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eIF4E S209 phosphorylation licenses myc- and stress-driven oncogenesis

  1. Hang Ruan
  2. Xiangyun Li
  3. Xiang Xu
  4. Brian J Leibowitz
  5. Jingshan Tong
  6. Lujia Chen
  7. Luoquan Ao
  8. Wei Xing
  9. Jianhua Luo
  10. Yanping Yu
  11. Robert E Schoen
  12. Nahum Sonenberg
  13. Xinghua Lu
  14. Lin Zhang
  15. Jian Yu  Is a corresponding author
  1. Department of Pathology, University of Pittsburgh School of Medicine, United States
  2. UPMC Hillman Cancer Center, United States
  3. Department of Stem cell and regenerative medicine, Daping Hospital, Army Medical University, China
  4. Central laboratory, State key laboratory of trauma, burn and combined Injury, Daping Hospital, China
  5. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, United States
  6. Department of Biomedical informatics, University of Pittsburgh School of Medicine, United States
  7. Departments of Medicine and Epidemiology, University of Pittsburgh, United States
  8. Department of Biochemistry, Goodman Cancer Research Centre, McGill University, Canada
Research Article
Cite this article as: eLife 2020;9:e60151 doi: 10.7554/eLife.60151
7 figures and 4 additional files

Figures

Figure 1 with 1 supplement
eIF4E S209 regulates CRC cell growth.

Isogenic HCT116 WT and two independent EIF4ES209A knockin (S209A/+, 4EKI1, and 4EKI2) clones were analyzed for growth. (A) The indicated proteins in log-phase cells were analyzed by western blotting. Actin is the loading control. (B) Genomic DNA sequencing confirmed T to G (S209A) change in 4EKI cells. (C) Cell growth monitored for 7 days by counting. (D–F) Representative images and quantification (bottom) of cell growth in clonogenic assay, soft agar, and spheroids on day 10, day 14, and day 7, respectively. (F) Scale bar: 10 μM. WT values were set at 100%. C, D, E, F, values are mean+s.d. (n = 3). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test, two tailed). WT vs. KI.

Figure 1—figure supplement 1
Generation and characterization of EIF4ES209A/+ KI HCT 116 cells.

(A) eIF4E and 4E-BP1 gene and protein expression analysis of colorectal adenocarcinoma in the published TCGA provisional cohort (n = 640) (cbioportal.org). (B) Schematic representation of the EIF4E genomic locus and the S209A targeting construct. (C) The KI allele confirmed by genomic PCR in two clones upon removal of floxed Neo. (D) The growth of log-phase cells was determined by ATP and (E) MTS assays, respectively. (F) Representative pictures and quantification (bottom) of BrdU incorporation following 15 min pulse in WT and 4EKI cells. D, E, F values represented mean+s.d. (n = 3). WT vs. KI, *p<0.05, **p<0.01 (Student’s t-test, two tailed).

Figure 2 with 1 supplement
eIF4E S209 regulates Myc translation in CRC.

WT and 4EKI cells were analyzed for translation. (A) Polysome profiling of log-phase cells. (B) Cap-dependent luciferase reporter activities were measured 24 hr after transfection with pcDNA-LUC reporter. (C) Translation of a bi-cistronic construct pYIC encoding cap-dependent and -independent reporters was analyzed by western blotting 24 hr after transfection. (D) The cells were subjected to serum starvation for 48 hr then stimulated with 10% FBS (+) for 4 hr. Binding of indicated eIF4F components (E, G, and 4E-BP1) to a synthetic cap analog (m7GTP) or their levels in the total lysate (input) were analyzed by western blotting. (E) The indicated eIF4E targets were analyzed by western blotting. Actin is the loading control. (F) The indicated transcripts in total cellular and (G) in polysomal RNAs were analyzed by RT-PCR. (H) The ratio of mRNA in polysome and total RNA normalized to WT (100%). N = 3. (B, F-H), values are mean+s.d. (n = 3). *p<0.05, **p<0.01 (Student’s t-test, two tailed). WT vs. KI. (I) Representative images of three CRC organoids treated with eFT508 (10 μM) for 48 hr. Scale bar: 100 µM. (J) The indicated proteins in CRC organoids treated with eFT508 at 24 hr were analyzed by western blotting.

Figure 2—figure supplement 1
Schematics of pCMV-Luc reporter and pYIC reporter encoding cap-dependent translation of Myc-tagged YFP and IRES-dependent HA-tagged CFP.
Figure 3 with 1 supplement
eIF4E S209 regulates CRC growth and the ISR in vivo.

