Aberrant methylation of GADD45A is associated with decreased radiosensitivity in cervical cancer through the PI3K/AKT signaling pathway

Lou,Meng; Li,Rong; Lang,Ting-Yuan; Zhang,Li-Ying; Zhou,Qi; Li,Li

doi:10.3892/ol.2020.12269

Aberrant methylation of GADD45A is associated with decreased radiosensitivity in cervical cancer through the PI3K/AKT signaling pathway

Authors:
- Meng Lou
- Rong Li
- Ting-Yuan Lang
- Li-Ying Zhang
- Qi Zhou
- Li Li
View Affiliations

Affiliations: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China, Department of Gynecologic Oncology, Chongqing University Cancer Hospital and Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing 400030, P.R. China, Department of Gynecology, The Second Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi 530000, P.R. China
Published online on: November 3, 2020 https://doi.org/10.3892/ol.2020.12269
Article Number: 8
Copyright: © Lou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Epigenetic inactivation of GADD45A is a common occurrence in different types of cancer. However, little is known regarding its association with radiosensitivity in cervical cancer (CC). Thus, the present study aimed to investigate the association between aberrant GADD45A methylation and radiosensitivity in CC. SiHa, HeLa and CaSki CC cells were treated with 5‑azacytidine (5‑azaC), with or without irradiation. The expression levels of GADD45A and AKT related molecules were detected via reverse transcription‑quantitative PCR and western blot analyses. The methylation status of GADD45A was assessed via methylation‑specific PCR and cell proliferation assays, while clonogenic assays and flow cytometric analysis were performed to assess the function of the genes (GADD45A and AKT) in the CC cell lines. The results demonstrated that methylation of GADD45A was significantly higher in the radioresistant tissues (63.16%) compared with the radiosensitive samples (33.33%). In addition, the surviving fraction of SiHa cells following irradiation with 2 Gy was demonstrated to be highest amongst the three CC cells (CaSki, 57±9.5%; HeLa, 70±4% and SiHa, 75±10%). The survival rate of SiHa cells following treatment with 5‑azaC and ionizing radiation (IR) significantly decreased as the radiation dose increased, compared with treatment with IR alone. Following overexpression of GADD45A or treatment with 5‑azaC, the radiosensitivity of SiHa cells significantly increased compared with both the control vector and PBS treated groups. In addition, the AKT inhibitor, MK‑2206, increased the radiosensitivity of SiHa cells. Notably, aberrant methylation of GADD45A was associated with decreased radiosensitivity in CC, and the PI3K/AKT signaling pathway was essential for radioresistance, which was mediated through downregulation of GADD45A.

