Mechanisms of Autophagy Initiation.

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17.

Class III PI3-kinase (PI3KC3) is essential for autophagy initiation, but whether PI3KC3 participates in other steps of autophagy remains unknown. The HOPS complex mediates the fusion of intracellular vesicles to lysosome, but how HOPS specifically tethers autophagosome to lysosome remains elusive. Here, we report Pacer (protein associated with UVRAG as autophagy enhancer) as a regulator of autophagy. Pacer localizes to autophagic structures and positively regulates autophagosome maturation. Mechanistically, Pacer antagonizes Rubicon to stimulate Vps34 kinase activity. Next, Pacer recruits PI3KC3 and HOPS complexes to the autophagosome for their site-specific activation by anchoring to the autophagosomal SNARE Stx17. Furthermore, Pacer is crucial for the degradation of hepatic lipid droplets, the suppression of Salmonella infection, and the clearance of protein aggregates. These results not only identify Pacer as a crucial multifunctional enhancer in autophagy but also uncover both the involvement of PI3KC3 and the mediators of HOPS's specific tethering activity in autophagosome maturation.

Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Recent advances of vacuolar protein-sorting 34 inhibitors targeting autophagy.

Autophagy is a ubiquitous pathological/physiological antioxidant cellular reaction in eukaryotic cells. Vacuolar protein sorting 34 (Vps34 or PIK3C3), which plays a crucial role in autophagy, has received much attention. As the only Class III phosphatidylinositol-3 kinase in mammals, Vps34 participates in vesicular transport, nutrient signaling and autophagy. Dysfunctionality of Vps34 induces carcinogenesis, and abnormal autophagy mediated by dysfunction of Vps34 is closely related to the pathological progression of various human diseases, which makes Vps34 a novel target for tumor immunotherapy. In this review, we summarize the molecular mechanisms underlying macroautophagy, and further discuss the structure-activity relationship of Vps34 inhibitors that have been reported in the past decade as well as their potential roles in anticancer immunotherapy to better understand the antitumor mechanism underlying the effects of these inhibitors.

Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination.

Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity.

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Identification of VPS34-PI(3)P-FEN1-mediated DNA repair pathway as a potential drug target to overcome chemoresistance.

Intrinsic or acquired chemoresistance represents a major obstacle in cancer treatment. Multiple mechanisms can contribute to cancer cells' resistance to chemotherapy. Among them, an aberrantly strengthened DNA repair mechanism is responsible for a large proportion of drug resistance to alkylating agents and radiation therapy. In cancer cells, damping overactivated DNA repair system can overcome survival advantages conferred by chromosomal translocations or mutations and lead to cytostatic effects or cytotoxic. Therefore, selectively targeting DNA repair system in cancer cells holds promise for overcoming chemoresistance. In this study, we revealed that the endonuclease Flap Endonuclease 1 (FEN1), essential for DNA replication and repair, directly interacts with phosphatidylinositol 3-phosphate [PI(3)P], and FEN1-R378 is the primary PI(3)P-binding site. PI(3)P-binding deficient FEN1 mutant (FEN1-R378A) cells exhibited abnormal chromosomal structures and were hypersensitized to DNA damage. The PI(3)P-mediated FEN1 functionality was essential for repairing DNA damages caused by multiple mechanisms. Furthermore, VPS34, the major PI(3)P synthesizing enzyme, was negatively associated with patients' survival in various cancer types, and VPS34 inhibitors significantly sensitized chemoresistant cancer cells to genotoxic agents. These findings open up an avenue for counteracting chemoresistance by targeting VPS34-PI(3)P-mediated DNA repair pathway, and call for assessing the efficacy of this strategy in patients suffering from chemoresistance-mediated cancer recurrence in clinical trials.

Class III PI3K Biology.

