RIOK3 promotes mTORC1 activation by facilitating SLC7A2-mediated arginine uptake in pancreatic ductal adenocarcinoma.

www.aging-us.com AGING 2023, Vol. 15, No. 4 Research Paper RIOK3 promotes mTORC1 activation by facilitating SLC7A2mediated arginine uptake in pancreatic ductal adenocarcinoma Henan Qin1,*, Rui Sun1,*, Xin Guo1,3,*, Lei Fang1, Mengyuan Xu2, Yibin Teng1, Ning Zhen1,3, Aman Wang1,3,&, Jiwei Liu1,3 1 The First Affiliated Hospital of Dalian Medical University, Dalian 116000, China Hangzhou Medical College Affiliated Lin’an People’s Hospital, Hangzhou 310000, China 3 Liaoning Key Laboratory of Molecular Targeted Drugs in Hepatobiliary and Pancreatic Cancer, Dalian 116000, China *Equal contribution 2 Correspondence to: Ning Zhen, Aman Wang, Jiwei Liu; email: , ; https://orcid.org/0000-0002-8856-4702, Keywords: RIOK3, SLC7A2, arginine metabolism, pancreatic ductal adenocarcinoma, Treg Received: October 14, 2022 Accepted: February 6, 2023 Published: February 24, 2023 Copyright: © 2023 Qin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor prognosis. Reprogramming of amino acid metabolism is one of the characteristics of PDAC, in which arginine metabolism is significantly altered in PDAC cells and is involved in important signaling pathways. Current studies have identified arginine deprivation as a potential strategy for PDAC treatment. In this study, we performed Liquid Chromatograph Mass Spectrometer (LC-MS)-based non-targeted metabolomic analysis on PDAC cell lines with stable Rio Kinase 3 (RIOK3) knockdown and PDAC tissues with different RIOK3 expressions and found that RIOK3 expression was significantly correlated with arginine metabolism in PDAC. Subsequent RNA sequencing (RNA-Seq) and Western blot analysis showed that RIOK3 knockdown significantly inhibited the expression of arginine transporter solute carrier family 7 member 2 (SLC7A2). Further studies revealed that RIOK3 promoted arginine uptake, mechanistic target of rapamycin complex 1 (mTORC1) activation, cell invasion, and metastasis in PDAC cells via SLC7A2. Finally, we found that patients with high expression of both RIOK3 and infiltrating Treg cells had a worse prognosis. Overall, our study found that RIOK3 in PDAC cells promotes arginine uptake and mTORC1 activation through upregulation of SLC7A2 expression, and also provides a new therapeutic target for therapeutic strategies targeting arginine metabolism. INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumors of high malignancy. The 2020 World Health Organization (WHO) GLOBOCAN project estimates that there will be more than 490,000 new cases of pancreatic cancer and more than 460,000 new deaths worldwide in a year, and the mortality to incidence ratio (M/I) is about 0.94, ranking first among all common tumor species [1, 2]. Currently, the overall 5-year survival rate for PDAC is only about 7%, and it www.aging-us.com 1039 is estimated that by 2030, PDAC deaths will be the 2nd leading cause of tumor-related death in the United States after non-small cell lung cancer [3]. Surgical resection is the only effective treatment for PDAC, and chemotherapy remains the first choice for patients with unresectable PDAC [4]. However, multiple treatments such as chemotherapy, radiotherapy, immunotherapy, and targeted therapy have not significantly improved the long-term survival rate of PDAC patients [5–7]. Therefore, a breakthrough is still needed in the treatment of PDAC. AGING Metabolic reprogramming is an important feature of tumors [8]. In addition to glucose and lipid metabolic reprogramming, abnormal amino acid metabolism also plays an important role in the rapid growth and proliferation of cancer cells [9–11]. Arginine is a nonessential or semi-essential amino acid that is involved in biological processes such as cell proliferation, survival, and protein synthesis. It is also the precursor of nitric oxide (NO), polyamine, proline, creatine, glutamate, and other metabolites [12]. Importantly, the mechanistic target of rapamycin complex 1 (mTORC1) can be activated by arginine sensing, which in turn releases essential amino acids from lysosomes and promotes PDAC cell growth. In contrast, arginine deprivation significantly inhibited the mTORC1 activation [13–16]. Current studies have found that arginine deprivation significantly inhibits the growth of PDAC cells with low Argininosuccinate synthase (ASS1) expression [17]. In addition, arginine deprivation promotes the sensitivity of PDAC to chemotherapy, radiotherapy, and other therapies [18, 19]. Therefore, the development of new therapeutic targets targeting arginine metabolism is expected to provide new options for the treatment of PDAC. In this study, we found that Rio Kinase 3 (RIOK3) was significantly correlated with arginine metabolism in PDAC by Liquid Chromatograph Mass Spectrometer (LC-MS)-based untargeted metabolomics analysis. High-throughput transcriptome analysis showed that RIOK3 knockdown significantly inhibited the expression of arginine transporter solute carrier family 7 member 2 (SLC7A2). Further studies showed that RIOK3 promoted arginine uptake, mechanistic target of rapamycin complex 1 (mTORC1) activation, cell invasion, and metastasis through SLC7A2 in PDAC cells. Finally, we found that the RIOK3 in PDAC tissues was significantly positively correlated with the number of infiltrating Regulatory T (Treg) cells, and the prognosis of PDAC patients with high expression of both RIOK3 and infiltrating Treg cells were worse. Our study revealed that RIOK3 is one of the kinesins responsible for arginine uptake by PDAC cells, which also provides a new therapeutic target for therapeutic strategies targeting arginine metabolism. RESULTS RIOK3 protein is highly expressed in PDAC tissues and associated with poor prognosis In our previous study, bioinformatics analysis of PDAC transcriptome data derived from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database revealed that RIOK3 mRNA was significantly overexpressed in PDAC tissues [20]. In this www.aging-us.com 1040 study, we used these data to perform Receiver operating characteristic (ROC) analysis and showed that RIOK3 mRNA expression had a good predictive ability to distinguish PDAC tissues from normal pancreatic tissues. The area under curve (AUC) was 0.945 (95% confidence interval [CI] = 0.921-0.969) (Figure 1A). To further clarify the protein expression of RIOK3 in PDAC tissues, we performed immunohistochemical (IHC) analysis on a tissue microarray containing 46 pairs of PDAC and adjacent normal tissues and found that the protein expression (H-score) of RIOK3 in PDAC tissues was significantly higher than that in adjacent normal tissues (Figure 1B). 46 PDAC patients’ baseline characteris (...truncated)