Glutamine Deprivation Regulates the Origin and Function of Cancer Cell Exosomes

Abstract

Exosomes are secreted extracellular vesicles (EVs) carrying diverse cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of these exosomes from cancer cells is increased by reducing Akt/mechanistic Target of Rapamycin (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. The resulting vesicles promote tumour cell proliferation and turnover, and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against Amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release of Rab11a-exosomes with protumorigenic functions, which we propose promote stress-induced tumour adaptation. KEY WORDSExosome / extracellular vesicle (EV) / mechanistic Target of Rapamycin (mTORC1) / recycling endosome / multivesicular body (MVB) 6 al., 2016). Fluorescent puncta were seen inside YFP-Rab11-positive SC compartments ( Figure 1C), but fewer than with CD63-GFP ( Figure 1B). Moreover, unlike CD63-GFP ( Figure S1B), the Rab11 fusion protein did not traffic to the plasma membrane ( Figure S1C, Movie S2). Sporadic YFP-Rab11-positive puncta were observed in the AG lumen, both in the gene trap line ( Figure S1C) and when YFP-Rab11 was specifically overexpressed in SCs ( Figure S1D). In contrast, a YFP-Rab7 gene trap fusion protein (Dunst et al., 2015) primarily trafficked to acidic LELs ( Figure 1D) and marked very few puncta in the AG lumen. We conclude that Rab11-labelled exosomes are formed at low levels in Rab11 SC compartments and secreted.In searching for other markers of these alternative exosomes, we found that an overexpressed GFP-tagged form of Breathless (Btl; a fly homologue of the human transmembrane FGF receptor), which is normally expressed in SCs ( Figure S1E), trafficked on to ILVs in Rab11compartments ( Figure 1E). Its expression did not affect large non-acidic compartments in SCs and had only a minor effect on acidic compartment number ( Figure S1H-J). In some SCs, Btl-GFP, like YFP-Rab11, was found in the lumen of an LEL, but, unlike CD63-GFP, these markers were rarely, if ever, observed on the LEL limiting membrane ( Figure S2), presumably because they reach LELs by sporadic fusion to Rab11-compartments (Corrigan et al., 2014) and not by endocytic trafficking. As with YFP-Rab11, transmembrane Btl-GFP protein was secreted in puncta into the AG lumen (Figure 2A). We, therefore, conclude that Rab11 and Btl are selective membrane-associated markers for exosomes generated in Rab11compartments of SCs...

Accessory ESCRT-III proteins selectively regulate Rab11-exosome biogenesis inDrosophilasecondary cells

“…The Drosophila accessory gland is composed of two cell types, the main cells and the secondary cells (SCs; Fig 1A). SCs have unusually large secretory and endosomal compartments, including between three and five large (> 2 µm) Rab7-positive late endosomes and lysosomes (LELs), which can be visualised with LysoTracker® and depending on the marker used, between ten and twenty large (> 2 µm) non-acidic compartments (Corrigan et al, 2014;Fan et al, 2019) Fig 1F,G). The latter can be fluorescently marked by a GFP-tagged form of Breathless (Btl), a fly FGF receptor homologue, when overexpressed in SCs, or by a YFP-Rab11 gene trap ( Fig 1B-E).…”

“…We have recently shown in both human cancer cell lines and in prostate-like secondary cells (SCs) of the male accessory gland in the fruit fly, Drosophila melanogaster, that exosomes are not only generated in late endosomes, but also in recycling endosomes marked by the trafficking regulator Rab11 (Fan et al, 2019).…”

GAPDH controls extracellular vesicle biogenesis and enhances therapeutic potential of EVs in silencing the Huntingtin gene in mice via siRNA delivery

“…For overexpression and knockdown experiments, the same strategy was employed, but in the presence of a UAS transgene. Six-day-old adult virgin males were used throughout this study to ensure that age-and mating-dependent effects on SC biology were mitigated 18,19,39 .…”

“…To assess the physiological role of GAPDH in EV biology, we exploited a Drosophila melanogaster model of exosome biogenesis. In secondary cells (SCs) of the Drosophila accessory gland (AG), exosomes are formed as intraluminal vesicles (ILVs) in highly enlarged Rab11-positive compartments and then secreted into the lumen of the AG, a storage site for seminal fluid 18,19 . These ILVs can be selectively marked by fluorescent transmembrane markers, such as a GFP-tagged form of the FGF receptor, Breathless (Btl-GFP).…”

“…These ILVs can be selectively marked by fluorescent transmembrane markers, such as a GFP-tagged form of the FGF receptor, Breathless (Btl-GFP). In SCs, ILVs form in clusters that surround a large dense-core granule (DCG) of aggregated protein and extend out to the limiting membrane of the Rab11 compartments 19 .…”