GI cancer ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2020-320652 on 20 November 2020. Downloaded from RNA- binding protein RALY reprogrammes mitochondrial metabolism via mediating miRNA processing in colorectal cancer Lei Sun,1 Arabella Wan,2 Zhuolong Zhou,3 Dongshi Chen,4 Heng Liang,1 Chuwei Liu,2 Shijia Yan,1 Yi Niu,1 Ziyou Lin,1 Siyue Zhan,1 Shanfeng Wang,5 Xianzhang Bu,1 Weiling He,2,6 Xiongbin Lu ,3,7 Anlong Xu ,8,9 Guohui Wan 1 For numbered affiliations see ABSTRACT end of article. Objective Dysregulated cellular metabolism is a Significance of this study distinct hallmark of human colorectal cancer (CRC). Correspondence to However, metabolic programme rewiring during tumour What is already known on this subject? Professor Guohui Wan, School ► Dysregulation of metabolism in cancer cells of Pharmaceutical Sciences, Sun progression has yet to be fully understood. Yat-Sen University, Guangzhou, Design We analysed altered gene signatures during is dynamically regulated at many cancer China; colorectal tumour progression, and used a complex of hallmarks. wanguoh@ mail. sysu. edu.cn, molecular and metabolic assays to study the regulation ► Metabolic reprogramming in cancer cells could Professor Anlong Xu, School of of metabolism in CRC cell lines, human patient-derived be impacted by post-translational events and Life Sciences, Beijing University epigenetic regulation of genes. of Chinese Medicine, Beijing, xenograft mouse models and tumour organoid models. China; Results We identified a novel RNA-binding protein, ► RALY was identified in the spliceosome complex xuanlong@ bucm. edu. cn and in the Drosha microprocessor complex. RALY (also known as hnRNPCL2), that is highly 6 and Professor Xiongbin Lu, associated with colorectal tumour aggressiveness. ► The m A modification is found to alter RNA Department of Medical and structure to enhance HNRNPC binding, and Molecular Genetics, Indiana RALY acts as a key regulatory component in the 6 University School of Medicine, Drosha complex, and promotes the post-tr anscriptional HNRNPA2B1 could bind to the m A marks in Indianapolis, IN, USA; processing of a specific subset of miRNAs (miR-483, some pri-miRNA transcripts to promote their xiolu@ iu. edu miR-676 and miR-877). These miRNAs systematically processing. LS and AW contributed equally. downregulate the expression of the metabolism- What are the new findings? associated genes (ATP5I, ATP5G1, ATP5G3 and CYC1) ► RALY is highly associated with metastasis of http://gut.bmj.com/ LS and AW are joint first and thereby reprogramme mitochondrial metabolism malignant colorectal cancer (CRC). authors. in the cancer cell. Analysis of The Cancer Genome ► RALY acts as a key regulatory component in Atlas (TCGA) reveals that increased levels of RALY are Received 10 January 2020 the Drosha complex, and promotes the post- Revised 8 October 2020 associated with poor prognosis in the patients with CRC transcriptional processing of a specific subset Accepted 27 October 2020 expressing low levels of mitochondrion-associated genes. of miRNAs. Mechanistically, induced processing of these miRNAs ► RALY- mediated miRNAs processing is is facilitated by their N6-methyladenosine switch under modulated by N6-methyladenosine switch, and on September 26, 2021 by guest. Protected copyright. reactive oxygen species (ROS) stress. Inhibition of the 6 reprogrammes mitochondrial metabolism by m A methylation abolishes the RALY recognition of the downregulating the electron transport chain terminal loop of the pri- miRNAs. Knockdown of RALY genes. inhibits colorectal tumour growth and progression in vivo ► Inhibition of RALY suppresses colorectal tumour and in organoid models. growth and progression in vivo and in the Conclusions Collectively, our results reveal a critical organoid models. metabolism- centric role of RALY in tumour progression, which may lead to cancer therapeutics targeting RALY for How might it impact on clinical practice in the treating CRC. foreseeable future? ► Our report first reveals a critical metabolism- centric role of RALY in tumour progression. It © Author(s) (or their provides a novel therapeutic strategy to target employer(s)) 2020. Re- use INTRODUCTION RALY for CRC treatment. permitted under CC BY- NC. No The acquired capability of unrestrained replicative commercial re- use. See rights potential has been a representative hallmark for and permissions. Published 1 by BMJ. cancer cells. To meet the requirement for hyper- cells is actively regulated at many cancer hallmarks, proliferation, cancer cells shift intracellular energy including proliferation, angiogenesis, invasion and To cite: Sun L, Wan A, metabolism and mainly rely on glycolysis pathways metastasis.3 4 In a recent study, we reported that Zhou Z, et