Repairing DNA damage by XRCC6/KU70 reverses TLR4-deficiency-worsened HCC development via restoring senescence and autophagic flux

Hepatocellular carcinoma (HCC) is among the most lethal and prevalent cancers in the human population. The initiation and progression of HCC is closely associated with chronic liver inflammation. Recent research indicates that nonhomologous end joining (NHEJ), one of the DNA repair mechanisms, autophagy and senescence are all involved in the pathogenesis of HCC induced by carcinogens or oxidative stress. DNA repair proteins including XRCC6/KU70 and XRCC5/KU80 are the critical NHEJ factors that play pivotal roles in genome-maintenance issues such as DNA replication and repair, telomere maintenance and chromosomal instability. Our studies indicate that a deficiency of toll-like receptor 4 (TLR4)-mediated immune activities results in a decreased expression of XRCC5 and XRCC6 in response to insult by the carcinogen diethylnitrosamine (DEN). This effect causes a failure in DNA repair, and promotes the transformation of precancerous hepatocytes and HCC development. Ectopic expression of XRCC6 protects against HCC initiation and progression by restoring the cellular senescent response and activation of immune networks, which induces an effective autophagic degradation, removes the accumulated reactive oxygen species (ROS), decreases DNA damage, attenuates proliferation, and promotes programmed cell death in TLR4-deficient livers. Our work indicates that repairing DNA damage by XRCC6 reverses TLR4-deficiency-worsened HCC development via restoring immunity to support senescence and autophagy in liver cells.     

Antioxidant N-Acetylcysteine Attenuates Hepatocarcinogenesis by Inhibiting ROS/ER Stress in TLR2 Deficient Mouse

Hepatocellular carcinoma (HCC) remains one of the most deadly solid tumor malignancies worldwide. We recently find that the loss of toll-like receptor 2 (TLR2) activities promotes the diethylnitrosamine (DEN) induced hepatocellular carcinogenesis and tumor progression, which associates with an abundant accumulation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. This finding suggests that the ROS/ER stress plays a role in TLR2 modulated carcinogenesis of HCC. To investigate the mechanism of TLR2 activity defending against hepatocarcinogenesis, the TLR2-deficient mice were treated with or without antioxidant N-acetylcysteine (NAC) before DEN administration. We found that pretreatment of these animals with NAC attenuated carcinogenesis and progression of HCC in the TLR2-deficient mice, declined ROS/ER stress, and alleviated the unfold protein response and inflammatory response in TLR2-deficient liver tissue. Moreover, the NAC treatment significantly reduced the enhanced aggregation of p62 and Mallory-Denk bodies in the DEN-induced HCC liver tissue, suggesting that NAC treatment improves the suppressive autophagic flux in the TLR2-deficient liver. These findings indicate that TLR2 activity defends against hepatocarcinogenesis through diminishing the accumulation of ROS and alleviating ER stress and unfold protein response mediated inflammatory response in the liver.     

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

Parkinsonian toxin-induced oxidative stress inhibits basal autophagy in astrocytes via NQO2/quinone oxidoreductase 2: Implications for neuroprotection

Oxidative stress (OS) stimulates autophagy in different cellular systems, but it remains controversial if this rule can be generalized. We have analyzed the effect of chronic OS induced by the parkinsonian toxin paraquat (PQ) on autophagy in astrocytoma cells and primary astrocytes, which represent the first cellular target of neurotoxins in the brain. PQ decreased the basal levels of LC3-II and LC3-positive vesicles, and its colocalization with lysosomal markers, both in the absence and presence of chloroquine. This was paralleled by increased number and size of SQSTM1/p62 aggregates. Downregulation of autophagy was also observed in cells chronically exposed to hydrogen peroxide or nonlethal concentrations of PQ, and it was associated with a reduced astrocyte capability to protect dopaminergic cells from OS in co-cultures. Surprisingly, PQ treatment led to inhibition of MTOR, activation of MAPK8/JNK1 and MAPK1/ERK2-MAPK3/ERK1 and upregulation of BECN1/Beclin 1 expression, all signals typically correlating with induction of autophagy. Reduction of OS by NMDPEF, a specific NQO2 inhibitor, but not by N-acetylcysteine, abrogated the inhibitory effect of PQ and restored autophagic flux. Activation of NQO2 by PQ or menadione and genetic manipulation of its expression confirmed the role of this enzyme in the inhibitory action of PQ on autophagy. PQ did not induce NFE2L2/NRF2, but when it was co-administered with NMDPEF NFE2L2 activity was enhanced in a SQSTM1-independent fashion. Thus, a prolonged OS in astrocytes inhibits LC3 lipidation and impairs autophagosome formation and autophagic flux, in spite of concomitant activation of several pro-autophagic signals. These findings outline an unanticipated neuroprotective role of astrocyte autophagy and identify in NQO2 a novel pharmacological target for its positive modulation.     

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.     

A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.     

SQSTM1/p62 (sequestosome 1) senses cellular ubiquitin stress through E2-mediated ubiquitination

The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub⁺ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub⁺ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub⁺ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.     

Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^?/? mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.     

Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway

In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.     

Research Article: Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^−/− mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Lipopolysaccaride induces autophagic signaling in macrophages via a TLR4, heme oxygenase-1 dependent pathway

Toll-like receptor (TLR) signaling is an important part of the innate immune response. One of the downstream responses to TLR4 signaling upon lipopolysaccharide (LPS) stimulation is the induction of autophagy, which is a key response to multiple stressors. An additional adaptive signaling molecule that is involved in the response to stress is heme oxygenase-1 (HO-1). HO-1 signaling is essential to limit inflammation and restore homeostasis. We found that LPS induced autophagic signaling in macrophages via a TLR4, HO-1 dependent pathway in macrophages. These data add to the developing contribution of autophagic signaling as part of the inflammatory response.     

Regulation of SQSTM1/p62 via UBA domain ubiquitination and its role in disease

Macroautophagy/autophagy can be a selective degradative process via the utilization of various autophagic receptor proteins. Autophagic receptors selectively recognize ubiquitinated cargoes and deliver them to phagophores, the precursors to autophagosomes, for their degradation. For example, SQSTM1/p62 directly binds to ubiquitinated protein aggregates via its UBA domain and sequesters them into inclusion bodies via its PB1 domain. SQSTM1also interacts with phagophores via its LC3-interacting (LIR) motif. However, a regulatory mechanism for autophagic receptors is not yet understood.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

Autophagy in regulation of Toll-like receptor signaling

Toll-like receptors (TLRs) serve as the major innate immune sensors for detection of specific molecular patterns on various pathogens. TLRs activate signaling events mainly by utilizing ubiquitin-dependent mechanisms. Recent research advances have provided evidence that TLR signaling is linked to induction of autophagy. Autophagy is currently known to affect both of the immune defense and suppression of inflammatory responses. In TLR-associated immune responses, autophagic lysis of intracellular microbes (called xenophagy) contributes to the former mechanism, while the latter seems to be mediated by the control of the mitochondrial integrity or selective autophagic clearance of aggregated signaling proteins (called aggrephagy). Several autophagy-related ubiquitin-binding proteins, such as SQSTM1/p62 and NDP52, mediate xenophagy and aggrephagy. In this review, we summarize the expanded knowledge regarding TLR signaling and autophagy signaling. After that, we will focus on autophagy-associated signaling downstream of TLRs and the effect of autophagy on TLR signaling, thus highlighting the signaling crosstalk between the TLR-associated innate immune responses and the regulation of innate immunity by xenophagy and aggrephagy.