1. Oncogenic MYC Activates a Feedforward Regulatory Loop Promoting Essential Amino Acid Metabolism and Tumorigenesis 
Ming Yue, Jue Jiang, Peng Gao, Hudan Liu, Guoliang Qing 
Cell Reports 
Volume 21, Issue 13, Pages (December 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 21, 3819-3832DOI: (10.1016/j.celrep.2017.12.002)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 Modulation of MYC Activities Affects EAA Uptake
(A) Immunoblot detection of N-MYC nuclear translocation in SHEP MYCN-ER cells upon 4-hydroxytamoxifen (4-OHT, 100 nM) treatment for 0, 24, and 48 hr. Histone H3 was used as a nuclear marker as well as a loading control.
(B) N-MYC induction activates aerobic glycolysis and glutamine consumption. SHEP MYCN-ER cells were treated with 4-OHT as shown in (A). Changes in glucose (Glc) consumption, lactate (Lac) secretion, and glutamine (Gln) consumption were analyzed and normalized to the same cell numbers and time (t) = 0 controls. The data shown represent the means (± SEM) of triplicates.
(C and D) N-MYC induction activates 3H-L-leucine (3H-Leu) and 3H-L-phenylalanine (3H-Phe) uptake. SHEP MYCN-ER cells were treated as indicated. 3H-Leu (C) or 3H-Phe (D) uptake (5 μCi/mL each) was measured, normalized to the same cell numbers and t = 0 controls, and presented as fold changes of the control uptake (0 hr and 0 mM Leu/Phe). The data shown represent the means (± SEM) of triplicates.
(E) Immunoblot analysis of C-MYC expression in P493 cells upon tetracycline (Tet, 100 ng/mL) treatment for 0, 24, and 48 hr. ACTIN was used as a loading control.
(F) C-MYC inactivation inhibits aerobic glycolysis and Gln consumption. P493 cells were treated as shown in (E). Changes in Glc consumption, Lac secretion, and Gln consumption were analyzed as shown in (B). The data shown represent the means (± SEM) of triplicates.
(G and H) C-MYC inactivation inhibits 3H-Leu and 3H-Phe uptake. P493 cells were treated as indicated. 3H-Leu (G) or 3H-Phe (H) uptake (5 μCi/mL each) was analyzed as shown in (C) and (D). The data shown represent the means (± SEM) of triplicates.
(I and J) Lysosomal recruitment of mTOR upon N-MYC induction (I) or C-MYC inhibition (J), analyzed by immunofluorescence against mTOR and the lysosomal marker LAMP2. SHEP MYCN-ER and P493 cells were pretreated with 4-OHT and Tet for 24 hr, respectively, starved of EAAs for 1 hr, and then placed in medium with or without 1× EAAs for 5 min. The scale bar represents 50 μm.
∗p < 0.05, ∗∗p < See also Figure S1.
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4. Figure 2 MYC Selectively Upregulates SLC7A5 and SLC43A1 Expression
(A–C) Time course analysis of Slc7a5, Slc43a1, Slc43a2, and Slc6a14 expression in 4-OHT-treated SHEP MYCN-ER (A), Tet-treated P493 (B), or JQ1-treated Daudi (C) cells by real-time qPCR. The data shown represent the means (± SEM) of triplicates. ∗∗p < 0.01.
(D and E) Representative immunoblots showing MYC, SLC7A5, and SLC43A1 expression in SHEP MYCN-ER (D) or in P493/Daudi (E) cells. ACTIN was used as a loading control. The asterisk denotes a non-specific band detected with C-MYC antibody in P493 cells.
(F) Tet-inducible inactivation of C-MYC in P493 xenografts caused a dramatic reduction in SLC7A5 and SLC43A1 expression in vivo. Left: tumor weights in mock- or Tet-treated mice. Six or eight tumors were analyzed per group. Right: immunoblots showing C-MYC, SLC7A5, and SLC43A1 levels in tumor lysates. ACTIN was used as a loading control.
See also Figure S2.
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5. Figure 3 MYC Directly Activates Slc7a5 and Slc43a1 Transcription
(A and B) shRNA knockdown of Mycn in BE-2C cells (A) or C-Myc in Daudi cells (B) selectively inhibits Slc7a5 and Slc43a1 expression. Relative Mycn, C-Myc, Slc7a5, Slc43a1, Slc43a2, and Slc6a14 mRNA levels were quantitated by real-time qPCR. The data shown represent the means (± SEM) of triplicates.
