Exercise-activated hepatic autophagy via the FN1-α5ß1 integrin pathway drives metabolic benefits of exercise.

How exercise elicits systemic metabolic benefits in both muscles and non-contractile tissues is unclear. Autophagy is a stress-induced lysosomal degradation pathway that mediates protein and organelle turnover and metabolic adaptation. Exercise activates autophagy in not only contracting muscles but also non-contractile tissues including the liver. However, the role and mechanism of exercise-activated autophagy in non-contractile tissues remain mysterious. Here, we show that hepatic autophagy activation is essential for exercise-induced metabolic benefits. Plasma or serum from exercised mice is sufficient to activate autophagy in cells. By proteomic studies, we identify fibronectin (FN1), which was previously considered as an extracellular matrix protein, as an exercise-induced, muscle-secreted, autophagy-inducing circulating factor. Muscle-secreted FN1 mediates exercise-induced hepatic autophagy and systemic insulin sensitization via the hepatic receptor α5ß1 integrin and the downstream IKKα/ß-JNK1-BECN1 pathway. Thus, we demonstrate that hepatic autophagy activation drives exercise-induced metabolic benefits against diabetes via muscle-secreted soluble FN1 and hepatic α5ß1 integrin signaling.

Degradative and Non-Degradative Roles of Autophagy Proteins in Metabolism and Metabolic Diseases.

Autophagy is a stress-induced lysosomal degradation pathway regulated by evolutionarily conserved autophagy-related (ATG) genes. Recent research has revealed that autophagy plays an important role in the regulation of energy metabolism, development of metabolic tissues, and pathogenesis of metabolic disorders. Bulk and selective degradation by autophagy helps maintain protein homeostasis and physiological function of cells. Aside from classical degradative roles, ATG proteins also carry out non-classical secretory functions of metabolic tissues. In this review, we summarize recent progresses and unanswered questions on the mechanisms of autophagy and ATG proteins in metabolic regulation, with a focus on organelle and nutrient storage degradation, as well as vesicular and hormonal secretion. Such knowledge broadens our understanding on the cause, pathophysiology, and prevention of metabolic diseases including obesity and diabetes.

The secretory function of BECN1 in metabolic regulation.

Macroautophagy/autophagy is primarily considered as a degradative pathway via the lysosome, yet the secretory functions of autophagy proteins have recently been unveiled. Autophagy proteins have been implicated in metabolic organ development, homeostasis and function, and deficiency in autophagy is associated with metabolic disorders. However, the molecular mechanisms by which autophagy proteins regulate energy metabolism and insulin sensitivity were unclear. We previously showed that systemic activation of autophagy by a hyperactive BECN1F121A mutant reduces insulin storage in islets but improves insulin sensitivity systemically. In our recent study, we found that BECN1 functions in adipose tissue to systemically regulate energy metabolism. Adipose-specific expression of BECN1F121A is sufficient to improve systemic insulin sensitivity without negatively affecting pancreatic insulin storage. We demonstrated that BECN1 interacts with exocyst subunit proteins and facilitates the secretion of an adipokine, ADIPOQ (adiponectin, C1Q and collagen domain containing), in adipose tissue. Thus, our findings suggest that BECN1 regulates insulin sensitivity in a non-degradative and non-cell autonomous manner by facilitating ADIPOQ secretion. Our study also highlighted the distinct functions of autophagy proteins in different metabolic tissues.

The autophagy protein Becn1 improves insulin sensitivity by promoting adiponectin secretion via exocyst binding.

Autophagy dysregulation is implicated in metabolic diseases, including type 2 diabetes. However, the mechanism by which the autophagy machinery regulates metabolism is largely unknown. Autophagy is generally considered a degradation process via lysosomes. Here, we unveil a metabolically important non-cell-autonomous, non-degradative mechanism regulated by the essential autophagy protein Becn1 in adipose tissue. Upon high-fat diet challenge, autophagy-hyperactive Becn1F121A mice show systemically improved insulin sensitivity and enhanced activation of AMP-activated protein kinase (AMPK), a central regulator of energy homeostasis, via a non-cell-autonomous mechanism mediated by adiponectin, an adipose-derived metabolic hormone. Adipose-specific Becn1F121A expression is sufficient to activate AMPK in non-adipose tissues and improve systemic insulin sensitivity by increasing adiponectin secretion. Further, Becn1 enhances adiponectin secretion by interacting with components of the exocyst complex via the coiled-coil domain. Together, our study demonstrates that Becn1 improves insulin sensitivity by facilitating adiponectin secretion through binding the exocyst in adipose tissue.

