DNA-PKcs/AKT1 inhibits epithelial-mesenchymal transition during radiation-induced pulmonary fibrosis by inducing ubiquitination and degradation of Twist1

Abstract

Introduction: Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial-mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits strong repair capacity when cells undergo radiation-induced damage; whether DNA-PKcs regulates EMT during RIPF remains unclear.

Objectives: To investigate the role and molecular mechanism of DNA-PKcs in RIPF and provide an important theoretical basis for utilising DNA-PKcs-targeted drugs for preventing RIPF.

Methods: DNA-PKcs knockout (DPK^−/−) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20 Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA-PKcs-targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR.

Results: The expression of DNA-PKcs is low in pulmonary fibrosis (PF) patients. DNA-PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA-PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA-PKcs and Twist1. High Twist1 expression and EMT-associated changes caused by DNA-PKcs deletion were blocked by insulin-like growth factor-1 (IGF-1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR-induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA-PKcs.

Conclusion: Our study clarified the critical role and mechanism of DNA-PKcs in RIPF and showed that it could be a potential target for preventing RIPF.

Keywords

DNA-PKcs / epithelial-mesenchymal transition / radiation-induced pulmonary fibrosis / Twist1 / VND3207

References

[1] Marks LB. The pulmonary effects of thoracic irradiation. Oncology (Williston Park). 1994;8(6):89-106. discussion 0, 3-4.

[2] Jin H, Yoo Y, Kim Y, Kim Y, Cho J, Lee YS. Radiation-induced lung fibrosis: preclinical animal models and therapeutic strategies. Cancers (Basel). 2020;12(6):1561.

[3] Chen Z, Wu Z, Ning W. Advances in molecular mechanisms and treatment of radiation-induced pulmonary fibrosis. Transl Oncol. 2019;12(1):162-169.

[4] Park HR, Jo SK, Jung U. Ionizing radiation promotes epithelial-to-mesenchymal transition in lung epithelial cells by TGF-β-producing M2 macrophages. In Vivo. 2019;33(6):1773-17784.

[5] Willis BC, duBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc. 2006;3(4):377-382.

[6] Corvol H, Flamein F, Epaud R, Clement A, Guillot L. Lung alveolar epithelium and interstitial lung disease. Int J Biochem Cell Biol. 2009;41(8-9):1643-1651.

[7] Degryse AL, Tanjore H, Xu XC, et al. Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2010;299(4):L442-L452.

[8] Felton VM, Borok Z, Willis BC. N-acetylcysteine inhibits alveolar epithelial‒mesenchymal transition. Am J Physiol Lung Cell Mol Physiol. 2009;297(5):L805-L812.

[9] Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A. 2006;103(35):13180-13185.

[10] Tanjore H, Xu XC, Polosukhin VV, et al. Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. Am J Respir Crit Care Med. 2009;180(7):657-665.

[11] Tanjore H, Cheng DS, Degryse AL, et al. Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress. J Biol Chem. 2015;290(6):3277.

[12] Chen S, Lee L, Naila T, Fishbain S, et al. Structural basis of long-range to short-range synaptic transition in NHEJ. Nature. 2021;593(7858):294-298.

[13] Ramsden DA, Carvajal-Garcia J, Gupta GP. Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining. Nat Rev Mol Cell Biol. 2022;23(2):125-140.

[14] Neal JA, Dang V, Douglas P, Wold MS, Lees-Miller SP, Meek K. Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation. Mol Cell Biol. 2011;31(8):1719-1733.

[15] Zhou Y, Paull TT. DNA-dependent protein kinase regulates DNA end resection in concert with Mre11-Rad50-Nbs1 (MRN) and ataxia telangiectasia-mutated (ATM). J Biol Chem. 2013;288(52):37112-37125.

[16] Ragunathan K, Upfold NLE, Oksenych V. Interaction between fibroblasts and immune cells following DNA damage induced by ionizing radiation. Int J Mol Sci. 2020;21(22):8635.

[17] Pearce KM, Okon I, Watson-Wright C. Induction of oxidative DNA damage and epithelial mesenchymal transitions in small airway epithelial cells exposed to cosmetic aerosols. Toxicol Sci. 2020;177(1):248-262.

[18] Alam SK, Yadav VK, Bajaj S, et al. DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53. Cell Death Differ. 2016;23(4):707-722.

[19] Yang Y, Liu S, Wang P, et al. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) drives chronic kidney disease progression in male mice. Nat Commun. 2023;14(1):1334.

[20] Habiel DM, Hohmann MS, Espindola MS, et al. DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis. BMC Pulm Med. 2019;19(1):165.

