Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth

Abstract

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

Fig 1. MiT/TFE transcription factors regulate mTORC1 activity both in vitro and in vivo

(A,B) Representative immuno-blotting analysis of TFEB, phospho-S6K and S6K in Tet-ON TFEB-CA cell line untreated (−DOX) or treated with Doxycycline (+DOX) for 24h. Cells were starved for a.a. 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 a Helper-Dependent adenovirus (HDad) expressing human TFEB under the control of a liver-specific promoter (TFEB-INJ.), or with PBS (CTRL) were starved for 22h (FASTED), and then reefed for 2h (FED). Liver lysates were analyzed for levels of indicated proteins. Actin was used as loading control. The plot shows ratio of phosphorylated S6K/pan-S6K (mean of three independent experiments). (D) Immunohistochemistry analysis 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 5μm 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. The plots show the ratios of phosphorylated S6K/pan-S6K and puromycin/actin expressed as relative to control mice (Tcfeb^flox/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. The plots show ratios of phosphorylated S6K/pan-S6K and puromycin/GAPDH. The plots in (C), (E) represent means of triplicates +/− SEM, Student t-test. The plot in (F) represent means +/− SEM; N=3; Anova (one-way) followed by Tukey’s test. In (C), (E), (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 were 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 (bp) from the transcriptional start site (TSS). Immuno-precipitated DNA was normalized to the input and plotted as relative enrichment over a mock control. (C) Luciferase assay analysis after transfection of increasing amounts of TFEB construct was performed in HeLa cells co-transfected with a 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 33bp 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-RagD^promedit) versus control HeLa and normalized relative to HPRT1 gene. (F) Immuno-blotting analysis of mTORC1 signaling in HeLa-RagD^promedit cells compared to control HeLa. The ratio of phosphorylated/total protein levels were shown for the indicated mTORC1 substrates. The plots in (A), (B), (C), (E), (F) represent mean ± SEM of 3 independent experiments (Student t test). (G) Mice with indicated genotypes were nutritionally synchronized and injected with puromycin 30 minutes prior sacrifice. Where indicated Tcfeb^flox/flox; Alb-Cre⁺ mice were injected with an AAV-vector carrying human RagD cDNA. Liver lysates were analyzed for phosphorylation of S6K and levels of puromycin incorporation. The plots show means of triplicates +/− SEM, Anova (one-way) expressed as ratio of phosphorylated S6K/pan-S6K and puromycin/actin. In (A), (B), (C), (E), (F), (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 additional 24 hours. (B) Representative immunofluorescence images of mTOR and LAMP2 in HeLa-RagD^promedit and in control HeLa cells. (A,B) Cells were deprived of a.a. for 50 minutes and then stimulated with a.a. for 15 minutes. The plots represent quantification of the data from 15 cells per condition from three independent experiments. Results are shown as means of co-localization coefficient of mTOR and LAMP1± SEM (Anova, one-way) in (A) of mTOR and LAMP2 ± SEM (Student t test) in (B). (**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 was shown as control unrelated gene. Gene expression was normalized relative to HPRT1. The plot represents means of three independent experiments ± SEM; Student t test. (B) Analysis of S6K phosphorylation at threonine 389 in HK-2 and HCR-59 cells 50 min starved for a.a. (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. The plot represents means of three independent experiments ± SEM; Anova (one way). (D) MITF-dependent melanoma patient-derived cells (501Mel) were analyzed for mRNA levels of RagD (B2M expression was shown as control unrelated gene). Values were expressed as relative to control melanoma cells (A375P). Gene expression was normalized relative to HPRT1. The plot represents means of three independent experiments ± SEM (Student 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 scramble (SCR), RagD or MITF siRNAs. The plot represents means of three independent experiments ± SEM; Anova (one way). (G,H) 501Mel cells were infected with a lentivirus expressing a short harpin RNA targeting the Luciferase (control, Sh-Luc) or RagD mRNAs and transplanted in NSG mice. (G) Representative picture of tumors isolated from both groups of mice. (H) Plot shows tumor volumes. Each dot represents a tumor. 12 tumors (n=12 mice) were analyzed per group; Student t test. In (A), (C), (D), (F), (H) **p<0.01, ***p < 0.001.