Highly conserved from yeast to mammals, vacuolar protein sorting 34 (Vps34) is the sole member of the third class of the phosphoinositide 3-kinase (PI3K) family. By producing phosphatidylinositol-3-monophosphate (PtdIns3P) through its scaffolding function essential for the catalytic lipid activity, Vps34 regulates endosomal trafficking, autophagy, phagocytosis, and nutrient-sensing signaling. The development of genetically modified mouse models and specific inhibitors has largely contributed over the past ten years to a better understanding of Vps34 functions in biological and physiological processes in mammals and, ultimately, its potential implications and targeting in human diseases. This chapter will summarize the current knowledge of the structure and regulation of Vps34 as well as its cellular and organismal functions.

Autophagy-related gene PXN as a prognostic marker: Promotion of ovarian cancer progression by suppressing the p110ß/Vps34/Beclin1 pathway.

Among gynecological malignancies, ovarian cancer has the highest mortality rate and has sparked widespread interest in studying the mechanisms underlying ovarian cancer development. Based on TCGA and GEO databases, we investigated the highly expressed autophagy-related genes that determine patient prognosis using limma differential expression and Kaplan-Meier survival analyses. The biological processes associated with these genes were also predicted using GO/KEGG functional enrichment analysis. CCK-8, cell scratch, and transwell assays were used to investigate the effects of PXN on the proliferation, migration, and invasion abilities of ovarian cancer cells. Transmission electron microscopy was used to observe the autophagosomes. The expression of autophagy proteins and the PI3K/Akt/mTOR and p110ß/Vps34/Beclin1 pathway proteins in ovarian cancer cells was detected using western blot; autophagy protein expression was further detected and localized using cellular immunofluorescence. A total of 724 autophagy-related genes were found to be overexpressed in ovarian -cancer tissues, with high expression of PEX3, PXN, and RB1 associated with poor prognosis in patients (p < .05). PXN activates and regulates signaling pathways related to cellular autophagy, ubiquitination, lysosomes, PI3K-Akt, and mTOR. Autophagosomes were observed in all cell groups. The increase in PXN gene expression promoted the proliferation, migration, and invasion of ovarian cancer cells, increased the expression of SQSTM1/p62 protein, decreased LC3II/LC3Ⅰ, inhibited the phosphorylation of Akt and mTOR proteins, and suppressed the expression of PI3K(p110ß) and Beclin1 proteins. The decrease in PXN expression also confirmed these changes. Thus, PXN is highly expressed during ovarian cancer and is associated with poor patient prognosis. It may promote ovarian cancer cell proliferation, migration, and invasion by inhibiting cellular autophagy via suppression of the p110ß/Vps34/Beclin1 pathway.

FBXL20-mediated Vps34 ubiquitination as a p53 controlled checkpoint in regulating autophagy and receptor degradation.

Vacuolar protein-sorting 34 (Vps34), the catalytic subunit in the class III PtdIns3 (phosphatidylinositol 3) kinase complexes, mediates the production of PtdIns3P, a key intracellular lipid involved in regulating autophagy and receptor degradation. However, the signal transduction pathways by which extracellular signals regulate Vps34 complexes and the downstream cellular mechanisms are not well understood. Here we show that DNA damage-activated mitotic arrest and CDK activation lead to the phosphorylation of Vps34, which provides a signal to promote its ubiquitination and proteasomal degradation mediated by FBXL20 (an F-box protein) and the associated Skp1 (S-phase kinase-associated protein-1)-Cullin1 complex, leading to inhibition of autophagy and receptor endocytosis. Furthermore, we show that the expression of FBXL20 is regulated by p53-dependent transcription. Our study provides a molecular pathway by which DNA damage regulates Vps34 complexes and its downstream mechanisms, including autophagy and receptor endocytosis, through SCF (Skp1-Cul1-F-box)-mediated ubiquitination and degradation. Since the expression of FBXL20 is regulated by p53-dependent transcription, the control of Vps34 ubiquitination and proteasomal degradation by FBXL20 and the associated SCF complex expression provides a novel checkpoint for p53 to regulate autophagy and receptor degradation in DNA damage response.

