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CRISPR/Cas9 library screening uncovered methylated PKP2 as a critical driver of lung cancer radioresistance by stabilizing β-catenin

Abstract

Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized β-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/β-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.

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Fig. 1: Genome-wide CRISPR/Cas9 screening identified PKP2 as a driver of lung cancer radioresistance.
Fig. 2: PKP2 stabilizes β-catenin to induce LIG4 transcription.
Fig. 3: LIG4 is a key player in PKP2-mediated radioresistance.
Fig. 4: PKP2 recruits USP7 to enhance the deubiquitination and stability of β-catenin.
Fig. 5: Arginine methylation of PKP2 by PRMT1 enhances the ability to bind to β-catenin.
Fig. 6: PKP2 R101 methylation promotes lung cancer radioresistance.
Fig. 7: The PRMT1 inhibitor C-7280948 contributes to overcoming radioresistance.

References

  1. 1.

    Rubin SM, Sage J. Manipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targets. Nature. 2019;569:343–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Nanavaty P, Alvarez MS, Alberts WM. Lung cancer screening: advantages, controversies, and applications. Cancer Control. 2014;21:9–14.

    PubMed  Google Scholar 

  3. 3.

    Kim BH, Kim YJ, Kim MH, Na YR, Jung D, Seok SH, et al. Identification of FES as a Novel Radiosensitizing Target in Human Cancers. Clin Cancer Res. 2020;26:265–73.

    CAS  PubMed  Google Scholar 

  4. 4.

    Coon D, Gokhale AS, Burton SA, Heron DE, Ozhasoglu C, Christie N. Fractionated stereotactic body radiation therapy in the treatment of primary, recurrent, and metastatic lung tumors: the role of positron emission tomography/computed tomography-based treatment planning. Clin Lung Cancer. 2008;9:217–21.

    PubMed  Google Scholar 

  5. 5.

    Chang JY, Liu YH, Zhu Z, Welsh JW, Gomez DR, Komaki R, et al. Stereotactic ablative radiotherapy: a potentially curable approach to early stage multiple primary lung cancer. Cancer. 2013;119:3402–10.

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Jeong YK, Oh JY, Yoo JK, Lim SH, Kim EH. The Biofunctional Effects of Mesima as a Radiosensitizer for Hepatocellular Carcinoma. Int J Mol Sci. 2020;21:871.

    PubMed Central  Google Scholar 

  7. 7.

    Laird JH, Lok BH, Ma J, Bell A, de Stanchina E, Poirier JT, et al. Talazoparib Is a Potent Radiosensitizer in Small Cell Lung Cancer Cell Lines and Xenografts. Clin Cancer Res. 2018;24:5143–52.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Lee SY, Jeong EK, Ju MK, Jeon HM, Kim MY, Kim CH, et al. Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation. Mol Cancer. 2017;16:10.

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Gul IS, Hulpiau P, Saeys Y, van Roy F. Metazoan evolution of the armadillo repeat superfamily. Cell Mol Life Sci. 2017;74:525–41.

    CAS  PubMed  Google Scholar 

  10. 10.

    Hatzfeld M. The armadillo family of structural proteins. Int Rev Cytol. 1999;186:179–224.

    CAS  PubMed  Google Scholar 

  11. 11.

    Tewari R, Bailes E, Bunting KA, Coates JC. Armadillo-repeat protein functions: questions for little creatures. Trends Cell Biol. 2010;20:470–81.

    CAS  PubMed  Google Scholar 

  12. 12.

    Coates JC. Armadillo repeat proteins: beyond the animal kingdom. Trends Cell Biol. 2003;13:463–71.

    CAS  PubMed  Google Scholar 

  13. 13.

    Martin-Padron J, Boyero L, Rodriguez MI, Andrades A, Diaz-Cano I, Peinado P, et al. Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer. Oncogene. 2020;39:5479–93.

    CAS  PubMed  Google Scholar 

  14. 14.

    Li D, Song H, Mei H, Fang E, Wang X, Yang F, et al. Armadillo repeat containing 12 promotes neuroblastoma progression through interaction with retinoblastoma binding protein 4. Nat Commun. 2018;9:2829.

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Niehrs C. The complex world of WNT receptor signalling. Nat Rev Mol Cell Biol. 2012;13:767–79.

    CAS  PubMed  Google Scholar 

  16. 16.

    Blanc RS, Richard S. Arginine Methylation: the Coming of Age. Mol Cell. 2017;65:8–24.

    CAS  PubMed  Google Scholar 

  17. 17.