[21] Yang Q, Zhang P, Liu T, et al. Magnesium isoglycyrrhizinate ameliorates radiation-induced pulmonary fibrosis by inhibiting fibroblast differentiation via the p38MAPK/Akt/Nox4 pathway. Biomed Pharmacother. 2019;115:108955.

[22] Tsoyi K, Chu SG, Patino-Jaramillo NG, et al. Syndecan-2 attenuates radiation-induced pulmonary fibrosis and inhibits fibroblast activation by regulating PI3K/Akt/ROCK pathway via CD148. Am J Respir Cell Mol Biol. 2018;58(2):208-215.

[23] Li CW, Xia W, Lim SO, et al. AKT1 inhibits epithelial-to-mesenchymal transition in breast cancer through phosphorylation-dependent twist1 degradation. Cancer Res. 2016;76(6):1451-1462.

[24] Pollom EL, Chin AL, Diehn M, Loo BW, Chang DT. Normal tissue constraints for abdominal and thoracic stereotactic body radiotherapy. Semin Radiat Oncol. 2017;27(3):197-208.

[25] Chargari C, Riet F, Mazevet M, Morel E, Lepechoux C, Deutsch E. Complications of thoracic radiotherapy. Presse Med. 2013;42(9 pt 2):e342-e351.

[26] Wang P, Yan Z, Zhou PK, Gu Y. The promising therapeutic approaches for radiation-induced pulmonary fibrosis: targeting radiation-induced mesenchymal transition of alveolar type II epithelial cells. Int J Mol Sci. 2022;23(23):15014.

[27] Shao L, Zhang Y, Shi W, et al. Mesenchymal stromal cells can repair radiation-induced pulmonary fibrosis via a DKK-1-mediated Wnt/β-catenin pathway. Cell Tissue Res. 2021;384(1):87-97.

[28] Hao H, Cohen DA, Jennings CD, Bryson JS, Kaplan AM. Bleomycin-induced pulmonary fibrosis is independent of eosinophils. J Leukoc Biol. 2000;68(4):515-521.

[29] Stone RC, Pastar I, Ojeh N, et al. Epithelial‒mesenchymal transition in tissue repair and fibrosis. Cell Tissue Res. 2016;365(3):495-506.

[30] Plygawko AT, Kan S, Campbell K. Epithelial‒mesenchymal plasticity: emerging parallels between tissue morphogenesis and cancer metastasis. Philos Trans R Soc Lond B Biol Sci. 2020;375(1809):20200087.

[31] Nagaraja SS, Nagarajan D. Radiation-induced pulmonary epithelial‒mesenchymal transition: a review on targeting molecular pathways and mediators. Curr Drug Targets. 2018;19(10):1191-1204.

[32] Nieto MA, Huang RY, Jackson RA, Thiery JP. EMT: 2016. Cell. 2016;166(1):21-45.

[33] Liu Z, Liang X, Li X, et al. MiRNA-21 functions in ionizing radiation-induced epithelium-to-mesenchymal transition (EMT) by downregulating PTEN. Toxicol Res (Camb). 2019;8(3):328-340.

[34] Wang D, Liu Z, Yan Z, et al. MiRNA-155-5p inhibits epithelium-to-mesenchymal transition (EMT) by targeting GSK-3β during radiation-induced pulmonary fibrosis. Arch Biochem Biophys. 2021;697:108699.

[35] Liang X, Yan Z, Wang P, et al. Irradiation activates MZF1 to inhibit miR-541-5p expression and promote epithelial‒mesenchymal transition (EMT) in radiation-induced pulmonary fibrosis (RIPF) by upregulating slug. Int J Mol Sci. 2021;22(21):11309.

[36] Yan Z, Ao X, Liang X, et al. Transcriptional inhibition of miR-486-3p by BCL6 upregulates Snail and induces epithelial‒mesenchymal transition during radiation-induced pulmonary fibrosis. Respir Res. 2022;23(1):104.

[37] Nagaraja SS, Subramanian U, Nagarajan D. Radiation-induced H3K9 methylation on E-cadherin promoter mediated by ROS/Snail axis : role of G9a signaling during lung epithelial‒mesenchymal transition. Toxicol In Vitro. 2021;70:105037.

[38] Ligresti G, Caporarello N, Meridew JA, et al. CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis. JCI Insight. 2019;5(12):e127111.

[39] Pyun BJ, Seo HR, Lee HJ, et al. Mutual regulation between DNA-PKcs and Snail1 leads to increased genomic instability and aggressive tumor characteristics. Cell Death Dis. 2013;4(2):e517.

[40] Zhang J, Jiang H, Xu D, Wu W-J, Chen H-D, He L. DNA-PKcs mediates an epithelial‒mesenchymal transition process promoting cutaneous squamous cell carcinoma invasion and metastasis by targeting the TGF-β1/Smad signaling pathway. Onco Targets Ther. 2019;12:9395-9405.

