Editor's evaluation: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis (2022)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa. Editor's evaluation This manuscript is an important body of work that addresses the role of the integrated stress response (ISR) and the role of the GCN2 protein kinase in prostate cancer. The studies comprehensively elucidate how GCN2 and amino acid transporters and uptake promote prostate cancer proliferation, as well as the therapeutic potential of inhibiting this pathway. This work, therefore, provides insights for both identification of new mechanisms and experimental therapeutics in prostate cancer. https://doi.org/10.7554/eLife.81083.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Prostate cancer is the fourth most common cancer worldwide, affecting over a million people each year. Existing drug treatments work by blocking the effects or reducing the levels of the hormone testosterone. However, these drug regimens are not always effective, so finding alternative treatments is an important area of research. One option is to target the 'integrated stress response', a pathway that acts as a genetic switch, turning on a group of genes that counteract cellular stress and are essential for the survival of cancer cells. The reason cancer cells are under stress is because they are hungry. They need to make a lot of proteins and other metabolic intermediates to grow and divide, which means they need plenty of amino acids, the building blocks that make up proteins and fuel metabolism. Amino acids enter cells through molecular gates called amino acid transporters, and scientists think the integrated stress response might play a role in this process. One of the integrated stress response components is a protein called General Control Nonderepressible 2, or GCN2 for short. In healthy cells, this protein helps to boost amino acid levels when supplies start to run low. Cordova et al. examined human prostate cancer cells to find out what role GCN2 plays in this cancer. In both lab-grown cells and tissue from patients, GCN2 was active and played a critical role in prostate tumor growth by turning on the genes for amino acid transporters to increase the levels of amino acids entering the cancer cells. Deleting the gene for GCN2, or blocking its effects with an experimental drug, slowed the growth of cultured prostate cancer cells and reduced tumor growth in mice. In these early experiments, Cordova et al. did not notice any toxic side effects to healthy tissues. If GCN2 works in the same way in humans as it does in mice, blocking it might help to control prostate cancer growth. The integrated stress response is also active in other cancer types, so the same logic might apply to different tumors. However, before GCN2 blockers can become treatments, researchers need a more complete understanding of their molecular effects. Introduction Prostate cancer (PCa) accounts for 20% of all newly diagnosed cancers and is the second leading cause of cancer-related deaths in men in the United States (Siegel et al., 2019). Many factors contribute to PCa, including genetics, diet, age, and race (Gann, 2002). The pathological grade of the PCa and whether it has disseminated into surrounding tissues is prognostic for risk and treatment efficacy (Klusa et al., 2020). Androgen receptor (AR) is considered the primary oncogenic driver for PCa (Heinlein and Chang, 2004; Huggins and Hodges, 1941), and therapies targeting AR activity are current standard of care, especially for those with metastatic PCa (Polotti et al., 2017; Virgo et al., 2017; Watson et al., 2015; Crawford et al., 2019). Unfortunately, patients develop resistance with time and fail therapy (Watson et al., 2015; Bubley and Balk, 2017). In addition, it remains unclear how additional pathways drive PCa through AR-dependent and AR-independent mechanisms. Therefore, the identification of novel targets for therapeutic intervention are critically needed and clinically relevant. The integrated stress response (ISR) is critical for cell adaptation and survival to environmental stresses. Central to the ISR is phosphorylation of the α subunit of eIF2 (p-eIF2α), which triggers reductions in global protein synthesis to conserve energy and nutrients, and concomitantly facilitates adaptive gene expression (Baird and Wek, 2012; Tian et al., 2021). Four eIF2α kinases respond to distinct stress conditions: The eIF2α kinase GCN2 (EIF2AK4) is induced by amino acid starvation and other conditions that trigger ribosomal collisions or stalling (Dong et al., 2000; Inglis et al., 2019; Wek et al., 1995). PERK (EIF2AK3) is responsive