Apoptotic stress causes mtDNA release during senescence and drives the SASP.

Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

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.

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.

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.

Senescent thyrocytes and thyroid tumor cells induce M2-like macrophage polarization of human monocytes via a PGE2-dependent mechanism.

BACKGROUND: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified. METHODS: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets. RESULTS: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors. CONCLUSIONS: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.

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.

Senescence Can Be BETter without the SASP?

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.Cancer Discov; 6(6); 576-8. ©2016 AACR.See related article by Tasdemir et al., p. 612.

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.

Oncogenic RAS-induced senescence in human primary thyrocytes: molecular effectors and inflammatory secretome involved.

Oncogene-induced senescence (OIS) is a robust and sustained antiproliferative response to oncogenic stress and constitutes an efficient barrier to tumour progression. We have recently proposed that OIS may be involved in the pathogenesis of thyroid carcinoma by restraining tumour progression as well as the transition of well differentiated to more aggressive variants. Here, an OIS inducible model was established and used for dissecting the molecular mechanisms and players regulating senescence in human primary thyrocytes. We show that oncogenic RAS induces senescence in thyrocytes as judged by changes in cell morphology, activation of p16INK4a and p53/p21CIP1 tumour suppressor pathways, senescence-associated ß-galactosidase (SA-ß-Gal) activity, and induction of proinflammatory components including IL-8 and its receptor CXCR2. Using RNA interference (RNAi) we demonstrate that p16INK4a is necessary for the onset of senescence in primary thyrocytes as its depletion rescues RAS-induced senescence. Furthermore, we found that IL-8/CXCR2 network reinforces the growth arrest triggered by oncogenic RAS, as its abrogation is enough to resume proliferation. Importantly, we observed that CXCR2 expression coexists with OIS markers in thyroid tumour samples, suggesting that CXCR2 contributes to senescence, thus limiting thyroid tumour progression.

S100A11 overexpression contributes to the malignant phenotype of papillary thyroid carcinoma.

CONTEXT: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels. OBJECTIVE: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology. DESIGN: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed. RESULTS: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation. CONCLUSION: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.

Evidence of oncogene-induced senescence in thyroid carcinogenesis.

Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-ß-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1). Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16(INK4a), p21(CIP1) and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis.

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.