    Suchankova J, Legartova S, Sehnalova P, Kozubek S, Valente S, Labella D, et al. PRMT1 arginine methyltransferase accumulates in cytoplasmic bodies that respond to selective inhibition and DNA damage. Eur J Histochem. 2014;58:2389.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Zhan T, Rindtorff N, Betge J, Ebert MP, Boutros M. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol. 2019;55:106–19.

    CAS  PubMed  Google Scholar 

  19. 19.

    Biau J, Chautard E, Verrelle P, Dutreix M, Altering DNA. Repair to Improve Radiation Therapy: specific and Multiple Pathway Targeting. Front Oncol. 2019;9:1009.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Jun S, Jung YS, Suh HN, Wang W, Kim MJ, Oh YS, et al. LIG4 mediates Wnt signalling-induced radioresistance. Nat Commun. 2016;7:10994.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Chen X, Bonne S, Hatzfeld M, van Roy F, Green KJ. Protein binding and functional characterization of plakophilin 2. Evidence for its diverse roles in desmosomes and beta -catenin signaling. J Biol Chem. 2002;277:10512–22.

    CAS  PubMed  Google Scholar 

  22. 22.

    Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 2005;123:1213–26.

    CAS  PubMed  Google Scholar 

  23. 23.

    Arimoto K, Burkart C, Yan M, Ran D, Weng S, Zhang DE. Plakophilin-2 promotes tumor development by enhancing ligand-dependent and -independent epidermal growth factor receptor dimerization and activation. Mol Cell Biol. 2014;34:3843–54.

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Zhang B, Wu J, Cai Y, Luo M, Wang B, Gu Y. TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer. Acta Biochim Biophys Sin (Shanghai). 2019;51:375–85.

    CAS  Google Scholar 

  25. 25.

    Wang Y, Chen X, Tang G, Liu D, Peng G, Ma W, et al. AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription. FEBS Lett. 2016;590:4586–93.

    CAS  PubMed  Google Scholar 

  26. 26.

    Yu-Ju WuC, Chen CH, Lin CY, Feng LY, Lin YC, Wei KC, et al. CCL5 of glioma-associated microglia/macrophages regulates glioma migration and invasion via calcium-dependent matrix metalloproteinase 2. Neuro Oncol. 2020;22:253–66.

    Google Scholar 

  27. 27.

    Zhou W, Wang K, Wang J, Qu J, Du G, Zhang Y. SOX17 Inhibits Tumor Metastasis Via Wnt Signaling In Endometrial Cancer. Onco Targets Ther. 2019;12:8275–86.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Novellasdemunt L, Foglizzo V, Cuadrado L, Antas P, Kucharska A, Encheva V, et al. USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating beta-Catenin Deubiquitination. Cell Rep. 2017;21:612–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Su D, Ma S, Shan L, Wang Y, Wang Y, Cao C, et al. Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis. J Clin Investig. 2018;128:4280–96.

    PubMed  Google Scholar 

  30. 30.

    Hsu JH, Hubbell-Engler B, Adelmant G, Huang J, Joyce CE, Vazquez F, et al. PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer. Cancer Res. 2017;77:4613–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Mertens C, Kuhn C, Franke WW. Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque. J Cell Biol. 1996;135:1009–25.

    CAS  PubMed  Google Scholar 

  32. 32.

    Fischer-Keso R, Breuninger S, Hofmann S, Henn M, Rohrig T, Strobel P, et al. Plakophilins 1 and 3 bind to FXR1 and thereby influence the mRNA stability of desmosomal proteins. Mol Cell Biol. 2014;34:4244–56.

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Zhang D, Qian Y, Liu X, Yu H, Zhao N, Wu Z. Up-regulation of plakophilin-2 is correlated with the progression of glioma. Neuropathology. 2017;37:207–16.

    CAS  PubMed  Google Scholar 

  34. 34.

    Niell N, Larriba MJ, Ferrer-Mayorga G, Sanchez-Perez I, Cantero R, Real FX, et al. The human PKP2/plakophilin-2 gene is induced by Wnt/beta-catenin in normal and colon cancer-associated fibroblasts. Int J Cancer. 2018;142:792–804.

    CAS  PubMed  Google Scholar 

  35. 35.

    Takahashi H, Nakatsuji H, Takahashi M, Avirmed S, Fukawa T, Takemura M, et al. Up-regulation of plakophilin-2 and Down-regulation of plakophilin-3 are correlated with invasiveness in bladder cancer. Urology. 2012;79:240 e241–248.

    Google Scholar 

  36. 36.

    Demirag GG, Sullu Y, Gurgenyatagi D, Okumus NO, Yucel I. Expression of plakophilins (PKP1, PKP2, and PKP3) in gastric cancers. Diagn Pathol. 2011;6:1.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Hao XL, Tian Z, Han F, Chen JP, Gao LY, Liu JY. Plakophilin-2 accelerates cell proliferation and migration through activating EGFR signaling in lung adenocarcinoma. Pathol Res Pr. 2019;215:152438.

