Methionyl-tRNA synthetase overexpression is associated with poor clinical outcomes in non-small cell lung cancer

Lung cancer is a major public health problem worldwide. This disease has a high prevalence and high mortality; specifically, lung cancer had the 3rd lowest survival rate among all cancers in 2014 [ 1, 2]. Thus, there is an urgent need for new diagnostic methods enabling early detection and for new treatment modalities for lung cancer.

Low-dose computerized tomography (LDCT) scans are currently used as a screening tool. Their use is supported by the National Lung Screening Trial (NLST), a randomized collected study involving more than 53,000 current or former heavy smokers [ 3]. However, lung cancer screening with LDCT has serious problems, such as false-positive rates exceeding 95%. This drawback leads to unnecessary repeated testing and increased costs [ 4]. Because of this and other limitations, new noninvasive methods for the early detection of lung cancer are needed. Multiple peripheral blood or body fluid matrix biomarkers in lung cancer have been proposed, such as protein biomolecules, miRNA, cell-free DNA, methylated DNA, circulating tumor cells, metabolites, and lipids [ 5]. However, protein markers are the only type of cancer biomarkers approved by the Food and Drug Administration (FDA) to date [ 6]. Protein biomarkers are easy to detect with high sensitivity and/or specificity in peripheral blood.

Aminoacyl-tRNA synthetases (ARSs) are housekeeping enzymes that catalyze the ligation of amino acids to their cognate transfer RNAs (tRNAs) with high fidelity [ 7]. Consuming one ATP in each reaction, these enzymes activate amino acids to aminoacyladenylates and then deliver the activated amino acids to the acceptor ends of tRNAs [ 8]. Mammalian ARSs have additional domains such as a glutathione S-transferase domain, a WHEP domain, leucine zipper domains, and α-helical appendices, which enable them to perform versatile intracellular and intercellular functions. Among the free-form ARSs, tryptophanyl-tRNA synthetase (WRS) and tyrosyl-tRNA synthetase (YRS) can be secreted and modified to control angiogenesis and immune responses in the tumor microenvironment. Truncation of the amino-terminal of WRS generates cytokines that suppress angiogenesis [ 9]. YRS is cleaved into N- and C-terminal domains, which have proangiogenic and immune activation functions, respectively [ 10, 11]. One the other hand, 8 different ARSs [bifunctional glutamyl-prolyl-tRNA synthetase (EPRS), isoleucyl-tRNA synthetase (IRS), leucyl-tRNA synthetase (LRS), glutaminyl-tRNA synthetase (QRS), lysyl-tRNA synthetase (KRS), arginyl-tRNA synthetase (RRS), aspartyl-tRNA synthetase (DRS), and methionyl tRNA synthetase (MRS)] form a complex with ARS-interacting multifunctional proteins (AIMPs) and play noncanonical roles [ 8]. EPRS is a translational silencer that suppresses vascular endothelial growth factor A [ 12]. KRS binds to microphthalmia-associated transcription factor (MITF) and is involved in the development of melanoma [ 13]. QRS interacts with apoptosis signal-regulating kinase 1 and suppresses apoptosis in a glutamine-dependent manner [ 14]. These reports suggested that ARS overexpression may impact cancer survival and progression and that ARS inhibitors are thus potential anticancer therapeutics. In addition, the multifunctional nature of ARSs and their localization to multiple areas suggest their potential as cancer diagnostic biomarkers in peripheral blood and tissue.

MRS is a critical enzyme in translation initiation and transfers Met to the initiator tRNA (tRNA _i ^Met), suggesting that this enzyme may play an important role in tumor growth [ 15]. MRS increases ribosomal RNA biogenesis in the nucleolus and interacts with various signaling molecules such as mTORC1, GCN2, CDK4, and VEGFR [ 7, 16, 17]. After phosphorylation of MRS at Ser662 by UV-mediated DNA damage, MRS dissociates from AIMP3 and links the DNA damage responses to global translation control [ 15]. For this reason, MRS has been considered a strong biomarker candidate for the therapy of lung cancer.

