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Supplementary Figure S10. The representative H&E staining photographs of human NASH-associated HCC.

Senescent cells exhibit several typical features, including the senescence-associated secretory phenotype (SASP), promoting the secretion of various inflammatory proteins and small extracellular vesicles (EVs). SASP factors cause chronic inflammation, leading to age-related diseases. Recently, therapeutic strategies targeting senescent cells, known as senolytics, have gained attention; however, noninvasive methods to detect senescent cells in living organisms have not been established. Therefore, the goal of this study was to identify novel senescent markers using small EVs (sEVs). sEVs were isolated from young and senescent fibroblasts using three different methods, including size-exclusion chromatography, affinity column for phosphatidylserine, and immunoprecipitation using antibodies against tetraspanin proteins, followed by mass spectrometry. Principal component analysis revealed that the protein composition of sEVs released from senescent cells was significantly different from that of young cells. Importantly, we identified ATP6V0D1 and RTN4 as novel markers that are frequently upregulated in sEVs from senescent and progeria cells derived from patients with Werner syndrome. Furthermore, these two proteins were significantly enriched in sEVs from the serum of aged mice. This study supports the potential use of senescent markers from sEVs to detect the presence of senescent cells in vivo.

研究グループは、肝臓に移行した腸内細菌叢の成分であるリポタイコ酸が、肝がん微小環境を変化させてがんの増殖進展を促進するSASP(Senescence-associated secretory phenotype、細胞老化随伴分泌現象)因子を老化肝星細胞から放出させるメカニズムを明らかにしました。がんの組織はがん細胞そのものだけでなく、線維芽細胞や免疫細胞など、様々な種類の細胞種が集まって「がん微小環境」を構成しています。進行したがん組織の微小環境ではがん細胞周囲の細胞ががんの増殖を助長していると考えられています。本グループは、脂肪肝を素地とする肝がん微小環境では、肝星細胞と呼ばれる線維芽細胞が細胞老化を引き起こしており、「細胞老化随伴分泌現象(SASP)」という様々な分泌因子を放出する現象が生じ、その分泌因子(SASP因子)ががんの増殖を促進することを以前から見出していました。しかしこれまでにSASP因子の放出メカニズムについては明らかにされていませんでした。本研究では高脂肪食摂取による肥満誘導性肝がんのマウスモデルを用い、老化肝星細胞の細胞膜上にガスダーミンDというタンパク質が酵素切断されて生じたN末端側の部分(以降GSDMD－Nと略記)が集合して形成される小孔を介して、SASP因子に含まれるサイトカインIL-1βとIL-33が細胞の外部に放出されることを明らかにしました。また、高脂肪食摂取マウスでは、腸管バリアが脆弱化しており、肝臓にグラム陽性腸内細菌の細胞壁成分であるリポタイコ酸が蓄積していました。さらに、蓄積したリポタイコ酸は老化肝星細胞にトル様受容体２(TLR2)を介した刺激を入れ続け、酵素切断で生じたGSDMD－Nによる細胞膜上の小孔形成とそれに続くIL-33やIL-1βの放出を促進していることもわかりました。老化肝星細胞から放出されたIL-33は、その受容体ST2が陽性の制御性T細胞(Treg細胞)に作用し、がんの増殖を促進させることがわかりました。また、GSDMD－NはヒトのNASH(Non-alcoholic steatohepatitis)肝がんの腫瘍部にある肝星細胞でもその存在が認められました。これらの結果から、ガスダーミンDによる小孔形成を阻害する薬剤は肝がんの予防や治療につながる可能性があります。, Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, ...

Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control., Senescent cells can influence the tumour microenvironment by secreting immunomodulatory factors and are thus a therapeutic target. Here, the authors identify a compound that degrades BET leading to DNA damage and activation of autophagy and a reduction in tumour growth.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

Significance During the aging process, senescent cells secrete inflammatory factors, causing various age-related pathologies. Thus, controlling the senescence-associated secretory phenotype (SASP) can tremendously benefit human health. Although SASP seems to be induced by the alteration of chromosomal organization, its underlying mechanism remains unclear. Here, it has been revealed that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive elements impairs the DNA binding of CCCTC-binding factor, resulting in the alteration of chromosomal accessibility and the activation of SASP-like inflammatory genes. Notably, the ncRNA was transferred into surrounding cells via small extracellular vesicles, acting as a tumorigenic SASP factor. Our study highlights a novel mechanism regulating chromatin interaction and inflammatory gene expression in senescence and cancer., Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non–cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Cellular senescence is historically regarded as a tumor suppression mechanism to prevent damaged cells from aberrant proliferation in benign and premalignant tumors. However, recent findings have suggested that senescent cells contribute to tumorigenesis and age‐associated pathologies through the senescence‐associated secretory phenotype (SASP). Therefore, to control age‐associated cancer, it is important to understand the molecular mechanisms of the SASP in the cancer microenvironment. New findings have suggested that the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling pathway, a critical indicator of innate immune response, triggers the SASP in response to accumulation of cytoplasmic DNA (cytoplasmic chromatin fragments, mtDNA and cDNA) in senescent cells. Notably, the cGAS‐STING signaling pathway promotes or inhibits tumorigenesis depending on the biological context in vivo, indicating that it may be a potential therapeutic target for cancer. Herein, we review the regulatory machinery and biological function of the SASP via the cGAS‐STING signaling pathway in cancer., This brief review discusses the role of the senescence‐associated secretory phenotype (SASP) and cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) signaling pathway on cellular senescence in cancer development. We suggest that the cGAS‐STING signaling pathway may be a novel target for induction of SASP.

