Crosstalk between autophagy inhibitors and endosome-related secretory pathways: a challenge for autophagy-based treatment of solid cancers

Abstract

Autophagy is best known for its role in organelle and protein turnover, cell quality control, and metabolism. The autophagic machinery has, however, also adapted to enable protein trafficking and unconventional secretory pathways so that organelles (such as autophagosomes and multivesicular bodies) delivering cargo to lysosomes for degradation can change their mission from fusion with lysosomes to fusion with the plasma membrane, followed by secretion of the cargo from the cell. Some factors with key signalling functions do not enter the conventional secretory pathway but can be secreted in an autophagy-mediated manner.Positive clinical results of some autophagy inhibitors are encouraging. Nevertheless, it is becoming clear that autophagy inhibition, even within the same cancer type, can affect cancer progression differently. Even next-generation inhibitors of autophagy can have significant non-specific effects, such as impacts on endosome-related secretory pathways and secretion of extracellular vesicles (EVs). Many studies suggest that cancer cells release higher amounts of EVs compared to non-malignant cells, which makes the effect of autophagy inhibitors on EVs secretion highly important and attractive for anticancer therapy. In this review article, we discuss how different inhibitors of autophagy may influence the secretion of EVs and summarize the non-specific effects of autophagy inhibitors with a focus on endosome-related secretory pathways. Modulation of autophagy significantly impacts not only the quantity of EVs but also their content, which can have a deep impact on the resulting pro-tumourigenic or anticancer effect of autophagy inhibitors used in the antineoplastic treatment of solid cancers.

Keywords: Amphisomes; Autophagy; Autophagy inhibitors; Cancer; Endosomes; Exosomes; Extracellular vesicles; Multivesicular bodies; Non-conventional secretory pathways.

Fig. 1

Autophagy and endocytic pathways can culminate in lysosomes. Endocytosis enables the transport of substances from the external environment and includes the clathrin-dependent pathway as well as clathrin-independent pathways such as phagocytosis. Phagocytosis is the endocytosis of large molecules or intact microorganisms. Protrusions of plasma membrane surround and internalize the extracellular cargo into single-membrane structures called phagosomes, which are then transported to the lysosome for degradation. Endocytosis (clathrin-mediated endocytosis is shown here) involves invagination of the plasma membrane and biogenesis of small intracellular vesicles that contain constituents of the plasma membrane and extracellular components. These small vesicles fuse and establish the compartment called the early endosome (EE). EE cargo can be recycled to the plasma membrane via recycling endosomes, transported to or from the Golgi apparatus via the retromer complex, or routed to lysosomes via multivesicular bodies (MVBs). During macroautophagy, double-membrane structures called autophagosomes are formed to deliver autophagic cargo to lysosomes or to fuse with MVBs. Autophagy and endocytic pathways cooperate at some stages and share many components of the molecular machinery

Fig. 2

Amino acid-based mTORC1 activation. By amino acid starvation, the inactive V-ATPase-Ragulator complex is unable to activate Rag GTPases on the lysosomal surface, thus mTORC1 is not recruited to the lysosome. The inactivation of mTORC1 leads to rapid translocation of transcription factors TFEB and TFE3 to the nucleus. Active TFEB upregulates the expression of lysosomal genes and critical regulators of autophagy. By amino acid abundancy, the V-ATPase undergoes conformational changes leading to the activation of Regulator, which in turn promotes the Rag heterodimer activation. Active Rag heterodimer (RagA/B(GTP)-RagC/D(GDP)) then recruits mTORC1 to the lysosomal surface where Rheb is present. Rheb can directly bind and activate mTORC1. TFEB is recruited on the lysosomal membrane, phosphorylated by active mTORC1, and then degraded by the proteasome

Fig. 3

Macroautophagy pathways. The autophagic process is divided into five stages including initiation, phagophore nucleation, phagophore formation, autophagosome-lysosome fusion, and cargo degradation in autolysosomes. Signals activating macroautophagy usually originate from starvation, hypoxia, oxidative stress, and stress of the endoplasmic reticulum (ER). These signals trigger the activity of Unc-51-like kinase 1 (ULK1) complex (consisting of ULK1, FIP200, ATG13, and ATG101), which then starts phosphorylation of components of the class III PI3K (PI3KC3) complex I (consisting of VPS34, VPS15, Beclin1, ATG14L, and NRBF2) enabling nucleation of the phagophore. VPS34 produces phosphatidylinositol-3-phosphate (PI3P) allowing the recruitment of autophagy-associated PI3P-binding proteins such as DFCP1 and WIPI mediating the initial stages of autophagosome formation by associating ATG2A stably to PI3P-containing areas. Expansion of the phagophore requires the ATG2A-WIPI complex mediating ER–phagophore association and establishing the transfer of lipid membranes from the ER and the vesicles to the phagophore. WIPI was also shown to bind ATG16L1, thus recruiting the ATG12–ATG5–ATG16L1 complex. Elongation of autophagosomes requires the ubiquitin-like conjugation system managing the orchestrated activity of ATG proteins and LC3 (microtubule-associated protein light chain 3) and/or GABARAP. The ATG12–ATG5–ATG16L1 complex enhances the final connection of phosphatidylethanolamine (PE) molecules resulting in the formation of membrane-bound LC3-II and/or GABARAP-PE. Cellular membranes, including the mitochondrial membrane, the plasma membrane, recycling endosomes, and the Golgi complex, contribute to the elongation of the phagophore by providing membrane material. Elongation of the phagophore gives rise to double-layered vesicles called autophagosomes. In addition to managing autophagy induction in complex I, complex VPS34-Beclin1 has also a role in the fusion of autophagosomes with lysosomes as complex II. UVRAG competes with ATG14L for binding to Beclin1. When bound to Beclin1, UVRAG stimulates RAB7 GTPase activity and autophagosome fusion with lysosomes. Autophagosome-lysosome fusion is managed by Syntaxin-17 (STX17) on autophagosomes, VAMP8 on lysosomes, and by accessory proteins such as ATG14 and homotypic fusion, and protein sorting (HOPS) tethering complex

Fig. 4

Autophagy with emphasis on the state of MAP1LC3B (LC3B). Newly translated LC3B, called pro-LC3, is cleaved by the cysteine protease ATG4B at the C-terminal end with subsequent exposure of glycine residues (G). A cleaved form of a protein (LC3-I; soluble LC3) is further processed by ATG7 and ATG3, which conjugates LC3-I to phosphatidylethanolamine (PE) molecules. The ATG12–ATG5–ATG16L1 complex enhances the final conjugation of LC3-I to PE molecules resulting in the formation of membrane-bound LC3-II specifically targeted to the elongating autophagosomal membrane. The two ends of the insulating membrane are subsequently joined together to form an autophagosome with a double membrane. STX17, which is located on the outer autophagosomal membrane (not on the isolation or lysosomal membrane), is required for the fusion of an autophagosome with a lysosome. In the resulting autolysosome, the material is cleaved by acid hydrolases. During internal degradation, STX17 proteins are released from the outer membrane of the autophagosome. LC3-II molecules conjugated to the inner autophagosomal membrane are degraded by acids hydrolases, while the LC3-II molecules on the outer membrane of the autophagosome are cleaved by ATG4B and recycled as LC3-I

Fig. 5

The transition from early to late endosomes. Binding and activation of VPS34 on endosomes are initiated through the recruitment of RAB5 to endosomes by the guanine nucleotide exchange factor Rabex5. VPS34 then produces PI3P increasing the binding of RAB5 and other downstream effectors. The transition from early to late endosomes is complicated by a positive feedback loop between Rabex5 and RAB5. MON1A is needed to interrupt this positive feedback loop by displacing Rabex5 from endosomal membranes. MON1A also manages the recruitment of RAB7. VPS34 recruits TBC1D2 protein to endosomes in a RAB7-dependent manner to further inactivate RAB5 and to facilitate early to late endosome maturation

Fig. 6

Multivesicular bodies and autophagy. After maturation of early endosomes to multivesicular bodies (MVBs), MVBs can fuse with the plasma membrane to release intraluminal vesicles (ILVs) to the extracellular space as exosomes. With the help of specific proteins, MVBs are trafficked towards the plasma membrane and/or lysosome. Under certain conditions, MVBs can fuse with autophagosomes to generate hybrid organelles called amphisomes. Amphisomes contain typical autophagosomal markers such as lipidated LC3, and due to their origin from endosomes, they also contain endosomal markers such as CD63, RAB5, RAB7, and RAB11. The fusion of MVBs with the lysosome (direct or via autophagosome) results in autophagic degradation. The MVBs-related secretion and autophagy pathways are connected via many proteins, including RAB GTPases, ESCRTs, SNAREs, Beclin1, ATG proteins, and LC3

Fig. 7

Modulators of autophagy and their effect on EVs release. In nutrient-rich conditions, mTORC1 constitutively blocks the ULK complex and autophagy. mTORC1 signals can be inhibited directly by C10, rapamycin, exercise, or starvation and indirectly by bafilomycin A1 (BAFA1) through lysosomal inhibition. Physical exercise was shown to induce the release of small extracellular vesicles (EVs) into the circulation [195]. The ULK complex activates the VPS34 complex. VPS34 is a class III phosphatidylinositol 3-phosphate-kinase (PI3KC3). A group of PI3K inhibitors, including 3-methyladenine (3-MA), wortmannin, and synthetic inhibitor LY294002, inhibits both class I as well as class III PI3Ks. VPS34 inhibitors include Spautin-1, autophinib, SAR405, and VPS34-IN1. Spautin-1 initiates the degradation of Beclin1 due to the inhibition of two of its deubiquitinases. SAR405 and VPS34-IN1 are highly potent inhibitors of VPS34 selective for the VPS34 and not affecting the closely related class I and class II PI3Ks. Autophinib is an ATP-competitive inhibitor of VPS34 decreasing the accumulation of the lipidated protein LC3 on the autophagosomal membrane. The late stages of the autophagic machinery include fusion and degradation. During fusion, the mature autophagosome fuses with lysosomes creating an autolysosome. PIKfyve (phosphoinositide kinase, FYVE-type zinc finger containing) inhibitors and EACC block autophagosome-lysosome fusion. BAFA1 inhibits the acidification of the autolysosome by blocking the V-ATPase while chloroquine (CQ) and 3-hydroxychloroquine (HCQ) impair the maturation of autolysosomes. All drugs are depicted within the rectangles. Effects of modulators activating autophagy are green, inhibitory effects are red