An autophagy-related protein Becn2 regulates cocaine reward behaviors in the dopaminergic system.

Drug abuse is a foremost public health problem. Cocaine is a widely abused drug worldwide that produces various reward-related behaviors. The mechanisms that underlie cocaine-induced disorders are unresolved, and effective treatments are lacking. Here, we found that an autophagy-related protein Becn2 is a previously unidentified regulator of cocaine reward behaviors. Becn2 deletion protects mice from cocaine-stimulated locomotion and reward behaviors, as well as cocaine-induced dopamine accumulation and signaling, by increasing presynaptic dopamine receptor 2 (D2R) autoreceptors in dopamine neurons. Becn2 regulates D2R endolysosomal trafficking, degradation, and cocaine-induced behaviors via interacting with a D2R-bound adaptor GASP1. Inactivating Becn2 by upstream autophagy inhibitors stabilizes striatal presynaptic D2R, reduces dopamine release and signaling, and prevents cocaine reward in normal mice. Thus, the autophagy protein Becn2 is essential for cocaine psychomotor stimulation and reward through regulating dopamine neurotransmission, and targeting Becn2 by autophagy inhibitors is a potential strategy to prevent cocaine-induced behaviors.

Inhibition of the Lipid Droplet-Peroxisome Proliferator-Activated Receptor α Axis Suppresses Cancer Stem Cell Properties.

Cancer stem cells (CSCs), having both self-renewal and tumorigenic capacity, utilize an energy metabolism system different from that of non-CSCs. Lipid droplets (LDs) are organelles that store neutral lipids, including triacylglycerol. Previous studies demonstrated that LDs are formed and store lipids as an energy source in some CSCs. LDs play central roles not only in lipid storage, but also as a source of endogenous lipid ligands, which are involved in numerous signaling pathways, including the peroxisome proliferator-activated receptor (PPAR) signaling pathway. However, it remains unclear whether LD-derived signal transduction is involved in the maintenance of the properties of CSCs. We investigated the roles of LDs in cancer stemness using pancreatic and colorectal CSCs and isogenic non-CSCs. PPARα was activated in CSCs in which LDs accumulated, but not in non-CSCs, and pharmacological and genetic inhibition of PPARα suppressed cancer stemness. In addition, inhibition of both re-esterification and lipolysis pathways suppressed cancer stemness. Our study suggested that LD metabolic turnover accompanying PPARα activation is a promising anti-CSC therapeutic target.

Verteporfin inhibits oxidative phosphorylation and induces cell death specifically in glioma stem cells.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumour in adults. Since glioma stem cells (GSCs) are associated with therapeutic resistance as well as the initiation and recurrence in GBM, therapies targeting GSCs are considered to be effective for long-term survival in GBM. Several reports suggested that oxidative phosphorylation (OXPHOS) of cancer stem cells is important for their survival; however, the requirement of OXPHOS in GSCs remains unclear. Few effective and safe agents that target GSC mitochondria are available in clinical settings. In this study, we demonstrated that GSCs had high OXPHOS activity compared with isogenic differentiated GSCs and that GSC survival depended on their OXPHOS activity. Remarkably, we showed that complexes III and IV had broad therapeutic windows and that the expression levels of mitochondrial DNA-coded components of complexes III and IV were elevated in GSCs compared with differentiated GSCs. Moreover, our search of the Food and Drug Administration-approved drugs for those targeting GSC mitochondria revealed that verteporfin (Visudyne® ), a drug approved for macular degeneration, was a novel GSC-specific cytotoxic compound that reduced OXPHOS activity. Importantly, the cytotoxic effect of verteporfin was specific to GSCs without any toxicity to normal cells, and the IC50 of approximately 200 nm was ten times less than its maximum blood concentration in humans. Overall, these findings indicated that high mitochondrial OXPHOS of GSCs is a potential GSC-specific vulnerability and that clinically available drugs, such as verteporfin, might become novel GSC-specific cytotoxic agents.