    CAS  Google Scholar 

  38. 38.

    Vartak SV, Swarup HA, Gopalakrishnan V, Gopinatha VK, Ropars V, Nambiar M, et al. Autocyclized and oxidized forms of SCR7 induce cancer cell death by inhibiting nonhomologous DNA end joining in a Ligase IV dependent manner. FEBS J. 2018;285:3959–76.

    CAS  PubMed  Google Scholar 

  39. 39.

    Seol JH, Shim EY, Lee SE. Microhomology-mediated end joining: good, bad and ugly. Mutat Res. 2018;809:81–87.

    CAS  PubMed  Google Scholar 

  40. 40.

    Williams GJ, Hammel M, Radhakrishnan SK, Ramsden D, Lees-Miller SP, Tainer JA. Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time. DNA Repair (Amst). 2014;17:110–20.

    CAS  Google Scholar 

  41. 41.

    Chen DJ, Nirodi CS. The epidermal growth factor receptor: a role in repair of radiation-induced DNA damage. Clin Cancer Res. 2007;13:6555–60.

    CAS  PubMed  Google Scholar 

  42. 42.

    Dittmann K, Mayer C, Fehrenbacher B, Schaller M, Raju U, Milas L, et al. Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase. J Biol Chem. 2005;280:31182–9.

    CAS  PubMed  Google Scholar 

  43. 43.

    Wu CT, Hsieh CC, Yen TC, Chen WC, Chen MF. TGF-beta1 mediates the radiation response of prostate cancer. J Mol Med (Berl). 2015;93:73–82.

    CAS  Google Scholar 

  44. 44.

    Kim MR, Lee J, An YS, Jin YB, Park IC, Chung E, et al. TGFbeta1 protects cells from gamma-IR by enhancing the activity of the NHEJ repair pathway. Mol Cancer Res. 2015;13:319–29.

    CAS  PubMed  Google Scholar 

  45. 45.

    Kanamoto T, Hellman U, Heldin CH, Souchelnytskyi S. Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair. EMBO J. 2002;21:1219–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Dubash AD, Kam CY, Aguado BA, Patel DM, Delmar M, Shea LD, et al. Plakophilin-2 loss promotes TGF-beta1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes. J Cell Biol. 2016;212:425–38.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Adams MM, Wang B, Xia Z, Morales JC, Lu X, Donehower LA, et al. 53BP1 oligomerization is independent of its methylation by PRMT1. Cell Cycle. 2005;4:1854–61.

    CAS  PubMed  Google Scholar 

  48. 48.

    Boisvert FM, Rhie A, Richard S, Doherty AJ. The GAR motif of 53BP1 is arginine methylated by PRMT1 and is necessary for 53BP1 DNA binding activity. Cell Cycle. 2005;4:1834–41.

    CAS  PubMed  Google Scholar 

  49. 49.

    Yang JH, Chiou YY, Fu SL, Shih IY, Weng TH, Lin WJ, et al. Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation. Nucleic Acids Res. 2014;42:9908–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Montenegro MF, Gonzalez-Guerrero R, Sanchez-del-Campo L, Pinero-Madrona A, Cabezas-Herrera J, Rodriguez-Lopez JN. Targeting the epigenetics of the DNA damage response in breast cancer. Cell Death Dis. 2016;7:e2180.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Montenegro MF, Gonzalez-Guerrero R, Sanchez-Del-Campo L, Pinero-Madrona A, Cabezas-Herrera J, Rodriguez-Lopez JN. PRMT1-dependent methylation of BRCA1 contributes to the epigenetic defense of breast cancer cells against ionizing radiation. Sci Rep.2020;10:13275.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Vadnais C, Chen R, Fraszczak J, Yu Z, Boulais J, Pinder J, et al. GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1. Nat Commun. 2018;9:1418.

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by grants from the special fund for Jiangxi Key Laboratory (20171BCD40026 to YS), the Jiangxi Provincial Natural Science Foundation of China (20192BAB215039 to YS) and the Natural Science Foundation of China (81772821 to KH).

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CC, XP, SL, JY, CL and JT performed all of the experiments and data analysis. CC and YS conceived the research design, experiments, and data analysis. KH, GH, WM and YS prepared and wrote the paper.

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Correspondence to Yi Sang.

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Cheng, C., Pei, X., Li, SW. et al. CRISPR/Cas9 library screening uncovered methylated PKP2 as a critical driver of lung cancer radioresistance by stabilizing β-catenin. Oncogene 40, 2842–2857 (2021). https://doi.org/10.1038/s41388-021-01692-x

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