GABARAPL1 is essential in extracellular vesicle cargo loading and metastasis development.

BACKGROUND AND PURPOSE: Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. MATERIALS AND METHODS: GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O2) or hypoxia (O2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. RESULTS: The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. CONCLUSION: GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade.

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.

Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.

Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles.

Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes.

Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers.

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis- and autophagy signalling cascades are listed.

Natural T-cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles.

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity.

Pathogen recognition by NK cells amplifies the pro-inflammatory cytokine production of monocyte-derived DC via IFN-γ.

BACKGROUND: Besides their prominent role in the elimination of infected or malignantly transformed cells, natural killer (NK) cells serve as modulators of adaptive immune responses. Enhancing bidirectional crosstalk between NK cells and dendritic cells (DC) is considered a promising tool to potentiate cancer vaccines. We investigated to what extent direct sensing of viral and bacterial motifs by NK cells contributes to the response of inflammatory DC against the same pathogenic stimulus. RESULTS: We demonstrated that sensing of bacterial and viral PAMPs by NK cells contributes to DC cytokine production via NK cell-derived soluble factors. This enhancement of DC cytokine production was dependent on the pattern recognition receptor (PRR) agonist but also on the cytokine environment in which NK cells recognized the pathogen, indicating the importance of accessory cell activation for this mechanism. We showed in blocking experiments that NK cell-mediated amplification of DC cytokine secretion is dependent on NK cell-derived IFN-γ irrespective of the PRR that is sensed by the NK cell. CONCLUSIONS: These findings illustrate the importance of bidirectional interaction between different PRR-expressing immune cells, which can have implications on the selection of adjuvants for vaccination strategies.

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.