NUTRIENT SENSING

Transcriptional activation of RagDGTPase controls mTORC1 andpromotes cancer growthChiara Di Malta,1 Diletta Siciliano,1 Alessia Calcagni,1 Jlenia Monfregola,1

Simona Punzi,2 Nunzia Pastore,3 Andrea N. Eastes,4 Oliver Davis,5 Rossella De Cegli,1

Angela Zampelli,1 Luca G. Di Giovannantonio,1 Edoardo Nusco,1 Nick Platt,6

Alessandro Guida,2 Margret Helga Ogmundsdottir,7 Luisa Lanfrancone,2

Rushika M. Perera,4 Roberto Zoncu,5 Pier Giuseppe Pelicci,2,8

Carmine Settembre,1,9,10 Andrea Ballabio1,3,10*

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome byRag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response tonutrients. We found that MiT/TFE transcription factors—master regulators of lysosomaland melanosomal biogenesis and autophagy—control mTORC1 lysosomal recruitment andactivity by directly regulating the expression of RagD. In mice, this mechanism mediatedadaptation to food availability after starvation and physical exercise and played animportant role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues frompatients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma,and melanoma triggered RagD-mediated mTORC1 induction, resulting in cellhyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanismenables cellular adaptation to nutrient availability and supports the energy-demandingmetabolism of cancer cells.

Amajordeterminant of species evolution is theability to switch between anabolic and cata-bolic pathways in response tonutrient avail-ability. Thenutrient-activatedkinase complextarget of rapamycin complex 1 (mTORC1)

promotes biosynthetic processes and inhibits cata-bolicpathways suchasautophagy (1,2), thusplayinga crucial role in the adaptation of the organism tothe environment (3, 4). The transcription factorsTFEB, TFE3, TFEC, andMITF belong to theMiT-TFE family and bind the same target sites in theproximal promoters of overlapping sets of genes(5, 6). TFEB and TFE3 aremaster transcriptionalregulators of lysosomal biogenesis and autophagy(6–8), whereasMITF regulatesmelanosomal bio-genesis (9).mTORC1negatively regulates the activ-ity of these transcription factors byphosphorylating

critical serine residues, leading to their cytoplasmicretention (8, 10–12). Conversely, during starvationor physical exercise, inhibition ofmTORC1 and acti-vationof thephosphatase calcineurin leads toTFEBand TFE3 dephosphorylation and nuclear trans-location (13, 14).We postulated the presence of a feedback loop

by which MiT-TFE transcription factors, whichare substrates ofmTORC1,may in turn influencemTORC1 activity. Small interferingRNA (siRNA)–mediateddepletionof eitherTFEBorTFE3 inHeLacells significantly decreasedmTORC1activity uponamino acid administration (fig. S1, A and B). Fur-thermore,mTORC1 reactivation upon prolongedstarvation (15) was inhibited inTFEB-silenced cells(fig. S1C). Overexpression of either wild-type or con-stitutivelyactiveTFEBandTFE3 (TFEB-CAandTFE3-CA) resulted in increasedmTORC1 activationuponstimulation with either a complete set of aminoacids (figs. S1, D to I, and S2) or solely leucine orarginine, thekey aminoacids that activatemTORC1(16) (Fig. 1, A and B). Consistently, viral-mediatedTFEB overexpression in the liver ofwild-typemiceincreasedmTORC1 signaling (Fig. 1, C andD). Con-versely, a significant reduction in the rate of pro-tein synthesis and impaired mTORC1 signalingwere detected in the livers fromTFEB liver-specificconditional knockout (KO)mice (Tcfebflox/flox;Alb-CRE+; hereafterTcfeb-LiKO) (Fig. 1E). In addition,exercisedmuscle-specificTFEBKOmice (Tcfebflox/flox;Mlc-CRE+; hereafter Tcfeb-MuKO) showed a reducedinductionofmTORC1activity andprotein synthesisin response to leucine after exercise (Fig. 1F). Thus,the effect of a proteinmeal after exercise onmuscleprotein synthesis requires TFEB-induced activa-tion of mTORC1 signaling.

