Background: Autophagy is elevated in metastatic tumors and is often associatedwith active epithelial-to-mesenchymal transition (EMT). However, the extent towhich EMT is dependent on autophagy is largely unknown. This ...Background: Autophagy is elevated in metastatic tumors and is often associatedwith active epithelial-to-mesenchymal transition (EMT). However, the extent towhich EMT is dependent on autophagy is largely unknown. This study aimed toidentify the mechanisms by which autophagy facilitates EMT.Methods: We employed a liquid chromatography-based metabolomic approachwith kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1)gene co-mutated (KL) cells that represent an autophagy/EMT-coactivatedinvasive lung cancer subtype for the identification of metabolites linked to autophagy-driven EMT activation. Molecular mechanisms of autophagy-drivenEMT activation were further investigated by quantitative real-time polymerasechain reaction (qRT-PCR), Western blotting analysis, immunoprecipitation,immunofluorescence staining, and metabolite assays. The effects of chemicaland genetic perturbations on autophagic flux were assessed by two orthogonalapproaches: microtubule-associated protein 1A/1B-light chain 3 (LC3) turnoveranalysis by Western blotting and monomeric red fluorescent protein-greenfluorescent protein (mRFP-GFP)-LC3 tandem fluorescent protein quenchingassay. Transcription factor EB (TFEB) activity was measured by coordinatedlysosomal expression and regulation (CLEAR) motif-driven luciferase reporterassay. Experimental metastasis (tail vein injection) mouse models were used toevaluate the impact of calcium/calmodulin-dependent protein kinase kinase 2(CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVISluciferase imaging system.Results: We found that autophagy in KL cancer cells increased acetyl-coenzymeA (acetyl-CoA), which facilitated the acetylation and stabilization of theEMT-inducing transcription factor Snail. The autophagy/acetyl-CoA/acetylSnail axis was further validated in tumor tissues and in autophagy-activatedpancreatic cancer cells. TFEB acetylation in KL cancer cells sustained prometastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)-independent manner. Pharmacological inhibition of this axis via CAMKK2inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KLcancer cells in vivo.Conclusions: This study demonstrates that autophagy-derived acetyl-CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRASLKB1 co-mutated lung cancer cells and that inhibition of the autophagy/acetylCoA/acetyl-Snail axis using CAMKK2 or ACLY inhibitors could be a potentialtherapeutic strategy to suppress metastasis of KL lung cancer.展开更多
基金Korea Health Technology R&D Project through the Korea Health Industry Development Institute,Grant/Award Number:HI14C1324National Research Foundation of Korea,Grant/Award Numbers:2020R1A2C3007792,2019R1A2C3004155,2019H1A2A1075632+2 种基金NCI Lung Cancer SPORE,Grant/Award Number:P50CA70907Cancer Prevention and Research Institute of Texas(CPRIT),Grant/Award Number:RP160652“Team Science Award”of Yonsei University College of Medicine,Grant/Award Number:6-2021-0194。
文摘Background: Autophagy is elevated in metastatic tumors and is often associatedwith active epithelial-to-mesenchymal transition (EMT). However, the extent towhich EMT is dependent on autophagy is largely unknown. This study aimed toidentify the mechanisms by which autophagy facilitates EMT.Methods: We employed a liquid chromatography-based metabolomic approachwith kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1)gene co-mutated (KL) cells that represent an autophagy/EMT-coactivatedinvasive lung cancer subtype for the identification of metabolites linked to autophagy-driven EMT activation. Molecular mechanisms of autophagy-drivenEMT activation were further investigated by quantitative real-time polymerasechain reaction (qRT-PCR), Western blotting analysis, immunoprecipitation,immunofluorescence staining, and metabolite assays. The effects of chemicaland genetic perturbations on autophagic flux were assessed by two orthogonalapproaches: microtubule-associated protein 1A/1B-light chain 3 (LC3) turnoveranalysis by Western blotting and monomeric red fluorescent protein-greenfluorescent protein (mRFP-GFP)-LC3 tandem fluorescent protein quenchingassay. Transcription factor EB (TFEB) activity was measured by coordinatedlysosomal expression and regulation (CLEAR) motif-driven luciferase reporterassay. Experimental metastasis (tail vein injection) mouse models were used toevaluate the impact of calcium/calmodulin-dependent protein kinase kinase 2(CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVISluciferase imaging system.Results: We found that autophagy in KL cancer cells increased acetyl-coenzymeA (acetyl-CoA), which facilitated the acetylation and stabilization of theEMT-inducing transcription factor Snail. The autophagy/acetyl-CoA/acetylSnail axis was further validated in tumor tissues and in autophagy-activatedpancreatic cancer cells. TFEB acetylation in KL cancer cells sustained prometastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)-independent manner. Pharmacological inhibition of this axis via CAMKK2inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KLcancer cells in vivo.Conclusions: This study demonstrates that autophagy-derived acetyl-CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRASLKB1 co-mutated lung cancer cells and that inhibition of the autophagy/acetylCoA/acetyl-Snail axis using CAMKK2 or ACLY inhibitors could be a potentialtherapeutic strategy to suppress metastasis of KL lung cancer.
