Role of Autophagy and AMPK in Cancer Stem Cells: Therapeutic Opportunities and Obstacles in Cancer.

Cancer stem cells represent a resilient subset within the tumor microenvironment capable of differentiation, regeneration, and resistance to chemotherapeutic agents, often using dormancy as a shield. Their unique properties, including drug resistance and metastatic potential, pose challenges for effective targeting. These cells exploit certain metabolic processes for their maintenance and survival. One of these processes is autophagy, which generally helps in energy homeostasis but when hijacked by CSCs can help maintain their stemness. Thus, it is often referred as an Achilles heel in CSCs, as certain cancers tend to depend on autophagy for survival. Autophagy, while crucial for maintaining stemness in cancer stem cells (CSCs), can also serve as a vulnerability in certain contexts, making it a complex target for therapy. Regulators of autophagy like AMPK (5' adenosine monophosphate-activated protein kinase) also play a crucial role in maintaining CSCs stemness by helping CSCs in metabolic reprogramming in harsh environments. The purpose of this review is to elucidate the interplay between autophagy and AMPK in CSCs, highlighting the challenges in targeting autophagy and discussing therapeutic strategies to overcome these limitations. This review focuses on previous research on autophagy and its regulators in cancer biology, particularly in CSCs, addresses the remaining unanswered questions, and potential targets for therapy are also brought to attention.

Association between dietary quality and the prevalence of periodontitis in older Korean adults aged 60 or over.

PURPOSE: This study investigated the association between dietary quality and the prevalence of periodontitis in older Korean adults (≥60 years of age) using data from the seventh Korea National Health and Nutrition Examination Survey (KNHANES VII, 2016-2018). METHODS: Among the 16,489 KNHANES participants from 2016-2018, those aged ≥60 years were selected as the eligible population. After applying our exclusion criteria, 3,527 participants were included in the final study population. Periodontal status was measured using the Community Periodontal Index (CPI). To determine the association between dietary quality and the prevalence of periodontitis, analysis of variance, the chi-square (χ²) test, and logistic regression analysis were performed. RESULTS: The population was divided into quartile groups and stratified by sex. The percentage of men and women with periodontitis was 54.34% and 42.74%, respectively. The quartile with higher Korean Healthy Eating Index scores had a lower percentage of people with periodontitis in both sexes. For men, only vegetable intake showed a significant difference between sub-groups with or without periodontitis, whereas, for women, the intake of fruits, milk, sweets, carbohydrates, and fats showed significant differences. There was a strong positive association between vegetable intake and periodontitis in men in the 3 models used in this study; model 3 had an adjusted odds ratio (aOR) of 1.367 (95% confidence interval [CI], 1.091-1.712). In women, a strong positive association with periodontitis was shown for sweets in all 3 models, with an aOR of 1.477 in model 3 (95% CI, 1.125-1.939). CONCLUSIONS: Dietary quality was inversely associated with the prevalence of periodontitis in Korean adults aged ≥60 years. Further comprehensive studies are needed to help establish nutrition and health policies for older adults in Korea.

AMPK-mTOR Signaling and Cellular Adaptations in Hypoxia.

Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I-IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed.

An alcoholic extract of Thuja orientalis L. leaves inhibits autophagy by specifically targeting pro-autophagy PIK3C3/VPS34 complex.

