Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Mitsuhiko Ikura,Sunil Sudarshan,Mary E. Ballestas,Arindam P. Ghosh,Tatjana Coric,Richard Kirkman,Christopher B. Marshall,Eun-hee Shim,Mary-Ann Bjornsti

doi:10.18632/oncotarget.4963

Abstract

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

Highlights

MTOR is a conserved serine/threonine kinase that integrates intracellular and extracellular signals to regulate vital cellular processes such as growth, proliferation and metabolism [1]
We analyzed cancer genomic sequence data from The Cancer Genome Atlas (TCGA) using the COSMIC and cBIO Cancer genomics portal [19,20,21] and found that mutations in MTOR were prevalent in about 6% of the patients with clear cell renal cell carcinoma (ccRCC) while mutations in RHEB are relatively uncommon and present in about 1% of the patients with RCC
MTOR deregulation is observed in multiple sporadic cancer types; it plays a causative role in various familial tumor syndromes such as Cowden syndrome, Peutz-Jeghers syndrome and Tuberous Sclerosis as recently summarized [49]

Summary

Introduction

MTOR is a conserved serine/threonine kinase that integrates intracellular and extracellular signals to regulate vital cellular processes such as growth, proliferation and metabolism [1]. RAPTOR www.impactjournals.com/oncotarget (regulatory associated protein of mTOR) and RICTOR (rapamycin-insensitive companion of mTOR) are unique scaffolding proteins that assemble the complexes and bind the substrates for mTORC1 and mTORC2, respectively [6, 7]. Unique components exist in each complex: mTORC1 comprises a negative regulator, PRAS40, whereas mTORC2 contains PROTOR (protein observed with rictor 1 and 2) and mSIN1(mammalian stress-activated map kinase-interacting protein 1) [8,9,10]. The complex in which mTOR participates dictates the substrate specificity of its kinase activity. S6K1 (S6 Kinase 1) and 4E-BP1 (eIF-4E binding protein 1) are two well-characterized mTORC1 substrates that associate with mRNAs and regulate both mRNA translation initiation and progression, enhancing protein synthesis [13, 14]. One of the most well characterized downstream targets of mTORC2 is AKT. mTORC2 directly activates AKT by phosphorylating its hydrophobic motif (Ser473), a site required for its maximal activation [18]

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