TFEB overexpression in cells lacking the essen-tial autophagy genes Atg5 or Atg7 still resulted inenhanced mTORC1 activity, similar to wild-typecells (fig. S3). Thus, MiT-TFE transcription factorsmay regulate mTORC1 by a mechanism that is dif-ferent from autophagy. To identify such a mech-anism, we searched for TFEB DNA binding sites,defined as “CLEAR elements” (6), in the promotersof 50 human genes known to play a role in the acti-vation of mTORC1. Among 20 TFEB/TFE3 puta-tive target genes (tables S1 and S2), the transcriptlevels of the guanosine triphosphatase (GTPase)RagDwere themost significantly decreased uponsingle or combinedTFEBorTFE3 silencing (fig. S4,A to C). Conversely,RagDwas strongly induced inTFEB-overexpressing cells both at themRNA (Fig.2A and fig. S4D)andprotein (fig. S4, EandF) levels.An inductionofRagDexpressionwas alsodetectedin liver samples frommice injected with helper-dependent adenovirus (HDad) containing TFEBorTFE3 (fig. S4G), and a reduction ofRagD expres-sionwas observed inTFEBLiKOandTFE3 full-KOmice (fig. S4H). To exert their activity, RagD andRagC need to heterodimerize withRagA or RagBand to be activated by folliculin (FLCN), aGTPaseactivating protein (GAP) (17). RagC and FLCN ex-pression levels were also influenced byMiT-TFEgenes, albeit to a lesser extent comparedwithRagD(Fig. 2A and fig. S4, A toD). RagD is expressed atvery low levels; thus, we postulate that RagD is alimiting factor for Rag GTPase activity.Chromatin immunoprecipitation (ChIP) and

luciferase assay experiments revealed thatRagDis a direct transcriptional target of TFEB (Fig.2, B and C). Thus, we used a clustered regularlyinterspaced short palindromic repeats–Cas9(CRISPR-Cas9)–mediatedgenome-editingapproachto delete the most responsive TFEB target sitein the RagD proximal promoter region in HeLacells (HeLa-RagDpromedit) (Fig. 2D). This cell lineshowed significantly reduced transcript and pro-tein levels of RagD—whereas other mTORC1-related genes were not affected (Fig. 2E and fig.S5A)—as well as a significant impairment ofmTORC1 activation upon amino acid stimulation(Fig. 2F and fig. S5, B and C). Overexpression ofexogenous RagD rescued mTORC1 signaling intheHeLa-RagDpromedit cell line (fig. S5D). Consist-ently, viral-mediated RagD gene delivery rescuedimpaired mTORC1 signaling and defective proteinsynthesis in the livers of Tcfeb‐LiKOmice (Fig. 2G).Thus, the transcriptional regulationofRagD expres-sion by MiT-TFE transcription factors plays animportant role in the control ofmTORC1 activity.TFEBandTFE3 are activated by starvation (7,8).

Consistently,we observed an increase ofRagDmRNAand protein levels during starvation, which wasbluntedby silencingof eitherTFEBorTFE3 (fig. S6,A and B). In addition, we found a significant corre-lation between starvation-inducedTFEB nuclearlocalization and RagD expression levels (fig. S6, Cand D). Accordingly, fasting and physical exerciseinmice inducedRagDexpression in liverandmuscle,which was blunted in TFEB LiKO andMuKOmice,respectively (fig. S6, E and F). Nutrients inducemTORC1 recruitment to the lysosomal surface viathe interaction of Rag GTPases with the Raptor

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Di Malta et al., Science 356, 1188–1192 (2017) 16 June 2017 1 of 5

