Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis.

Robert W Button,Joseph H Vincent,Shouqing Luo,Conor J Strang

doi:10.18632/oncotarget.6986

Abstract

The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy.

Highlights

Macroautophagy, is a bulk degradation system that mediates clearance of unwanted cytoplasmic constituents, including aberrant long-lived cytoplasmic proteins, intracellular pathogens and damaged organelles such as mitochondria [1,2,3]
The effect was only slightly enhanced by the lysosomal inhibitor Bafilomycin A1 (Baf), but markedly reduced by the PI-3 kinase (PI-3K) inhibitor 3-methyladenine (3-MA), and phosphorylation of p70S6 kinase (p70S6K), a prominent mammalian target of rapamycin (mTOR) substrate, was inhibited by PI103, confirming that PI-103 strongly inhibited mTOR kinase activity in our system (Figure 1B)
We show that two dual inhibitors of mTOR and PI-3K, PI-103 and NVP-BEZ-235, inhibit autophagy, suggesting that this effect is common across all such agents

Summary

Introduction

Macroautophagy (autophagy hereafter), is a bulk degradation system that mediates clearance of unwanted cytoplasmic constituents, including aberrant long-lived cytoplasmic proteins, intracellular pathogens and damaged organelles such as mitochondria [1,2,3]. Autophagy involves the formation of double-membrane structures called autophagosomes which engulf portions of cytoplasm. They are trafficked to lysosomes where their contents are degraded after fusion [1]. Autophagosomes may directly fuse with lysosomes, or initially fuse with late endosomes to form amphisomes, which subsequently fuse with lysosomes [4]. The ULK1/2-Atg13-FIP200 complex senses the signals for autophagosome initiation and stimulates the downstream class-III PI-3 kinase (PI-3K), Vps34 [5, 6]. Beclin 1-Vps complex generates PtdIns3P (PI(3) P), which is required for autophagosome/phagophore nucleation [7]. LC3-II, the lipidated form of LC3, is required for the expansion and completion of pre-autophagosomal membranes [12,13,14]

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