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. 2015 Jul 20;6(20):17895-910.
doi: 10.18632/oncotarget.4963.

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

Arindam P Ghosh  1 , Christopher B Marshall  2 , Tatjana Coric  3 , Eun-Hee Shim  1 , Richard Kirkman  1 , Mary E Ballestas  4 , Mitsuhiko Ikura  2 , Mary-Ann Bjornsti  3 , Sunil Sudarshan  1
Affiliations

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

Arindam P Ghosh et al. Oncotarget. .

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.

Keywords: RHEB; mTOR; mutations; rapamycin; renal cancer.

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Conflict of interest statement

CONFLICTS OF INTERESTS

The authors declare that there are no competing financial interests in relation to the work described.

Figures

Figure 1
Figure 1. Point mutations in MTOR are clustered in various regulatory domains in ccRCCC and are associated with poor prognosis
a. Analysis of ccRCC cases from the COSMIC and cBIO databases show that mTOR mutations are present in about 6% cases of cases. Clusters of mTOR mutations are represented in the various domains of mTOR. b. Sites of mTOR mutations reported in RCC are indicated as grey spheres on the structure of mTORΔN (PDB ID code: 4JSN). The catalytic site is represented with pink spheres, and the domains are colored as indicated previously. KD-N and KD-C represent the N and C terminal lobes of the kinase domain. Inset: Structure of the TRD (Tetratricopeptide repeat domains)1/2 regions (1385-1666) of the FAT domain with the sites of mutations characterized in this study represented as spheres and coloured by atom type (C, grey; O, red; N, blue; S, orange). These mutations were clustered in the kinase and FAT domains of mTOR. c. Survival of RCC patients as a function of MTOR mutation.
Figure 2
Figure 2. Mutations in the FAT domain promote mTORC1 activation
HEK293T cell lysates expressing mutant or wild-type mTOR in the presence or absence of nutrients were immunoblotted for levels of a. phosphorylated S6K(Thr389) b. phosphorylated S6 (Ser 235/236) c. phosphorylated 4E-BP1 (Ser 65) and d. phosphorylated 4E-BP1(Thr 37/46).
Figure 3
Figure 3. Mutations in the FAT domain promote mTORC2 kinase activity
a. HEK293T cell lysates expressing mutant or wild-type mTOR in the presence or absence of nutrients were immunoblotted for levels of phosphorylated AKT (Ser 473). b. Immunoprecipitates from HEK293T cells expressing wild-type or mutant mTOR was subjected to an in vitro kinase assay using recombinant insect AKT as a substrate. HEK293T cells transfected with wild-type mTOR, immunoprecipitated with normal mouse IgG was used as a control.
Figure 4
Figure 4. Mutations in the FAT domain of mTOR promote increased cell proliferation relative to wild-type
Proliferation in cells expressing wild-type or mutant mTOR was assessed in HEK293T cells. Cells were transfected and replated at 24 hrs. Proliferation was determined 24 hours after replating. The rate of proliferation in the mutant mTOR expressing cells from biologic replicates was normalized and expressed as a fraction of the average rate of proliferation of cells expressing wild-type mTOR.
Figure 5
Figure 5. Mutations in the FAT domain of mTOR lead to altered DEPTOR and PRAS40 binding
Wild-type and mutant mTOR complexes were immunoprecipitated using a myc antibody, washed and analyzed for relative levels of mTOR complex proteins. HEK293T cells transfected with wild-type mTOR, immunoprecipitated with normal mouse IgG was used as a control. The input controls comprised 10% of the lysates used for IP.
Figure 6
Figure 6. Mutations in the FAT domain of mTOR lead to decreased sensitivity to the inhibitory effects of rapamycin at clinically relevant doses
a. HEK293T cells overexpressing wild-type or mutant mTOR were treated with 10nM rapamycin for 30 mins and protein lysates were immunoblotted for the indicated proteins. b. HEK293T cells overexpressing wild-type or mutant mTOR were treated with 20nM rapamycin for 60 mins and protein lysates were immunoblotted for the indicated proteins. c. Cell proliferation was assessed as described previously in cells expressing wild-type or mutant mTOR in the presence of rapamycin (10nM for 24hrs).
Figure 7
Figure 7. Y35N RHEB mutation in ccRCC causes mTORC1 hyperactivation by resistance to TSC2 GAP activity
a. HEK293T cells expressing FLAG-tagged wild-type or mutant RHEB were analyzed for levels of phosphorylated mTORC1 downstream targets in the presence and absence of nutrients. b. Immunoprecipitates from wild-type or mutant RHEB overexpressing cells were analyzed for relative levels of RICTOR, RAPTOR and TSC2. HEK293T cells transfected with wild-type RHEB, immunoprecipitated with normal mouse IgG was used as a control c. Hydrolysis of GTP in the presence of absence of TSC2 GAP domain in wild-type and Y35N RHEB. Results from three independent experiments were normalized to the rate of GTP hydrolysis of wild-type RHEB. # represents a p value of ≤ 0.05 in the GTP hydrolysis rate of Y35N RHEB in comparison to wild-type RHEB in the presence of TSC2 GAP domain. d. Cells expressing FLAG-tagged wild-type or mutant RHEB were treated with rapamycin (10nM) for 30 mins and protein lysates were immunoblotted for the indicated proteins.
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