ATF4 promotes glutaminolysis and glycolysis in colorectal cancer by transcriptionally inducing SLC1A5

Abstract

Glutaminolysis and glycolysis promote the malignant progression of colorectal cancer. The role of activating transcription factor 4 (ATF4) in solute carrier family 1 member 5 (SLC1A5)-mediated glutaminolysis and glycolysis remains to be elucidated. SLC1A5 and ATF4 expression levels are detected in colorectal cancer tissues. ATF4 is knocked down or overexpressed to assess its role in cell viability, migration and invasion. SLC1A5 is knocked down to evaluate its role in cell viability, migration, invasion, and metastasis and the metabolism of glutamine and glucose. The regulatory effect of the transcription factor ATF4 on SLC1A5 transcription and expression is determined using a luciferase reporter assay and chromatin immunoprecipitation (ChIP) techniques. Upregulated ATF4 and SLC1A5 expressions are observed in tumor tissue, which is positively correlated with the tumor, node, and metastasis (TNM) stages. ATF4-overexpressing SW480 cells show the increased cell viability, migration and invasion. Conversely, ATF4 knockdown decreases the viability, migration and invasion of HCT-116 cells. SLC1A5 knockdown inhibits viability, migration, invasion, and metastasis and the metabolism of glutamine and glucose in HT-29 cells, as well as the expressions of two key glycolytic enzymes, hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2). The luciferase activity of the SLC1A5 promoter is increased by ATF4 overexpression. SLC1A5 promoter enrichment is increased by anti-ATF4 antibody immunoprecipitation in ATF4-overexpressing colorectal cells, indicating that ATF4 targets SLC1A5 to promote glutamine and glucose metabolism in these cells. In summary, the ATF4/SLC1A5 axis plays a significant role in the progression of colorectal cancer by regulating glutamine metabolism and glycolysis.

Keywords: ATF4; SLC1A5; colorectal cancer; glycolysis.

Figure 1

ATF4 and SLC1A5 expressions in colorectal cancer tissues and cell lines (A) ATF4 protein expression in colorectal cancer tissue was analyzed via immunohistochemistry in clinical colorectal cancer tissue microarrays. (B) The relative protein expression of ATF4 was quantitatively analyzed with ImageJ software. (C) SLC1A5 protein expression in colorectal cancer tissue was analyzed via immunohistochemistry in clinical colorectal cancer tissue microarrays. (D) Relative protein expression of ATF4 in clinical colorectal cancer tissues with or without lymph node metastasis (LNM). (E) The relative protein expression of ATF4 in colorectal cancer tissues with or without LNM was quantitatively analyzed with NIH ImageJ software. (F) Kaplan-Meier plot of the cumulative probability of remaining event free for patients with colorectal cancer. * P < 0.05. (G) ATF4 mRNA expression in colon adenocarcinoma (COAD) samples in the Gene Expression Profiling Interactive Analysis (GEPIA) database was determined. (H) SLC1A5 mRNA expression in COAD samples in the GEPIA database was determined.

Figure 2

ATF4 knockdown inhibits the viability, migration, invasion, and metastasis of colorectal cancer cells (A,B) The relative mRNA (A) and protein (B) expression levels of ATF4 were examined in colorectal cancer cell lines. (C,D) Wound healing assay (C) and transwell assay (D) of ATF4-transfected SW480 cells. (E,F) HCT-116 cells transfected with ATF4 shRNA were assayed via a wound healing assay (E) and a transwell assay (F). (G,H) The cell viability of ATF-overexpressing SW480 cells (G) and ATF-knockdown HCT-116 cells (H) was determined with a Cell Counting Kit-8 assay. (I,J) Apoptosis in ATF-overexpressing SW480 cells (I) and ATF-knockdown HCT-116 cells (J) was detected by Annexin-V-FITC/propidium iodide staining. *P < 0.05, *** P < 0.001.

Figure 3

SLC1A5 knockdown inhibits the viability, migration, invasion, and metastasis of colorectal cancer cells (A,B) SLC1A5 mRNA (A) and protein (B) expression was examined in HT-29 cells expressing SLC1A5 shRNA-1, -2, or -3 and compared with that in cells expressing nonspecific shRNA as the negative control (shNC). (C–F) Cell viability (C), wound healing (D,E), and invasion (F) of HT-29 cells expressing shSLC1A5-1, -2, or shNC. BALB/c nude mice were injected with HT-29 cells expressing shSLC1A5-1 or shNC. (G,H) Lung tissues were isolated for H&E staining (G) and metastatic node counting (H). Scale bar: 500 μm. **P < 0.01, ***P < 0.001 vs shNC; ###P < 0.001, shSLC1A5-1 vs shSLC1A5-2.

Figure 4

ATF4 promotes SLC1A5 expression (A–D) ATF4 (A,B) and SLC1A5 (C,D) expressions in SW480 cells transfected with an ATF expression vector (oeATF4) or a blank vector. (E) The binding sites of ATF4 and SLC1A5 were predicted by JASPAR. (F) Luciferase activity of the wild-type (WT) or mutant (MUT) SLC1A5 promoter in SW480 cells transfected with oeATF4 or the empty vector. (G) The enrichment of the SLC1A5 promoter in anti-ATF4 immunoprecipitates from cells transfected with the WT or MUT SLC1A5 promoter and the oeATF4 or blank vector was determined by qPCR. **P < 0.01, ***P < 0.001 vs vector.

Figure 5

ATF4 promotes the growth and metastasis of colorectal cancer by targeting SLC1A5 in vivo (A) Representative images were taken on the 14th day (left panel), and quantification of metastatic cells in the mouse body was performed via bioluminescence analysis (right panel). (B,C) Mice were sacrificed, and colorectal tissue and lungs were removed from the mice. Images showing metastases in the lungs. (D,E) ATF4 and SLC1A5 expressions in primary lesions in colorectal tissue. (F,G) ATF4 and SLC1A5 expressions in lung metastases. *P < 0.05, **P < 0.01, ***P < 0.001 vs shNC; ##P < 0.01, ###P < 0.001 vs shATF4; &&&P < 0.001 vs shATF4 + Rotenone.

Figure 6

SLC1A5 knockdown inhibits glutamine and glucose metabolism in colorectal cancer cells (A–E) Glutamine uptake (A), glucose uptake (B), ATP level (C), lactate content (D), and expression of HK2 and PKM2 (E) in colorectal cancer cells expressing shSLC1A5-1, -2, or shNC. ***P < 0.001 vs shNC.

Figure 7

ATF4 promotes glutamine and glucose metabolism in colorectal cancer cells by targeting SLC1A5 (A–F) SLC1A5 expression (A), cell viability (B), glutamine uptake (C), glucose uptake (D), ATP (E), and lactate content (F) in colorectal cancer cells expressing shSLC1A5 and/or oeATF4. **P < 0.01, ***P < 0.001 vs vector + shNC. ###P < 0.001 vs oeATF4 + shNC.

Figure 8

ATF4 promotes glutaminolysis and glycolysis in colorectal cancer by targeting SLC1A5 in vivo (A,B) Immunofluorescence of Ki67 in primary and metastatic lesions. (C) Glutamine uptake. (D) Glucose uptake. (E) Lactate content of metastases in the lungs. *P < 0.05, **P < 0.01, ***P < 0.001 vs shNC; #P < 0.05, ##P < 0.01, ###P < 0.001 vs shATF4; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs shATF4 + Rotenone.