16INK4a did not have any impact on oncogene-induced senescence suggesting the possibility that p16INK4a may cooperate with other factors in triggering senescence. A major consequence of oncogene activation is the secretion of a plethora of proteins including growth factors, cytokines, and chemokines. Some of these secreted proteins are known to be regulators of OIS. For example, IGFBP7 is recently proposed as a mediator of BRAFV600E-induced senescence in melanocytes. We showed that IGFBP7 gene is frequently downregulated in Papillary thyroid tumour (PTC); restoration of its expression in a PTC-derived, IGFBP7-negative cell line affects growth properties, migration and invasion, and this is associated to the induction of apoptosis. Thus, our results implicate IGFBP7 as a tumour suppressor protein in thyroid tumours. Additionally, we investigated the possible involvement of IGFBP7 in our cellular senescence model. Our data indicate that IGFBP7 expression is not changed during OIS. We also established and characterized OIS inducible system in our cellular model; we transduced human primary thyrocytes with a retroviral vector carrying an activated form of RAS oncogene coupled to the ligand binding domain of the estrogen receptor. Upon addition of 4-hydroxytamoxifen (4-OHT), we observed a robust induction of senescence, including significantly reduced incorporation of BrdU and high levels of p16INK4a p21CIP1, and p53. Moreover, we assessed the relative importance of tumour suppressor proteins, p16INK4a and p53 in mediating OIS in H-RASG12V transfected thyrocytes. Surprisingly, silencing of p53 expression did not alter the growth arrest induced by H-RASG12V, whereas p16INK4a seems to be the dominant effector of senescence in this experimental setting, as its specific inactivation delays the onset of senescence. We also found that senescence induced by H-RASG12V is associated with activation of inflammatory transcriptome including IL8 and its receptor CXCR2. We reported that knocking down the chemokine receptor CXCR2 either IL8 prevents H-RASG12V cells from undergoing OIS. To translate information from in vitroexperiments to a clinic setting, senescence markers identified in cultured cells were used to detect OIS in a panel of thyroid tumours characterized by different aggressiveness. Our immunohistochemical analysis showed that the expression of OIS markers is upregulated at early stages of the thyroid tumour and lost during tumour progression. These evidences support the notion that OIS may counteract oncogenic activity in thyroid tumours, and its escape allows thyroid tumour progression

Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth

Cytoplasmic chromatin triggers inflammation in senescence and cancer

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders

Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP

Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease

Senescence can be BETter without the SASP?

Summary: Global remodeling of the chromatin landscape occurs during senescence, although its functional consequence is still unclear. In this issue, Tasdemir and colleagues show that the epigenetic regulator BRD4 is required for expression of the proinflammatory senescence-associated secretory phenotype and immune clearance of senescent cells in vitro and in vivo. Their results could be useful in the design of novel therapies to treat aging-related diseases, including cancer

Cytoplasmic chromatin fragments—from mechanisms to therapeutic potential

Senescent cells, damaged cells that permanently exit the cell cycle, play important roles in development, tissue homeostasis, and tumorigenesis. Although many of these roles are beneficial in acute responses to stress and damage, the persistent accumulation of senescent cells is associated with many chronic diseases through their proinflammatory senescence-associated secretory phenotype (SASP). SASP expression is linked to DNA damage; however, the mechanisms that control the SASP are incompletely understood. More recently, it has been shown that senescent cells shed fragments of nuclear chromatin into the cytoplasm, so called cytoplasmic chromatin fragments (CCF). Here, we provide an overview of the current evidence linking DNA damage to the SASP through the formation of CCF. We describe mechanisms of CCF generation and their functional role in senescent cells, with emphasis on therapeutic potential

Role of Stat3 in TRK-induced cell growth of NIH3T3 derived foci.

<p>(<b>A</b>) Phase contrast images of NF861, NF797 and NIH3T3 cells treated or not with S3I-201 (100 µM, 48 hr). (<b>B</b>) Cell viability of NF861 and NF797 cells treated with DMSO (0.3%) or S3I-201 (100 µM) determined by the Alamar Blue cell viability assay. (<b>C</b>) Western blot analysis of NIH3T3, NF861 and NF797 cells treated with S3I-201 (100 µM, 48 hr). Samples were immunoblotted with the indicated antibodies.</p

Reduction of Stat3 level is associated to morphological transformation induced by TRK oncogenes activity.

<p>(<b>A</b>) NIH3T3, NF861 and NF797 cells were treated with K252a (200nM) for the indicated time; cell lysates were processed and immunoblotted with the indicated antibodies. Densitometric analysis of the bands is reported in the bottom panel. Images were acquired by Biorad ChemiDoc and analysed with Image Quant software. Data are reported as ratio of STAT3/vinculin and normalized over untreated samples for any cell line. (<b>B</b>) Cell extracts from NIH3T3, NF861 and NF797 cells treated with K252a (200nM, 16 hr) were analyzed by Western blot with the indicated antibodies. (<b>C</b>) Cell extracts from NIH3T3, NF861 and NF797 cells treated with UO126 (10 µM, 16 hr) were analyzed by Western blot with the indicated antibodies. (<b>D</b>) Phase contrast images (top) and α-tubulin immunostaining (bottom) of NIH3T3 cells, NF797 cells treated or not with K252a (200nM, overnight) or UO126 (10 µM).</p

STAT3 activation by TRK oncogenes.

<p>(<b>A</b>) Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRKA, TRK-T3, and TRK cDNAs. NGF treatment (50ng/ml, 10′) is indicated. Cell lysates and immunocomplexes were separated by SDS PAGE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009446#s4" target="_blank">Material and Methods</a> and immunoblotted with indicated antibodies. In the bottom panel two distinct exposures of the same blot were used for documenting TRKA or TRK oncoproteins phosphorylation. (<b>B</b>) HeLa cells were co-transfected with pM67 and pRL-TK in combination with the indicated TRK oncogene cDNAs in the absence (left graph) or presence (right graph) of STAT3 cDNA and assayed for STAT3-dependent luciferase activity 48 hours later. Activity is expressed as the ratio of luciferase/renilla activity, and reported as fold-inductions over empty vector (left panel) or STAT3 (right panel) (RLA: Relative Luciferase Activity). The data represent the mean values ± SD of triplicate samples. Similar results were obtained in three independent experiments. (<b>C</b>) MAPK involvement in TRK-induced Stat3 phosphorylation. Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRK-T3 or TRK cDNAs. NGF (10′, 50ng/ml) and UO126 (16 hr, 10 µM) treatments are indicated. Immunoblotting was performed with the indicated antibodies.</p