View Figures

View References

1	Cohen PA, Jhingran A, Oaknin A and Denny L: Cervical cancer. Lancet. 393:169–182. 2019. View Article : Google Scholar : PubMed/NCBI
2	Dickinson JA: Age of initiation of cervical cancer screening. JAMA. 321:611–612. 2019. View Article : Google Scholar : PubMed/NCBI
3	Petrelli F, De Stefani A, Raspagliesi F, Lorusso D and Barni S: Radiotherapy with concurrent cisplatin-based doublet or weekly cisplatin for cervical cancer: A systematic review and meta-analysis. Gynecol Oncol. 134:166–171. 2014. View Article : Google Scholar : PubMed/NCBI
4	Global Burden of Disease Cancer Collaboration, ; Fitzmaurice C, Dicker D, Pain A, Hamavid H, Moradi-Lakeh M, MacIntyre MF, Allen C, Hansen G, Woodbrook R, et al: The Global Burden of Cancer 2013. JAMA Oncol. 1:505–527. 2015. View Article : Google Scholar : PubMed/NCBI
5	Begg AC, Stewart FA and Vens C: Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 11:239–253. 2011. View Article : Google Scholar : PubMed/NCBI
6	Wang W, Zhang F, Hu K and Hou X: Image-guided, intensity-modulated radiation therapy in definitive radiotherapy for 1,433 patients with cervical cancer. Gynecol Oncol. 151:444–448. 2018. View Article : Google Scholar : PubMed/NCBI
7	Ishikawa K, Koyama-Saegusa K, Otsuka Y, Ishikawa A, Kawai S, Yasuda K, Suga T, Michikawa Y, Suzuki M, Iwakawa M and Imai T: Gene expression profile changes correlating with radioresistance in human cell lines. Int J Radiat Oncol Biol Phys. 65:234–245. 2006. View Article : Google Scholar : PubMed/NCBI
8	Theys J, Jutten B, Habets R, Paesmans K, Groot AJ, Lambin P, Wouters BG, Lammering G and Vooijs M: E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells. Radiother Oncol. 99:392–397. 2011. View Article : Google Scholar : PubMed/NCBI
9	Carlson DJ, Yenice KM and Orton CG: Tumor hypoxia is an important mechanism of radioresistance in hypofractionated radiotherapy and must be considered in the treatment planning process. Med Phys. 38:6347–6350. 2011. View Article : Google Scholar : PubMed/NCBI
10	Grana TM, Rusyn EV, Zhou H, Sartor CI and Cox AD: Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways. Cancer Res. 62:4142–4150. 2002.PubMed/NCBI
11	Skvortsova I, Skvortsov S, Stasyk T, Raju U, Popper BA, Schiestl B, von Guggenberg E, Neher A, Bonn GK, Huber LA and Lukas P: Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells. Proteomics. 8:4521–4533. 2008. View Article : Google Scholar : PubMed/NCBI
12	Bristow RG and Hill RP: Hypoxia and metabolism. Hypoxia, DNA repair and genetic instability. Nat Rev Cancer. 8:180–192. 2008. View Article : Google Scholar : PubMed/NCBI
13	Fabbrizi MR, Warshowsky KE, Zobel CL, Hallahan DE and Sharma GG: Molecular and epigenetic regulatory mechanisms of normal stem cell radiosensitivity. Cell Death Discov. 18:4:1172018. View Article : Google Scholar
14	Wei W, Dong Z, Gao H, Zhang YY, Shao LH, Jin LL, Lv YH, Zhao G, Shen YN and Jin SZ: MicroRNA-9 enhanced radiosensitivity and its mechanism of DNA methylation in non-small cell lung cancer. Gene. 710:178–185. 2019. View Article : Google Scholar : PubMed/NCBI
15	Sutton LP, Jeffreys SA, Phillips JL, Taberlay PC, Holloway AF, Ambrose M, Joo JE, Young A, Berry R, Skala M and Brettingham-Moore KH: DNA methylation changes following DNA damage in prostate cancer cells. Epigenetics. 14:989–1002. 2019. View Article : Google Scholar : PubMed/NCBI
16	Hervouet E, Cheray M, Vallette FM and Cartron PF: DNA methylation and apoptosis resistance in cancer cells. Cells. 2:545–573. 2013. View Article : Google Scholar : PubMed/NCBI
17	Desjobert C, El Maï M, Gérard-Hirne T, Guianvarc'h D, Carrier A, Pottier C, Arimondo PB and Riond J: Combined analysis of DNA methylation and cell cycle in cancer cells. Epigenetics. 10:82–91. 2015. View Article : Google Scholar : PubMed/NCBI
18	Pfeifer GP: Defining driver DNA methylation changes in human cancer. Int J Mol Sci. 19:11662018. View Article : Google Scholar