Involvement of GLUT1-mediated glucose transport and metabolism in gefitinib resistance of non-small-cell lung cancer cells.

Use of epidermal growth factor receptor (EGFR) inhibitors represented by gefitinib and erlotinib has become the standard of treatment for non-small-cell lung cancers (NSCLCs) with activating EGFR mutations. However, the majority of NSCLCs, which overexpress EGFR without such mutations, are resistant to EGFR inhibitors, and the mechanism(s) behind such primary resistance of NSCLCs without activating EGFR mutations to EGFR inhibitors still remains poorly understood. Here in this study, we show that glucose metabolism mediated by GLUT1, a facilitative glucose transporter, is involved in gefitinib resistance of NSCLC cells. We found that GLUT1 expression and glucose uptake were increased in resistant NSCLC cells after gefitinib treatment and that genetic as well as pharmacological inhibition of GLUT1 sensitized not only NSCLC cells with primary resistance but also those with acquired resistance to gefitinib. In vivo, the combination of systemic gefitinib and a GLUT1 inhibitor, both of which failed to inhibit tumor growth when administered alone, significantly inhibited the growth of xenograft tumors formed by the implantation of NSCLC cells with wild-type EGFR (wt-EGFR). Since our data indicated that GLUT1 was similarly involved in erlotinib resistance, our findings suggest that the activity of GLUT1-mediated glucose metabolism could be a critical determinant for the sensitivity of NSCLC cells to EGFR inhibitors and that concurrent GLUT1 inhibition may therefore be a mechanism-based approach to treating NSCLCs resistant to EGFR inhibitors, including those with wt-EGFR.

AS602801, an Anti-Cancer Stem Cell Drug Candidate, Suppresses Gap-junction Communication Between Lung Cancer Stem Cells and Astrocytes.

BACKGROUND/AIM: Cancer stem cells (CSCs) are associated with tumorigenesis, recurrence, and metastasis. Cell-cell communication via gap junctions (GJs) between metastatic cancer cells and astrocytes is necessary for brain metastasis. Agents targeting communication between CSCs and astrocytes are expected to suppress brain metastasis. MATERIALS AND METHODS: Using the A549 CSC, a cancer stem-like cell derived from A549, we examined the effect of AS602801, an anti-cancer stem cell agent whose safety has been confirmed in a phase 2 clinical trial, on GJ communication and connexin expression using a dye-transfer assay and immunoblot analysis, respectively. RESULTS: AS602801 specifically suppressed cell-cell communication in A549 CSCs without any suppression of GJ communication in astrocytes; it also decreased the expression of connexin 43, a constituent of GJs, in A549 CSCs. CONCLUSION: The anti-cancer stem cell agent, AS602801, is a potential drug candidate against brain metastasis.

The BECN1-BCL2 complex regulates insulin secretion and storage in mice.

Macroautophagy/autophagy abnormality has been recently associated with metabolic disorders, such as type 2 diabetes (T2D). However, the effect of autophagy activation in systemic energy metabolism was poorly understood. In our recent study, we demonstrated that autophagy plays different roles in distinct metabolic tissues, using an autophagy-hyperactive mouse model. In insulin-producing ß cells, excess autophagy degrades insulin-containing vesicles (a process termed vesicophagy), resulting in decreased insulin contents and systemic glucose intolerance; whereas in insulin-responsive cells, activating autophagy decreases endoplasmic reticulum (ER) stress and improves insulin sensitivity.

Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity.

Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing ß cells versus insulin-responsive cells, utilizing knockin (Becn1F121A) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in ß cells, a process we term "vesicophagy." The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, ß cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development.