1Telethon Institute of Genetics and Medicine (TIGEM), ViaCampi Flegrei 34, 80078 Pozzuoli, Naples, Italy. 2Departmentof Experimental Oncology, European Institute of Oncology,20139 Milan, Italy. 3Department of Molecular and HumanGenetics and Neurological Research Institute, Baylor Collegeof Medicine, Houston, TX 77030, USA. 4Department ofAnatomy and Helen Diller Family Comprehensive CancerCenter, University of California San Francisco, San Francisco,CA 94143, USA. 5Department of Molecular and Cellular Biologyand Paul F. Glenn Center for Aging Research, University ofCalifornia, Berkeley, Berkeley, CA 94720, USA. 6Department ofPharmacology, University of Oxford, Oxford, UK. 7Departmentof Biochemistry and Molecular Biology, University of Iceland,Vatnsmyrarvegur 16, Reykjavik 101, Iceland. 8Department ofOncology, University of Milan, 20139 Milan, Italy. 9DulbeccoTelethon Institute, Via Campi Flegrei 34, 80078 Pozzuoli,Naples, Italy. 10Medical Genetics Unit, Department of Medicaland Translational Science, Federico II University, Via Pansini 5,80131 Naples, Italy.*Corresponding author. Email: ballabio@tigem.it

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Fig. 1. MiT/TFE transcription factors regulate mTORC1 activity bothin vitro and in vivo. (A and B) Representative immunoblots of TFEB,phospho-S6K, and S6K in Tet-ON TFEB-CA cell line untreated (–DOX) ortreated with doxycycline (+DOX) for 24 hours. Cells were starved for aminoacids for 50 min (0) and stimulated with decreasing levels (expressed as% of concentration in RPMImedium) of leucine (A) or arginine (B) for 20min.(C) C57BL6 mice injected with HDad expressing human TFEB under thecontrol of a liver-specific promoter (TFEB-INJ) or with phosphate-bufferedsaline (PBS) (CTRL) were starved for 22 hours (FASTED) and then refed for2 hours (FED). Liver lysates were analyzed for levels of indicated proteins.Actin was used as loading control. Plot shows ratio of phosphorylatedS6K/pan-S6K (mean of three independent experiments). (D) Immunohisto-chemistry of liver sections from mice injected with saline PBS (CTRL)or HDAd-TFEB (TFEB-INJ).Tissues were stained for serine 240/244

phosphorylated-S6 (P-S6). Insets show overlapping P-S6 and TFEBimmunostainings in two consecutive 5-mm liver sections isolated from HDad-TFEB–injected mice. (E) Liver samples from mice with indicated genotypeswere analyzed for the levels of S6K phosphorylation and puromycinincorporation. Plots show the ratios of phosphorylated S6K/pan-S6K andpuromycin/actin expressed as relative to control mice (Tcfebflox/flox).(F) Phosphorylation of S6K and levels of puromycin incorporation analysis inmuscle samples from mice with indicated genotypes after oral gavage ofleucine. Mice were exercised where indicated. Plots show ratios ofphosphorylated S6K/pan-S6K and puromycin/glyceraldehyde phosphatedehydrogenase (GAPDH). Plots in (C) and (E) represent means of triplicates± SEM, Student’s t test. Plots in (F) represent means ± SEM;N = 3/condition;one-way analysis of variance (ANOVA) followed by Tukey’s test. [(C), (E),and (F)]: *P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 2. MiT/TFE transcription factors control mTORC1 activitythrough RagD. (A) mRNA levels of mTORC1-related genes in TFEB-CA HeLa cells treated with doxycycline. Values normalized relative to HPRT1and expressed as fold change relative to untreated cells. (B) ChIP analysisof TFEB binding to RagD promoter in doxycycline-treated HeLa TFEB-CAcells. Squares represent CLEAR sites in RagD promoter and numbers refer totheir distance [in base pairs (bp)] from the transcriptional start site (TSS).Immunoprecipitated DNAwas normalized to the input and plotted as relativeenrichment over a mock control. (C) Luciferase assay after transfectionof increasing amounts of TFEB construct was performed in HeLa cellscotransfected with wild-type (RAGD-wt) or mutated (RAGD-mut) RagD-promoter luciferase reporter plasmids. (D) Scheme of CRISPR-Cas9–mediatedmutation in the endogenous RagD promoter of HeLa cells. A regionof 33 bp containing the CLEAR site at position –284 (in red) was ablated.

