Ionizing Radiation Promotes Epithelial-Mesenchymal Transition Phenotype and Stem Cell Marker in The Lung adenocarcinoma: In Vitro and Bioinformatic Studiesc

Document Type : Original Article


1 Health Research Center, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Student Research Committee, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Department of Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran

4 Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran


Objective: Ionizing radiation (IR) is one of the major therapeutic approaches in the non-small cell lung cancer (NSCLC);
however, it can paradoxically result in cancer progression likely through promoting epithelial-mesenchymal transition
(EMT) and the cancer stem cell phenotype. Therefore, we aimed to determine whether IR promote EMT/CSC and to
investigate the clinical relevance of EMT/CSC hallmark genes.
Materials and Methods: In this experimental and bioinformatic study, A549 cell line was irradiated with a high dosage
(6 Gy) or a fractionated regimen (2 Gy/day for 15 fractions). The EMT-related features, including cellular morphology,
migratory and invasive capacities were evaluated using scratch assay and transwell migration/invasion assays. The
mRNA levels of EMT-related genes (CDH1, CDH2, SNAI1 and TWIST1), stemness-related markers (CD44, PROM1,
and ALDH1A1) and the CDH2/CDH1 ratio were evaluated via real-time polymerase chain reaction (PCR). The clinical
significance of these genes was assessed in the lung adenocarcinoma (LUAD) samples using online databases.
Results: Irradiation resulted in a dramatic elongation of cell shape and enhanced invasion and migration capabilities. These EMT-like alterations were accompanied with enhanced levels of SNAI1, CDH2, TWIST1, CD44, PROM1, and ALDH1A1 as well as an enhanced CDH2/CDH1 ratio. TCGA analysis revealed that, TWIST1, CDH1, PROM1 and CDH2 were upregulated; whereas, CD44, SNAI1 and ALDH1A1 were downregulated. Additionally, correlations between SNAI1-TWIST1, CDH2- TWIST1, CDH2-SNAI1, and ALDH1A1-PROM1 was positive. Kaplan-Meier survival analysis identified lower expression of CDH1, PROM1 and ALDH1A1 and increased expression of CDH2, SNAI1, and TWIST1 as well as CDH2/CDH1 ratio predict overall survival. Additionally, downregulation of ALDH1A1 and upregulation of CDH2, SNAI1 and TWIST1 could predict a shorter first progression.
Conclusion: Altogether, these findings demonstrated that IR promotes EMT phenotype and stem cell markers in A549
cell line and these genes could function as diagnostic or prognostic indicators in LUAD samples.


