GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

  1. Ricardo A Cordova
  2. Jagannath Misra
  3. Parth H Amin
  4. Anglea J Klunk
  5. Nur P Damayanti
  6. Kenneth R Carlson
  7. Andrew J Elmendorf
  8. Hyeong-Geug Kim
  9. Emily T Mirek
  10. Bennet D Elzey
  11. Marcus J Miller
  12. X Charlie Dong
  13. Liang Cheng
  14. Tracy G Anthony
  15. Robero Pili  Is a corresponding author
  16. Ronald C Wek  Is a corresponding author
  17. Kirk A Staschke  Is a corresponding author
  1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, United States
  2. Indiana University Melvin and Bren Simon Comprehensive Cancer Center, United States
  3. Department of Neurological Surgery, Indiana University School of Medicine, United States
  4. Department of Nutritional Sciences, Rutgers University, United States
  5. Department of Comparative Pathology, Purdue University, United States
  6. Department of Urology, Indiana University School of Medicine, United States
  7. Department of Medical and Molecular Genetics, Indiana University School of Medicine, United States
  8. Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, United States
  9. Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, United States
7 figures and 11 additional files

Figures

Figure 1 with 6 supplements
GCN2 promotes growth of prostate cancer (PCa) cells.

(A) Expression of the indicated eIF2α kinase was reduced in LNCaP cells using gene-specific siRNAs. Two different siRNAs were used for knockdown of each eIF2α kinase and compared to scrambled siRNA …

Figure 1—figure supplement 1
Knockdown of eIF2α kinases and ATF4 in prostate cancer (PCa) cell lines.

(A) LNCaP cells were transfected with the indicated gene-specific siRNAs or a scramble control and the protein levels of HRI, PERK, or PKR and p-eIF2α, total eIF2α, and ATF4 were measured by …

Figure 1—figure supplement 2
CRISPR/Cas9 knockout of GCN2 in 22Rv1 and PC-3 cells.

(A) Immunoblot analyses for 22Rv1, PC-3, and GCN2 KO clones. Protein lysates were analyzed by immunoblot to measure the levels of GCN2, ATF4, p-eIF2α, total eIF2α, or actin. Molecular weight markers …

Figure 1—figure supplement 3
Inhibition of the integrated stress response (ISR) by GCN2iB in prostate cancer (PCa) cell lines.

(A) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured (N

Figure 1—figure supplement 4
Knockdown of ATF4 reduces growth in prostate cancer (PCa) cell lines.

ATF4 expression was reduced in LNCaP, MR49F, C4-2B, or PC-3 cells using ATF4-specific siRNAs. Cell growth was measured in replicate wells (N = 5) for up to 6 days and is plotted as fold change (mean …

Figure 1—figure supplement 5
Inhibition of GCN2 in non-cancerous prostate cell line BPH-1.

(A) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, …

Figure 1—figure supplement 6
p-GCN2 staining of prostate core needle specimens.

H&E and IHC staining using p-GCN2-T899 antibody in prostatic needle biopsy specimens from patients with prostate cancer with Gleason scores 4 + 4 = 8 (top) or 4 + 5 = 9 (bottom). Non-malignant …

Figure 2 with 1 supplement
GCN2 induces integrated stress response (ISR) transcriptome featuring expression of amino acid transporters.

(A) Volcano plot illustrating log2 fold change in gene transcript levels with adjusted p value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. …

Figure 2—figure supplement 1
Inhibition of GCN2 in prostate cancer (PCa) cells reduces expression of SLC genes involved in amino acid transport.

(A) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative …

Figure 3 with 5 supplements
GCN2 is critical for maintenance of free amino acids.

(A) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. Bar graphs in the top panel show high abundance amino acids and the lower panel those with …

Figure 3—figure supplement 1
Amino acid measurements of LNCaP cells treated with vehicle (DMSO), 2 µM GCN2iB, or 2 µM GCN2iB + essential amino acid (EAA) for 48 hr.

Bar graphs in the top panel show high abundance amino acids and the lower panel highlights those with lower levels. Statistical significance was determined using an unpaired two-tailed t-test. Error …

Figure 3—figure supplement 2
Effect of AA supplementation on growth of LNCaP cells.

LNCaP cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) in standard growth media, media supplemented with non-essential amino acids (NEAA), supplemented with essential amino acids …

Figure 3—figure supplement 3
Loss of GCN2 or ATF4 expression in prostate cancer (PCa) cell lines induces cell cycle arrest.

(A) LNCaP, MR49F, C4-2B, 22Rv1, or PC-3 cells were transfected with GCN2 siRNA, ATF4 siRNA, or scrambled siRNA control for 48 hr and subjected to cell cycle analysis as described in the Materials …

Figure 3—figure supplement 4
GCN2 inhibition and supplementation with essential amino acids (EAA) affect charging of tRNAHIS and global translation.

(A) LNCaP cells were treated with 2 µM GCN2iB or vehicle (DMSO) control in the absence or presence of EAA for 8 hr and tRNAHis charging levels were determined by qRT-PCR as described in the …

Figure 3—figure supplement 5
Growth of MR49F and 22Rv1 can be rescued with amino acid supplements.