Autophagy is a lysosome-dependent degradation program to maintain cellular homeostasis in response to a variety of stressful conditions, such as long-lived or non-functional subcellular organelles, protein aggregates, nutrient limitation, and virus/bacteria infection. Accordingly, dysregulation of autophagy is closely associated with many human pathophysiological conditions, such as neurodegenerative diseases, aging, and cancer, and autophagy is highlighted as an important therapeutic target for these human diseases. In autophagy process, PIK3C3/VPS34 complex plays important roles in autophagosome biogenesis. Accumulating evidences that inhibition of PIK3C3/VPS34 complex successfully blocks autophagy make the complex as an attractive target for the development of autophagy-specific inhibitors. However, considering that various forms of PIK3C3/VPS34 complex exist and they are involved in many different cellular functions, the targeting of the pro-autophagy PIK3C3/VPS34 complex is required to specifically inhibit autophagy. To identify autophagy inhibitors targeting the pro-autophagy complex, we have performed the screening of a customized natural product library consisting of 35 herbal extracts which are widely used in the oriental medicine as anti-inflammation and/or anti-tumor reagents. We discovered that an alcoholic extract of Thuja orientalis L. leaves inhibits pro-autophagy complex formation by disrupting the interaction between autophagy-specific factor, ATG14L, and the complex core unit Vps34-Beclin 1 in vitro. Also, it inhibits the nutrient starvation induced autophagy and diminished pro-autophagy PIK3C3/VPS34 complex containing either ATG14L or UVRAG in several cell lines. Our results strongly suggest that Thuja orientalis L. leave extract functions as an autophagy-specific inhibitor not decreasing the complex activity nor the protein level, but preventing protein-protein interaction between autophagy-specific factor (ATG14L and UVRAG) and PIK3C3/VPS34 complex core unit, Vps34-Beclin 1, thereby specifically depleting the pro-autophagy complex to inhibit autophagy.

mTOR as a central hub of nutrient signalling and cell growth.

The highly conserved protein kinase mechanistic target of rapamycin (mTOR; originally known as mammalian target of rapamycin) is a central cell growth regulator connecting cellular metabolism and growth with a wide range of environmental inputs as part of mTOR complex 1 (mTORC1) and mTORC2. In this Review, we introduce the landmark discoveries in the mTOR field, starting from the isolation of rapamycin to the molecular characterizations of key components of the mTORC signalling network with an emphasis on amino acid sensing, and discuss the perspectives of mTORC inhibitors in therapeutic applications.

Autophagy: An Essential Degradation Program for Cellular Homeostasis and Life.

Autophagy is a lysosome-dependent cellular degradation program that responds to a variety of environmental and cellular stresses. It is an evolutionarily well-conserved and essential pathway to maintain cellular homeostasis, therefore, dysfunction of autophagy is closely associated with a wide spectrum of human pathophysiological conditions including cancers and neurodegenerative diseases. The discovery and characterization of the kingdom of autophagy proteins have uncovered the molecular basis of the autophagy process. In addition, recent advances on the various post-translational modifications of autophagy proteins have shed light on the multiple layers of autophagy regulatory mechanisms, and provide novel therapeutic targets for the treatment of the diseases.

The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation.

The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13HORMA complex is responsible for the binding of the PtdIns3K complex. This region adopts a ß-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13HORMA complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13HORMA: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13HORMA: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13HORMA: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Mitochondrial ROS-derived PTEN oxidation activates PI3K pathway for mTOR-induced myogenic autophagy.

Muscle differentiation is a crucial process controlling muscle development and homeostasis. Mitochondrial reactive oxygen species (mtROS) rapidly increase and function as critical cell signaling intermediates during the muscle differentiation. However, it has not yet been elucidated how they control myogenic signaling. Autophagy, a lysosome-mediated degradation pathway, is importantly recognized as intracellular remodeling mechanism of cellular organelles during muscle differentiation. Here, we demonstrated that the mtROS stimulated phosphatidylinositol 3 kinase/AKT/mammalian target of rapamycin (mTOR) cascade, and the activated mTORC1 subsequently induced autophagic signaling via phosphorylation of uncoordinated-51-like kinase 1 (ULK1) at serine 317 and upregulation of Atg proteins to prompt muscle differentiation. Treatment with MitoQ or rapamycin impaired both phosphorylation of ULK1 and expression of Atg proteins. Therefore, we propose a novel regulatory paradigm in which mtROS are required to initiate autophagic reconstruction of cellular organization through mTOR activation in muscle differentiation.

Ursolic Acid Causes Cell Death in PC-12 Cells by Inducing Apoptosis and Impairing Autophagy.