al. Gut Epub ahead of print: [please instead of mitochondrial oxidative phosphorylation USP13 as a key regulator rewires energy metabo- include Day Month Year]. (OXPHOS) to support their energy demand and lism through deubiquitinating ATP citrate synthase doi:10.1136/ biomass synthesis, even when the oxygen level is (ACLY) and 2-o xoglutarate dehydrogenase (OGDH) gutjnl-2020-320652 sufficient.1 2 Dysregulation of metabolism in cancer and promotes ovarian tumour progression.5 Sun L, et al. Gut 2020;0:1–15. doi:10.1136/gutjnl-2020-320652 1 GI cancer Emerging evidence has shown that metabolic reprogramming in oligomycin and 50 mM 2-deo xyglucose). For OCR analysis, cells cancer cells could be impacted by post- translational events and were added with Mito stress- test base medium (10 mM glucose, Gut: first published as 10.1136/gutjnl-2020-320652 on 20 November 2020. Downloaded from epigenetic modulation of gene expression.6–8 Identification of 2 mM glutamine, 1 mM pyruvate, pH 7.4) and monitored every key regulators in mediating cancer cell metabolism could lead to 3 min following successive administration of inhibitors (2 µM a new therapeutic approach for cancer treatment. oligomycin, 2 µM FCCP, or 0.5 µM rotenone/antimycin A).11 As ‘energy factories’, mitochondria control the bioenergetics, A detailed description of the materials and methods can be biosynthetic and signalling organelles of cancer cells and serve found in online supplemental materials and methods. as an attractive target for cancer therapy.9–11 Previous reports showed that inhibition of electron transport chain (ETC) genes RESULTS could sensitise cancer cells to glucose depletion and reduce Upregulation of RALY is associated with aggressiveness in tumour progression.12 13 However, regulation of ETC gene CRC expression in tumour progression remains largely unknown. Specific focal gene amplifications, gene losses and altered gene- Recent studies indicate that epigenetic regulation has an essential expression levels have been identified as signature events for role in controlling cellular signalling pathways during tumouri- CRC progression.24 We conducted intensive analysis of gene genesis.14 15 Delineation of multifaceted epigenetic changes in alterations in CRC from the TCGA database by using genomic mitochondrial metabolism during tumour progression would set enrichment analysis (GSEA), and identified a specific group provide insights into the crucial mechanism by which cancer of genes related to RNA alternative splicing that is highly asso- cells adapt to a changing microenvironment. ciated with tumour metastasis (figure 1A). Among those genes, a Recently, miRNAs have been found to act as master gene novel RNA binding protein, RALY was identified as a molecular regulators, similar to transcription factors, to mediate gene signature associated with tumour aggressiveness from the inte- expression patterns in various cellular activities, including cell grative analysis of tumour stages, lymph node invasion, vascular metabolism.16 17 C- Myc suppression of miR- 23a and miR- invasion and distant metastasis status at the time of surgery 23b enhances mitochondrial glutaminase expression, leading (figure 1B). Amplification of the RALY gene occurs frequently in to elevated glutamine catabolism in prostate cancer and B human cancers and locates in the proximity of E2F1 (figure 1C lymphoma.18 The RNA binding protein Lin28 inhibits let-7 and D). Upregulation of RALY is found in 56% of CRC cases biogenesis to promote the insulin- PI3K- mTOR pathway and (figure 1E). We demonstrate that RALY is overexpressed in nine increase glucose uptake.19 MiR-103, miR-107 and miR-143 are human CRC cell lines as compared with the normal human colon involved in regulating insulin- mediated glucose metabolism,20 21 epithelial cell line (figure 1F–H). By using CRC patient tissue while miR-33 cooperates with SREBPs to target ABCA1 and samples locally procured, we further validated the upregulation coordinate cholesterol biosynthesis and trafficking.22 MiRNAs of RALY in the CRC tumours in comparison with their adjacent have been known to provide a functional link between metabolic normal tissues (figure 1I and J and online supplemental figure 1) reprogramming and tumourigenesis. In this study, we identify and (online supplemental table 1). Analysis of TCGA database a novel RNA- binding protein, RALY (HnRNP-Associated with and the local CRC database revealed that expression of RALY Lethal Yellow, also called hnRNPCL2), as a crucial indicator for is tightly associated with its gene copy number (figure 1K) and tumour progression in colorectal cancer (CRC). We present here high expression of RALY correlates with poor clinical outcome http://gut.bmj.com/ a role of RALY in reprogramming