to disruptions in the endoplasmic reticulum (Walter and Ron, 2011; Wek and Cavener, 2007). PKR (EIF2AK2) is activated by viral infection and inflammation (Chukwurah et al., 2021; García et al., 2006), and HRI (EIF2AK1) is enhanced by restricted heme or iron supply, oxidative stress, and disruptions in mitochondrial function (Fessler et al., 2020; Guo et al., 2020; Suragani et al., 2012). Activation of these ISR regulators may also occur in response to oncogenic drivers, such as MYC amplification and loss of the PTEN tumor suppressor, frequently observed in prostate tumors. Therefore, both extrinsic and intrinsic cell stresses can invoke the ISR in tumors and pharmacological inhibition of translational control is reported to trigger cytotoxicity in different cancer models (Staschke and Wek, 2019; Tameire et al., 2019; Nguyen et al., 2018). To reprogram gene expression toward recovery from stress damage, the ISR also induces preferential translation of specific ISR gene transcripts, such as activating transcription factor 4 (ATF4), a transcriptional regulator of genes involved in amino acid metabolism and proteostasis control, oxidative stress defenses, along with feedback control of the ISR (Harding et al., 1999; Young and Wek, 2016; Hinnebusch et al., 2016). Therefore, p-eIF2α induces both translational and transcriptional modes of gene expression to achieve stress adaptation. Targeted inhibition of ATF4 impedes progression of MYC-driven cancers, emphasizing the important role of this ISR effector in tumor progression (Staschke and Wek, 2019; Tameire et al., 2019; Nguyen et al., 2018). Here, we dissect the role of eIF2α kinase(s) induced in PCa and the mechanisms by which the ISR serves to promote growth of this cancer. Using androgen-sensitive and castration-resistant human PCa cell lines, primary human PCa tissues, and mouse xenograft models, we show that the ISR regulator GCN2 is activated in PCa. GCN2 induces expression of key ISR effector genes involved in nutrient transport that are critical for appropriate maintenance of free amino acid pools. Genetic or pharmacological inhibition of GCN2 dysregulates expression of these transporters and inhibits PCa growth. These results point to GCN2 as a potential therapeutic target for PCa. Results GCN2 activity supports PCa growth To determine the role of individual eIF2α kinases and their importance for proliferation of PCa cells in culture, androgen-sensitive LNCaP cells cultured in androgen replete media conditions were individually depleted of HRI, PKR, PERK, or GCN2 using target-specific siRNAs. There was robust reduction of protein levels of each of the eIF2α kinases; however, only cells depleted for GCN2 or PKR showed a significant decrease in proliferation (Figure 1A, B, Figure 1—figure supplement 1A, and Supplementary file 1). Whereas GCN2 depletion lowered p-eIF2α levels and ATF4 expression, knockdown of PKR, HRI, or PERK had no effect on p-eIF2α and ATF4 protein levels (Figure 1B and Figure 1—figure supplement 1A). These results suggest that LNCaP cell proliferation requires GCN2 as an ISR regulator. Figure 1 with 6 supplements see all Download asset Open asset 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 control. Cell growth was measured in replicate wells (N = 5) for up to 6 days and is plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in Supplementary file 1; *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. (B) LNCaP cells were transfected with two different siRNAs targeting GCN2 or a scramble siRNA control and cell lysates were prepared and immunoblotted for the indicated proteins. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to scramble siRNA control are indicated. (C) Expression of GCN2 was knocked-down in LAPC-4, C4-2B, MR49F, 22Rv1, or PC-3 cells using two different siRNAs and compared to scrambled siRNA control. Cell growth was measured for up to 6 days in replicate wells (N = 5) as described in A. Statistical significance was determined using a two-way ANOVA as described in Supplementary file 1; ***p ≤ 0.001, ****p ≤ 0.0001. (D) LNCaP cells were treated with indicated concentrations of GCN2iB and cell growth was measured for up to 6 days in replicate wells (N = 5) as described in A. Statistical significance was determined using a two-way ANOVA as described in Supplementary file 1; ****p ≤0.0001. (E) LNCaP cells were treated with GCN2iB (2 µM) or DMSO control for 24 hr and protein lysates were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin as indicated. Relative levels of p-eIF2α normalized to total eIF2α are shown. (F) Levels of p-GCN2 were measured in prostate tumor microarrays (Biomax PR1921b and PR807c) using immunohistochemistry (IHC). Staining for p-GCN2-T899 from non-malignant (N = 33) and malignant PCa tissue (N = 88) from patients >50 years old was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t-test; *p ≤ 0.05. Representative images showing p-GCN2-T899 staining of normal and malignant prostate tissues are shown. Scale bars shown are 200 µm (main image) and 20 µm (insert). To broaden our investigation to other PCa cell lines, we knocked down GCN2 in the androgen-sensitive line LAPC-4 and in the castration-resistant cell lines C4-2B, MR49F, 22Rv1, and PC-3. MR49F cells express AR, but are resistant to enzalutamide (Kuruma et al., 2013), 22Rv1 cells express the ARv7 splice variant and are also resistant to enzalutamide (DeSantis et al., 2016; Li et al., 2013; Sramkoski et al., 1999), PC-3 is AR null (Tilley et al., 1990). In each of these PCa cell lines, depletion of GCN2 reduced proliferation and lowered ATF4 expression (Figure 1C, Figure 1—figure supplement 1B, and Supplementary file 1). In 22Rv1 and PC-3 cells, we were also able to achieve a deletion of EIF2AK4 (GCN2) using CRISPR/Cas9, which reduced p-eIF2α and ATF4 levels (Figure 1—figure supplement 2A). In the case of 22Rv1, we determined a similar slow growth phenotype as observed with siRNA knockdown of GCN2 (Figure 1—figure supplement 2B and Supplementary file 1). Reintroduction of GCN2 into the GCN2 KO cells rescued the slow growth phenotype, and expression of ATF4 and its target gene ASNS (Figure 1—figure supplement 2B, Figure 1—figure supplement 2C, and Supplementary file 1). The small molecule GCN2iB is a selective inhibitor of GCN2 eIF2α kinase activity (Nakamura et al., 2018). Using the inhibitory concentration range of 500 nM to 10 µM of GCN2iB, we determined that pharmacological inhibition of GCN2 also caused significant growth inhibition of LNCaP cells, and similarly reduced the growth of C4-2B, 22Rv1, and PC-3 cells (Figure 1D, Figure 1—figure supplement 3A, and Supplementary file 1). Treatment of GCN2 KO cells with up to 10 µM GCN2iB had no effect on growth demonstrating the specificity of this inhibitor (Figure 1—figure supplement 3B and Supplementary file 1). As expected, treatment of the LNCaP, 22Rv1, C4-2B, or PC-3 cells with GCN2iB lowered activation of GCN2 as measured by autophosphorylation of the activation loop (p-GCN2-T899), reduced p-eIF2α levels, and lowered expression of ATF4, and its target genes ASNS and TRIB3. Importantly, treatment with GCN2iB did not alter protein levels of AR (Figure 1E and Figure 1—figure supplement 3C). To further test the importance of the ISR in PCa, we selectively depleted ATF4 using siRNA in LNCaP, MR49F, C4-2B, and PC-3 cells and observed similar growth inhibition for each cell line (Figure 1—figure supplement 1B, Figure 1—figure supplement 4, and Supplementary file 1). Importantly, GCN2 is not active in the non-cancerous prostate epithelial cell line BPH-1 (Hayward et al., 1995), and loss of GCN2 or ATF4, or pharmacological inhibition of GCN2, did not reduce p-eIF2α or ATF4 levels, or significantly impact the growth of these cells (Figure 1—figure supplement 5A, Figure 1—figure supplement 5B, Figure 1—figure supplement 5C, and Supplementary file 1). These results support the hypothesis that the eIF2α kinase activity of GCN2 and the ISR support PCa proliferation. We next sought to determine whether GCN2 is activated in human malignant PCa tissues. Using immunohistochemistry and prostate tumor microarrays featuring normal and non-malignant prostate tissue (N = 33) and malignant tumor tissues (N = 88), we determined that there was enhanced GCN2 activity in the tumor samples compared to normal tissues as measured by p-GCN2-T899 staining (Figure 1F). In addition, increased p-GCN2 (Thr-899) staining was also observed in prostate tumors from patient core needle biopsies as compared to adjacent non-malignant tissue (Figure 1—figure supplement 6). Collectively, these results indicate that GCN2 is active basally in both androgen-sensitive and castration-resistant PCa models without appreciable extrinsic stress treatments, confers growth advantages, and is activated in PCa patient tissues. GCN2 induces ISR transcriptome featuring expression of amino acid transporters To address the mechanisms by which GCN2 confers growth advantages in PCa, we cultured LNCaP cells in the presence or absence of GCN2iB and then measured changes in the transcriptome by RNA-seq. With 24 hr of GCN2iB treatment, 239 transcripts showed a significant ≥twofold increase, whereas 374 transcripts were decreased ≥twofold (fdr ≤0.05) compared to vehicle-treated cells (Figure 2A, Supplementary file 5). We carried out a gene set enrichment analysis (GSEA) of the differentially expressed transcripts to distinguish the molecular functions that were altered by GCN2 inhibition. Included among the functional gene groups showing significant dependence on GCN2 for expression were those linked to amino acid deprivation and transport (Figure 2B, C). These genes are representative of ISR-targeted gene groups and include genes regulated by ATF4 such as DDIT3 (CHOP), ASNS, and TRIB3, those involved in glycine–serine biosynthesis (PHGDH, PSAT1, PSPH, and SHMT2), aminoacyl-tRNA synthetases, and a large number of SLC transporters, many of which function in the transport of amino acids (Figure 2B and Supplementary file 2). As illustrated in the heat map, expression of the SLC genes was significantly lowered after 6 hr of GCN2iB treatment, with further reductions following 24 hr (Figure 2C). It is noteworthy that of the 61 known SLC genes involved in the transport of amino acids, 27 were measurably expressed in LNCaP cells, and 20 of these expressed transcripts were significantly decreased following treatment with GCN2iB (Figure 2B and Supplementary file 2). Figure 2 with 1 supplement see all Download asset Open asset 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. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. (C) Heat map displaying significantly downregulated SLC genes as indicated in panel A. The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t-test (N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Lowered expression of SLC proteins upon GCN2 inhibition was further validated by immunoblot analyses. xCT (SLC7A11), CAT-1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), and 4F2 (SLC3A2) protein levels were sharply lowered with GCN2iB treatment of LNCaP cells, along with reduced expression of ATF4 and its target gene ASNS (Figure 2D and Figure 2—figure supplement 1A). Similarly, treatment of C4-2B, 22Rv1, and PC-3 cells with GCN2iB also reduced LAT-1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) (Figure 1—figure supplement 3C). We also measured transporter proteins in 22Rv1 and PC-3 cells and showed that deletion of EIF2AK4 (GCN2) also diminished protein levels of xCT (SLC7A11), LAT-1 (SLC7A5) (LAT1), and 4F2 (SLC3A2) (Figure 2E, Figure 2—figure supplement 1B). Furthermore, the reintroduction of GCN2 fully restored expression of ATF4 and ASNS and partially restored expression of all three SLC proteins in 22Rv1 cells (Figure 2E). To more directly address the effects of GCN2 inhibition on amino acid transport, we measured uptake of leucine, lysine, and arginine in LNCaP and 22Rv1 cells. Treatment of LNCaP cells with GCN2iB or deletion of GCN2 in 22Rv1 cells significantly lowered the uptake of each of these amino acids (Figure 2F, G), suggesting that GCN2-directed gene expression is important for uptake of amino acids in PCa cells. GCN2 is critical for maintenance of free amino acid pools Given that GCN2 is important for expression of SLC genes and amino acid uptake, we next addressed whether the intracellular levels of free amino acids are disrupted upon inhibition of GCN2. LNCaP cells were cultured in the presence or absence of GCN2iB for 8 hr and free amino acid levels were measured. With pharmacological inhibition of GCN2, there was a sharp reduction in the amounts of essential amino acids (EAA), including histidine, the branched-chain amino acids, threonine, phenylalanine, and methionine, and non-essential amino acids (NEAA), such as glutamate and glutamine, aspartate, asparagine, alanine, and (Figure We next considered whether the with additional amino acids the growth that with the inhibition of GCN2. GCN2iB treatment of LNCaP cells cultured in standard reduced of additional into the this proliferation and increased free amino acid pools in treated cells (Figure Figure supplement Figure supplement 2, and Supplementary file 1). with additional did not the LNCaP growth by GCN2iB treatment (Figure supplement 2 and Supplementary file 1). Figure with supplements see all Download asset Open asset GCN2 is critical for maintenance of free amino (A) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. in the panel show amino acids and the panel those with levels. The heat map on the fold change in amino acid for each biological replicate of LNCaP cells versus the vehicle with the showing the fold change in and in Statistical significance was determined using an unpaired two-tailed bars indicate standard deviation (N = *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (B) LNCaP cells were treated with GCN2iB (2 vehicle essential amino acids (EAA), or GCN2iB (2 µM) and cell growth was measured for up to 6 bars indicate (N = 5). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in Supplementary file 1; ****p ≤ 0.0001. (C) Cell of LNCaP cells treated with GCN2iB (2 vehicle or GCN2iB (2 µM) for hr. Statistical significance was determined using a ANOVA with bars indicate (N = ***p ≤ 0.001, ****p ≤ 0.0001. (D) analysis of LNCaP cells treated with vehicle (DMSO), GCN2iB (2 or GCN2iB (2 µM) for 8 hr. The is shown as a with fold change compared to measured in LNCaP cells are shown. bars indicate (N = (E) for in LNCaP cells treated with GCN2iB, or GCN2iB Statistical significance was determine using a ANOVA with (N = 4); ***p ≤ 0.001, ****p ≤ 0.0001. (F) LNCaP cells were treated with GCN2iB (2 GCN2iB (2 µM) or GCN2iB (2 µM) with the indicated individual amino Cell growth was measured 4 days in wells (N = Statistical significance was determined using a ANOVA with bars indicate ****p ≤ 0.0001. (G) Cell analysis of LNCaP cells were treated with GCN2iB (2 GCN2iB (2 µM) or with media for hr. Statistical significance was determined using a ANOVA with bars indicate (N = ***p ≤ 0.001, ****p ≤ 0.0001. LNCaP cells were cultured in normal media with or media with µM) for 24 hr. Lysates were analyzed by using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin. Molecular weight markers are in for each immunoblot The relative levels of p-eIF2α normalized to total eIF2α compared to normal media control are indicated. To address the effect of GCN2 inhibition and on the cell in cultured PCa cells, LNCaP cells were treated with GCN2iB or GCN2 expression was reduced with methods of GCN2 depletion caused an increase in the of the cell with a reduction in of control of the cell that can occur with amino acid depletion et al., 2015; et al., 2004; Figure and Figure supplement of LNCaP cells with the of cells in by GCN2iB (Figure 3C). These results suggest that inhibition of GCN2 can cause cell which was with siRNA knockdown of GCN2 or ATF4 (Figure supplement were observed in 22Rv1 cells for GCN2 or in other PCa cell lines when GCN2 or ATF4 levels were reduced with siRNA (Figure supplement and Figure supplement upon depletion of amino acids and is a primary activating GCN2 et al., 2017). We cultured LNCaP cells in the presence or absence of GCN2iB and measured using et al., 2020). There was a reduction of upon GCN2iB treatment that was restored when additional were to the growth (Figure and Supplementary file These results were further by measurements of levels by Addition of to cultured LNCaP cells significantly increased levels treatment with GCN2iB reduced levels which was by the of (Figure supplement These results support the that depletion and the of can to activation of GCN2. treatment with GCN2iB, which lowered p-eIF2α and appropriate of the there was depletion of by of in a slow growth To further address the role of availability in the slow growth phenotype with GCN2 we cultured LNCaP cells in with supplements of individual amino In with our only the of to the the growth inhibition by GCN2iB (Figure and Figure supplement 2). of from the growth increased similar to GCN2iB treatment, of additional to the the cell by GCN2iB treatment (Figure with the of or significantly lowered and ATF4 expression, it is that only of the ISR compared to of (Figure this reduction in activation of GCN2, with sharply increased of (Figure and there was a toward enhanced global protein synthesis as measured by It is that there was a toward decreased translation upon GCN2iB treatment that was restored with the of (Figure supplement As GCN2 inhibition for hr the cell and amino acid levels, which are suggested to reduce translation upon GCN2iB treatment lowered p-eIF2α levels. We next determined whether in other PCa cell lines also the slow growth phenotype by loss of GCN2 Addition of or also the slow growth phenotype of MR49F cells treated with GCN2iB (Figure supplement However, in 22Rv1 cells, or the slow growth with deletion of EIF2AK4 (GCN2) (Figure supplement These growth studies suggest that GCN2 is for in PCa cells, the by which GCN2 out these functions can among PCa cell lines to in their genetic and mechanisms specific gene expression and metabolic of GCN2 in LNCaP and MR49F cells to whereas deletion of GCN2 22Rv1 cells to SLC transporters are critical for PCa Given that GCN2 has a primary role for expression of SLC genes, we sought to determine the of these transporters for PCa growth and To their in an we a reported targeting human SLC genes and et al., 2020). of the SLC genes was with specific along with for target specificity and for cell