Paired HCT116 WT and 4EKI cells were characterized in vitro and in vivo. (A) Reactome Pathway analysis of genes elevated in WT cells (3-fold or more) visualized by ClueGO. (B) RT-PCR of indicated transcripts in total and (C) in polysomal RNAs. (D) The indicated transcripts were analyzed by RT-PCR in HCT116 cells transfected with the indicated siRNAs for 24 hr. N = 3. (E–K) Xenograft tumors. (E) The growth curve of WT and KI (KI1 and KI2) cells (four million) injected s.c. into different flanks of nude mice by day 23. (F) Representative images of WT and KI (KI1) xenografts on day 23. (G) Representative IF/IHC of the indicated makers in randomly chosen WT and KI tumors. Scale bar: 50 µm and (H) quantification of markers in (G) using 400x fields. (H) Representative PLA of eIF4E-4E-BP1 binding, scale bar: 25 µm, and (J) quantification (dots/cell). (K) The indicated transcripts were detected by RT-PCR. WT values were set at 100%. (B-D, F, H, J-K), values represent mean+s.d. (n = 3 or as indicated). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test, two tailed). WT vs. KI. +p<0.05, ++p<0.01, (one-way ANOVA with TUKEY post-hoc test), or scrambled vs. specific siRNAs.

Figure 3—figure supplement 1
eIF4E S209 regulates the ISR in vitro and in vivo.

(A) Scatter plot of microarray data from log-phase cells. Significantly elevated genes in WT (red) (89) or 4E KI cells (blue) (Croft et al., 2014), 3-fold or more, *p<0.05. (B) Reactome pathway analysis of genes elevated in WT cells from (A). (C) Reactome pathway analysis of genes elevated in 4EKI visualized by ClueGO. (D) Reactome pathway analysis of genes elevated in 4EKI cells from (A). (E) Indicated transcripts in translation, (F) cell death and (G) other Myc metabolic targets (nucleotide synthesis, fatty acid, and glucose metabolism) were analyzed by RT-PCR. (H) Enlarged p-GCN2 and p-eIF2a IF in the indicated xenografts. Scale bar: 25 µM. (I) Representative p-S6 IF in the indicated xenografts. Scale bar: 50 µM. (J) Indicated transcripts involved in cell death were analyzed in xenografts by RT-PCR. (K) GEPIA expression analysis of indicated ISR targets in TCGA COAD and READ cohorts with matched TCGA normal and GTEX data. (E-G, J), values represent mean+s.d. (n = 3). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test, two tailed).

Figure 4 with 1 supplement
eIF4E S209 promotes Myc-driven oncogenesis in mice and human.

(A) Representative IF of indicated markers in polyps and adjacent ‘normal’ crypts in ApcMin/+ mice. Scale bar: 100 µm. (B) Representative images of polyps and (C) quantification in the small intestine of 3-month-old ApcMin/+ (4E+/+) (n = 5), ApcMin/+;4EKI/+ (n = 8), and ApcMin/+;4EKI/KI (n = 4) mice. ++p<0.01, +++p<0.001 (One-Way ANOVA with TUKEY Post-hoc test). (D) Representative IF of indicated markers and (E) quantitation in the polyps in indicated mice. Scale bar: 100 µM. (F) Myc mRNA levels in the polyps from indicated mice. (G) p-4E (S209) and total eIF4E in human adenomas and normal colon analyzed by western blotting. Actin is a loading control.

Figure 4—figure supplement 1
eIF4E S209 promotes polyposis in ApcMin/+ mice.

(A) Representative images of 4EKI genotypes in ApcMin/+ mice. (B) Tumor burden in the proximal (PI), middle (MI) and distal intestine (DI), and C, colon. Values represent mean+s.d. +p<0.05, ++p<0.001 (One-Way ANOVA with TUKEY Post-hoc test). (C) Enlarged p-4E and KI-67 IF in the ‘normal’ crypts of the indicated mice. Scale bar: 50 µM. (D) Representative Ki-67 IF in ‘normal’ intestinal crypts in the indicated mice. Scale bar: 100 µm, and (E) Quantification. (F) Indicated transcripts in the mucosa of 6-week-old (pre-cancerous) ApcMin/+, and ApcMin/+, 4EKI were analyzed by RT-PCR and normalized to that in ApcMin/+ (Siegel et al., 2019). N = 3. Values are mean+s.d. *p<0.05, **p<0.01 (Student’s t-test, two tailed). WT vs. KI. (G) Representative IF of indicated markers in the polyps and H and E image of polyps. Scale bar: 100 µM, and (H) quantitation.

Figure 5 with 1 supplement
p-4E promotes ISR-dependent glutamine addiction.