(E) Transcript levels of Rags and Flcn genes were analyzed in the mutatedHeLa cell line (HeLa-RagDpromedit) versus control HeLa and normalizedrelative to HPRT1 gene. (F) Immunoblots of mTORC1 signaling in HeLa-RagDpromedit cells compared with control HeLa.The ratio of phosphorylated/total protein levels shown for the indicated mTORC1 substrates. Plots in(A), (B), (C), (E), and (F) represent mean ± SEM of three independentexperiments (Student’s t test). (G) Mice with indicated genotypes werenutritionally synchronized and injected with puromycin 30 min beforesacrifice.Where indicated,Tcfebflox/flox;Alb-Cre+ mice were injected with anadeno-associated virus vector carrying human RagD cDNA. Liver lysateswere analyzed for phosphorylation of S6K and levels of puromycinincorporation. Plots show means of triplicates ± SEM, one-way ANOVAexpressed as ratio of phosphorylated S6K/pan-S6K and puromycin/actin.[(A), (B), (C), (E), (F), and (G)]: *P < 0.05; **P < 0.01; ***P < 0.001.

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subunit of the mTORC1 complex (18, 19). We de-tected an increase of amino acid–inducedmTORC1recruitment to the lysosome in TFEB-CA overex-pressing cells comparedwithwild-type cells (fig.S7), whereas an opposite effect was observedin TFEB-depleted cells, as well as in the HeLa-RagDpromedit cell line (Fig. 3, A and B), which wasrescued by RagD overexpression (Fig. 3A). Thus,TFEB-mediated control of RagD promotes the ef-ficient recruitment of mTORC1 to the lysosome.MiT-TFE are known oncogenes overexpressed

in a variety of tumors such as renal cell carci-noma (RCC),melanoma, sarcoma, and pancreaticductal adenocarcinoma (20–22). TFEB kidney-specific conditional overexpressingmice displaya phenotype that closely recapitulates humanRCC(23).We observed hyperactivation ofmTORC1 sig-naling and increasedRagD transcript levels in bothkidney tissues and primary kidney cells from thesemice (fig. S8, A to C). Treatment with the mTORC1inhibitor Torin 1 fully rescued the hyperprolifer-ative phenotype of primary kidney cells (fig. S8D).

Similarly, kidney cells derived fromapatientwithRCC carrying a chromosomal translocation thatinvolves theTFE3gene (HCR-59) showed increasedRagD transcript levels and enhanced mTORC1 sig-naling (Fig. 4, A and B). Notably, silencing of eitherTFE3 orRagD rescuedmTORC1 hyperactivationand reduced tumor cell proliferation (fig. S8, E andF, and Fig. 4C). Furthermore, a survey of RNA se-quencing data obtained from patients with RCCcarryingTFE3 chromosomal translocations revealeda consistent increase ofRagD expression levels (fig.S8G) (24).We also analyzed cell lines frompatientswith pancreatic ductal adenocarcinoma (PDA) inwhichMiT/TFE genes are up-regulated (22) andfound increased RagD levels (fig. S9, A and B).Silencing of TFE3 in two of these cell lines de-creasedRagD levels and rescuedmTORC1 hyper-activation (fig. S9, C to F).MITF, anothermember of theMiT/TFE family,

is an establishedoncogene inmelanoma (25). Tran-sient overexpression ofMITF inHeLa cells inducedup-regulationofRagD transcript levels and increased