(A) MR49F cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) for 96 hr in normal growth media, growth media supplemented with essential amino acid (EAA), or growth media supplemented …

Figure 4 with 3 supplements
GCN2 is critical for expression of 4F2 (SLC3A2) and facilitates amino acid transport.

(A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x-axis. Significantly depleted genes (p ≤ 0.05) in …

Figure 4—figure supplement 1
SLC-specific CRISPR/Cas9 KO library reveals that 4F2 (SLC3A2) is critical for cell fitness in LNCaP and 22Rv1 cells.

LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) …

Figure 4—figure supplement 2
GCN2 regulates 4F2 (SLC3A2) mRNA levels in LNCaP cells.

(A) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), (B) cultured in …

Figure 4—figure supplement 3
GCN2 and the integrated stress response (ISR) control expression of 4F2 (SLC3A2) which provides a growth advantage in prostate cancer (PCa) cell lines.

(A) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using …

Model for GCN2 regulation of amino acid homeostasis in prostate cancer.

Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of …

Figure 6 with 4 supplements
GCN2 regulates amino acid transporters ensuring sufficient amino acids for tumor growth in mouse prostate cancer (PCa) xenograft models.

(A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is …

Figure 6—figure supplement 1
Deletion of GCN2 reduces tumor growth and proliferation in 22Rv1 and PC-3 tumors.

(A) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) …

Figure 6—figure supplement 2
Deletion of GCN2 or ATF4 reduces the expression of SLC genes associated with amino acid transport in 22Rv1 tumors.

(A) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), …

Figure 6—figure supplement 3
Effect of essential amino acid (EAA) supplementation on mouse weight, amino acid levels, and expression of SLC genes associated with amino acid transport in 22Rv1 WT and GCN2 KO tumors.

(A) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in Figure 6G. Statistical significance was …

Figure 6—figure supplement 4
Effect of overexpression of 4F2 (SLC3A2) on amino acid levels in 22Rv1 WT and GCN2 KO tumors.

(A) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. …

Figure 7 with 3 supplements
Pharmacological inhibition of GCN2 reduces tumor growth in cell line-derived and patient-derived xenograft models.

Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N = 4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). …

Figure 7—figure supplement 1
GCN2iB reduces proliferation in prostate cancer (PCa) cell line-derived and patient-derived xenograft models and has no effect on mouse body weight.

Male NSG mice were injected subcutaneously with LNCaP (N = 5) or 22Rv1 (N = 4) cells, or implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) or the castration-resistant …

Figure 7—figure supplement 2
Treatment with GCN2 inhibitor, GCN2iB, reduces the expression of SLC genes associated with amino acid transport in 22Rv1 and TM00298 tumors.

Immunoblot analysis of 22Rv1 (A) or TM00298 (B) tumors from mice treated with GCN2iB as described in Figure 7. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The …

Figure 7—figure supplement 3
4F2 (SLC3A2) protein levels are higher in malignant prostate cancer tissue compared to normal prostate tissue.

(A) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue (N = 10) …

Additional files

Supplementary file 1

Statistical analysis for growth assays.

https://cdn.elifesciences.org/articles/81083/elife-81083-supp1-v2.xlsx
Supplementary file 2

Normalized read counts and analysis of RNA-seq data.

https://cdn.elifesciences.org/articles/81083/elife-81083-supp2-v2.xlsx
Supplementary file 3

Normalized read counts and analysis of Charge-seq data.

https://cdn.elifesciences.org/articles/81083/elife-81083-supp3-v2.xlsx
Supplementary file 4

CRISPR screen analysis of depleted genes and single guide RNAs (sgRNAs).

https://cdn.elifesciences.org/articles/81083/elife-81083-supp4-v2.xlsx
Supplementary file 5

Supplementary tables.

https://cdn.elifesciences.org/articles/81083/elife-81083-supp5-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/81083/elife-81083-mdarchecklist1-v2.docx
Source data 1

Western blot source data for main figures, including all individual uncropped western blot images.

https://cdn.elifesciences.org/articles/81083/elife-81083-data1-v2.zip
Source data 2

Western blot source data for Figure 1—figure supplement 1 and Figure 1—figure supplement 2, including all individual uncropped western blot images.

https://cdn.elifesciences.org/articles/81083/elife-81083-data2-v2.zip
Source data 3

Western blot source data for Figure 1—figure supplement 3, including all individual uncropped western blot images.

https://cdn.elifesciences.org/articles/81083/elife-81083-data3-v2.zip
Source data 4

Western blot source data for Figure 1—figure supplement 5, Figure 3—figure supplement 2, Figure 3—figure supplement 4, Figure 4—figure supplement 3, and Figure 6—figure supplement 2, including all individual uncropped western blot images.

https://cdn.elifesciences.org/articles/81083/elife-81083-data4-v2.zip
Source data 5

Western blot source data for Figure 6—figure supplement 3, Figure 6—figure supplement 4, and Figure 7—figure supplement 2, including all individual uncropped western blot images.

https://cdn.elifesciences.org/articles/81083/elife-81083-data5-v2.zip

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