Isogenic HCT 116 WT and 4E KI cells were subjected to glutamine deprivation (2 to 0 mM), glucose deprivation (4 to 0 mM) or GLS inhibitor (CB-839, 40 µM) treatment, and analyzed at indicated times (0–48 hr). (A) Attached cells were stained with crystal violet at 48 hr. (B) Cell death were analyzed at 48 hr by flow cytometry and (C) quantification of AnnexinV+ cells. Representative flow cytometry plots and results are shown. (D) The indicated proteins were analyzed by western blotting at indicated times after Gln deprivation. (E) Heatmap of indicated transcripts in polysomes at 24 hr were analyzed by RT-PCR, normalized to WT 0 hr (Siegel et al., 2019). (F–G) WT cells were transfected the indicated siRNAs for 24 hr, followed by 24 hr recovery, and subjected to glutamine deprivation. (F) Heatmaps of indicated transcripts in total RNA at 24 hr were analyzed by RT-PCR, normalized to WT 0 hr (Siegel et al., 2019). (G) Cell death at 48 hr was quantitated by Annexin V+ cells. (H) 4EKI cells were transfected with control, Myc or ATF4 expression construct and subjected to glutamine deprivation. Cell death at 48 hr was quantitated by Annexin V+ cells. (I) Cell death 48 hr after GLSi was quantitated by Annexin V+ cells.

Figure 5—figure supplement 1
p-4E drives Myc and ISR-dependent glutamine addiction.

HCT 116 WT or 4E KI cells were subjected to glutamine deprivation or GLS inhibitor (CB-839, 40 µM) treatment, and analyzed at indicated times (0–48 hr). (A) WT cells were transfected the indicated siRNAs for 24 hr, and followed by 24 hr recovery, then subjected to glutamine deprivation. The indicated proteins at 24 hr were analyzed western blotting. (B) 4EKI cells were transfected with control, Myc or ATF4 (Flag-tagged) expression constructs for 24 hr, followed by 24 hr recovery and subjected to glutamine deprivation. The indicated proteins at 24 hr were analyzed by western blotting. (C) Attached cells were stained with crystal violet at 48 hr, and (D) quantitation of Annex V+ cells by flow cytometry. (E–G) WT and 4EKI2 cells were analyzed for cell growth and death at 48 hr, and the indicated proteins at 24 hr. (I) The indicated protein and (H) transcripts were analyzed 24 hr after GLSi (CB) treatment. Values represent mean+s.d. (n = 3). **p<0.01 (Student’s t-test, two tailed). WT vs. KI.

Figure 6 with 1 supplement
Mutant KRAS and Myc promotes p-4E-dependent glutamine addiction.

Indicated WT and mutant KRAS isogenic CRC lines were subject to glutamine deprivation and analyzed. (A) The basal levels of indicated proteins were analyzed by western blotting. (B) Attached cells at 48 hr were stained with crystal violet. (C) Cell death at 48 hr was quantitated by flow cytometry. Representative results were shown. (D) The indicated proteins at 24 hr in two pairs of cell lines were analyzed by western blotting. *, non-specific band. (E) Heat map of indicated transcripts at 24 hr were detected by RT-PCR and normalized to the control (WT, Gln+, 1). (F) Numbers of differential genes upon Gln deprivation in the isogenic HCT 116 pair, 2-fold or more, *p<0.05. (G) Venn diagram of induced genes from (F). (H–J) Reactome pathway analysis of (H) shared, WT (I), and 4EKI-specific upregulated genes(J). Top five enriched pathways are shown.

Figure 6—figure supplement 1
Mutant KRAS promotes glutamine addiction.

Two pairs of wt and mutant KRAS isogenic CRC cells were subjected to glutamine deprivation. (A and B) Cells at 48 hr were analyzed by flow cytometry with quantification of PI+ and PI- AnnexinV+ populations. (C) The indicated proteins at 24 hr were analyzed by western blotting. (D) Scatter plot of microarray data from isogenic HCT 116 cells 24 hr after Gln deprivation.

Figure 7 with 1 supplement
p-4E controls metabolic stress-induced cell death and transcriptomic heterogeneity.

Indicated CRC lines were subject to glutamine deprivation and analyzed. (A) Attached cells at 48 hr were visualized by crystal violet. (B) The indicated proteins were analyzed by western blotting. (C) Transcripts at 24 hr were analyzed by RT-PCR and normalized to respective cell line controls (Gln+, 1). (D) Venn diagram of Gln deprivation induced genes in three sensitive cell lines. Two fold or more, *p<0.05. (E) Reactome Pathway analysis of shared genes (Silva-Almeida et al., 2020) in (D). (F) A model of p-4E in CRC development. CRC drivers converge on increased p-4E (S209) and p-4E-BP1(S65/T70) to promote Myc- and ISR (p-eIF2a/ATF4)-dependent adaptation and AKT activation via increased glutamine (Gln) metabolism. Acute metabolic stress (i.e. Gln deprivation) disrupts adaptation and triggers crisis in such cells due to rapid loss of p-4E/4E-BP1 and Myc and hyperactivation of ISR and p-AKT, leading to increased cell death and transcriptional heterogeneity.

Figure 7—figure supplement 1
Glutamine deprivation leads to ISR hyperactivation in mutant KRAS/BRAF CRC cells.

RKO and HT29 cells were subjected to glutamine deprivation for 24 hr. The indicated proteins were analyzed by western blotting. *, non specific band.

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