Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth

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Science  16 Jun 2017:
Vol. 356, Issue 6343, pp. 1188-1192
DOI: 10.1126/science.aag2553
  • Fig. 1 MiT/TFE transcription factors regulate mTORC1 activity both in vitro and in vivo.

    (A and B) Representative immunoblots of TFEB, phospho-S6K, and S6K in Tet-ON TFEB-CA cell line untreated (–DOX) or treated with doxycycline (+DOX) for 24 hours. Cells were starved for amino acids for 50 min (0) and stimulated with decreasing levels (expressed as % of concentration in RPMI medium) of leucine (A) or arginine (B) for 20 min. (C) C57BL6 mice injected with HDad expressing human TFEB under the control of a liver-specific promoter (TFEB-INJ) or with phosphate-buffered saline (PBS) (CTRL) were starved for 22 hours (FASTED) and then refed for 2 hours (FED). Liver lysates were analyzed for levels of indicated proteins. Actin was used as loading control. Plot shows ratio of phosphorylated S6K/pan-S6K (mean of three independent experiments). (D) Immunohistochemistry 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 TFEB immunostainings in two consecutive 5-μm liver sections isolated from HDad-TFEB–injected mice. (E) Liver samples from mice with indicated genotypes were analyzed for the levels of S6K phosphorylation and puromycin incorporation. Plots show the ratios of phosphorylated S6K/pan-S6K and puromycin/actin expressed as relative to control mice (Tcfebflox/flox). (F) Phosphorylation of S6K and levels of puromycin incorporation analysis in muscle samples from mice with indicated genotypes after oral gavage of leucine. Mice were exercised where indicated. Plots show ratios of phosphorylated S6K/pan-S6K and puromycin/glyceraldehyde phosphate dehydrogenase (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.

  • Fig. 2 MiT/TFE transcription factors control mTORC1 activity through RagD.

    (A) mRNA levels of mTORC1-related genes in TFEB-CA HeLa cells treated with doxycycline. Values normalized relative to HPRT1 and expressed as fold change relative to untreated cells. (B) ChIP analysis of TFEB binding to RagD promoter in doxycycline-treated HeLa TFEB-CA cells. Squares represent CLEAR sites in RagD promoter and numbers refer to their distance [in base pairs (bp)] from the transcriptional start site (TSS). Immunoprecipitated DNA was normalized to the input and plotted as relative enrichment over a mock control. (C) Luciferase assay after transfection of increasing amounts of TFEB construct was performed in HeLa cells cotransfected with wild-type (RAGD-wt) or mutated (RAGD-mut) RagD-promoter luciferase reporter plasmids. (D) Scheme of CRISPR-Cas9–mediated mutation in the endogenous RagD promoter of HeLa cells. A region of 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 mutated HeLa cell line (HeLa-RagDpromedit) versus control HeLa and normalized relative 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 independent experiments (Student’s t test). (G) Mice with indicated genotypes were nutritionally synchronized and injected with puromycin 30 min before sacrifice. Where indicated, Tcfebflox/flox;Alb-Cre+ mice were injected with an adeno-associated virus vector carrying human RagD cDNA. Liver lysates were analyzed for phosphorylation of S6K and levels of puromycin incorporation. Plots show means of triplicates ± SEM, one-way ANOVA expressed 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.

  • Fig. 3 MiT/TFE transcription factors promote lysosomal recruitment of mTOR upon nutrient loading.

    (A) Representative immunofluorescence images of endogenous mTOR, LAMP1-GFP (visualized as red) and RAGD-HA in HeLa cells. Cells were transfected with scramble (CTRL) or with TFEB siRNA (siTFEB) and after 48 hours with LAMP1-GFP and with RagD-HA plasmids for an additional 24 hours. (B) Representative immunofluorescence images of mTOR and LAMP2 in HeLa-RagDpromedit and in control HeLa cells. [(A) and (B)] Cells were deprived of amino acids for 50 min and then stimulated with amino acids for 15 min. Plots represent quantification of the data from 15 cells per condition from three independent experiments. Results are shown as means of colocalization coefficient of (A) mTOR and LAMP1 ± SEM (one-way ANOVA) and (B) mTOR and LAMP2 ± SEM (Student’s t test). **P < 0.01; ***P < 0.001. Scale bars, 10 μm.

  • Fig. 4 Deregulation of the MiT/TFE-RagD-mTORC1 regulatory axis supports cancer growth.

    (A) mRNA levels of RagD in a cell line from a patient with RCC (HCR-59) relative to control kidney cells (HK-2). B2M expression shown as control unrelated gene. Gene expression was normalized relative to HPRT1. Plot represents means of three independent experiments ± SEM; Student’s t test. (B) Analysis of S6K phosphorylation at threonine 389 in HK-2 and HCR-59 cells 50 min starved for amino acids (0) and then stimulated with increasing levels of amino acids for 20 min. (C) Proliferation levels of HCR-59 cells transfected with scramble (SCR), RagD, or TFE3 siRNAs. Plot represents means of three independent experiments ± SEM; one-way ANOVA. (D) MITF-dependent melanoma patient–derived cells (501Mel) were analyzed for mRNA levels of RagD (B2M expression was shown as control unrelated gene). Values expressed as relative to control melanoma cells (A375P). Gene expression was normalized relative to HPRT1. Plot represents means of three independent experiments ± SEM (Student’s t test). (E) Representative immunoblotting analysis for the indicated proteins in control (A375P) and MITF-dependent melanoma (501Mel) cells stimulated with increased levels of amino acids. (F) Proliferation index of 501Mel cells transfected with SCR, RagD, or MITF siRNAs. Plot represents means of three independent experiments ± SEM; one-way ANOVA. (G and H) 501Mel cells were infected with a lentivirus expressing a short hairpin RNA targeting the Luciferase (control, Sh-Luc) or RagD mRNAs and transplanted in NOD scid gamma (NSG) mice. (G) Representative picture of tumors isolated from both groups of mice. (H) Plot shows tumor volumes. Each dot represents a tumor. Twelve tumors (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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