Ursolic acid (UA) is a natural pentacyclic triterpene that has various biological activities, including anticancer and anti-inflammatory effects. This study investigated the ability of UA to cause cell death in pheochromocytoma (PC-12) cells. UA was cytotoxic to PC-12 cells (half-maximum inhibitory concentration=53.2 µM) and significantly reduced the clonogenic ability of PC-12 cells. It also triggered apoptosis by reducing the level of B-cell lymphoma 2 (BCL2), activating caspase-3, and inducing cleavage of poly (ADP-ribosyl) polymerase. To investigate the effects of UA treatment on the induction and progression of autophagy, the levels of p62 and the conversion of the microtubule-associated protein light chain 3 (LC3)-I to LC3-II, which are important markers of autophagic flux, were monitored. UA treatment induced the accumulation of p62 and increased the LC3-II/LC3-I ratio. These results demonstrate that UA treatment induced autophagy, but the downstream signaling pathway was blocked. In summary, this study shows that UA kills PC-12 cells by inducing apoptosis and impairing autophagy progression.

Induction of Cell Death by Betulinic Acid through Induction of Apoptosis and Inhibition of Autophagic Flux in Microglia BV-2 Cells.

Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC50 of approximately 2.0 µM. Treatment of BA resulted in a dose-dependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy.

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.

Targeting of AMP-activated protein kinase: prospects for computer-aided drug design.

INTRODUCTION: Dysregulation of energy homeostasis has been implicated in a number of human chronic diseases including diabetes, obesity, cancer, and inflammation. Given the functional attributes as a central regulator of energy homeostasis, AMP-activated protein kinase (AMPK) is emerging as a therapeutic target for these diseases, and lines of evidence have highlighted the need for rational and robust screening systems for identifying specific AMPK modulators with a therapeutic potential for preventing and/or curing these diseases. Areas covered: Here, the authors review the recent advances in the understanding of three-dimensional structures of AMPK in relationship with the regulatory mechanisms, potentials of AMPK as a therapeutic target in human chronic diseases, and prospects of computer-based drug design for AMPK. Expert opinion: Accumulating information of AMPK structure has provided us with deep insight into the molecular basis underlying the regulatory mechanisms, and further discloses several structural domains, which can be served for a target site for computer-based drug design. Molecular docking and simulations provides useful information about the binding sites between potent drugs and AMPK as well as a rational screening format to discover isoform-specific AMPK modulators. For these reasons, the authors suggest that computer-aided virtual screening methods hold promise as a rational approach for discovering more specific AMPK modulators.

Ezetimibe, an NPC1L1 inhibitor, is a potent Nrf2 activator that protects mice from diet-induced nonalcoholic steatohepatitis.

Oxidative stress is important for the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a chronic disease that ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The nuclear factor erythroid 2-related factor 2-Kelch-like ECH associated protein 1 (Nrf2-Keap1) pathway is essential for cytoprotection against oxidative stress. In this study, we found that oxidative stress or inflammatory biomarkers and TUNEL positive cells were markedly increased in NASH patients compared to normal or simple steatosis. In addition, we identified that the hepatic mRNA levels of Nrf2 target genes such as Nqo-1 and GSTA-1 were significantly increased in NASH patients. Ezetimibe, a drug approved by the Food and Drug Administration for the treatment of hypercholesterolemia, improves NAFLD and alleviates oxidative stress. However, the precise mechanism of its antioxidant function remains largely unknown. We now demonstrate that ezetimibe activates Nrf2-Keap1 pathway which was dependent of autophagy adaptor protein p62, without causing cytotoxicity. Ezetimibe activates AMP-activated protein kinase (AMPK), which in turn phosphorylates p62 (p-S351) via their direct interaction. Correspondingly, Ezetimibe protected liver cells from saturated fatty acid-induced apoptotic cell death through p62-dependent Nrf2 activation. Furthermore, its role as an Nrf2 activator was supported by methione- and choline- deficient (MCD) diet-induced NASH mouse model, showing that ezetimibe decreased the susceptibility of the liver to oxidative injury. These data demonstrate that the molecular mechanisms underlying ezetimibe's antioxidant role in the pathogenesis of NASH.

Novel pharmacological modulators of autophagy: an updated patent review (2012-2015).

