N⁶-methyladenosine-mediated upregulation of LNCAROD confers radioresistance in esophageal squamous cell carcinoma through stabilizing PARP1

Abstract

Background: Radiotherapy is a primary therapeutic modality for esophageal squamous cell carcinoma (ESCC), but its effectiveness is still restricted due to the resistance of cancer cells to radiation. Long non-coding RNAs (lncRNAs) and N⁶-methyladenosine (m6A) have been shown to play significant roles in tumour radioresistance. However, the precise manifestation and role of m6A-modified lncRNAs in ESCC radioresistance remain unclear.

Methods: Bioinformatics analysis was conducted to identify m6A-modified lncRNAs implicated in the radioresistance of ESCC. A series of functional experiments were performed to investigate the function of LNCAROD in ESCC. Methylated RNA immunoprecipitation, chromatin isolation by RNA purification-mass spectrometry, RNA immunoprecipitation, and co-immunoprecipitation experiments were performed to explore the mechanism of m6A-mediated upregulation of LNCAROD expression and the downstream mechanism enhancing the radioresistance of ESCC. The efficacy of LNCAROD in vivo was assessed using murine xenograft models.

Results: Herein, we identified LNCAROD as a novel METTL3-mediated lncRNA that enhanced radioresistance in ESCC cells and was post-transcriptionally stabilised by YTHDC1. Moreover, we confirmed that LNCAROD prevented ubiquitin-proteasome degradation of PARP1 protein by facilitating PARP1-NPM1 interaction, thereby contributing to homologous recombination-mediated DNA double-strand breaks repair and enhancing the radiation resistance of ESCC cells. Silencing LNCAROD in a nude mouse model of ESCC in vivo resulted in slower tumour growth and increased radiosensitivity.

Conclusion: Our findings enhance the understanding of m6A-modified lncRNA-driven machinery in ESCC radioresistance and underscore the significance of LNCAROD in this context, thereby contributing to the development of a potential therapeutic target for ESCC patients.

Keywords

esophageal squamous cell carcinoma / LNCAROD / N6-methyladenosine / PARP1 / radioresistance

References

[1] Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-249.

[2] Chen R, Zheng R, Zhang S, et al. Patterns and trends in esophageal cancer incidence and mortality in China: an analysis based on cancer registry data. J Nat Cancer Center. 2023; 3(1): 21-27.

[3] He Y, Liang D, Du L, et al. Clinical characteristics and survival of 5283 esophageal cancer patients: a multicenter study from eighteen hospitals across six regions in China. Cancer Commun (Lond, Engl). 2020; 40(10): 531-544.

[4] Lagergren J, Smyth E, Cunningham D, et al. Oesophageal cancer. Lancet (London, England). 2017; 390(10110): 2383-2396.

[5] Thrift AP. Global burden and epidemiology of Barrett oesophagus and oesophageal cancer. Nat Rev Gastroenterol Hepatol. 2021; 18(6): 432-443.

[6] Jiang X, Liu B, Nie Z, et al. The role of m6A modification in the biological functions and diseases. Signal Transduct Targeted Ther. 2021; 6(1): 74.

[7] Yan X, Pei K, Guan Z, et al. AI-empowered integrative structural characterization of m(6)A methyltransferase complex. Cell Res. 2022; 32(12): 1124-1127.

[8] Liu J, Yue Y, Han D, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014; 10(2): 93-95.

[9] Frye M, Harada BT, Behm M, et al. RNA modifications modulate gene expression during development. Science. 2018; 361(6409): 1346-1349.

[10] Zheng G, Dahl JA, Niu Y, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013; 49(1): 18-29.

[11] Jia G, Fu Y, Zhao X, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011; 7(12): 885-887.

[12] Patil DP, Pickering BF, Jaffrey SR. Reading m(6)A in the transcriptome: m(6)A-binding proteins. Trends Cell Biol. 2018; 28(2): 113-127.

[13] Meyer KD, Jaffrey SR. Rethinking m(6)A readers, writers, and erasers. Annu Rev Cell Dev Biol. 2017; 33: 319-342.

[14] Visvanathan A, Patil V, Arora A, et al. Essential role of METTL3-mediated m(6)A modification in glioma stem-like cells maintenance and radioresistance. Oncogene. 2018; 37(4): 522-533.

[15] Zhang C, Chen L, Peng D, et al. METTL3 and N6-methyladenosine promote homologous recombination-mediated repair of DSBs by modulating DNA-RNA hybrid accumulation. Mol Cell. 2020; 79(3): 425-442.

[16] Wang Y, Zhang L, Sun XL, et al. NRP1 contributes to stemness and potentiates radioresistance via WTAP-mediated m6A methylation of Bcl-2 mRNA in breast cancer. Apoptosis. 2023; 28(1-2): 233-246.

[17] Shi X, Liu X, Pan S, et al. A novel autophagy-related long non-coding RNA signature to predict prognosis and therapeutic response in esophageal squamous cell carcinoma. Int J Gen Med. 2021; 14: 8325-8339.

[18] Shi X, Liu X, Huang S, et al. miR-4443 promotes radiation resistance of esophageal squamous cell carcinoma via targeting PTPRJ. J Transl Med. 2022; 20(1): 626.

