Glycan degradation promotes macroautophagy.

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.

Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

mTORC1 Activation Requires DRAM-1 by Facilitating Lysosomal Amino Acid Efflux.

Sensing nutrient availability is essential for appropriate cellular growth, and mTORC1 is a major regulator of this process. Mechanisms causing mTORC1 activation are, however, complex and diverse. We report here an additional important step in the activation of mTORC1, which regulates the efflux of amino acids from lysosomes into the cytoplasm. This process requires DRAM-1, which binds the membrane carrier protein SCAMP3 and the amino acid transporters SLC1A5 and LAT1, directing them to lysosomes and permitting efficient mTORC1 activation. Consequently, we show that loss of DRAM-1 also impacts pathways regulated by mTORC1, including insulin signaling, glycemic balance, and adipocyte differentiation. Interestingly, although DRAM-1 can promote autophagy, this effect on mTORC1 is autophagy independent, and autophagy only becomes important for mTORC1 activation when DRAM-1 is deleted. These findings provide important insights into mTORC1 activation and highlight the importance of DRAM-1 in growth control, metabolic homeostasis, and differentiation.

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis.

During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signalling pathway. Using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK-dependent MOMP Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.

Autophagy Inhibition Mediates Apoptosis Sensitization in Cancer Therapy by Relieving FOXO3a Turnover.

Macroautophagy (autophagy) is intimately linked with cell death and allows cells to evade apoptosis. This has prompted clinical trials to combine autophagy inhibitors with other drugs with the aim of increasing the likelihood of cancer cells dying. However, the molecular basis for such effects is unknown. Here, we describe a transcriptional mechanism that connects autophagy to apoptosis. The autophagy-regulating transcription factor, FOXO3a, is itself turned over by basal autophagy creating a potential feedback loop. Increased FOXO3a upon autophagy inhibition stimulates transcription of the pro-apoptotic BBC3/PUMA gene to cause apoptosis sensitization. This mechanism explains how autophagy inhibition can sensitize tumor cells to chemotherapy drugs and allows an autophagy inhibitor to change the action of an MDM2-targeted drug from growth inhibition to apoptosis, reducing tumor burden in vivo. Thus, a link between two processes mediated via a single transcription factor binding site in the genome can be leveraged to improve anti-cancer therapies.

Targeting BCR-ABL-Independent TKI Resistance in Chronic Myeloid Leukemia by mTOR and Autophagy Inhibition.

Background: Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods: Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4-6 mice per group). All statistical tests were two-sided. Results: We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion: Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

Verapamil, an L-type calcium channel blocker, has been used successfully to treat cardiovascular diseases. Interestingly, we have recently shown that treatment of cancer cells with verapamil causes an effect on autophagy. As autophagy is known to modulate chemotherapy responses, this prompted us to explore the impact of verapamil on autophagy and cell viability in greater detail. We report here that verapamil causes an induction of autophagic flux in a number or tumor cells and immortalized normal cells. Moreover, we found that inhibition of autophagy in COLO 205 cells, via treatment with the chloroquine (CQ) or by CRISPR/Cas9-mediated disruption of the autophagy genes Atg7 and Atg5, causes an upregulation of apoptotic markers in response to verapamil. In search of a mechanism for this effect and because autophagy can often mitigate metabolic stress, we examined the impact of verapamil on cellular metabolism. This revealed that in normal prostate cells, verapamil diminishes glucose and glycolytic intermediate levels leading to adenosine 5'-triphosphate (ATP) depletion. In contrast, in COLO 205 cells it enhances aerobic glycolysis and maintains ATP. Importantly, we found that the autophagic response in these cells is related to the activity of l-lactate dehydrogenase A (LDHA, EC 1.1.1.27), as inhibition of LDHA reduces both basal and verapamil-induced autophagy and consequently decreases cell viability. In summary, these findings not only identify a novel mechanism of cytoprotective autophagy induction but they also highlight the potential of using verapamil together with inhibitors of autophagy for the treatment of malignant disease. ENZYMES: l-lactate dehydrogenase (LDHA, EC 1.1.1.27).

p53 directly regulates the glycosidase FUCA1 to promote chemotherapy-induced cell death.

