RESUMO
BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.
Assuntos
Neoplasias dos Ductos Biliares , Caseína Quinase II , Colangiocarcinoma , Lisossomos , Mutação , Naftiridinas , Fenazinas , Pinocitose , Piperazinas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Pinocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/antagonistas & inibidores , Piperazinas/farmacologia , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , proteínas de unión al GTP Rab7/metabolismo , Morte Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers.