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Theranostics 2022; 12(7):3518-3533. doi:10.7150/thno.72581 This issue Cite

Research Paper

mTOR regulates aerobic glycolysis through NEAT1 and nuclear paraspeckle-mediated mechanism in hepatocellular carcinoma

Hong Zhang^1,2, Xiaoyang Su^1,3, Stephen K. Burley^1,4,5,6, X.F. Steven Zheng^1,2✉

1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA.
2. Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA.
3. Department of Medicine, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, 125 Paterson Street, New Brunswick, NJ 08901.
4. RCSB Protein Data Bank and Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, 174 Frelinghuysen Road, NJ 08854 USA.
5. Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 174 Frelinghuysen Road, Piscataway, NJ 08854 USA.
6. RCSB Protein Data Bank, Skaggs School of Pharmacy and Pharmaceutical Sciences and San Diego Supercomputing Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 USA.

✉ Corresponding author: Prof. X.F. Steven Zheng, Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel.: 732-235-6879; E-mail: zhengstrutgers.edu.

Abstract

Background: Hepatocellular Carcinoma (HCC) is a major form of liver cancer and a leading cause of cancer-related death worldwide. New insights into HCC pathobiology and mechanism of drug actions are urgently needed to improve patient outcomes. HCC undergoes metabolic reprogramming of glucose metabolism from respiration to aerobic glycolysis, a phenomenon known as the 'Warburg Effect' that supports rapid cancer cell growth, survival, and invasion. mTOR is known to promote Warburg Effect, but the underlying mechanism(s) remains poorly defined. The aim of this study is to understand the mechanism(s) and significance of mTOR regulation of aerobic glycolysis in HCC.

Methods: We profiled mTORC1-dependent long non-coding RNAs (lncRNAs) by RNA-seq of HCC cells treated with rapamycin. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to explore the transcriptional regulation of NEAT1 by mTORC1. [U-¹³C]-glucose labeling and metabolomic analysis, extracellular acidification Rate (ECAR) by Seahorse XF Analyzer, and glucose uptake assay were used to investigate the role of mTOR-NEAT1-NONO signaling in the regulation of aerobic glycolysis. RNA immunoprecipitation (RIP) and NONO-binding motif scanning were performed to identify the regulatory mechanism of pre-mRNA splicing by mTOR-NEAT1. Myristoylated AKT1 (mAKT1)/NRAS^V12-driven HCC model developed by hydrodynamic transfection (HDT) was employed to explore the significance of mTOR-NEAT1 signaling in HCC tumorigenesis and mTOR-targeted therapy.

Results: mTOR regulates lncRNA transcriptome in HCC and that NEAT1 is a major mTOR transcriptional target. Interestingly, although both NEAT1_1 and NEAT1_2 are down-regulated in HCC, only NEAT1_2 is significantly correlated with poor overall survival of HCC patients. NEAT1_2 is the organizer of nuclear paraspeckles that sequester the RNA-binding proteins NONO and SFPQ. We show that upon oncogenic activation, mTORC1 suppresses NEAT1_2 expression and paraspeckle biogenesis, liberating NONO/SFPQ, which in turn, binds to U5 within the spliceosome, stimulating mRNA splicing and expression of key glycolytic enzymes. This series of actions lead to enhanced glucose transport, aerobic glycolytic flux, lactate production, and HCC growth both in vitro and in vivo. Furthermore, the paraspeckle-mediated mechanism is important for the anticancer action of US FDA-approved drugs rapamycin/temsirolimus.

Conclusions: These findings reveal a molecular mechanism by which mTOR promotes the 'Warburg Effect', which is important for the metabolism and development of HCC, and anticancer response of mTOR-targeted therapy.

Keywords: mTOR, NEAT1, Paraspeckles, Splicing, Rapamycin, Normoxia, Hypoxia, Warburg Effect, HIF1, Aerobic Glycolysis, Hepatocellular carcinoma

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