DNA damage, caused by various oncogenic stresses, can induce cell death or cellular senescence as an important tumor suppressor mechanism. Senescent cells display the features of a senescence-associated secretory phenotype (SASP), secreting inflammatory proteins into surrounding tissues, and contributing to various age-related pathologies. In addition to this inflammatory protein secretion, the release of extracellular vesicles (EVs) is also upregulated in senescent cells. However, the molecular mechanism underlying this phenomenon remains unclear. Here, we show that DNA damage activates the ceramide synthetic pathway, via the downregulation of sphingomyelin synthase 2 (SMS2) and the upregulation of neutral sphingomyelinase 2 (nSMase2), leading to an increase in senescence-associated EV (SA-EV) biogenesis. The EV biogenesis pathway, together with the autophagy-mediated degradation pathway, functions to block apoptosis by removing cytoplasmic DNA fragments derived from chromosomal DNA or bacterial infections. Our data suggest that this SA-EV pathway may play a prominent role in cellular homeostasis, particularly in senescent cells. In summary, DNA damage provokes SA-EV release by activating the ceramide pathway to protect cells from excessive inflammatory responses.

Accumulating evidence indicates that the senescence-associated secretory phenotype (SASP) contributes to many aspects of physiology and disease. Thus, controlling the SASP will have tremendous impacts on our health. However, our understanding of SASP regulation is far from complete. Here, we show that cytoplasmic accumulation of nuclear DNA plays key roles in the onset of SASP. Although both DNase2 and TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in pre-senescent cells, the expression of these DNases is downregulated in senescent cells, resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through induction of interferon-β. Notably, the blockage of this pathway prevents SASP in senescent hepatic stellate cells, accompanied by a decline of obesity-associated hepatocellular carcinoma development in mice. These findings provide valuable new insights into the roles and mechanisms of SASP and possibilities for their control., Activation of DNA damage response induces the acquisition of senescence-associated secretory phenotype (SASP) in senescent cells, but precise mechanisms remain unclear. Here, the authors show that the cytoplasmic accumulation of nuclear DNA activated cytoplasmic DNA sensors to provoke SASP.

Obesity increases the risk of cancers, including hepatocellular carcinomas (HCC). However, the precise molecular mechanisms through which obesity promotes HCC development are still unclear. Recent studies have shown that gut microbiota may influence liver diseases by transferring its metabolites and components. Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA), a Gram-positive gut microbial component, promotes HCC development by creating a tumor-promoting microenvironment. LTA enhances the senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) collaboratively with an obesity-induced gut microbial metabolite, deoxycholic acid, to upregulate the expression of SASP factors and COX2 through Toll-like receptor 2. Interestingly, COX2-mediated prostaglandin E2 (PGE2) production suppresses the antitumor immunity through a PTGER4 receptor, thereby contributing to HCC progression. Moreover, COX2 overexpression and excess PGE2 production were detected in HSCs in human HCCs with noncirrhotic, nonalcoholic steatohepatitis (NASH), indicating that a similar mechanism could function in humans. Significance: We showed the importance of the gut–liver axis in obesity-associated HCC. The gut microbiota–driven COX2 pathway produced the lipid mediator PGE2 in senescent HSCs in the tumor microenvironment, which plays a pivotal role in suppressing antitumor immunity, suggesting that PGE2 and its receptor may be novel therapeutic targets for noncirrhotic NASH-associated HCC. Cancer Discov; 7(5); 522–38. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 443

Cellular senescence caused by oncogenic stimuli is related to the development of various age-related diseases via senescence-associated secretory phenotype (SASP). Recent studies have revealed that the SASP is induced via the cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway. However, the molecular mechanism of nucleotide ligand production for these cytoplasmic sensors remains unclear. Here, we discover that the expression of RNaseH2A is regulated by E2F transcription factor and decreases during cellular senescence, thus abnormally incorporated ribonucleoside monophosphates (rNMPs) cause genomic DNA fragmentation, while excess R-loop structures lead to RNA-DNA hybrid accumulation in the nucleoli and cytoplasm of senescent cells. Consequently, both chromosomal DNA fragments and RNA-DNA hybrids induce aberrant activation of the cGAS-STING pathway and SASP-factor gene expression. Furthermore, RNaseH2A reduction was associated with pathological features and poor prognoses in individuals with Werner syndrome and cervical, ovarian and colorectal cancers. Our findings provide new insights into how nucleotide ligand production relates to SASP induction.

Obesity increases the risk of cancers, including hepatocellular carcinomas (HCC). However, the precise molecular mechanisms through which obesity promotes HCC development are still unclear. Recent studies have shown that gut microbiota may influence liver diseases by transferring its metabolites and components. Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA), a Gram-positive gut microbial component, promotes HCC development by creating a tumor-promoting microenvironment. LTA enhances the senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) collaboratively with an obesity-induced gut microbial metabolite, deoxycholic acid, to upregulate the expression of SASP factors and COX2 through Toll-like receptor 2. Interestingly, COX2-mediated prostaglandin E

Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer. As the worldwide obesity epidemic has shown no signs of abating, better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.

Nature 499, 97–101 (2013); doi:10.1038/nature12347 In this Letter, the forename of author Masahira Hattori was spelled incorrectly as ‘Masahisa’. It has been corrected in the HTML and PDF versions of the manuscript.