Repositioning CEP-1347, a chemical agent originally developed for the treatment of Parkinson's disease, as an anti-cancer stem cell drug.

CEP-1347 is a mixed lineage kinase inhibitor tested in a large-scale phase 2/3 clinical trial in early Parkinson's disease, in which its safety and tolerability, but nevertheless not efficacy, was demonstrated. Here we identify by drug repositioning CEP-1347 as a potential anti-cancer stem cell drug. In vitro, CEP-1347 efficiently induced differentiation and inhibited the self-renewal and tumor-initiating capacities of human cancer stem cells from glioblastoma as well as from pancreatic and ovarian cancers at clinically-relevant concentrations, without impairing the viability of normal fibroblasts and neural stem cells. In vivo, a 10-day systemic administration of CEP-1347 at a dose that was less than 1/10 the mouse equivalent of the dose safely given to humans for 2 years was sufficient to effectively reduce tumor-initiating cancer stem cells within established tumors in mice. Furthermore, the same treatment protocol significantly extended the survival of mice receiving orthotopic implantation of glioma stem cells. Together, our findings suggest that CEP-1347 is a promising candidate for cancer stem cell-targeting therapy and that further clinical and preclinical studies are warranted to evaluate its efficacy in cancer treatment.

Antitumor activity of gemcitabine against high-grade meningioma in vitro and in vivo.

Currently, there is no established therapeutic option for high-grade meningioma recurring after surgery and radiotherapy, and few chemotherapeutic agents are in development for the treatment of high-grade meningioma. Here in this study, we screened a panel of chemotherapeutic agents for their possible antitumor activity in high-grade meningioma and discovered that high-grade meningioma cells show a preferential sensitivity to antimetabolites, in particular, to gemcitabine. In vitro, gemcitabine inhibited the growth of high-grade meningioma cells effectively by inducing S-phase arrest and apoptotic cell death. In vivo, systemic gemcitabine chemotherapy suppressed not only tumor initiation but also inhibited the growth and achieved a long-term control of established tumors in xenograft models of high-grade meningioma. Histological analysis indicated that systemic gemcitabine blocks cell cycle progression and promotes apoptotic cell death in tumor cells in vivo. Together, our data demonstrate that gemcitabine exerts potent antitumor activity against high-grade meningioma through cytostatic and cytotoxic mechanisms. We therefore propose gemcitabine is a promising chemotherapeutic agent that warrants further investigation as a treatment option for high-grade meningioma.

Olanzapine, an Atypical Antipsychotic, Inhibits Survivin Expression and Sensitizes Cancer Cells to Chemotherapeutic Agents.

BACKGROUND/AIM: Olanzapine, an atypical antipsychotic, is now increasingly used as an off-label indication for the management of cancer patients with chemotherapy-induced nausea and vomiting (CINV). However, how olanzapine affects cancer cells per se remains poorly understood. MATERIALS AND METHODS: The effects of olanzapine treatment and survivin knockdown, alone or in combination with chemotherapeutic agents, on survivin expression and cell viability were investigated in human cancer cell lines. RESULTS: Olanzapine reduced survivin expression in lung and pancreatic cancer stem cell (CSC) lines and sensitized them to chemotherapeutic agents such as 5-fluorouracil, gemcitabine, and cisplatin in a survivin expression-dependent manner. Olanzapine also reduced survivin expression and chemosensitized serum-cultured, non-CSC ovarian cancer cells that expressed survivin. CONCLUSION: Olanzapine may benefit cancer patients not only as an antiemetic for CINV, but also by enhancing the effects of chemotherapeutic agents through down-regulation of survivin, which has been implicated in multidrug chemoresistance.

Licochalcone A specifically induces cell death in glioma stem cells via mitochondrial dysfunction.

Glioblastoma multiforme is the most malignant primary intrinsic brain tumor. Glioma stem cells (GSCs) are associated with chemoradiotherapy resistance and the recurrence of glioblastomas after conventional therapy. The targeting of GSCs is potentially an effective treatment for the long-term survival of glioblastoma patients. Licochalcone A, a natural chalconoid from licorice root, exerts anticancer effects; however, its effect on GSCs remains unknown. We found that Licochalcone A induced massive caspase-dependent death in GSCs but not in differentiated GSCs nor normal somatic and neural stem cells. Prior to cell death, Licochalcone A caused mitochondrial fragmentation and reduced the membrane potential and ATP production in GSCs. Thus, Licochalcone A induces mitochondrial dysfunction and shows promise as an anticancer stem cell drug.

Rho-Associated Protein Kinase (ROCK) Inhibitors Inhibit Survivin Expression and Sensitize Pancreatic Cancer Stem Cells to Gemcitabine.

BACKGROUND: Targeting pathways regulating survivin expression, which has been implicated in multidrug resistance of cancer cells, is a promising strategy to overcome cancer chemoresistance. To date, the role of rho-associated protein kinases (ROCKs) in survivin expression remains largely unknown. MATERIALS AND METHODS: The effects of ROCK inhibitors Y-27632 and fasudil on survivin expression and cell viability were determined by immunoblot analysis and dye exclusion, respectively, in PANC-1 CSLC, a cancer stem cell line derived from a serum-cultured, gemcitabine-sensitive pancreatic cancer cell line, PANC-1. RESULTS: siRNA-mediated knockdown of survivin revealed that the gemcitabine resistance of PANC-1 CSLC was dependent on survivin expression. Both Y-27632 and fasudil, reduced survivin expression in PANC-1 CSLC cells and sensitized them to gemcitabine. ROCK inhibition also reduced survivin expression in various other human cancer cell lines. CONCLUSION: Small molecule inhibitor-mediated targeting of ROCK may be a viable strategy to overcome cancer chemoresistance through down-regulation of survivin.

Aripiprazole, an Antipsychotic and Partial Dopamine Agonist, Inhibits Cancer Stem Cells and Reverses Chemoresistance.

BACKGROUND: There is a growing interest in repurposing antipsychotic dopamine antagonists for cancer treatment; however, antipsychotics are often associated with an increased risk of fatal events. The anticancer activities of aripiprazole, an antipsychotic drug with partial dopamine agonist activity and an excellent safety profile, remain unknown. MATERIALS AND METHODS: The effects of aripiprazole alone or in combination with chemotherapeutic agents on the growth, sphere-forming ability and stem cell/differentiation/chemoresistance marker expression of cancer stem cells, serum-cultured cancer cells from which they were derived, and normal cells were examined. RESULTS: At concentrations non-toxic to normal cells, aripiprazole inhibited the growth of serum-cultured cancer cells and cancer stem cells. Furthermore, aripiprazole induced differentiation and inhibited sphere formation, as well as stem cell marker expression of cancer stem cells while inhibiting their survivin expression and sensitizing them to chemotherapeutic agents. CONCLUSION: Repurposing aripiprazole as an anticancer stem cell drug may merit further consideration.

Impact of H3K27 Demethylase Inhibitor GSKJ4 on NSCLC Cells Alone and in Combination with Metformin.

BACKGROUND: GSKJ4, an H3K27 demethylase inhibitor, reportedly exhibits antitumor activity against specific cancers harboring genetic alterations in genes encoding chromatin modulators. However, its potential as an anticancer agent against human cancers not associated with such genetic alterations, including non-small cell lung cancer (NSCLC), remains unknown. MATERIALS AND METHODS: The effect of GSKJ4 on the growth of three NSCLC cell lines and normal lung fibroblasts was investigated using the WST-8, dye exclusion, and colony formation assays. RESULTS: GSKJ4, alone and in combination with an anti-diabetic drug metformin, induced cell death and inhibited the growth of NSCLC cell lines efficiently at concentrations non-toxic to normal cells, irrespective of their genetic backgrounds (mutations in the KRAS, TP53 and EGFR genes) and also of their resistance to cisplatin and paclitaxel. CONCLUSION: GSKJ4, being a promising anticancer agent for NSCLC, may be effective against a wider spectrum of cancers than previously thought.

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.

Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.