1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; 68(1): 7-30.
2. Chen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK. Nonsmall-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer. 2014; 14(8): 535-546.
3. Bradley JD, Paulus R, Komaki R, Masters G, Blumenschein G, Schild S, et al. Standard-dose versus high-dose conformal radiotherapy with concurrent and consolidation carboplatin plus paclitaxel with or without cetuximab for patients with stage IIIA or IIIB non-small-cell lung cancer (RTOG 0617): a randomised, two-bytwo factorial phase 3 study. Lancet Oncol. 2015; 16(2): 187-199.
4. Ghasemi Z, Tahmasebi-Birgani MJ, Ghafari Novin A, Motlagh PE, Teimoori A, Ghadiri A, et al. Fractionated radiation promotes proliferation and radioresistance in bystander A549 cells but not in bystander HT29 cells. Life Sci. 2020; 257: 118087.
5. Gomez-Casal R, Bhattacharya C, Ganesh N, Bailey L, Basse P, Gibson M, et al. Non-small cell lung cancer cells survived ionizing radiation treatment display cancer stem cell and epithelial-mesenchymal transition phenotypes. Mol Cancer. 2013; 12(1): 94.
6. Tahmasebi-Birgani MJ, Teimoori A, Ghadiri A, Mansoury-Asl H, Danyaei A, Khanbabaei H. Fractionated radiotherapy might induce epithelial-mesenchymal transition and radioresistance in a cellular context manner. J Cell Biochem. 2018; 120(5): 8601-8610.
7. Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019; 20(2): 69-84.
8. Ye X, Weinberg RA. Epithelial-mesenchymal plasticity: a central regulator of cancer progression. Trends Cell Biol. 2015; 25(11): 675-686.
9. Brower JV, Amini A, Chen S, Hullett CR, Kimple RJ, Wojcieszynski AP, et al. Improved survival with dose-escalated radiotherapy in stage III non-small-cell lung cancer: analysis of the National Cancer Database. Ann Oncol. 2016; 27(10): 1887-1894.
10. Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Ponce-Rodriguez I, Chakravarthi B, et al. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia. 2017; 19(8): 649-658.
11. Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017; 45(W1): W98-W102.
12. Schober P, Boer C, Schwarte LA. Correlation coefficients: appropriate use and interpretation. Anesth Analg. 2018; 126(5): 1763-1768.
13. Győrffy B, Surowiak P, Budczies J, Lánczky A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One. 2013; 8(12): e82241.
14. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, et al. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016; 66(4): 271-289.
15. Krause M, Dubrovska A, Linge A, Baumann M. Cancer stem cells: Radioresistance, prediction of radiotherapy outcome and specific targets for combined treatments. Adv Drug Deliv Rev. 2017; 109: 63-73.
16. Moding EJ, Kastan MB, Kirsch DG. Strategies for optimizing the response of cancer and normal tissues to radiation. Nat Rev Drug Discov. 2013; 12(7): 526-542.
17. Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol. 2017; 14(10): 611-629.
18. Marcucci F, Stassi G, De Maria R. Epithelial-mesenchymal transition: a new target in anticancer drug discovery. Nat Rev Drug Discov. 2016; 15(5): 311-325.
19. Choi YJ, Baek GY, Park HR, Jo SK, Jung U. Smad2/3-regulated expression of DLX2 is associated with radiation-induced epithelial-mesenchymal transition and radioresistance of A549 and MDA-MB-231 human cancer cell lines. PLoS One. 2016; 11(1): e0147343.
20. Lu J, Zhong Y, Chen J, Lin X, Lin Z, Wang N, et al. Radiation enhances the epithelial- mesenchymal transition of A549 cells via miR3591-5p/USP33/PPM1A. Cell Physiol Biochem. 2018; 50(2): 721-733.
21. Ogata T, Teshima T, Inaoka M, Minami K, Tsuchiya T, Isono M, et al. Carbon ion irradiation suppresses metastatic potential of human non-small cell lung cancer A549 cells through the phosphatidylinositol-
3-kinase/Akt signaling pathway. J Radiat Res. 2011; 52(3): 374-379.
22. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelialmesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15(3): 178-196.
23. Theveneau E, Mayor R. Cadherins in collective cell migration of mesenchymal cells. Curr Opin Cell Biol. 2012; 24(5): 677-684.
24. Yang F, Sun L, Li Q, Han X, Lei L, Zhang H, et al. SET8 promotes epithelial-mesenchymal transition and confers TWIST dual transcriptional activities. EMBO J. 2012; 31(1): 110-123.
25. Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, et al. Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat Cell Biol. 2010; 12(10): 982-992.
26. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, et al. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol. 2008; 10(3): 295-305.
27. Araki K, Shimura T, Suzuki H, Tsutsumi S, Wada W, Yajima T, et al. E/N-cadherin switch mediates cancer progression via TGF-β-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma. Br J Cancer. 2011; 105(12): 1885-1893.
28. Gravdal K, Halvorsen OJ, Haukaas SA, Akslen LA. A switch from E-cadherin to N-cadherin expression indicates epithelial to mesenchymal transition and is of strong and independent importance for the progress of prostate cancer. Clin Cancer Res. 2007; 13(23): 7003-7011.
29. Hui L, Zhang S, Dong X, Tian D, Cui Z, Qiu X. Prognostic significance of twist and N-cadherin expression in NSCLC. PLoS One. 2013; 8(4): e62171.
30. Tran PT, Shroff EH, Burns TF, Thiyagarajan S, Das ST, Zabuawala T, et al. Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis. PLoS Genet. 2012; 8(5): e1002650.
31. Chen C, Zhao S, Karnad A, Freeman JW. The biology and role of CD44 in cancer progression: therapeutic implications. J Hematol Oncol. 2018; 11(1): 64.
32. Jiang H, Zhao W, Shao W. Prognostic value of CD44 and CD44v6 expression in patients with non-small cell lung cancer: meta-analysis. Tumour Biol. 2014; 35(8): 7383-7389.
33. Hirata T, Fukuse T, Naiki H, Hitomi S, Wada H. Expression of CD44 variant exon 6 in stage I non-small cell lung carcinoma as a prognostic factor. Cancer Res. 1998; 58(6): 1108-1110.
34. Luo Z, Wu RR, Lv L, Li P, Zhang LY, Hao QL, et al. Prognostic value of CD44 expression in non-small cell lung cancer: a systematic review. Int J Clin Exp Pathol. 2014; 7(7): 3632-3646.
35. Leung EL, Fiscus RR, Tung JW, Tin VP, Cheng LC, Sihoe AD, et al. Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One. 2010; 5(11): e14062.
36. Louderbough JM, Schroeder JA. Understanding the dual nature of CD44 in breast cancer progression. Mol Cancer Res. 2011; 9(12): 1573-1586.
37. Fuchs K, Hippe A, Schmaus A, Homey B, Sleeman JP, Orian-Rousseau V. Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44. Cell Death Dis. 2013; 4(10): e819.
38. Zhang H, Brown RL, Wei Y, Zhao P, Liu S, Liu X, et al. CD44 splice isoform switching determines breast cancer stem cell state. Genes Dev. 2019; 33(3-4): 166-179.
39. Zhao S, Chen C, Chang K, Karnad A, Jagirdar J, Kumar AP, et al. CD44 expression level and isoform contributes to pancreatic cancer cell plasticity, invasiveness, and response to therapy. Clin Cancer Res. 2016; 22(22): 5592-5604.