mTORC1 activation (fig. S9, G andH), indicatingthatMITF is also able to positively regulateRagDexpression. Consistently, a cell line from a patientwith melanoma, associated with high levels ofMITF, 501Mel, showed induction ofRagD expres-sion and increased mTORC1 activation (Fig. 4, DandE).Notably, silencing ofRagDwas sufficient tosignificantly revert thehyperproliferativephenotypeof this tumor cell line (Fig. 4F). In addition, a surveyof published microarray data available for mela-nomametastatic patients (The Cancer GenomeAtlas data set) and melanoma cell lines (GeneExpressionOmnibus database) revealed a signif-icant correlation betweenMITF and RagD geneexpression levels (fig. S9, I and J). Importantly,xenotransplantation experimentsperformedusingthe 501Melmelanoma cell line showedmarkedly re-duced xenograft tumor growth upon RagD silenc-ing, demonstrating a key role ofRagD inpromotingtumor growth (Fig. 4, G and H). In conclusion,we identified anMiT/TFE-RagD-mTORC1-MiT/TFE feedback circuit, whose fine modulation is

Di Malta et al., Science 356, 1188–1192 (2017) 16 June 2017 4 of 5

Fig. 3. MiT/TFE tran-scription factorspromote lysosomalrecruitment of mTORupon nutrient loading.(A) Representative immu-nofluorescence imagesof endogenous mTOR,LAMP1-GFP (visualizedas red) and RAGD-HA inHeLa cells. Cells weretransfected with scramble(CTRL) or with TFEBsiRNA (siTFEB) and after48 hours with LAMP1-GFPand with RagD-HA plas-mids for an additional24 hours. (B) Represent-ative immunofluorescenceimages of mTORand LAMP2 in HeLa-RagDpromedit and in controlHeLa cells. [(A) and (B)]Cells were deprived ofamino acids for 50 minand then stimulated withamino acids for 15 min.Plots represent quantifi-cation of the data from15 cells per condition fromthree independent experi-ments. Results areshown as means of co-localization coefficient of(A) mTOR and LAMP1 ±SEM (one-way ANOVA)and (B) mTOR andLAMP2 ± SEM (Student’st test). **P < 0.01; ***P <0.001. Scale bars, 10 mm.

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critical formetabolic adaptation to nutrient avail-ability. Deregulation of this mechanism supportscancermetabolism, thus promoting tumor growth(fig. S10).

REFERENCES AND NOTES

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12. J. A. Martina et al., Autophagy 8, 903–914 (2012).13. C. Settembre, A. Fraldi, D. L. Medina, A. Ballabio, Nat. Rev. Mol.

Cell Biol. 14, 283–296 (2013).14. D. L. Medina et al., Nat. Cell Biol. 17, 288–299 (2015).15. L. Yu et al., Nature 465, 942–946 (2010).16. H. Ban et al., Int. J. Mol. Med. 13, 537–543 (2004).17. Z.-Y. Tsun et al., Mol. Cell 52, 495–505 (2013).18. Y. Sancak et al., Science 320, 1496–1501 (2008).19. E. Kim, P. Goraksha-Hicks, L. Li, T. P. Neufeld, K.-L. Guan,

Nat. Cell Biol. 10, 935–945 (2008).20. R. Haq, D. E. Fisher, J. Clin. Oncol. 29, 3474–3482 (2011).21. E. C. Kauffman et al., Nat. Rev. Urol. 11, 465–475 (2014).22. R. M. Perera et al., Nature 524, 361–365 (2015).23. A. Calcagnì et al., eLife 5, e17047 (2016).24. G. G. Malouf et al., Clin. Cancer Res. 20, 4129–4140 (2014).25. H. Tsao, L. Chin, L. A. Garraway, D. E. Fisher, Genes Dev. 26,

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ACKNOWLEDGMENTS

We are grateful to M. A. De Matteis, G. Diez-Roux, andG. Napolitano for helpful suggestions. We thank A. Salzano and

