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.

1. Introduction

Colorectal cancer is one of the most common digestive cancers in the human cancer spectrum; it has an annual incidence of 1.8 million cases and the second-highest mortality rate worldwide [1,2]. Owing to the low rate of early diagnosis and the lack of effective therapeutic targets, the 5-year survival rate of affected patients is very low [3,4]. Colorectal cancer is highly anabolic with increased glutaminolysis and glycolysis, which can induce a hypercatabolic state in patients. Mechanistically, both glutamine metabolism and glycolysis (the Warburg effect) play pivotal roles in promoting malignant proliferation and metastasis [5]. During treatment, cancer cells can exploit metabolic plasticity to resist metabolic and energetic blockade. Therefore, the inhibition of both glutaminolysis and glycolysis has become an emerging and promising drug discovery approach to combat cancer [6].

Notably, glutamine metabolism can interact with glycolysis to provide a carbon source for the glycolytic tricarboxylic acid cycle [7–9]. In terms of mechanism, the intracellular signaling pathways of glycolysis and glutaminolysis are altered by oncogene activation and tumor suppressor gene inactivation [10]. The conversion of glutamine to glutamate is catalyzed by the mitochondrial glutaminase [11]. However, the molecular mechanisms by which glycolysis and glutaminolysis interact are not well defined.

Activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor, is ubiquitously expressed throughout the body. Moreover, the ATF4 protein is induced in response to amino acid limitation or hypoxia to control life-death decisions. Once induced, ATF4 can directly control the transcription of several adaptive genes (metabolic or redox balance enzymes) and proapoptotic genes. On the other hand, ATF4 indirectly modulates autophagy and protein synthesis to influence survival [12,13]. Therefore, identifying the mechanisms that modulate the effects of ATF4 on cellular metabolism may reveal new targets for the treatment of colorectal cancer.

Solute carrier family 1 member 5 (SLC1A5), also known as ASCT2, is a transporter required for high-affinity uptake of glutamine by rapidly proliferating tumor cells. Glutamine uptake and subsequent glutaminolysis are vital for the activation of the mechanistic target of the rapamycin complex 1 (mTORC1) nutrient-sensing pathway, which can regulate cancer cell growth [14]. In colorectal cancer cells, SLC1A5 expression is upregulated to promote tumor cell proliferation, and the upregulation of SLC1A5 is significantly correlated with vascular invasion, depth of invasion, and distant metastasis [15]. Notably, ATF4 is considered a possible regulator of SLC1A5, and ATF4-controlled survival mechanisms confer synthetic vulnerability to targeting glutaminolysis and glycolysis [16]. However, it is currently unclear how ATF4 regulates SLC1A5 in colorectal cancer.