[41] Zhou S, Zhu J, Zhou PK, Gu Y. Alveolar type 2 epithelial cell senescence and radiation-induced pulmonary fibrosis. Front Cell Dev Biol. 2022;10:999600.

[42] Otoupalova E, Smith S, Cheng G, Thannickal VJ. Oxidative stress in pulmonary fibrosis. Compr Physiol. 2020;10(2):509-547.

[43] Heitmeir B, Deniz M, Janni W, Rack B, Schochter F, Wiesmüller L. Circulating tumor cells in breast cancer patients: a balancing act between stemness, EMT features and DNA damage responses. Cancers (Basel). 2022;14(4):997.

[44] Zhang J, Tian XJ, Zhang H, et al. TGF-β-induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci Signal. 2014;7(345):ra91.

[45] Lindley LE, Briegel KJ. Molecular characterization of TGFbeta-induced epithelial‒mesenchymal transition in normal finite lifespan human mammary epithelial cells. Biochem Biophys Res Commun. 2010;399(4):659-664.

[46] Taube JH, Herschkowitz JI, Komurov K, et al. Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci U S A. 2010;107(35):15449-15454.

[47] Liu Q, Ma L, Jones T, et al. Subjugation of TGFβ signaling by human papilloma virus in head and neck squamous cell carcinoma shifts DNA repair from homologous recombination to alternative end joining. Clin Cancer Res. 2018;24(23):6001-6014.

[48] Pustovalova M, Alhaddad L, Blokhina T, et al. The CD44high subpopulation of multifraction irradiation-surviving NSCLC cells exhibits partial EMT-program activation and DNA damage response depending on their p53 status. Int J Mol Sci. 2021;22(5):2369.

[49] Chen H, Chen N, Li F, et al. Repeated radon exposure induced lung injury and epithelial‒mesenchymal transition through the PI3K/AKT/mTOR pathway in human bronchial epithelial cells and mice. Toxicol Lett. 2020;334:4-13.

[50] Oeck S, Al-Refae K, Riffkin H, et al. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity. Sci Rep. 2017;7:42700.

[51] Kumar SS, Ghosh A, Devasagayam TP, Chauhan PS. Effect of vanillin on methylene blue plus light-induced single-strand breaks in plasmid pBR322 DNA. Mutat Res. 2000;469(2):207-214.

[52] Sinigaglia M, Lehmann M, Baumgardt P, et al. Vanillin as a modulator agent in SMART test: inhibition in the steps that precede N-methyl-N-nitrosourea-, N-ethyl-N-nitrosourea-, ethylmethanesulphonate- and bleomycin-genotoxicity. Mutat Res. 2006;607(2):225-230.

[53] Keshava C, Keshava N, Ong TM, Nath J. Protective effect of vanillin on radiation-induced micronuclei and chromosomal aberrations in V79 cells. Mutat Res. 1998;397(2):149-159.

[54] Sasaki YF, Imanishi H, Watanabe M, Ohta T, Shirasu Y. Suppressing effect of antimutagenic flavorings on chromosome aberrations induced by UV-light or X-rays in cultured Chinese hamster cells. Mutat Res. 1990;229(1):1-10.

[55] Zheng H, Chen ZW, Wang L, et al. Radioprotection of 4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells is associated with minimizing DNA damage and activating Akt. Eur J Pharm Sci. 2008;33(1):52-59.

[56] Novais EJ, Tran VA, Johnston SN, et al. Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice. Nat Commun. 2021;12(1):5213.

[57] Hickson LJ, Langhi Prata LGP, Bobart SA, et al. Senolytics decrease senescent cells in humans: preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease. EBioMedicine. 2019;47:446-456.

[58] Yang H, Wang H, Ren J, Chen Q, Chen ZJ. cGAS is essential for cellular senescence. Proc Natl Acad Sci U S A. 2017;114(23):E4612-E4620.

[59] Zhang D, Liu Y, Zhu Y, et al. A non-canonical cGAS-STING-PERK pathway facilitates the translational program critical for senescence and organ fibrosis. Nat Cell Biol. 2022;24(5):766-782.

[60] Wang L, Zhang Y, Ren Y, et al. Pharmacological targeting of cGAS/STING-YAP axis suppresses pathological angiogenesis and ameliorates organ fibrosis. Eur J Pharmacol. 2022;932:175241.

[61] Sun X, Liu T, Zhao J, et al. DNA-PK deficiency potentiates cGAS-mediated antiviral innate immunity. Nat Commun. 2020;11(1):6182.

[62] Hristova DB, Oliveira M, Wagner E, et al. DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells. iScience. 2024;27(1):108760.

RIGHTS & PERMISSIONS

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