EGF-activated PI3K/Akt signalling coordinates leucine uptake by regulating LAT3 expression in prostate cancer

Blake K Zhang,Jeff Holst,Charles G Bailey,Anne M Moran,Qian Wang,John E J Rasko

doi:10.1186/s12964-019-0400-0

Abstract

BackgroundGrowth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling.MethodsTo investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay.ResultsInhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.ConclusionGrowth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.

Highlights

Growth factors, such as epidermal growth factor (EGF), activate the phosphoinositide 3 kinase (PI3K)/Akt/mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth
ShRNA knockdown of L-type amino acid transporter 3 (LAT3) blocks leucine uptake and cell growth in prostate cancer cell lines both in vitro [23] and in vivo [24]. Since both the growth factor-activated PI3K/Akt signalling pathway and amino acid transporters are required for mTORC1 activation, we investigated whether there were any direct links between PI3K/Akt signalling and LAT3 activity
PI3K/Akt signalling pathway regulates leucine transport To model the effects of growth factors on prostate cancer, we utilized the androgen-sensitive prostate adenocarcinoma cell line LNCaP, which contains one deleted allele and one mutated allele of phosphatase and tensin homolog (PTEN) [27], and the androgen-insensitive cell line PC-3, which has a homozygous deletion of PTEN [27, 28], leading to the hyperactivation of the PI3K/Akt pathway

Summary

Introduction

Growth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. Binding of growth factors to the extracellular ligand binding domain of their membrane-bound receptors leads to a conformational change of the receptors, thereby activating tyrosine or serine/threonine kinase domains This activation enables the recruitment of diverse substrates, propagating signals that mediate a Growth factors and their receptors are commonly increased in a variety of cancers, with expression of epidermal growth factor (EGF) and its receptor (EGFR) significantly increased in prostate cancer [6]. MTORC1 integrates upstream signalling pathways as well as amino acid availability to mediate protein synthesis, cell growth and proliferation

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