Oncogenes and Tumor Suppressors p53 Deletion or Hotspot Mutations Enhance mTORC1Activity byAltering LysosomalDynamics of TSC2 and Rheb

The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distributionof TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR associationwith Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53null cells didnotchange lysosomalmTOR. In summary, lossof the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. Implications: This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype. Mol Cancer Res; 14(1); 1–12. 2015 AACR. The mammalian target of rapamycin (mTOR) is a highly conserved regulator of cell growth andproliferation in eukaryotes. mTOR is present as two functionally distinct complexes: mTORC1, which has kinase activity toward two proteins ratelimiting to cap-dependent protein synthesis initiation, 4EBP1 and p70-S6K1 (S6K1), andmTORC2, that phosphorylates Akt at S473 (1–3). The mTORC1 complex includes the catalytic subunit mTOR, PRAS40, an mTOR inhibitor inactivated by phosphorylation by Akt at T246, and Raptor, which acts as a scaffold for the recruitment of the substrates 4EBP1 and S6K1 (4). mTORC1 activity is localized on a subcellular membrane compartment (5) recently determined to be lysosomal membranes (6), which acts as the assembly site of the several components of this complex. This subcellular organization is critical for the regulation of mTORC1 activity: the activation of mTORC1 by amino acids involves the partitioning of mTOR onto lysosomes (7–11) and stimulation of mTORC1 by insulin involves induction of the dissociation of tuberin (TSC2) from proximity to mTOR on lysosomal membranes (5, 6). TSC2 facilitates the conversion of the obligatorymTORC1-activator Rheb-GTP to the inactive RhebGDP (12–14). Although the TSC1 and TBC1D7 components of the tuberous sclerosis complex (TSC) appear fixed at the lysosomes, TSC2 dynamically partitions onto and off these membranes, and it is this partitioning that is currently thought to be involved in the effect of TSC2 on the ability of Rheb to activate mTORC1 (6). The Sestrin proteins have been shown to inhibit mTORC1 kinase activity and they, likewise, suppress the translocation of mTOR to the lysosome (7, 15–17). Akt and, thereby, the downstream mTORC1 are frequently activated in human carcinomas due to mutation of upstream K-Ras andPI3K that impact control of this pathway.However, p53 is themost commonly mutated or lost gene inmany tumor types, including colon and lung carcinomas. We sought to clarify the effect of p53 loss or mutation on control of mTORC1. Both TSC2 and the Sestrins have been shown to be targets for p53-directed transcription (18–20). It has been reported that the p53 response to DNA damaging agents diminishes mTORC1 activity in carcinoma cells; this effect requires the intermediate activation of the AMP-dependent protein kinase (AMPK; ref. 21). We now report that both loss of p53 or mutation of key residues in the DNA binding domain of this tumor suppressor also substantially upregulatemTORC1 activity by an effect that is downstream from AMPK and therefore separate from the effect of DNA-damage Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia. Department of Microbiology and Immunology and Massey Cancer Center,Virginia Commonwealth University, Richmond, Virginia. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). S. Agarwal and C.M. Bell contributed equally to this article. Corresponding Author: Richard G. Moran, Virginia Commonwealth University, 401 College Street, Goodwin Building, Room 123, Richmond, VA 23298-0035. Phone: 804-828-5783; Fax: 804-827-0810; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-15-0159 2015 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1 on April 19, 2017. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst September 18, 2015; DOI: 10.1158/1541-7786.MCR-15-0159