19	Russo G, Landi R, Pezone A, Morano A, Zuchegna C, Romano A, Muller MT, Gottesman ME, Porcellini A and Avvedimento EV: DNA damage and repair modify DNA methylation and chromatin domain of the targeted locus: Mechanism of allele methylation polymorphism. Sci Rep. 6:332222016. View Article : Google Scholar : PubMed/NCBI
20	Borràs-Fresneda M, Barquinero JF, Gomolka M, Hornhardt S, Rössler U, Armengol G and Barrios L: Differences in DNA repair capacity, cell death and transcriptional response after irradiation between a radiosensitive and a radioresistant cell line. Sci Rep. 6:270432016. View Article : Google Scholar : PubMed/NCBI
21	Dukaew N, Konishi T, Chairatvit K, Autsavapromporn N, Soonthornchareonnon N and Wongnoppavich A: Enhancement of radiosensitivity by eurycomalactone in human NSCLC cells through G₂/M cell cycle arrest and delayed DNA double-strand break repair. Oncol Res. 28:161–175. 2020. View Article : Google Scholar : PubMed/NCBI
22	Feng Q, Hawes SE, Stern JE, Dem A, Sow PS, Dembele B, Toure P, Sova P, Laird PW and Kiviat NB: Promoter hypermethylation of tumor suppressor genes in urine from patients with cervical neoplasia. Cancer Epidemiol Biomarkers Prev. 16:1178–1184. 2007. View Article : Google Scholar : PubMed/NCBI
23	Snoek BC, Splunter APV, Bleeker MCG, Ruiten MCV, Heideman DAM, Rurup WF, Verlaat W, Schotman H, Gent MV, Trommel NEV and Steenbergen RDM: Cervical cancer detection by DNA methylation analysis in urine. Sci Rep. 9:30882019. View Article : Google Scholar : PubMed/NCBI
24	Kazim N, Adhikari A, Oh TJ and Davie J: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma. Cell Death Dis. 11:672020. View Article : Google Scholar : PubMed/NCBI
25	Chi HC, Tsai CY, Tsai MM and Lin KH: Impact of DNA and RNA methylation on radiobiology and cancer progression. Int J Mol Sci. 19:5552018. View Article : Google Scholar
26	Jiao X, Zhang S, Jiao J, Zhang T, Qu W, Muloye GM, Kong B, Zhang Q and Cui B: Promoter methylation of SEPT9 as a potential biomarker for early detection of cervical cancer and its overexpression predicts radioresistance. Clin Epigenetics. 11:1202019. View Article : Google Scholar : PubMed/NCBI
27	Guerrero-Setas D, Pérez-Janices N, Blanco-Fernandez L, Ojer A, Cambra K, Berdasco M, Esteller M, Maria-Ruiz S, Torrea N and Guarch R: LRASSF hypermethylation is present and related to shorter survival in squamous cervical cancer. Mod Pathol. 26:1111–1122. 2013. View Article : Google Scholar : PubMed/NCBI
28	Zerbini LF and Libermann TA: GADD45 deregulation in cancer: Frequently methylated tumor suppressors and potential therapeutic targets. Clin Cancer Res. 11:6409–6413. 2005. View Article : Google Scholar : PubMed/NCBI
29	Ying J, Srivastava G, Hsieh WS, Gao Z, Murray P, Liao SK, Ambinder R and Tao Q: The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors. Clin Cancer Res. 11:6442–6449. 2005. View Article : Google Scholar : PubMed/NCBI
30	Zackrisson B: Radiobiological cell survival models. A methodological overview. Acta Oncol. 31:433–441. 1992. View Article : Google Scholar : PubMed/NCBI
31	Na YK, Lee SM, Hong HS, Kim JB, Park JY and Kim DS: Hypermethylation of growth arrest DNA-damage-inducible gene 45 in non-small cell lung cancer and its relationship with clinicopathologic features. Mol Cells. 30:89–92. 2010. View Article : Google Scholar : PubMed/NCBI
32	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
33	Tsikouras P, Zervoudis S, Manav B, Tomara E, Iatrakis G, Romanidis C, Bothou A and Galazios G: Cervical cancer: Screening, diagnosis and staging. J BUON. 21:320–325. 2016.PubMed/NCBI
34	Egger G, Liang G, Aparicio A and Jones PA: Epigenetics in human disease and prospects for epigenetic therapy. Nature. 429:457–463. 2004. View Article : Google Scholar : PubMed/NCBI
35	Zhang W, Li T, Shao Y, Zhang C, Wu Q, Yang H, Zhang J, Guan M, Yu B and Wan J: Semi-quantitative detection of GADD45-gamma methylation levels in gastric, colorectal and pancreatic cancers using methylation-sensitive high-resolution melting analysis. J Cancer Res Clin Oncol. 136:1267–1273. 2010. View Article : Google Scholar : PubMed/NCBI
