Replication factor C1, the large subunit of replication factor C, is proteolytically truncated in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder because of a LMNA gene mutation that produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here, we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ~ 75 kDa, which appears to be defective in loading proliferating cell nuclear antigen (PCNA) and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Because of the crucial role of RFC in DNA replication, our findings provide a mechanistic interpretation for the observed early replicative arrest and premature aging phenotypes of HPGS and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS.     

DNA损伤监测及修复相关酶与细胞衰老

衰老是细胞的重要生命现象之一,衰老假说之一认为细胞中残留DNA损伤的积累可加速细胞的衰老.因此,细胞内DNA损伤监测及修复系统的正常运行与细胞衰老调控密切相关,DNA损伤监测及修复相关酶如PARP、DNA-PK、ATM、p53等在细胞衰老中的调控作用日益受到广泛关注.研究这些蛋白质分子间的相互作用及其在细胞衰老过程中的调控功能,有利于揭示DNA损伤应激、损伤修复调控与细胞衰老之间的内在联系,为抗衰老研究及从衰老角度治疗肿瘤提供新的思路.     

Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage

Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.     

Viral subversion of autophagy impairs oncogene-induced senescence

Many viruses have evolved elegant strategies to co-opt cellular autophagic responses to facilitate viral propagation and evasion of immune surveillance. Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a life-long persistent infection in its human host, and is etiologically linked to several cancers. KSHV gene products have been shown to modulate autophagy but their contribution to pathogenesis remains unclear. Our recent study demonstrated that KSHV subversion of autophagy promotes bypass of oncogene-induced senescence (OIS), an important host barrier to tumor initiation. These findings suggest that KSHV has evolved to subvert autophagy, at least in part, to establish an optimal niche for infection, concurrently dampening host antiviral defenses and allowing the ongoing proliferation of infected cells.     

Viral subversion of autophagy impairs oncogene-induced senescence

Many viruses have evolved elegant strategies to co-opt cellular autophagic responses to facilitate viral propagation and evasion of immune surveillance. Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a life-long persistent infection in its human host, and is etiologically linked to several cancers. KSHV gene products have been shown to modulate autophagy but their contribution to pathogenesis remains unclear. Our recent study demonstrated that KSHV subversion of autophagy promotes bypass of oncogene-induced senescence (OIS), an important host barrier to tumor initiation. These findings suggest that KSHV has evolved to subvert autophagy, at least in part, to establish an optimal niche for infection, concurrently dampening host antiviral defenses and allowing the ongoing proliferation of infected cells.     

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G₂/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.     

DNA double strand break responses and chromatin alterations within the aging cell

Cellular senescence is a state of permanent replicative arrest that allows cells to stay viable and metabolically active but resistant to apoptotic and mitogenic stimuli. Specific, validated markers can identify senescent cells, including senescence-associated β galactosidase activity, chromatin alterations, cell morphology changes, activated p16- and p53-dependent signaling and permanent cell cycle arrest. Senescence is a natural consequence of DNA replication-associated telomere erosion, but can also be induced prematurely by telomere-independent events such as failure to repair DNA double strand breaks. Here, we review the molecular pathways of senescence onset, focussing on the changes in chromatin organization that are associated with cellular senescence, particularly senescence-associated heterochromatin foci formation. We also discuss the altered dynamics of the DNA double strand break response within the context of aging cells. Appreciating how, mechanistically, cellular senescence is induced, and how changes to chromatin organization and DNA repair contributes to this, is fundamental to our understanding of the normal and premature human aging processes associated with loss of organ and tissue function in humans.     

Manipulation of autophagy by MIR375 generates antitumor effects in liver cancer

The exploration into the roles of autophagy in tumorigenesis, either as tumor suppressor or tumor promoter, has led to a great increase in the knowledge of cancer development, progression and treatment. However, there is currently no consensus on how to manipulate autophagy to improve antitumor effects. In this study, we investigated the role of autophagy in established liver cancer cells in response to hypoxia. Hypoxia not only is the most pervasive microenvironmental stress in solid tumors but is also a canonical stimulus for autophagy. The involvement of dysregulated microRNAs in hypoxia-induced autophagy and their therapeutic potential in advanced liver cancer were examined.     

Autophagy-dependent senescence in response to DNA damage and chronic apoptotic stress

Autophagy regulates cell survival and cell death upon various cellular stresses, yet the molecular signaling events involved are not well defined. Here, we established the function of a proteolytic Cyclin E fragment (p18-CycE) in DNA damage-induced autophagy, apoptosis, and senescence. p18-CycE was identified in hematopoietic cells undergoing DNA damage-induced apoptosis. In epithelial cells exposed to DNA damage, chronic but not transient expression of p18-CycE leads to higher turnover of LC3 I/II and increased emergence of autophagosomes and autolysosomes. Levels of p18-CycE, which was generated by proteolytic cleavage of endogenous Cyclin E, were greatly increased by chloroquine and correlated with LC 3II conversion. Preventing p18-CycE genesis blocked conversion of LC3 I to LC3 II. Upon DNA damage, cytoplasmic ataxia-telangiectasia-mutated (ATM) was phosphorylated in p18-CycE-expressing cells resulting in sustained activation of the adenosine-mono-phosphate-dependent kinase (AMPK). These lead to sustained activation of mammalian autophagy-initiating kinase ULK1, which was abrogated upon inhibiting ATM and AMPK phosphorylation. Moreover, p18-CycE was degraded via autophagy followed by induction of senescence. Both autophagy and senescence were prevented by inhibiting autophagy, which leads to increased apoptosis in p18-CycE-expressing cells by stabilizing p18-CycE expression. Senescence was further associated with cytoplasmic co-localization and degradation of p18-CycE and Ku70. In brief, chronic p18-CycE expression-induced autophagy leads to clearance of p18-CycE following DNA damage and induction of senescence. Autophagy inhibition stabilized the cytoplasmic p18-CycE-Ku70 complex leading to apoptosis. Thus, our findings define how chronic apoptotic stress and DNA damage initiate autophagy and regulate cell survival through senescence and/or apoptosis.     

How autophagy both activates and inhibits cellular senescence

Autophagy and cellular senescence are stress responses essential for homeostasis. While recent studies indicate a genetic relationship between autophagy and senescence, whether autophagy acts positively or negatively on senescence is still subject to debate. Although autophagy was originally recognized as a nonspecific lysosomal degradation pathway (general autophagy), increasing evidence supports a selective form of autophagy that mediates the degradation of specific targets (selective autophagy). Our recent study revealed distinctive roles of selective autophagy and general autophagy in the regulation of senescence, at least in part resolving apparently contradictory reports regarding the relationship between these 2 important homeostatic stress responses.     

Ku70 is stabilized by increased cellular SUMO

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.     

Neratinib augments the lethality of [regorafenib + sildenafil]

Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein–tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)–AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.