p53 is a central factor in tumor suppression as exemplified by its frequent loss in human cancer. p53 exerts its tumor suppressive effects in multiple ways, but the ability to invoke the eradication of damaged cells by programmed cell death is considered a key factor. The ways in which p53 promotes cell death can involve direct activation or engagement of the cell death machinery, or can be via indirect mechanisms, for example though regulation of ER stress and autophagy. We present here another level of control in p53-mediated tumor suppression by showing that p53 activates the glycosidase, FUCA1, a modulator of N-linked glycosylation. We show that p53 transcriptionally activates FUCA1 and that p53 modulates fucosidase activity via FUCA1 up-regulation. Importantly, we also report that chemotherapeutic drugs induce FUCA1 and fucosidase activity in a p53-dependent manner. In this context, while we found that over-expression of FUCA1 does not induce cell death, RNAi-mediated knockdown of endogenous FUCA1 significantly attenuates p53-dependent, chemotherapy-induced apoptotic death. In summary, these findings add an additional component to p53s tumor suppressive response and highlight another mechanism by which the tumor suppressor controls programmed cell death that could potentially be exploited for cancer therapy.

Loss of autophagy causes a synthetic lethal deficiency in DNA repair.

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

p53 status determines the role of autophagy in pancreatic tumour development.

Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

Does androgen-ablation therapy (AAT) associated autophagy have a pro-survival effect in LNCaP human prostate cancer cells?

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Androgen-ablation therapy (AAT) and chemotherapy are commonly used to treat incurable prostate cancer. To improve outcome, there is major on-going research to develop more effective treatments with less toxicity. Autophagy has been suggested from previous studies to play a potential role in cell survival and may be associated with resistance to chemotherapy. Autophagy is known to be upregulated by nutrient starvation or AAT in prostate cancer. However, its functional impact is not fully known. The present study describes the potential synergism between the blockade of autophagy and AAT alone or AAT combined with taxane chemotherapy. Hence, future combined treatment options are warranted to further investigate the clinical impact of autophagy suppression as a treatment strategy. OBJECTIVE: To study the cellular effects of the anti-androgen bicalutamide on autophagy and its potential impact on response to androgen-ablation therapy (AAT) alone or combined with docetaxel chemotherapy in human prostate cancer LNCaP cells. MATERIALS AND METHODS: LNCaP cells were treated with bicalutamide ± docetaxel, and cellular effects were assayed: lipidated LC3 (a microtubule-associated protein) for autophagy and its trafficking to fuse with lysosome; flow cytometry using propidium iodide or caspase 3 for cell death; and sulforhodamine B assay for cell growth. RESULTS: Bicalutamide treatment enhanced autophagy in LNCaP cells with increased level of autophagosome coupled with an altered cellular morphology reminiscent of neuroendocrine differentiation. Consistent with the literature on the interaction between androgen receptor activation and taxane chemotherapy, bicalutamide diminished docetaxel mediated cytotoxicity. Significantly, pharmacological inhibition of autophagy with 3-methyladenine significantly enhanced the efficacy cell kill mediated by AAT ± docetaxel. CONCLUSION: Autophagy associated with bicalutamide treatment in LNCaP cells may have a pro-survival effect and strategy to modulate autophagy may have a potential therapeutic value.

DRAM-1 encodes multiple isoforms that regulate autophagy.

Macro(autophagy) is a cellular mechanism which delivers cytoplasmic constituents to lysosomes for degradation. Due to its role in maintaining cellular integrity, autophagy protects against various diseases including cancer. p53 is a major tumor suppressor gene which can modulate autophagy both positively and negatively. p53 induces autophagy via transcriptional activation of Damage-Regulated Autophagy Modulator (DRAM-1). We report here that DRAM-1 encodes not just one mRNA, but a series of p53-inducible splice variants which are expressed at varying levels in multiple human and mouse cell lines. Two of these new splice variants, termed SV4 and SV5, result in mature mRNA species. Different to 'full-length' DRAM-1 (SV1), SV4 and SV5 do not localise to lysosomes or endosomes, but instead partially localise to peroxisomes and autophagosomes respectively. In addition, SV4 and SV5 can also be found co-localised with certain markers of the endoplasmic reticulum. Similar to SV1, SV4 and SV5 do not appear to be inducers of programmed cell death, but they do modulate autophagy. In summary, these findings identify new autophagy regulators that provide insight into the control of autophagy downstream of p53.

The cyclin-dependent kinase PITSLRE/CDK11 is required for successful autophagy.

(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the aetiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclin-dependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE orthologue, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.

Oncogene-induced sensitization to chemotherapy-induced death requires induction as well as deregulation of E2F1.

