Fig 2: MEK/ERK-MYC-MiT/TFE-lysosomal gene axis in renal cell carcinomas (RCCs). (A) MYC and ATP6V1B1 levels in several types of renal tumors and adjacent normal tissues. The expression of each gene in primary renal tumor (red) and normal tissue (blue) was analyzed (TCGA data set). Boxes represent the interquartile range and line inside the box represents median value. (B) Gene set variation analysis of MYC and lysosome-related gene expression in MSigDB 7.1. (C) Immunohistochemistry for pERK, c-MYC, TFEB, TFE3, MITF, ATP6V1B1, and LAMP1 in RCC and matched-adjacent normal renal tissues (magnification: ×20). Inset: ×160 magnified view. Representative images from 24 samples are shown. Scale bars, 100 µm. (D) Kaplan-Meier plots of MYC and ATP6V1B1 in renal cancer (TCGA data set). (E) Schematic model of MEK/ERK pathway role in regulating the fate of MVBs and control EV secretion. In normal cells, the MEK/ERK pathway is inactivated, presumably preserving the expression of lysosomal genes, including vacuolar-type H+-ATPase, and maintaining lysosome function. MVBs are degraded and therefore the number of MVBs fusing with the plasma membrane is low, leading to suppression of EV secretion. In cancer cells, when the MEK/ERK pathway is activated, MYC expression is increased and MiT/TFEs transcription factors are retained in the cytoplasm, inactivating the transcription of lysosomal genes and disrupting lysosome function. A large amount of MVBs can therefore fuse with the plasma membrane, promoting EV secretion. Statistical analysis was performed using one-way ANOVA. ***p < 0.001

Fig 3: Effect of TFEB S142 phosphorylation on PLX4720-resistant BRAFV600E melanoma cells. a Colony formation assay of PLX4720-resistant A375R melanoma cells stably expressing vector, WT TFEB, S142A, or S142E TFEB mutants as indicated. Bars are mean ± SD percentage of colonies for each group after 21 days. n = 3 independent experiments. b Western blot analysis of TFEB expression in cells shown in (a). Actin served as a loading control. Data are from one experiment that is representative of three independent experiments. c Tumor volume of xenografts formed after subcutaneous injection of NOD/SCID mice with A375R melanoma cells stably expressing the indicated TFEB constructs. Results are the mean volume ± SD for 5–6 mice per group per time point. d Tumor weight from experiment shown in (c) upon autopsy at day 30. Representative images of tumor size of the indicated A375R xenograft tumor genotypes are shown below. e Western blot analysis of autophagy (p62 and LC3-I/II), lysosome (CTSD), TGF-ß/Smad2/3 signaling, EMT-related [E-cadherin (E-Cad.), N-cadherin (N-Cad.), and Vimentin, Twist1, ZEB1, Snail, and Slug], ?-H2AX, ERK and mTORC1 activation in the indicated xenografts (three randomly chosen samples per group; similar results observed in all 10–12 samples per condition). Actin served as a loading control. See Supplementary Fig. 13 for uncropped data. For all quantification, data represent the mean ± SD derived from indicated number of independent experiments. Comparisons were made using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: TFEB S142 phosphorylation confers BRAFi resistance in melanoma cells. a Representative images (left) and quantification (right) of the colonogenic survival of A375 cells stably expressing control shRNA (pGIPZ) or TFEB shRNA complemented with empty vector, WT TFEB, and S142A or S142E TFEB mutants that are shRNA-resistant, after treatment with DMSO or PLX4720 at the indicated concentrations. Endogenous and reconstituted TFEB expression was confirmed by immunoblotting (bottom right). n = 3 independent experiments. b Bioluminescence images (top) of tumor regression of the indicated A375R xenograft tumor genotype in live NOD/SCID mice at the indicated time after inoculation. Radiant efficiency expressed as p/s/cm2/sr/(µW/cm2) was quantified (bottom). c Effect of PLX4720 on tumor response of xenografts formed by the indicated A375 cell lines. After tumor establishment (~ 500 mm3), mice bearing A375 xenografts were treated with PLX4720 (20 mg/kg, i.p.) daily for 21 days. Values are the mean tumor volume ± SD per time point for 6–7 mice per group. d, e Representative gross images (d) of livers with metastatic nodules (top), H&E-stained sections (second row) and IHC analysis of TGF-ß and p-Smad3 of the indicated A375 xenograft genotypes 21 days after PLX4720 treatment (20 mg/kg, i.p., daily). Scale bars, 100 µm. The numbers of metastatic nodules in the liver in (d) was quantified in (e) (n = 5–6 mice per group). For all quantification, data represent the mean ± SD derived from indicated number of independent experiments. Comparisons were made using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant

Fig 5: TFEB S142 phosphorylation in BRAFV600E melanoma cells. a PLX4720 inhibits TFEB-ERK interaction and TFEB S142 phosphorylation. A375 and MeWo cells expressing TFEB-GFP were treated with PLX4720 (1 µM, 12 h), and WCLs were IP with anti-GFP-Trap beads, followed by IB with the indicated antibodies. n = 3. b PLX4720 releases ERK and TFEB from the lysosomes. n = 3. c Quantification of nuclear localization of TFEB-GFP in A375 cells expressing indicated TFEB mutants. n = 3. d In vitro phosphorylation of WT or TFEB mutants by ERK (CA). Phosphorylated TFEB were detected by autoradiography (top). The same gel was stained with Coomassie Blue to visualize total proteins (middle). Relative fold change in TFEB phosphorylation was quantified (bottom). e Representative images and quantification of cytoplasmic retention of WT, S142A, and S211A TFEB in A375 cells expressing ERK(CA) w/ or w/o treatment of PLX4720, Leptomycin B (LMB; 20 nM, 2 h), Raptor- or CRM1-shRNA. n = 3. f TFEBS142E associates with the lysosomes regardless of the treatment of PLX4720 or Raptor-shRNA. n = 3. g S142A mutation reduces phosphorylation of the 14-3-3-binding motif in TFEB. WT or mutant TFEB-GFP was expressed in A375 cells and IP with GFP-Trap beads, followed by IB with the indicated antibody. n = 3. h The predominant role of S142 phosphorylation over S211 phosphorylation in TFEB interaction with 14-3-3. A375 cells as indicated were treated with PLX4720, followed by IP with GFP-Trap beads and IB for the indicated proteins. n = 3. i-k IB (i) and quantification of the LC3-II/LC3-I (j) and p62/Actin ratios (k) in A375 cells expressing WT/mutant TFEB w/ or w/o PLX4720 treatment. n = 3. Scale bars, 10 µm. Data in a, d, g, h, and i are from one that is representative of three independent experiments. For all quantification, data represent the mean ± SD derived from the indicated number of independent experiments. Comparisons were made using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant. See Supplementary Fig. 13 for uncropped data of a, e, h, i