(C) Representative immunoblots detecting MYC (N-MYC or C-MYC), SLC7A5, and SLC43A1 expression with ACTIN as a loading control.
(D) Schematic of the MYC consensus binding site, the MYC response elements (MYC REwt), and their mutants (REmut) within the promoter of Slc7a5 or the first intron of the Slc43a1 gene.
(E) Luciferase assays performed using MYC REwt and MYC REmut constructs in the presence or absence of ectopically expressed MYC (C-MYC or N-MYC) in 293T cells. The data shown represent the means (± SEM) of triplicates.
(F and G) Binding of MYC to Slc7a5, Slc43a1, Ncl (encodes nucleolin), and Actin, analyzed by ChIP assays in BE-2C (F) and P493 (G) cells, respectively, with N-MYC- and C-MYC-specific antibodies. The data shown represent the means (± SEM) of triplicates between MYC ChIP and the isotype IgG controls.
(H) Relative expression of Mycn, Slc7a5, and Slc43a1 in 643 primary neuroblastoma tumors (GSE45547). non-Amp, Mycn-nonamplified tumors (n = 550); Amp, Mycn-amplified tumors (n = 93).
(I) Correlation between mRNA levels of Slc7a5 versus Mycn or Slc43a1 versus Mycn in 643 primary neuroblastoma tumors.
(J) Relative expression of C-Myc, Slc7a5, and Slc43a1 in 221 aggressive lymphomas (GSE4475). Non-BL, non-Burkitt’s lymphoma (n = 129); Intermediate, 48; BL, Burkitt’s lymphoma (n = 44).
(K) Correlation between mRNA levels of Slc7a5 versus C-Myc or Slc43a1 versus C-Myc in 221 lymphoma tumors.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S3.
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6. Figure 4
SLC7A5/SLC43A1 Sustains the Oncogenic Capacities of MYC-Overexpressing Tumor Cells
(A) 3H-Leu uptake by BE-2C and Daudi cells upon SLC7A5 or SLC43A1 depletion. BE-2C and Daudi cells were infected with two specific lentiviral shRNAs targeting human Slc7a5 or Slc43a1, respectively, for 48 hr. 3H-Leu transport (5 μCi/mL) was analyzed as shown in Figure 1C. The data shown represent the means (± SEM) of triplicates.
(B and C) Proliferation of BE-2C (B) and Daudi (C) cells over 7 days, as measured by serial cell counts upon SLC7A5 or SLC43A1 depletion. The data shown represent the means (± SEM) of triplicates.
(D and E) Xenograft tumor growth (D) and weight (E) upon SLC7A5 depletion in BE-2C cells. 6–8 tumors were analyzed in each group.
(F) Ectopic SLC7A5 expression promotes BE-2C cell proliferation at low amino acid (AA) levels. 2× 105 BE-2C cells stably expressing the control vector or Flag-SLC7A5 were seeded in RPMI medium containing 1× (regular RPMI), 0.5×, or 0.25× AA concentration, respectively, and cell proliferation was analyzed by serial cell counts over 7 days. The data shown represent the means (± SEM) of triplicates.
(G and H) Xenograft tumor growth (G) and weight (H) upon SLC7A5 overexpression in 1 million BE-2C cells. 6 tumors were analyzed in each group.
(I) Left: representative SLC7A5 and Ki67 immunochemistry staining on paraffin-embedded tumor sections shown in Figure 4H. Right: quantitation of SLC7A5 and Ki67 immunostaining. 6–8 tumors were analyzed per group. The scale bar represents 50 μm.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S4.
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7. Figure 5
SLC7A5 Depletion Activates the GCN2-eIF2α Stress Response Pathway, Leading to Inhibition of C-Myc or Mycn mRNA Translation
(A) Immunoblot analysis of MYC expression upon SLC7A5 depletion. MCL1 was used as a negative control, and ACTIN was used as a loading control.
(B) Representative SLC7A5, N-MYC, and Ki67 immunochemistry staining on paraffin-embedded tumor sections shown in Figure 4E. The scale bar represents 50 μm. ∗∗p < 0.01.