Exploring the mechanism of trehalose: dual functions of PI3K/Akt and VPS34/mTOR pathways in porcine oocytes and cumulus cells†.

Autophagy, an intracellular recycling system, is essential for the meiotic maturation of porcine oocytes. Trehalose has been reported as a novel mammalian target of rapamycin (mTOR)-independent autophagy inducer in many cells. Furthermore, we previously have demonstrated that trehalose supplementation during in vitro maturation of porcine oocytes improves the developmental competence of parthenogenetic embryos, possibly via autophagic activation, whereas the underlying mechanisms remain unclear. Therefore, the aim of this study was to address this issue. We found that trehalose plays a role as an autophagy activator by autophagic flux assay and determined that it promotes phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) inhibition and vacuolar protein sorting 34 (VPS34)/mTOR activation by immunoblotting, both in cumulus cells (CCs) and oocytes. However, interestingly, the effects and the mechanisms regulated by trehalose were different in them, respectively. In CCs, the autophagy was activated through the improvement of lysosomal function/autophagic clearance viability by upregulation of coordinated lysosomal expression and regulation genes via PI3K/Akt inhibition. Whereas in oocytes, autophagy was activated via induction of VPS34, which directly influences autophagosome formation, and the precise meiotic process was ensured via Akt inhibition and mTOR activation. Taken together, this study furtherly elucidates the novel detailed mechanism of trehalose during porcine oocyte maturation, thus laying the biological foundations for pharmacological application.

Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues.

Autophagy is thought to be involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, how SARS-CoV-2 interferes with the autophagic pathway and whether autophagy contributes to virus infection in vivo is unclear. In this study, we identified SARS-CoV-2-triggered autophagy in animal models, including the long-tailed or crab-eating macaque (Macaca fascicularis), human angiotensin-converting enzyme 2 (hACE2) transgenic mice, and xenografted human lung tissues. In Vero E6 and Huh-7 cells, SARS-CoV-2 induces autophagosome formation, accompanied by consistent autophagic events, including inhibition of the Akt-mTOR pathway and activation of the ULK-1-Atg13 and VPS34-VPS15-Beclin1 complexes, but it blocks autophagosome-lysosome fusion. Modulation of autophagic elements, including the VPS34 complex and Atg14, but not Atg5, inhibits SARS-CoV-2 replication. Moreover, this study represents the first to demonstrate that the mouse bearing xenografted human lung tissue is a suitable model for SARS-CoV-2 infection and that autophagy inhibition suppresses SARS-CoV-2 replication and ameliorates virus-associated pneumonia in human lung tissues. We also observed a critical role of autophagy in SARS-CoV-2 infection in an hACE2 transgenic mouse model. This study, therefore, gives insights into the mechanisms by which SARS-CoV-2 manipulates autophagosome formation, and we suggest that autophagy-inhibiting agents might be useful as therapeutic agents against SARS-CoV-2 infection. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with limited therapeutics. Insights into the virus-host interactions contribute substantially to the development of anti-SARS-CoV-2 therapeutics. The novelty of this study is the use of a new animal model: mice xenografted with human lung tissues. Using a combination of in vitro and in vivo studies, we have obtained experimental evidence that induction of autophagy contributes to SARS-CoV-2 infection and improves our understanding of potential therapeutic targets for SARS-CoV-2.

Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase.

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival.

BACKGROUND: Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. METHODS: Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA-protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. RESULTS: We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-ß/Smad2 pathway. CONCLUSIONS: Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.

Inhibition of Vps34 and p110δ PI3K Impairs Migration, Invasion and Three-Dimensional Spheroid Growth in Breast Cancer Cells.

