Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells.

Seokmin Jeong,Keun-Gyu Park,Jungwook Chin,Sung Cho,In-Kyu Lee,Jun-Kyu Byun,Yeon-Kyung Choi

doi:10.3390/nu10111638

Abstract

Macropinocytosis is a regulated form of endocytosis that mediates the nonselective uptake of nutrients to support growth under nutrient-deprived conditions. KRAS-mutant cancer cells upregulate macropinocytosis to import extracellular proteins, which subsequently undergo proteolytic degradation in the lysosome. Although transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and function, its role in the degradation of extracellular protein from macropinocytosis in KRAS-mutant cells has not previously been elucidated. In this study, we investigated the role of TFEB in the recovery of macropinocytosis-mediated mTORC1 activity and cell growth under nutrient depletion. Mouse embryonic fibroblasts (MEFs) expressing KrasG12D and KRAS-mutant human cancer cells took up markedly higher levels of tetramethylrhodamine (TMR)-dextran than the corresponding wild-type cells. siRNA-mediated inhibition of TFEB did not influence extracellular TMR-dextran uptake, but significantly attenuated lysosomal degradation of extracellular protein. Bovine serum albumin (BSA) treatment restored p-S6K levels and cell proliferation suppressed by leucine deprivation, and these effects were blocked by siTFEB. Collectively, our results show that TFEB plays a role in macropinocytosis-mediated KRAS-mutant cell growth under nutrient deprivation by promoting lysosomal degradation of extracellular proteins.

Highlights

Macropinocytosis is a fluid-phase endocytic process whereby extracellular fluid and its content are internalized into cells through large uncoated vacuoles called [1]
Increased rates of macropinocytosis in KrasG12D Mouse embryonic fibroblasts (MEFs) and KRAS-mutant human cancer cells were significantly attenuated by knockdown of KRAS or treatment with a pharmacological inhibitor of macropinocytosis, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) (Figure 1D,E)
We demonstrated that Transcription factor EB (TFEB) plays a pivotal role in acquisition of nutrients

Summary

Introduction

Macropinocytosis is a fluid-phase endocytic process whereby extracellular fluid and its content are internalized into cells through large uncoated vacuoles called [1]. In addition to oncogenic mutations and growth factors, membrane-bound phospholipids, in particular phosphoinositides and other protein kinases such as p21-activated kinase 1 (Pak1) and protein kinase C, have been implicated in the process of macropinocytosis [3,4]. Lysosomal delivery of fluid-phase cargo, including protein, and lysosomal degradation are both important for maintaining cell growth under amino acid starvation [6]. Transcription factor EB (TFEB), a member of the MiTF/TFE family, is a master regulator of multiple cellular processes, including lysosomal biogenesis and autophagy [7]. TFEB regulates the expression of a large number of target genes, thereby orchestrating cellular degradative pathways and intracellular clearance in response to environmental cues [8]. Altered TFEB expression is associated with initiation and progression of multiple types of cancer, including lung and pancreatic cancers [11,12,13]

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