RESUMO
Autophagy normally involves the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation. Here we report the modification of single-membrane vacuoles in cells by autophagy proteins. LC3 (Light chain 3) a component of autophagosomes, is recruited to single-membrane entotic vacuoles, macropinosomes and phagosomes harbouring apoptotic cells, in a manner dependent on the lipidation machinery including ATG5 and ATG7, and the class III phosphatidylinositol-3-kinase VPS34. These downstream components of the autophagy machinery, but not the upstream mammalian Tor (mTor)-regulated ULK-ATG13-FIP200 complex, facilitate lysosome fusion to single membranes and the degradation of internalized cargo. For entosis, a live-cell-engulfment program, the autophagy-protein-dependent fusion of lysosomes to vacuolar membranes leads to the death of internalized cells. As pathogen-containing phagosomes can be targeted in a similar manner, the death of epithelial cells by this mechanism mimics pathogen destruction. These data demonstrate that proteins of the autophagy pathway can target single-membrane vacuoles in cells in the absence of pathogenic organisms.
Assuntos
Autofagia , Endocitose , Membranas Intracelulares/metabolismo , Vacúolos/metabolismo , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Membranas Intracelulares/patologia , Lisossomos/metabolismo , Fusão de Membrana , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Fatores de Tempo , Transfecção , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Vacúolos/patologiaRESUMO
The laminin-binding integrin α6ß1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6ß1 integrin to produce the structural variant α6pß1 on tumor cell surfaces is mediated by the serine protease urokinase plasminogen activator (uPA). Cleavage of α6ß1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pß1 production. In human tumors, uPA and uPAR are expressed in tumor cells and tumor-associated macrophages (TAM). TAMs localize to solid tumors and contribute to increased tumor growth and the metastatic phenotype. In this study, we utilized a coculture system of PC-3 prostate tumor cells and macrophages [12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human leukemia HL-60 cells] to investigate the hypothesis that macrophages stimulate the production of the prometastatic variant α6pß1 on human prostate cancer cells via the uPA/uPAR axis. Our results indicate that adherent macrophages cocultured with PC-3 cells increased PC-3 uPAR mRNA, uPAR cell surface protein expression and α6 integrin cleavage. The stimulation does not require macrophage/tumor cell contact because macrophage conditioned medium is sufficient for increased uPAR transcription and α6 cleavage-dependent PC-3 cell invasion. The increased cleavage was dependent on uPAR because production was blocked by silencing RNA-targeting uPAR. These results indicate that macrophages can stimulate uPA/uPAR production in tumor cells which results in α6 integrin cleavage. These data suggest that TAMs promote prometastatic integrin-dependent pericellular proteolysis.