36	Kim JS, Kim SY, Lee M, Kim SH, Kim SM and Kim EJ: Radioresistance in a human laryngeal squamous cell carcinoma cell line is associated with DNA methylation changes and topoisomerase II α. Cancer Biol Ther. 16:558–566. 2015. View Article : Google Scholar : PubMed/NCBI
37	Smits KM, Melotte V, Niessen HE, Dubois L, Oberije C, Troost EG, Starmans MH, Boutros PC, Vooijs M, van Engeland M and Lambin P: Epigenetics in radiotherapy: Where are we heading? Radiother Oncol. 111:168–177. 2014. View Article : Google Scholar : PubMed/NCBI
38	Dote H, Cerna D, Burgan WE, Carter DJ, Cerra MA, Hollingshead MG, Camphausen K and Tofilon PJ: Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine. Clin Cancer Res. 11:4571–4579. 2005. View Article : Google Scholar : PubMed/NCBI
39	Kim EH, Park AK, Dong SM, Ahn JH and Park WY: Global analysis of CpG methylation reveals epigenetic control of the radiosensitivity in lung cancer cell lines. Oncogene. 29:4725–4731. 2010. View Article : Google Scholar : PubMed/NCBI
40	Muschel RJ, Soto DE, McKenna WG and Bernhard EJ: Radiosensitization and apoptosis. Oncogene. 17:3359–3363. 1998. View Article : Google Scholar : PubMed/NCBI
41	Yuan YH, Wang HY, Lai Y, Zhong W, Liang WL, Yan FD, Yu Z, Chen JK and Lin Y: Epigenetic inactivation of HOXD10 is associated with human colon cancer via inhibiting the RHOC/AKT/MAPK signaling pathway. Cell Commun Signal. 17:92019. View Article : Google Scholar : PubMed/NCBI
42	Schuurbiers OC, Kaanders JH, van der Heijden HF, Dekhuijzen RP, Oyen WJ and Bussink J: The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol. 4:761–767. 2009. View Article : Google Scholar : PubMed/NCBI
43	Zhou HM, Sun QX and Cheng Y: Paeonol enhances the sensitivity of human ovarian cancer cells to radiotherapy-induced apoptosis due to downregulation of the phosphatidylinositol-3-kinase/Akt/phosphatase and tensin homolog pathway and inhibition of vascular endothelial growth factor. Exp Ther Med. 14:3213–3220. 2017. View Article : Google Scholar : PubMed/NCBI
44	Man J, Shoemake JD, Ma T, Rizzo AE, Godley AR, Wu Q, Mohammadi AM, Bao S, Rich JN and Yu JS: Hyperthermia sensitizes glioma stem-like cells to radiation by inhibiting AKT signaling. Cancer Res. 75:1760–1769. 2015. View Article : Google Scholar : PubMed/NCBI
45	Otani K, Naito Y, Sakaguchi Y, Seo Y, Takahashi Y, Kikuta J, Ogawa K and Ishii M: Cell-cycle-controlled radiation therapy was effective for treating a murine malignant melanoma cell line in vitro and in vivo. Sci Rep. 6:306892016. View Article : Google Scholar : PubMed/NCBI
46	Kim JG, Bae JH, Kim JA, Heo K, Yang K and Yi JM: Combination effect of epigenetic regulation and ionizing radiation in colorectal cancer cells. PLoS One. 9:e1054052014. View Article : Google Scholar : PubMed/NCBI
47	Jiang W, Li YQ, Liu N, Sun Y, He QM, Jiang N, Xu YF, Chen L and Ma J: 5-Azacytidine enhances the radiosensitivity of CNE2 and SUNE1 cells in vitro and in vivo possibly by altering DNA methylation. PLoS One. 9:e932732014. View Article : Google Scholar : PubMed/NCBI
48	Momparler RL: Epigenetic therapy of non-small cell lung cancer using decitabine (5-aza-2′-deoxycytidine). Front Oncol. 3:1882013. View Article : Google Scholar : PubMed/NCBI
49	Kim HJ, Kim JH, Chie EK, Young PD, Kim IA and Kim IH: DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity. Radiat Oncol. 7:392012. View Article : Google Scholar : PubMed/NCBI
50	Wang L, Zhang Y, Li R, Chen Y, Pan X, Li G, Dai F and Yang J: 5-aza-2′-Deoxycytidine enhances the radiosensitivity of breast cancer cells. Cancer Biother Radiopharm. 28:34–44. 2013. View Article : Google Scholar : PubMed/NCBI
51	Steiner M, Clark B, Tang JZ, Zhu T and Lobie PE: 14-3-3σ mediates G2-M arrest produced by 5-aza-2′-deoxycytidine and possesses a tumor suppressor role in endometrial carcinoma cells. Gynecol Oncol. 127:231–240. 2012. View Article : Google Scholar : PubMed/NCBI