Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer

Abstract

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a variety of cellular functions such as growth, proliferation and autophagy. In a variety of cancer cells, overactivation of mTOR has been reported. In addition, mTOR inhibitors, such as rapamycin and its derivatives, are being evaluated in clinical trials as anticancer drugs. However, no active mutants of mTOR have been identified in human cancer. Here, we report that two different point mutations, S2215Y and R2505P, identified in human cancer genome database confer constitutive activation of mTOR signaling even under nutrient starvation conditions. S2215Y was identified in large intestine adenocarcinoma whereas R2505P was identified in renal cell carcinoma. mTOR complex 1 prepared from cells expressing the mutant mTOR after nutrient starvation still retains the activity to phosphorylate 4E-BP1 in vitro. The cells expressing the mTOR mutant show increased percentage of S-phase cells and exhibit resistance to cell size decrease by amino-acid starvation. The activated mutants are still sensitive to rapamycin. However, they show increased resistance to 1-butanol. Our study points to the idea that mTOR activating mutations can be identified in a wide range of human cancer.

Figure 1

Mammalian target of rapamycin (mTOR) mutations found in cancer genome. mTOR mutations were identified by searching the COSMIC Library (Catalogue of Somatic Mutations in Cancer) as described in text. The mTOR mutations identified in cancer genome are indicated above the linear representation of mTOR. The tissues where the mTOR mutations in cancer are identified are represented in parentheses. Cancer types and the COSMIC ID numbers of mTOR mutations are indicated at the bottom.

Figure 2

mTOR^S2215Y and mTOR^R2505P cancer mutations confer constitutive activation of mTOR complex 1 (mTORC1) but not mTORC2. (a) Mutations in mTOR were introduced by two-step polymerase chain reaction (PCR) into pcDNA3-AU1-mTOR as described previously (Urano et al., 2007). HEK293T cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin at 37°C and 5% CO₂ were co-transfected with either the empty vector (control) or pcDNA3 containing the indicated AU1-tagged mTOR together with pCMV5-FLAG-S6K1 by using Polyfect (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. At 24 h after the transfection, the cells were cultured in DMEM supplemented with 0.1% bovine serum albumin (BSA) overnight and then incubated in Dulbecco’s modified phosphate-buffered saline (D-PBS) containing 1mM Mg²⁺ and Ca²⁺ (Invitrogen, Carlsbad, CA, USA) for 1 h to achieve nutrient starvation. The cells were lysed in 1 × SDS sample buffer by sonication at 4°C. The proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with anti-AU1 (Covance, Berkeley, CA, USA), anti-phospho-p70 S6 kinase (T389) (Cell Signaling Technology, Danvers, MA, USA) or anti-FLAG M2 (Sigma, St Louis, MO, USA) antibodies. (b) HEK293T cells were transfected with either the empty vector (control) or pcDNA3 containing the indicated AU1-tagged mTOR. Lysates of the nutrient-starved cells were divided into two aliquots. The first aliquot was heated at 95°C for 5 min, and the soluble heat-stable fractions were resolved by SDS–PAGE and immunoblotted with anti-phospho-4E-BP1 (T37/46) (Cell Signaling Technology) and then with anti-4E-BP1 (Cell Signaling Technology). The second aliquot was resolved by SDS–PAGE and then immunoblotted with anti-AU1. (c) HEK293T cells were co-transfected with either the empty vector (control) or with pcDNA3 containing the indicated AU1-tagged mTOR and pCMV-myc-Akt. After serum starvation, the cells were lysed as described in a. Proteins were immunoblotted with anti-AU1, anti-phospho-Akt (S473) (Cell Signaling Technology) or anti-Akt (Cell Signaling Technology).

Figure 3

mTOR^S2215Y increases the catalytic activity of mTOR complex 1 (mTORC1) but not mTORC2 in vitro. (a) HEK293T cells were transfected with either the empty vector (control) or pcDNA3-AU1-mTOR (wild type or S2215Y). Cell extracts were prepared from the serum and nutrient-starved cells and then immunoprecipitated with anti-AU1 antibody as described previously (Urano et al. 2007). Immunoprecipitates were detected by western blotting using anti-AU1, anti-raptor (Bethyl, Montgomery, TX, USA), anti-rictor (Bethyl), or anti-mLST8 (Bethyl) antibodies. (b, c) HEK293T cells transfected with either the empty vector (control) or pcDNA3-AU1-mTOR (wild type, E2419K or S2215Y) were subjected to serum starvation and nutrient starvation. mTORC1 was immunoprecipitated with anti-Raptor and used for the in vitro kinase assay using recombinant 4E-BP1, whereas mTORC2 was immunoprecipitated with anti-rictor antibody and used for the assay with Akt as described previously (Sato et al., 2009). After the reaction, the proteins were immunoblotted with the indicated antibodies.

Figure 4

mTOR^S2215Y effects on cell cycle and cell size under nutrient starvation and sensitivity to rapamycin, osmotic stress and 1-butanol. (a) HEK293T cells transfected with pcDNA3-AU1-mTOR (wild type or S2215Y) were subjected to serum starvation. After trypsinization, the cells were washed twice with Dulbecco’s modified phosphate-buffered saline (D-PBS) and suspended in propidium iodide (PI) solution (0.1% sodium citrate, 0.3% Triton-X 100, 0.1 mg/ml PI, 20 mg/ml RNase A). The cells were immediately analyzed on a Becton Dickson FACScan (Becton Dickson, San Jose, CA, USA) for PI (cell cycle). (b) HEK293T cells transfected with pcDNA3-AU1-mTOR (wild type or S2215Y) were subjected to serum starvation and nutrient starvation. After trypsinization, the cells were washed twice with D-PBS and suspended in PI solution. The cells were immediately analyzed on a Becton Dickson FACScan for forward scatter (cell size). (c) HEK293T cells were co-transfected with either the empty vector (control) or pcDNA3 containing the indicated AU1-tagged mTOR together with pCMV5-FLAG-S6K. After serum starvation, the cells were cultured in D-PBS containing Mg²⁺ and Ca²⁺ in the presence or absence of 50 nM rapamycin (Calbiochem, San Diego, CA, USA) for 1 h. The proteins were resolved by SDS–PAGE and immunoblotted using indicated antibodies. (d) HEK293T cells co-transfected with pcDNA3-AU1-mTOR (wild type or S2215Y) and pCMV5-FLAG-S6K1 were serum-starved and then treated with 50 nM rapamycin, 1mM H₂O₂, 0.8% 1-butanol, 0.4M sorbitol or without reagent (control) in DMEM supplemented with 0.1% bovine serum albumin (BSA) for 1 h. The proteins were resolved by SDS–PAGE and immunoblotted using indicated antibodies.