Amino Acid Starvation-induced Autophagy Research Articles

Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.

Amino acid starvation promotes podocyte autophagy through mammalian target of rapamycin inhibition and transcription factor EB activation.

Autophagy is important for maintaining normal physiological functions and podocyte cell homeostasis. Amino acid signaling is an important upstream signaling pathway for autophagy regulation. However, the function and the associated mechanism of amino acid signaling in podocyte autophagy is unclear. The present study used normal culture medium and amino acid deprivation medium to culture podocytes in vitro. Multiple methods were utilized to detect autophagic activity including western blot analysis to measure the levels of microtubule-associated protein 1 light chain 3 (LC3) II and beclin1, reverse transcription-quantitative polymerase chain reaction was performed to evaluate the levels of LC3 mRNA and transmission electron microscopy was conducted to observe autophagosomes. In addition, tandem green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3 adenoviruses were employed to transduce podocytes to observe autophagic flux. Furthermore, the present study examined the effects of amino acid signaling on mammalian target of rapamycin (mTOR) activity and the nuclear translocation of transcription factor EB (TFEB), a core regulator of autophagy, using western blotting and immunofluorescence. The results revealed that amino acid starvation promoted the expression of LC3II and beclin1, and increased the number of autophagosomes and autolysosomes. Amino acid starvation inhibited mTOR activity, and promoted nuclear translocation and TFEB activity. Inhibition of TFEB blocked amino acid starvation-induced autophagy. These results indicated that amino acid starvation stimulated podocyte autophagy, and thus suggested that mTOR suppression and TFEB activation may mediate amino acid starvation-induced autophagy in podocytes.

Amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in weaning piglets derived from sows fed low-protein diet during gestation and lactation : Autophagy is involved in reduced fat deposition in maternal low-protein piglets.

The study aimed to determine the effects of maternal low-protein (LP) diet on subcutaneous fat deposition of weaning piglets and the potential mechanism. Sows were fed either a standard protein (SP, 15 and 18% crude protein) or a LP diet (50% protein levels of SP) throughout pregnancy and lactation. Subcutaneous fat and blood were sampled from male piglets at 28 days of age. Serum biochemical metabolites and hormone concentrations were detected with the enzymatic colorimetric methods. Serum-free amino acid (FAA) levels were measured by amino acid auto-analyzer. The mRNA and protein were measured by qRT-PCR and Western blot. Body weight, back fat thickness, triglycerides concentrations in subcutaneous fat tissue, and serum, as well as FFA concentrations were significantly reduced in LP piglets when compared with SP piglets. Further studies showed that mRNA and protein expression of acetyl-CoA carboxylase and fatty acid synthetase, two key enzymes of de novo lipogenesis, were significantly reduced in LP piglets, while mRNA expression and the lipolytic enzymes activities of lipolysis genes, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased. Furthermore, expression of autophagy-related gene 7 and autophagy maker gene microtubule-associated protein 1A/1B-light chain 3 (LC 3) as well as the conversion of LC3I to LC3II were significantly elevated, along with the expression of activating transcription factor-4 and eukaryotic translation initiation factor-2a. These results indicate that amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in maternal LP weaning piglets, demonstrating links between maternal protein restriction and offspring fat deposition.

Telomerase reverse transcriptase induces basal and amino acid starvation-induced autophagy through mTORC1

Telomerase is a reverse transcriptase that consists of the telomerase RNA component (TERC) and the reverse transcriptase catalytic subunit (TERT) and specializes in the elongation of telomere ends. New evidence suggests that beyond classical telomere maintenance, TERT also possesses telomere length-independent functions that are executed via interaction with other binding proteins. One such reported TERT-interacting proteins is mTOR, a master nutrient sensor that is upregulated in several cancers; however, the physiological implications of the TERT-mTOR interaction in normal cellular processes as well as in tumorigenesis are poorly understood. Here, we report that TERT inhibits the kinase activity of mTOR complex 1 (mTORC1) in multiple cell lines, resulting in the activation of autophagy under both basal and amino acid-deprived conditions. Furthermore, TERT-deficient cells display the inability to properly execute the autophagy flux. Functionally, TERT-induced autophagy provides a survival advantage to cells in nutrient-deprived conditions. Collectively, these findings support a model in which gain of TERT function modulates mTORC1 activity and induces autophagy, which is required for metabolic rewiring to scavenge the nutrients necessary for fueling cancer cell growth in challenging tumor microenvironments.