The analysis of DNA tumor viruses has provided landmark insights into the molecular pathogenesis of cancer. A paradigm for this field has been the study of the adenoviral E1a protein, which has led to the identification of proteins such as p300, p400, and members of the retinoblastoma family. Through binding Rb family members, E1a causes deregulation of E2F proteins--an event common to most human cancers and a central pathway in which oncogenes, including E1a, sensitize cells to chemotherapy-induced programmed cell death. We report here, however, that E1a not only causes deregulation of E2F, but importantly that it also causes the posttranscriptional upregulation of E2F1 protein levels. This effect is distinct from the deregulation of E2F1, however, as mutants of E2F1 impaired in pRb binding are induced by E1a and E2F1 induction can also be observed in Rb-null cells. Analysis of E1a mutants selectively deficient in cellular protein binding revealed that induction of E2F1 is instead intrinsically linked to p400. Mutants unable to bind p400, despite being able to deregulate E2F1, do not increase E2F1 protein levels and they do not sensitize cells to apoptotic death. These mutants can, however, be complemented by either the knockdown of p400, resulting in the restoration of the ability to induce E2F1, or by the overexpression of E2F1, with both events reenabling sensitization to chemotherapy-induced death. Due to the frequent deregulation of E2F1 in human cancer, these studies reveal potentially important insights into E2F1-mediated chemotherapeutic responses that may aid the development of novel targeted therapies for malignant disease.

p53-mediated induction of Noxa and p53AIP1 requires NFkappaB.

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFkappaB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFkappaB. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NFkappaB inhibits cell death from p53, but at the same time sensitizes cells to death by TNFalpha. We found in agreement with a recent report showing that NFkappaB is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53's ability to induce Noxa is also impeded by inhibition of NFkappaB. In contrast to the regulation by p73, however, blockade of NFkappaB downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NFkappaB inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NFkappaB, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NFkappaB. These studies therefore add further insight into the complex relationship of p53 and NFkappaB. In addition, since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NFkappaB plays a pro-apoptotic role downstream of p53.

Analysis of DRAM-related proteins reveals evolutionarily conserved and divergent roles in the control of autophagy.

Autophagy is a membrane-trafficking process that serves to deliver cytoplasmic proteins and organelles to the lysosome for degradation. The process is genetically defined and many of the factors involved are conserved from yeast to man. Recently, a number of new autophagy regulators have been defined, including the Damage-Regulated Autophagy Modulator (DRAM), which is a lysosomal protein that links autophagy and the tumor suppressor, p53. We describe here analysis of DRAM-related proteins which reveals evolutionary conservation and divergence of DRAM's role in autophagy. We report that humans have 5 other proteins that show significant homology to DRAM. The closest of these, which we have termed DRAM2, displays 45% identity and 67% conservation when compared to DRAM. Interestingly, although similar to DRAM in terms of homology, DRAM2 is different from DRAM as it not induced by p53 or p73. DRAM2 is also a lysosomal protein, but again unlike DRAM its overexpression does not modulate autophagy. In contrast to humans, the Drosophila genome only encodes one DRAM-like protein, which is approximately equal in similarity to human DRAM and DRAM2. This questions, therefore, whether DRAM function is conserved from fly to man or whether DRAM's capacity to regulate autophagy has evolved in higher eukaryotes. Expression of DmDRAM, however, clearly revealed an ability to modulate autophagy. This points, therefore, to a conserved role of DRAM in this process and that additional human proteins have more recently evolved which, while potentially sharing some similarities with DRAM, may not be as intrinsically connected to autophagy regulation.

Hypoxia-selective macroautophagy and cell survival signaled by autocrine PDGFR activity.

The selective regulation of macroautophagy remains poorly defined. Here we report that PDGFR signaling is an essential selective promoter of hypoxia-induced macroautophagy. Hypoxia-induced macroautophagy in tumor cells is also HIF1alpha-dependent, with HIF1alpha integrating signals from PDGFRs and oxygen tension. Inhibition of PDGFR signaling reduces HIF1alpha half-life, despite buffering of steady-state protein levels by a compensatory increase in HIF1alpha mRNA. This markedly changes HIF1alpha protein pool dynamics, and consequently reduces the HIF1alpha transcriptome. As autocrine growth factor signaling is a hallmark of many cancers, cell-autonomous enhancement of HIF1alpha-mediated macroautophagy may represent a mechanism for augmenting tumor cell survival under hypoxic conditions.