E. De Gennaro for technical assistance. This work was supportedby grants from the Italian Telethon Foundation (TGM11CB6); MIURFIRB RBAP11Z3YA (A.B.); European Research Council AdvancedInvestigator grant no. 250154 (CLEAR) and no. 694282(LYSOSOMICS) (A.B.) and no. 341131 (InMec) (P.G.P.); U.S.National Institutes of Health (R01-NS078072) (A.B.); and theAssociazione Italiana per la Ricerca sul Cancro (A.I.R.C.) to A.B(IG 2015 Id 17639) and C.S. (IG 2015 Id 17717). All data needed toevaluate the conclusions in the paper are present in the paperand/or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/356/6343/1188/suppl/DC1Materials and MethodsFigs. S1 to S10Tables S1 and S2References (26–30)

31 May 2016; resubmitted 13 April 2017Accepted 23 May 201710.1126/science.aag2553

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Fig. 4. Deregulation of the MiT/TFE-RagD-mTORC1 regulatory axis supports cancergrowth. (A) mRNA levels of RagD in a cell line froma patient with RCC (HCR-59) relative to controlkidney cells (HK-2). B2M expression shown ascontrol unrelated gene. Gene expression wasnormalized relative to HPRT1. Plot representsmeans of three independent experiments ± SEM;Student’s t test. (B) Analysis of S6K phosphoryl-ation at threonine 389 in HK-2 and HCR-59 cells50 min starved for amino acids (0) and thenstimulated with increasing levels of amino acids for20 min. (C) Proliferation levels of HCR-59 cellstransfected with scramble (SCR), RagD, or TFE3siRNAs. Plot represents means of threeindependent experiments ± SEM; one-wayANOVA. (D) MITF-dependent melanoma patient–derived cells (501Mel) were analyzed for mRNAlevels of RagD (B2M expression was shown ascontrol unrelated gene). Values expressed asrelative to control melanoma cells (A375P). Geneexpression was normalized relative to HPRT1. Plotrepresents means of three independentexperiments ± SEM (Student’s t test). (E) Repre-sentative immunoblotting analysis for the indicatedproteins in control (A375P) and MITF-dependentmelanoma (501Mel) cells stimulated with increasedlevels of amino acids. (F) Proliferation index of501Mel cells transfected with SCR, RagD, orMITF siRNAs. Plot represents means of threeindependent experiments ± SEM; one-way ANOVA.(G and H) 501Mel cells were infected with alentivirus expressing a short hairpin RNA targetingthe Luciferase (control, Sh-Luc) or RagDmRNAsand transplanted in NOD scid gamma (NSG) mice.(G) Representative picture of tumors isolated fromboth groups of mice. (H) Plot shows tumorvolumes. Each dot represents a tumor.Twelvetumors (n = 12 mice) were analyzed per group;Student’s t test. [(A), (C), (D), (F), and (H)]:**P < 0.01, ***P < 0.001.

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Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth

Settembre and Andrea BallabioMargret Helga Ogmundsdottir, Luisa Lanfrancone, Rushika M. Perera, Roberto Zoncu, Pier Giuseppe Pelicci, CarmineOliver Davis, Rossella De Cegli, Angela Zampelli, Luca G. Di Giovannantonio, Edoardo Nusco, Nick Platt, Alessandro Guida, Chiara Di Malta, Diletta Siciliano, Alessia Calcagni, Jlenia Monfregola, Simona Punzi, Nunzia Pastore, Andrea N. Eastes,

DOI: 10.1126/science.aag2553 (6343), 1188-1192.356Science 

, this issue p. 1188Sciencemechanism is particularly important in cancer cells.process so that on refeeding, it efficiently reactivates mTORC1, which is recruited to the lysosomal surface. Thisactivity also requires amino acids, which are lacking during starvation. Nevertheless, the cell becomes ''primed'' by this triphosphatases that are required for mTORC1 activity and for its recruitment to the lysosome. However, mTORC1transition. During starvation, a pair of transcription factors promotes the expression of a pair of guanosine

elucidated a mechanism that regulates the metabolic changes that are necessary during the fast-to-feedet al.Malta A protein kinase complex known as mTORC1 plays a key role in cellular metabolism and nutrient sensing. Di

Preparing for the feast during the fast

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REFERENCES

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