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    MicroRNA-1291-5p sensitizes pancreatic carcinoma cells to arginine deprivation and

    chemotherapy through the regulation of arginolysis and glycolysis

    Mei-Juan Tu, Zhijian Duan, Zhenzhen Liu, Chao Zhang, Richard J. Bold, Frank J.

    Gonzalez, Edward J. Kim, Ai-Ming Yu

    Department of Biochemistry & Molecular Medicine, UC Davis School of Medicine,

    Sacramento, CA 95817, USA ((M.-J. T., Z. D., Z. L., C. Z., A.-M. Y.)); Division of

    Hematology and Oncology, Department of Internal Medicine, UC Davis School of

    Medicine, Sacramento, CA 95817, USA (R. J B., E. J K.); Laboratory of Metabolism,

    National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA (F. J

    G.)

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    Running title: MiR-1291 regulates cancer metabolism

    Address correspondence to: Dr. Edward J. Kim, Division of Hematology and Oncology,

    Department of Internal Medicine, UC Davis School of Medicine, Sacramento, CA 95817;

    Email: [email protected]; or Prof. Dr. Ai-Ming Yu, Department of Biochemistry &

    Molecular Medicine, UC Davis School of Medicine, Sacramento, CA 95817, USA; Email:

    [email protected]

    Number of Text Pages: 25

    Number of Figures: 6

    Number of References: 55

    Number of Words in Abstract: 214

    Number of Words in Introduction: 817

    Number of Words in Discussion: 1174

    Abbreviations: 2-DG, 2-deoxy-D-glucose; 2-NBDG,

    2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2 Deoxyglucose; AA, antimycin A;

    ACN, acetonitrile; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase;

    ECAR, extracellular acidification rate; ESI, electrospray ionization; FA, formic acid;

    FPLC, fast protein liquid chromatography; GLUT1, glucose transporter protein type 1;

    glycoPER, glycolytic proton efflux rate; HPLC, high-performance liquid chromatography;

    hsa-pre-miR-1291, human miR-1291 precursor; IS, internal standard; KRB, Krebs Ringer

    bicarbonate; LC-MS/MS, liquid chromatography-tandem mass spectroscopy; LLOQ,

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    lower limit of quantification; miRNA or miR, microRNA; MRM, multiple reaction

    monitoring; MSA, sephadex aptamer tagged methionyl-tRNA; OCR, oxygen consumption

    rate; OXPHOS, oxidative phosphorylation; PAGE, polyacrylamide gel electrophoresis; PC,

    pancreatic cancer; PDX, patient derived xenograft; PEG-ADI, pegylated arginine

    deiminase; ROT, rotenone; RT-qPCR, reverse transcription quantitative real-time PCR.

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    Abstract

    Cancer cells are dysregulated and addicted to continuous supply and metabolism of

    nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical

    macromolecules for uncontrolled growth. Recent studies have revealed that

    genome-derived microRNA-1291-5p (miR-1291-5p or miR-1291) may modulate the

    expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1

    (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents

    for research, which are distinguished from conventional chemo-engineered miRNA

    mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was

    selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC)

    cells. Following the suppression of ASS1 protein levels, miR-1291 perturbed arginine

    homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation

    therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both

    AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and

    glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved

    cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that

    miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and

    chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis,

    respectively, which may provide insights into understanding miRNA signaling underlying

    cancer cell metabolism and development of new strategies for the treatment of lethal PC.

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    Significance Statement

    Many anticancer drugs in clinical use and under investigations exert pharmacological

    effects or improve efficacy of co-administered medications by targeting cancer cell

    metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a

    metabolism modulator in pancreatic carcinoma cells through the regulation of ASS1- and

    GLUT1-mediated arginolysis and glycolysis. Consequently, miR-1291 was effective to

    enhance the efficacy of arginine deprivation (PEG-ADI) and chemotherapy (cisplatin),

    offering new insights into development of rational combination therapies.

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    Introduction

    Metabolism reprogramming is one of the major hallmarks of cancer cells, with higher

    rates of nutrients (e.g. glucose and amino acids) transport and metabolism than normal

    cells essential for rapid proliferation (Counihan et al., 2018; DeBerardinis and Chandel,

    2016). Cells predominately metabolize glucose through glycolysis in cytoplasm and

    oxidative phosphorylation (OXPHOS) in mitochondria to produce ATP

    (Martinez-Outschoorn et al., 2017; Pavlova and Thompson, 2016). Different from normal

    cells, many carcinoma cells prefer the utilization of glycolysis-derived ATP rather than

    OXPHOS-produced ATP, which is called ‘‘Warburg effect’’ or aerobic glycolysis (Asgari

    et al., 2015; Hsu and Sabatini, 2008). To meet a high demand of glucose, glucose

    transporter protein type 1 (GLUT1) is commonly upregulated in various cancer types (Ooi

    and Gomperts, 2015). As such, there are growing interests in developing new strategies to

    target glycolysis for the treatment of cancers or improve the efficacy of existing therapies

    (Counihan et al., 2018; Dai et al., 2020; Xi et al., 2020).

    Aberrant aminolyses are well recognized in cancer cells (Martinez-Outschoorn et al., 2017;

    Pavlova and Thompson, 2016) while amino acids are vital components for protein

    synthesis and signaling transduction (Wu, 2013). Arginine is a semi-essential amino acid

    that can be synthesized within cells from citrulline via argininosuccinate lyase (ASL) and

    the rate limiting enzyme argininosuccinate synthase (ASS1) but not necessarily at

    sufficient quantities and therefore may need to be supplemented by extracellular stores

    This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 13, 2020 as DOI: 10.1124/molpharm.120.000130

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    generated by diet. Synthesis of arginine within cells is often downregulated in many kinds

    of cancers including pancreatic cancer (PC) and thus these cancer cells become more

    dependent on the extracellular stores of arginine from the diet (Delage et al., 2010).

    Therefore, arginine deprivation therapy has emerged as a new strategy to combat tumor

    cells that are auxotrophic for arginine due to ASS1 downregulation (Delage et al., 2010;

    Patil et al., 2016). Arginine deprivation can be achieved by using

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