Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis

Harilaos Filippakis,Nicola Alesi,Brian Siroky,Amine Belaid,Elizabeth P Henske,Constance Wu,Thomas Hougard,Hilaire C Lam,Julie Nijmeh

doi:10.1038/s41598-018-32256-x

Abstract

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5−/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

Highlights

Tuberous Sclerosis Complex (TSC) is a multisystem hamartomatous disease in which tumors develop in multiple organs, including the brain, heart, kidney, skin and lung[1,2]
In parallel with these data on the impact of intracellular metabolic reprogramming on TSC2-deficient cells, the uptake and utilization of extracellular nutrients is altered by mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. mTORC1-hyperactive cells have been shown to be highly dependent on the uptake of extracellular glucose and glutamine, leading to sensitivity to inhibitors of glycolysis and glutaminolysis[13,14]
To examine whether macropinocytosis is regulated by Tsc[2], Tsc2+/+ and Tsc2−/− mouse embryonic fibroblasts (MEF) were treated with rapamycin (20 nM) to inhibit mTORC1, Torin[1] (250 nM) to inhibit mTORC1 and mTORC2, or vehicle (DMSO) for 24 hours

Summary

Introduction

Tuberous Sclerosis Complex (TSC) is a multisystem hamartomatous disease in which tumors develop in multiple organs, including the brain (subependymal giant cell astrocytomas), heart (rhabdomyomas), kidney (angiomyolipomas and renal cell carcinoma), skin (angiofibromas) and lung (lymphangioleiomyomatosis)[1,2]. Inhibitor, while inhibition of mTORC1-dependent autophagy in Tsc2-deficient cells leads to specific sensitivity to further autophagy/lysosomal inhibition and to inhibition of the pentose phosphate pathway[11,12]. In parallel with these data on the impact of intracellular metabolic reprogramming on TSC2-deficient cells, the uptake and utilization of extracellular nutrients is altered by mTORC1 hyperactivity. In tumor cells with oncogenic Ras mutations, macropinocytosis allows the internalization of extracellular protein, which is delivered to the lysosome for degradation[16,17]. The role of Vps[34] in macropinocytic uptake of nutrients is poorly understood in mTORC1-hyperactive tumors

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Conclusion