ZNF471 modulates EMT and functions as methylation regulated tumor suppressor with diagnostic and prognostic significance in cervical cancer
Tóm tắt
Cervical cancer (CC) is a leading cause of cancer-related death among women in developing countries. However, the underlying mechanisms and molecular targets for therapy remain to be fully understood. We investigated the epigenetic regulation, biological functions, and clinical utility of zinc-finger protein 471 (ZNF471) in CC. Analysis of cervical tissues and five independent public datasets of CC showed significant hypermethylation of the ZNF471 gene promoter. In CC cell lines, promoter DNA methylation was inversely correlated with ZNF471 expression. The sensitivity and specificity of the ZNF471 hypermethylation for squamous intraepithelial lesion (SIL) vs tumor and normal vs tumor was above 85% with AUC of 0.937. High methylation and low ZNF471 expression predicted poor overall and recurrence-free survival. We identified −686 to +114 bp as ZNF471 promoter, regulated by methylation using transient transfection and luciferase assays. The promoter CpG site methylation of ZNF471 was significantly different among cancer types and tumor grades. Gal4-based heterologous luciferase reporter gene assays revealed that ZNF471 acts as a transcriptional repressor. The retroviral mediated overexpression of ZNF471 in SiHa and CaSki cells inhibited growth, proliferation, cell migration, invasion; delayed cell cycle progression in vitro by increasing cell doubling time; and reduced tumor growth in vivo in nude mice. ZNF471 overexpression inhibited key members of epithelial-mesenchymal transition (EMT), Wnt, and PI3K-AKT signaling pathways. ZNF471 inhibited EMT by directly targeting vimentin as analyzed by bioinformatic analysis, ChIP-PCR, and western blotting. Thus, ZNF471 CpG specific promoter methylation may determine the prognosis of CC and could function as a potential tumor suppressor by targeting EMT signaling.
Tài liệu tham khảo
Agarwal SK, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY, et al. Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell. 1999;96(1):143–52 https://linkinghub.elsevier.com/retrieve/pii/S0092867400809678.
Ambrosini G, Groux R, Bucher P. PWMScan: a fast tool for scanning entire genomes with a position-specific weight matrix. Hancock J, editor. Bioinformatics. 2018;34(14):2483–4 https://academic.oup.com/bioinformatics/article/34/14/2483/4921176.
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(Web Server):W202–8. https://doi.org/10.1093/nar/gkp335.
Bhat S, Kabekkodu SP, Noronha A, Satyamoorthy K. Biological implications and therapeutic significance of DNA methylation regulated genes in cervical cancer. Biochimie. 2016;121:298–311 https://linkinghub.elsevier.com/retrieve/pii/S0300908415004320.
Bhat S, Kabekkodu SP, Jayaprakash C, Radhakrishnan R, Ray S, Satyamoorthy K. Gene promoter-associated CpG island hypermethylation in squamous cell carcinoma of the tongue. Virchows Arch. 2017a;470(4):445–54. https://doi.org/10.1007/s00428-017-2094-2.
Bhat S, Kabekkodu SP, Varghese VK, Chakrabarty S, Mallya SP, Rotti H, et al. Aberrant gene-specific DNA methylation signature analysis in cervical cancer. Tumor Biol. 2017b;39(3):101042831769457. https://doi.org/10.1177/1010428317694573.
Burk RD, Chen Z, Saller C, Tarvin K, Carvalho AL, Scapulatempo-Neto C, et al. Integrated genomic and molecular characterization of cervical cancer. Nature. 2017;543(7645):378–84.
Cao L, Wang S, Zhang Y, Wong K-C, Nakatsu G, Wang X, et al. Zinc-finger protein 471 suppresses gastric cancer through transcriptionally repressing downstream oncogenic PLS3 and TFAP2A. Oncogene. 2018;37(26):3601–16 http://www.nature.com/articles/s41388-018-0220-5.