Here we evaluated MRS expression in mouse tissue and in human lung cancer tissue to determine its clinical implications. In addition, we evaluated the relationship between MRS expression and the mTOR pathway, which plays a critical role in cancer growth and proliferation, to examine the relationship between tumor growth and MRS expression.

Besides its canonical role as a translation initiator by transferring Met to initiator tRNA _i ^Met, MRS has multiple noncanonical functions. MRS senses intracellular Met, leading to the activation of mTORC1 signaling [ 18], and also stabilizes CDK4, thereby inducing cell cycle progression (unpublished data). MRS also detects intracellular oxidative stress, defends cells from DNA damage, and controls protein synthesis [ 15]. In addition to these roles, the discovery of MRS genetic variations in the late-onset autosomal dominant Charcot–Marie–Tooth neuropathy indicates that MRS has additional uncharacterized roles [ 19].

We evaluated MRS expression in the organs of wild type C57BL/6 mice and observed only low levels of expression. Remarkably, MRS was strongly expressed in heart tissue, whereas MRS expression was lower in hepatic tissues, where protein is more actively synthesized than in the spleen and intestine. MRS expression was weakly correlated with mTOR signaling components in wild type mouse tissue samples; however, the correlation between mTORC1 signaling and MRS overexpression was more prominent in the NSCLC tissue samples. These findings suggest that the regulatory mechanisms are working properly in normal tissue, but that in cancer tissue these mechanisms are dysregulated and the cancer cells are dependent on mTOR signaling. However, more evidence is required to prove that activation of the mTOR pathway induces MRS overexpression.

The majority of the NSCLC tissue samples showed clear expression of MRS, with cytoplasmic expression more frequent than nuclear expression. Nuclear MRS has unique functions in growth stimulating conditions related to ribosomal RNA biosynthesis [ 20]. In this study, a small percentage (31.6%) of NSCLC cells showed nuclear MRS expression, which was not statistically significant. This finding might be due to the fact that few cases exhibited nuclear MRS overexpression, meaning that the sample size was too small to detect significance.

When the Kaplan-Myer estimator was used to assess the clinical implications of MRS expression, MRS expression was not found to be an independent prognostic factor for DFS. This finding may be due to the positive correlation between the degree of MRS expression and the age of the study participant (Pearson correlation coefficient = 0.145, P = 0.027) and the strong relationship between pStage and MRS expression. Evaluation of the individual TNM staging components revealed that maximal tumor diameter and T staging component were associated with MRS expression. Moreover, MRS expression was significantly higher with each N stage increment ( P = 0.007, Pearson’s χ ²-test), suggesting that MRS may play a role in lymphangitic metastasis. Using the gene expression datasets of lung adenocarcinoma and lung squamous cell carcinoma from the TCGA, the effect of MRS gene expression level on clinical outcome was analyzed. In the lung cancer dataset that includes all stages, there were no prominent differences in clinical outcome according to MRS level, whereas in the subgroup of stage III ~ IV the patients with elevated MRS level tended to show a poor clinical outcome (Additional file 4 A-C).

We anticipate that this initial confirmation of MRS expression in cancer tissue and demonstration of its clinical implications will lead into future investigations of the potential of MRS expression a therapeutic target for drug development. To be developed as a diagnostic biomarker, MRS expression needs to be easily evaluated in readily obtainable bodily fluid. Also, the mechanisms driving MRS overexpression need to be elucidated. One possible explanation of MRS overexpression is copy number gain at the MRS gene locus (COSMIC); however, only limited cases showed copy number gain. In order to develop MRS as a therapeutic target, a targetable site also needs to be identified in MRS.

In conclusion, MRS is specifically and frequently overexpressed in NSCLC tissue. MRS overexpression in NSCLC tissue was related to mTORC1 activity and poor clinical outcomes, suggesting that further studies on MRS overexpression are warranted to pursue MRS as a therapeutic target.