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NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

ABSTRACT: In mammals, autophagosome formation is initiated by ULK1 via the posttranslational modification of this protein. However, the precise role of ULK1 ubiquitination in modulating autophagy is unknown. Here, we show that NEDD4L, an E3 ubiquitin ligase, binds ULK1 in pancreatic cancer cells. ULK1 expression was stabilized in NEDD4L knockdown cells compared to that in control cells, suggesting that NEDD4L is involved in ULK1 ubiquitination and its subsequent degradation. Autophagy activity was enhanced in NEDD4L knockdown cells compared to control cells. NEDD4L-depleted cells exhibited an increase in the cellular oxygen consumption rate (OCR) and mitochondrial membrane potential, and maintained mitochondrial fusion status in response to metabolic stress. Enhanced OCR and mitochondrial fusion morphology in NEDD4L knockdown cells were repressed by siRNA targeting ULK1. In addition to ULK1, ASCT2, a glutamine transporter, was accumulated in NEDD4L-depleted cells; this is important for maintaining autophagy activation and mitochondrial metabolic function. Finally, the cellular growth and survival rate increased in NEDD4L knockdown cells compared to control cells. However, the genetic or pharmacological blockade of either ULK1 or ASCT2 in NEDD4L-depleted cells sensitized pancreatic cancer cells, particularly in response to nutrient deprivation. In a mouse xenograft model of pancreatic cancer, the use of autophagy inhibitors suppressed tumor growth more in NEDD4L-depleted cells than in tumors from control cells. NEDD4L and ULK1 levels were inversely correlated in two different pancreatic cancer mouse models-xenograft mouse and KPC mouse models. These results suggest that NEDD4L suppressed autophagy and mitochondrial metabolism by reducing cellular ULK1 or ASCT2 levels, and thus could repress the growth and survival of pancreatic cancer cells. Therefore, ubiquitin ligase-mediated autophagy plays a critical role in regulating mitochondrial metabolism, thereby contributing to the growth and survival of certain cancers with low NEDD4L levels.

SUBMITTER: Lee DE

PROVIDER: S-EPMC6971022 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

Lee Dong-Eun DE Yoo Ju Eun JE Kim Jiyea J Kim Seungki S Kim Sunshin S Lee Ho H Cheong Heesun H

Cell death & disease 20200120 1

In mammals, autophagosome formation is initiated by ULK1 via the posttranslational modification of this protein. However, the precise role of ULK1 ubiquitination in modulating autophagy is unknown. Here, we show that NEDD4L, an E3 ubiquitin ligase, binds ULK1 in pancreatic cancer cells. ULK1 expression was stabilized in NEDD4L knockdown cells compared to that in control cells, suggesting that NEDD4L is involved in ULK1 ubiquitination and its subsequent degradation. Autophagy activity was enhance ...[more]

PMID: 31959741

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Project description:BackgroundHepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC.MethodsThe expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC.ResultsOur in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results.ConclusionOur study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC.

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NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

Publications

NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

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