Breast cancer is a heterogeneous disease that represents the most common cancer around the world; it comprises 12% of new cases according to the World Health Organization. Despite new approaches in early diagnosis and current treatment, breast cancer is still the leading cause of death for cancer mortality. New targeted therapies against key signalling transduction molecules are required. Phosphoinositide 3-kinase (PI3K) regulates multiple biological functions such as proliferation, survival, migration, and growth. It is well established that PI3K isoform-selective inhibitors show fewer toxic side effects compared to broad spectrum inhibition of PI3K (pan-PI3K inhibitors). Therefore, we tested the PI3K p110δ-selective inhibitor, IC87114, and Vps34-selective inhibitor, Vps34-IN1, on the breast cancer cell lines MCF-7 and MDA-MB-231, representing hormone-responsive and triple-negative breast cancer cells, respectively. Our data show that both inhibitors decreased migration of MCF-7 and MDA-MB-231 cells, and Vps34 also significantly impacted MCF-7 cell proliferation. Three-dimensional (3D) in vitro culture models show that IC87114 and Vps34-IN1 treatment reduced the growth of MCF-7 and MDA-MB-231 cells in 3D tumour spheroid cultures. This study identifies IC87114 and Vps34-IN1 as potential therapeutic approaches in breast cancer.

Impact of Autophagy Impairment on Experience- and Diet-Related Synaptic Plasticity.

The beneficial effects of diet and exercise on brain function are traditionally attributed to the enhancement of autophagy, which plays a key role in neuroprotection via the degradation of potentially harmful intracellular structures. The molecular machinery of autophagy has also been suggested to influence synaptic signaling via interaction with trafficking and endocytosis of synaptic vesicles and proteins. Still, the role of autophagy in the regulation of synaptic plasticity remains elusive, especially in the mammalian brain. We explored the impact of autophagy on synaptic transmission and homeostatic and acute synaptic plasticity using transgenic mice with induced deletion of the Beclin1 protein. We observed down-regulation of glutamatergic and up-regulation of GABAergic synaptic currents and impairment of long-term plasticity in the neocortex and hippocampus of Beclin1-deficient mice. Beclin1 deficiency also significantly reduced the effects of environmental enrichment, caloric restriction and its pharmacological mimetics (metformin and resveratrol) on synaptic transmission and plasticity. Taken together, our data strongly support the importance of autophagy in the regulation of excitatory and inhibitory synaptic transmission and synaptic plasticity in the neocortex and hippocampus. Our results also strongly suggest that the positive modulatory actions of metformin and resveratrol in acute and homeostatic synaptic plasticity, and therefore their beneficial effects on brain function, occur via the modulation of autophagy.

Suppression of SARS-CoV-2 infection in ex-vivo human lung tissues by targeting class III phosphoinositide 3-kinase.

The novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the coronavirus disease 19 (COVID-19) pandemic due to its high transmissibility and early immunosuppression. Previous studies on other betacoronaviruses suggested that betacoronavirus infection is associated with the host autophagy pathway. However, it is unclear whether any components of autophagy or virophagy can be therapeutic targets for COVID-19 treatment. In this report, we examined the antiviral effect of four well-characterized small molecule inhibitors that target the key cellular factors involved in key steps of the autophagy pathway. They include small molecules targeting the ULK1/Atg1 complex involved in the induction stage of autophagy (ULK1 inhibitor SBI0206965), the ATG14/Beclin1/VPS34 complex involved in the nucleation step of autophagy (class III PI3-kinase inhibitor VPS34-IN1), and a widely-used autophagy inhibitor that persistently inhibits class I and temporary inhibits class III PI3-kinase (3-MA) and a clinically approved autophagy inhibitor that suppresses autophagy by inhibiting lysosomal acidification and prevents the formation of autophagolysosome (HCQ). Surprisingly, not all the tested autophagy inhibitors suppressed SARS-CoV-2 infection. We showed that inhibition of class III PI3-kinase involved in the initiation step of both canonical and noncanonical autophagy potently suppressed SARS-CoV-2 at a nano-molar level. In addition, this specific kinase inhibitor VPS34-IN1, and its bioavailable analogue VVPS34-IN1, potently inhibited SARS-CoV-2 infection in ex vivo human lung tissues. Taken together, class III PI3-kinase may be a possible target for COVID-19 therapeutic development.

Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.

BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.