(C) Immunoblot analysis of N-MYC expression in tumor lysates shown in Figure 4H.
(D) Ectopic SLC7A5 expression rescued N-MYC loss resulting from inhibition of endogenous Slc7a5 by specific shRNAs targeting its 3′ UTRs. ACTIN was used as a loading control.
(E) BE-2C cells were infected with control or Slc7a5-specific shRNA as shown. 48 hr later, cells were treated with cycloheximide (CHX, 20 μg/mL) and harvested at the indicated time points. Protein levels were analyzed by immunoblot, with ACTIN as a loading control.
(F) Induction of GCN2-eIF2α stress response pathway upon SLC7A5 depletion. ATF4, p-GCN2, GCN2, p-eIF2α, and eIF2α expression was analyzed by immunoblot.
(G) Autoradiogram of 35S-methionine (Met)-labeled proteins resolved by SDS-PAGE after a 30-min pulse labeling of control or Slc7a5 shRNA-infected BE-2C cells as indicated. To analyze 35S-Met-labeled MYC proteins, equal amounts of total cellular lysates were immunoprecipitated with specific N-MYC and C-MYC antibodies, respectively, before being subjected to SDS-PAGE and autoradiography. 35S-Met-labeled signals were quantitated and normalized to the Coomassie-stained proteins.
See also Figure S5.
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8. Figure 6 SLC7A5 Depletion Inhibits MYC-Dependent Transcription
(A) Venn diagram of MYC- and SLC7A5-regulated genes.
(B) Heatmap of the top 50 down- and upregulated genes upon Slc7a5 depletion in Daudi cells based on signal-to-noise ratio (SNR) score; p < The relative expression levels of these genes in C-Myc-depleted cells are also shown. The data are presented as row-normalized.
(C) GESA of MYC-dependent gene sets in transcriptional profiles of Daudi cells infected with control shRNA (Ctrl-sh), C-Myc shRNA (C-Myc-sh), or Slc7a5 shRNA (Slc7a5-sh).
(D) Real-time qPCR analysis of representative MYC targets in Daudi cells upon SLC7A5 depletion. The data shown represent the means (± SEM) of triplicates.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S6.
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9. Figure 7
Inhibition of SLC7A5/SLC43A1 Compromises Metabolic Reprogramming in MYC-Overexpressing Tumor Cells
(A) BE-2C cells were infected with the indicated shRNAs, selected with puromycin for 24 hr, and then placed in fresh medium for another 24 hr. Glc consumption, Lac secretion, and Gln consumption were analyzed and normalized to the same cell number. The data shown represent the means (± SEM) of triplicates.
(B–D) BE-2C cells were starved of all EAAs, Leu, or Phe as indicated. Glc consumption (B), Lac secretion (C), and Gln consumption (D) were analyzed as shown in (A).
(E) BE-2C cells were treated with vehicle control or BCH (2-aminobicyclo-2,2,1-heptane-2-carboxylic acid, 20 mM) as indicated. Glc consumption, Lac secretion, and Gln consumption were analyzed as shown in (A).
(F) Analysis of the extracellular acidification rate (ECAR) in vehicle- or BCH-treated BE-2C cells. The data shown represent the means (± SEM) of triplicates. 2-DG, 2-deoxy-D-glucose.
(G) Analysis of the oxygen consumption rate (OCR) in vehicle- or BCH-treated BE-2C cells. The data shown represent the means (± SEM) of triplicates.
(H) Heatmap of changes in metabolite concentrations between control and Slc7a5/Slc43a1-depleted BE-2C cells based on p < BE-2C cells infected with Slc7a5 and Slc43a1 shRNAs were selected with puromycin for 48 hr and then placed in fresh medium for another 24 hr. Cells were harvested, and intracellular metabolites were obtained and analyzed by gas chromatography-mass spectrometry (GC-MS) (n = 6 biological replicates). Colors indicate changes in metabolite abundance relative to mock treatment: yellow, increased metabolite abundance; blue, decreased metabolite abundance.
(I) Model depicting the MYC-SLC7A5/SLC43A1 signaling circuit in promotion of tumorigenesis. See the text for more details.
∗p < 0.05, ∗∗p < See also Figure S7.
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