INTRODUCTION: Autophagy is a lysosome-dependent degradation pathway that maintains cellular homeostasis in response to a variety of cellular stresses. Accumulating reports based on animal models have indicated the importance of this catabolic program in many human pathophysiological conditions, including diabetes, neurodegenerative diseases, aging, and cancers. Therefore, autophagy has been highlighted as a novel therapeutic target with a wide range of beneficial effects on human diseases. Here, we review the recent advances of our knowledge toward autophagy, as well as the efforts for developing autophagy modulators. Areas covered: The relevant patents (published at 2012-2015) and the research literature claiming the pharmacological modulation of autophagy are reviewed. Also, their molecular mechanisms and potential therapeutic utilities are discussed. Expert opinion: Considering the molecular machinery involved in autophagy induction, the targeting of autophagy-specific protein is very important to design the therapeutic interventions for specifically treating a variety of autophagy-associated disorders. Many patents and the research literature described in this review have shown promising applications of the relevant autophagy modulators for cancer or neurodegeneration treatments, a few of which are already being considered for clinical evaluation. However, most patents have claimed the modulators of autophagy with little information regarding their mechanisms of action. To design highly potent therapeutics, further work, such as developing compounds that specifically target the autophagy-specific machinery, are required.

Bacterial Overexpression and Denaturing Purification of VPS34-Binding Domain of Beclin 1.

As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Escherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.

AMPK activators: mechanisms of action and physiological activities.

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, which coordinates metabolic pathways and thus balances nutrient supply with energy demand. Because of the favorable physiological outcomes of AMPK activation on metabolism, AMPK has been considered to be an important therapeutic target for controlling human diseases including metabolic syndrome and cancer. Thus, activators of AMPK may have potential as novel therapeutics for these diseases. In this review, we provide a comprehensive summary of both indirect and direct AMPK activators and their modes of action in relation to the structure of AMPK. We discuss the functional differences among isoform-specific AMPK complexes and their significance regarding the development of novel AMPK activators and the potential for combining different AMPK activators in the treatment of human disease.

Rag GTPase in amino acid signaling.

Rag small GTPases were identified as the sixth subfamily of Ras-related GTPases. Compelling evidence suggests that Rag heterodimer (RagA/B and RagC/D) plays an important role in amino acid signaling toward mechanistic target of rapamycin complex 1 (mTORC1), which is a central player in the control of cell growth in response to a variety of environmental cues, including growth factors, cellular energy/oxygen status, and amino acids. Upon amino acid stimulation, active Rag heterodimer (RagA/B(GTP)-RagC/D(GDP)) recruits mTORC1 to the lysosomal membrane where Rheb resides. In this review, we provide a current understanding on the amino acid-regulated cell growth control via Rag-mTORC1 with recently identified key players, including Ragulator, v-ATPase, and GATOR complexes. Moreover, the functions of Rag in physiological systems and in autophagy are discussed.

Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions.

AMPK and autophagy in glucose/glycogen metabolism.

Glucose/glycogen metabolism is a primary metabolic pathway acting on a variety of cellular needs, such as proliferation, growth, and survival against stresses. The multiple regulatory mechanisms underlying a specific metabolic fate have been documented and explained the molecular basis of various pathophysiological conditions, including metabolic disorders and cancers. AMP-activated protein kinase (AMPK) has been appreciated for many years as a central metabolic regulator to inhibit energy-consuming pathways as well as to activate the compensating energy-producing programs. In fact, glucose starvation is a potent physiological AMPK activating condition, in which AMPK triggers various subsequent metabolic events depending on cells or tissues. Of note, the recent studies show bidirectional interplay between AMPK and glycogen. A growing number of studies have proposed additional level of metabolic regulation by a lysosome-dependent catabolic program, autophagy. Autophagy is a critical degradative pathway not only for maintenance of cellular homeostasis to remove potentially dangerous constituents, such as protein aggregates and dysfunctional subcellular organelles, but also for adaptive responses to metabolic stress, such as nutrient starvation. Importantly, many lines of evidence indicate that autophagy is closely connected with nutrient signaling modules, including AMPK, to fine-tune the metabolic pathways in response to many different cellular cues. In this review, we introduce the studies demonstrating the role of AMPK and autophagy in glucose/glycogen metabolism. Also, we describe the recent advances on their contributions to the metabolic disorders.