Abstract 3665: WAC: A candidate tumor suppressor gene in colorectal cancer

Abstract Our lab recently performed a DNA transposon forward genetic screen in mice that was designed to identify low-frequency mutations that contribute to colorectal cancer (CRC) initiation and progression. Results from this screen identified the WW domain-containing adaptor with coiled-coil (WAC) gene as a top DNA transposon insertion site. WAC has previously been implicated in several cellular processes including amino acid starvation-induced autophagy, golgi biogenesis, and transcription associated histone modification but has never before been linked to tumorigenesis. Transposon mutagenesis screens performed by others (Takeda et al. Nature Genetics 2015) have also identified Wac as a common insertion site, a result that further implicates WAC as a candidate CRC driver gene. Analyses of transposon insertion patterns within Wac predict loss of gene function and a role as a tumor suppressor. Soft agar colony formation assays reveal that shRNA mediated silencing of Wac cooperates with Apc mutations in mouse colorectal cells to promote cellular transformation. Additional colony formation assays using immortalized human colonic epithelial cells and the adenoma derived AAC1 cell line also shows that silencing WAC is protumorigenic. Using a zebrafish model we demonstrated that overexpression of wild type but not cancer-associated mutant forms of WAC induce expression of the cell cycle inhibitor p21, which suggests that loss of WAC may lead to uncontrolled cellular proliferation. Finally, using publicly available mutation data we determined that WAC is somatically mutated in both breast and lung cancers; a finding that indicates WAC may serve a critical tumor suppressive role in several tissues. Currently we are developing a conditional knockout mouse to further investigate the role of WAC in CRC tumor formation. Citation Format: Christopher R. Clark, Caitlin Conboy, Makayla Maile, Callie Janik, Julia Hatler, Robert Cormier, David Largaespada, Timothy K. Starr. WAC: A candidate tumor suppressor gene in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3665.

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.

Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex–dependent and –independent autophagy

Autophagy is a finely orchestrated cellular catabolic process that requires multiple autophagy-related gene products (ATG). The ULK1 complex functions to integrate upstream signals to downstream ATG proteins through an unknown mechanism. Here, we identified an interaction between mammalian FIP200 and ATG16L1, essential components of the ULK1 and ATG5 complexes, respectively. Further analyses demonstrate that this is a direct interaction mediated by a short domain of ATG16L1 which we term the FIP200-Binding Domain (FBD). The FBD is not required for ATG16L1 self-dimerization or interaction with ATG5. Importantly, FBD-deleted ATG16L1 mutant is defective in mediating amino acid starvation-induced autophagy, which requires the ULK1 complex. Intriguingly, this mutant retains its function in supporting glucose deprivation-induced autophagy, a ULK1 complex-independent process. Our study has therefore identified a novel interaction between the ULK1 and ATG5 complexes that can distinguish ULK1-dependent and -independent autophagy processes.

Baculovirus Infection Triggers a Shift from Amino Acid Starvation-Induced Autophagy to Apoptosis

Autophagy plays a central role in regulating important cellular functions such as cell survival during starvation and control of infectious pathogens. On the other hand, many pathogens have evolved mechanisms of inhibition of autophagy such as blockage of the formation of autophagosomes or the fusion of autophagosomes with lysosomes. Baculoviruses are important insect pathogens for pest control, and autophagy activity increases significantly during insect metamorphosis. However, it is not clear whether baculovirus infection has effects on the increased autophagy. In the present study, we investigated the effects of the Autographa californica nucleopolyhedrovirus (AcMNPV) infection on autophagy in SL-HP cell line from Spodoptera litura induced under amino acid deprivation. The results revealed that AcMNPV infection did not inhibit autophagy but triggered apoptosis under starvation pressure. In the early stage of infection under starvation, mitochondrial dysfunction was detected, suggesting the organelles might be involved in cell apoptosis. The semi-quantitative PCR assay revealed that the expression of both p35 and ie-1 genes of AcMNPV had no significant difference between the starved and unstarved SL-HP cells. The western blot analysis showed that no cleavage of endogenous Atg6 occurred during the process of apoptosis in SL-HP cells. These data demonstrated that some permissive insect cells may defend baculovirus infection via apoptosis under starvation and apoptosis is independent of the cleavage of Atg6 in SL-HP cells.

Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP-LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.

14-3-3Tau regulates Beclin 1 and is required for autophagy.