[19] Gu Z, Zhu W, Wang W, et al. Anlotinib inhibits tumor angiogenesis and promotes the anticancer effect of radiotherapy on esophageal cancer through inhibiting EphA2. J Oncol. 2022; 2022: 5632744.

[20] Zhou Y, Zeng P, Li YH, et al. SRAMP: prediction of mammalian N6-methyladenosine (m6A) sites based on sequence-derived features. Nucleic Acids Res. 2016; 44(10): e91.

[21] Zhao X, Ma Y, Li J, et al. The AEG-1-USP10-PARP1 axis confers radioresistance in esophageal squamous cell carcinoma via facilitating homologous recombination-dependent DNA damage repair. Cancer Lett. 2023; 577: 216440.

[22] Wang W, Shao F, Yang X, et al. METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N(6)-methyladenosine-dependent YTHDF binding. Nat Commun. 2021; 12(1): 3803.

[23] Han H, Yang C, Zhang S, et al. METTL3-mediated m(6)A mRNA modification promotes esophageal cancer initiation and progression via Notch signaling pathway. Mol Ther Nucl Acids. 2021; 26: 333-346.

[24] Chen L, Zhang C, Ma W, et al. METTL3-mediated m6A modification stabilizes TERRA and maintains telomere stability. Nucleic Acids Res. 2022; 50(20): 11619-11634.

[25] Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010; 24(4): 679-686.

[26] Takano S, Shibamoto Y, Wang Z, et al. Optimal timing of a γH2AX analysis to predict cellular lethal damage in cultured tumor cell lines after exposure to diagnostic and therapeutic radiation doses. J Radiat Res (Tokyo). 2023; 64(2): 317-327.

[27] Zong C, Zhu T, He J, et al. PARP mediated DNA damage response, genomic stability and immune responses. Int J Cancer. 2022; 150(11): 1745-1759.

[28] Sekhar KR, Freeman ML. Nucleophosmin plays a role in repairing DNA damage and is a target for cancer treatment. Cancer Res. 2023; 83(10): 1573-1580.

[29] Goldstein M, Kastan MB. The DNA damage response: implications for tumor responses to radiation and chemotherapy. Annu Rev Med. 2015; 66: 129-143.

[30] Groelly FJ, Fawkes M, Dagg RA, et al. Targeting DNA damage response pathways in cancer. Nat Rev Cancer. 2023; 23(2): 78-94.

[31] Ray Chaudhuri A, Nussenzweig A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat Rev Mol Cell Biol. 2017; 18(10): 610-621.

[32] Xu Y, Dong B, Zhu W, et al. A Phase III multicenter randomized clinical trial of 60 Gy versus 50 Gy radiation dose in concurrent chemoradiotherapy for inoperable esophageal squamous cell carcinoma. Clin Cancer. 2022; 28(9): 1792-1799.

[33] An L, Li M, Jia Q. Mechanisms of radiotherapy resistance and radiosensitization strategies for esophageal squamous cell carcinoma. Mol Cancer. 2023; 22(1): 140.

[34] Sun Y, Wang J, Ma Y, et al. Radiation induces NORAD expression to promote ESCC radiotherapy resistance via EEPD1/ATR/Chk1 signalling and by inhibiting pri-miR-199a1 processing and the exosomal transfer of miR-199a-5p. J Exp Clin Cancer Res: CR. 2021; 40(1): 306.

[35] Zhou Y, Shao Y, Hu W, et al. A novel long noncoding RNA SP100-AS1 induces radioresistance of colorectal cancer via sponging miR-622 and stabilizing ATG3. Cell Death Differ. 2023; 30(1): 111-124.

[36] Ban Y, Tan P, Cai J, et al. LNCAROD is stabilized by m6A methylation and promotes cancer progression via forming a ternary complex with HSPA1A and YBX1 in head and neck squamous cell carcinoma. Mol Oncol. 2020; 14(6): 1282-1296.

[37] Ansari S, Nikpour P. LNCAROD promotes the proliferation and migration of gastric cancer: a bioinformatics analysis and experimental validation. Funct Integr Genomics. 2023; 23(1): 34.

[38] Roundtree IA, Evans ME, Pan T, et al. Dynamic RNA modifications in gene expression regulation. Cell. 2017; 169(7): 1187-1200.

[39] Liu WW, Zhang ZY, Wang F, et al. Emerging roles of m6A RNA modification in cancer therapeutic resistance. Exp Hematol Oncol. 2023; 12(1): 21.

[40] Xu X, Zhang P, Huang Y, et al. METTL3-mediated m6A mRNA contributes to the resistance of carbon-ion radiotherapy in non-small-cell lung cancer. Cancer Sci. 2023; 114(1): 105-114.

[41] Alemasova EE, Lavrik OI. Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins. Nucleic Acids Res. 2019; 47(8): 3811-3827.

[42] Ziv O, Zeisel A, Mirlas-Neisberg N, et al. Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin. Nat Commun. 2014; 5: 5437.

[43] Xu R, Yu S, Zhu D, et al. hCINAP regulates the DNA-damage response and mediates the resistance of acute myelocytic leukemia cells to therapy. Nat Commun. 2019; 10(1): 3812.

[44] Qin G, Wang X, Ye S, et al. NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. Nat Commun. 2020; 11(1): 1669.

RIGHTS & PERMISSIONS

2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.