ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer.

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition.

Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII+) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII+ cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII+ tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII+ cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII+ tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII+ glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII+ glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII+ glioblastoma.

GABARAPL1 is required for increased EGFR membrane expression during hypoxia.

BACKGROUND AND PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors and hypoxia is a common feature of solid tumors. Both EGFR and hypoxia are associated with therapy resistance and poor treatment outcome. To survive hypoxia, cells adapt by activation of hypoxia responsive pathways and expression of hypoxia-induced plasma membrane proteins. We observed that GABAA receptor associated protein like1 (GABARAPL1) and plasma membrane expression of EGFR were increased during hypoxia. Here we explored the role of the GABARAPL1 in EGFR membrane expression during hypoxia. MATERIAL AND METHODS: Quantitative qPCR, immunoblot analysis, flow cytometry and cytochemistry were used to assess this interplay. RESULTS: GABARAPL1 mRNA and protein levels are increased during hypoxia in vitro and correlate with tumor hypoxia in a panel of primary HNSCC xenografts. High GABARAPL1 mRNA is associated with poor outcome of HNSCC patients. During hypoxia, EGFR membrane expression is increased and GABARAPL1 and EGFR colocalize at the plasma membrane. GABARAPL1 knockdown inhibits EGFR membrane expression during hypoxia. CONCLUSION: GABARAPL1 is required for increased membrane expression of EGFR during hypoxia, suggesting a role for GABARAPL1 in the trafficking of these membrane proteins.

Canonical autophagy does not contribute to cellular radioresistance.

BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.

The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia.

BACKGROUND AND PURPOSE: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. MATERIAL AND METHODS: Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. RESULTS: ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. CONCLUSIONS: We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.

Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction.

BACKGROUND AND PURPOSE: Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation. MATERIAL AND METHODS: The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells. RESULTS: We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation. CONCLUSIONS: Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.

Autophagy is required during cycling hypoxia to lower production of reactive oxygen species.

BACKGROUND AND PURPOSE: Human tumors are characterized by the presence of cells that experience periodic episodes of hypoxia followed by reoxygenation. These cells are exposed to reactive oxygen species (ROS) upon reoxygenation and require adaptation to this stress by lowering ROS production or enhancing ROS-clearance for their survival. We hypothesized that autophagy, a lysosomal degradation pathway, may be involved in reducing ROS during periodic hypoxia through removal of ROS producing species. MATERIALS AND METHODS: Human tumor cells (MCF-7, HT29, U373) were exposed to cycles of hypoxia (O(2)<0.02%) and reoxygenation in the absence or presence of the autophagy inhibitor chloroquine (CQ). Clonogenic survival, ROS production and mitochondrial-DNA content were assessed. In addition, A549 cells overexpressing wild-type or K63-mutated ubiquitin (K63R) were analyzed for ROS production. RESULTS: Our data indicate that CQ treatment sensitizes cells to cycling hypoxia, due to increased production of ROS, associated with an incapacity to reduce mitochondrial content. Addition of the ROS-scavenger N-acetyl-cysteine increased cell viability and neutralized CQ-effects. Additionally, genetic prevention of K63-linked ubiquitin chains that are required for the removal of toxic protein aggregates by autophagy, resulted in increased ROS production. CONCLUSIONS: Inhibition of autophagy substantially increases cell death induced by cycling hypoxia through increased ROS production, providing an opportunity to decrease the hypoxic fraction within tumors and enhance tumor therapy.

The rat cytomegalovirus R78 G protein-coupled receptor gene is required for production of infectious virus in the spleen.

The rat cytomegalovirus (RCMV) R33 and R78 genes are conserved within members of the subfamily Betaherpesvirinae and encode proteins (pR33 and pR78, respectively) that show sequence similarity with G protein-coupled receptors. Previously, the biological relevance of these genes was demonstrated by the finding that R33- and R78-deleted RCMV strains are severely attenuated in vivo. In addition, R78-deleted strains were found to replicate less efficiently in cell culture. To monitor of the expression of R33- and R78-encoded proteins, recombinant RCMV strains, designated RCMV33G and RCMV78G, were generated. These recombinants expressed enhanced green fluorescent protein (EGFP)-tagged versions of pR33 and pR78 instead of native pR33 and pR78, respectively. Here it is reported that, although RCMV33G replicates as efficiently as wt virus in rat embryo fibroblast cultures, strain RCMV78G produces virus titres that are 3- to 4-fold lower than wt RCMV in the culture medium. This result indicates that the pR78-EGFP protein, as expressed by RCMV78G, does not completely functionally replace its native counterpart (pR78) in vitro. Interestingly, in infected rats, infectious RCMV33G was produced in significantly lower amounts than infectious wt RCMV, as well as RCMV78G, in the salivary glands. Conversely, although RCMV33G replicated to similar levels as wt virus in the spleen, both RCMV78G and an R78 knock-out strain (RCMV Delta R78a) replicated poorly in this organ. Together, these data indicate that R78 is crucial for the production of infectious RCMV in the spleen of infected rats.