BackgroundBeclin 1 plays an essential role in autophagy; however, the regulation of Beclin 1 expression remains largely unexplored. An earlier ChIP-on-chip study suggested Beclin 1 could be an E2F target. Previously, we also reported that 14-3-3τ regulates E2F1 stability, and is required for the expression of several E2F1 target genes. 14-3-3 proteins mediate many cellular signaling processes, but its role in autophagy has not been investigated. We hypothesize that 14-3-3τ could regulate Beclin 1 expression through E2F1 and thus regulate autophagy.Methods and FindingsUsing the RNAi technique we demonstrate a novel role for one of 14-3-3 isoforms, 14-3-3τ, in the regulation of Beclin 1 expression and autophagy. Depletion of 14-3-3τ inhibits the expression of Beclin 1 in many different cell lines; whereas, upregulation of 14-3-3τ induces Beclin 1. The regulation is physiologically relevant as an extracellular matrix protein tenascin-C, a known 14-3-3τ inducer, can induce Beclin 1 through 14-3-3τ. Moreover, rapamycin-induced, serum free-induced and amino acid starvation-induced autophagy depends on 14-3-3τ. We also show the expression of Beclin 1 depends on E2F, and E2F can transactivate the Beclin 1 promoter in a promoter reporter assay. Upregulation of Beclin 1 by 14-3-3τ requires E2F1. Depletion of E2F1, like 14-3-3τ, also inhibits autophagy.ConclusionTaken together, this study uncovers a role for 14-3-3τ in Beclin 1 and autophagy regulation probably through regulation of E2F1.

Ordered Organelle Degradation during Starvation-induced Autophagy

Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route. Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.

Molecular Mechanisms of the mTOR Inhibitor Temsirolimus (CCI-779) Antiproliferative Effects in Mantle Cell Lymphoma: Induction of Cell Cycle Arrest, Autophagy, and Synergy with Vorinostat (SAHA).

Aberrant activation of the PI3-Kinase/Akt/mTOR survival pathway has been implicated in promoting the growth and survival of a variety of cancers, including lymphoma, and is currently being explored for cancer therapy. Importantly, the small molecule mTOR inhibitor temsirolimus (CCI-779) recently demonstrated significant clinical activity in patients with relapsed mantle cell lymphoma (MCL). However, the mechanism of action of temsirolimus in MCL cells is unknown. In this study, we demonstrated that temsirolimus induced cell growth inhibition in three MCL cell lines in a time-dependent and dose-dependent manner. The activity of temsirolimus was determined in 3 mantle cell lymphoma cell lines (Jeko-1, Mino, SP53). Temsirolimus upregulated p27 without altering cyclin D1 levels, resulting in cell cycle arrest in the G0/G1 phase. The Akt/mTOR pathway has been implicated in regulating cellular autophagy in yeasts and in mammalian cells. Thus, we examined whether temsirolimus may also induce autophagy in MCL cells, which is identified by the sequestering of cytoplasmic proteins into the lytic autophagosomes and autolysosome, and the formation of acidic vesicular organelles (AVOs). Temsirolimus induced AVOs formation indicative of autophagy in all MCL cell lines at doses ranging between 1 and 1000 nM in a time-dependent manner, with the highest activity observed between 72 and 96 hours of incubation. LC3 is essential for amino acid starvation-induced autophagy in yeasts. LC3-I is the cytoplasmic form, which is processed into the LC3-II form that is associated with the autophagosome membrane. Incubation of the SP53 cells with temsirolimus (1,000 nM) for 96 hours, resulted in processing LC3-I into LC3-II, indicative of autophagy induction. To further confirm induction of autophagy, SP53 cells expressing LC3-fused green fluorescent protein (GFP-LC3) were treated with temsirolimus and the pattern of LC3 distribution was compared with untreated cells using fluorescence microscopy. Untreated control cells showed a diffuse cytoplasmic distribution of LC3, whereas temsirolimus -treated cells showed a punctate pattern of green fluorescence, indicative of its association with autophagosomes. Furthermore, temsirolimus increased acidic vesicular organelles and microtubule-associated protein 1 light chain 3 (LC3) processing as determined by Western blot, which are characteristic of autophagy. In contrast, temsirolimus had minimal induction of apoptosis. Moreover, temsirolimus inhibited ribosomal S6 phosphorylation, an mTOR downstream target. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) demonstrated antiproliferative activity in a dose and time dependent manner in all three MCL cell lines. SAHA enhanced the activity of temsirolimus, which was associated with ERK dephosphorylation and caspase 3 activation. In contrast, temsirolimus did not potentiate the antitumor effects of bortezomib, doxorubicin, or gemcitabine. Our results demonstrate that in short-term culture, temsirolimus is primarily a cytostatic drug, and suggest that SAHA may potentiate the clinical efficacy of temsirolimus patients with MCL.

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells.

Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 microM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.