Castellano E, Downward J. RAS interaction with PI3K: more than just another effector pathway. genes. Cancer. 2011;2(3):261–74. https://doi.org/10.1177/1947601911408079.
Das PM, Singal R. DNA methylation and cancer. J. Clin. Oncol. 2004;22(22):4632–42. https://doi.org/10.1200/JCO.2004.07.151.
Davidowitz RA, Selfors LM, Iwanicki MP, Elias KM, Karst A, Piao H, et al. Mesenchymal gene program–expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance. J. Clin. Invest. 2014;124(6):2611–25 http://www.jci.org/articles/view/69815.
Farkas SA, Milutin-Gašperov N, Grce M, Nilsson TK. Genome-wide DNA methylation assay reveals novel candidate biomarker genes in cervical cancer. Epigenetics. 2013;8(11):1213–25. https://doi.org/10.4161/epi.26346.
Garg M. Epithelial-mesenchymal transition - activating transcription factors - multifunctional regulators in cancer. World J. Stem Cells. 2013;5(4):188. http://www.wjgnet.com/1948-0210/full/v5/i4/188.htm–95.
Giftson Senapathy J, Umadevi P, Kannika PS. The present scenario of cervical cancer control and HPV epidemiology in India: an outline. Asian Pacific J. Cancer Prev. 2011;12(5):1107–15.
Gomih A, Smith JS, North KE, Hudgens MG, Brewster WR, Huang Z, et al. DNA methylation of imprinted gene control regions in the regression of low-grade cervical lesions. Int. J. Cancer. 2018;143(3):552–60. https://doi.org/10.1002/ijc.31350.
Hanley MP, Hahn MA, Li AX, Wu X, Lin J, Wang J, et al. Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia. Oncogene. 2017;36(35):5035–44 http://www.nature.com/articles/onc2017130.
Hu X, Sui X, Li L, Huang X, Rong R, Su X, et al. Protocadherin 17 acts as a tumour suppressor inducing tumour cell apoptosis and autophagy, and is frequently methylated in gastric and colorectal cancers. J. Pathol. 2013;229(1):62–73. https://doi.org/10.1002/path.4093.
Huang X, Yuan W, Huang W, Bai Y, Deng Y, Zhu C, et al. ZNF569, a novel KRAB-containing zinc finger protein, suppresses MAPK signaling pathway. Biochem. Biophys. Res. Commun. 2006;346(3):621–8 https://linkinghub.elsevier.com/retrieve/pii/S0006291X06010916.
Kabekkodu SP, Bhat S, Radhakrishnan R, Aithal A, Mascarenhas R, Pandey D, et al. DNA promoter methylation-dependent transcription of the double C2-like domain β (DOC2B) gene regulates tumor growth in human cervical cancer. J. Biol. Chem. 2014;289(15):10637–49. https://doi.org/10.1074/jbc.M113.491506.
Kaneda A, Wakazono K, Tsukamoto T, Watanabe N, Yagi Y, Tatematsu M, et al. Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers. Cancer Res. 2004;64(18):6410–5. https://doi.org/10.1158/0008-5472.CAN-04-1543.
Karayan-Tapon L, Quillien V, Guilhot J, Wager M, Fromont G, Saikali S, et al. Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J. Neurooncol. 2010;97(3):311–22. https://doi.org/10.1007/s11060-009-0031-1.
Khaki-khatibi F, Ghorbani M, Sabzichi M, Ramezani F, Mohammadian J. Adjuvant therapy with stattic enriches the anti-proliferative effect of doxorubicin in human ZR-75-1 breast cancer cells via arresting cell cycle and inducing apoptosis. Biomed Pharmacother. 2019;109:1240–8 https://linkinghub.elsevier.com/retrieve/pii/S0753332218357287.
Kolsch V, Charest PG, Firtel RA. The regulation of cell motility and chemotaxis by phospholipid signaling. J. Cell Sci. 2008;121(5):551–9. https://doi.org/10.1242/jcs.023333.
Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial–mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15(3):178–96 http://www.nature.com/articles/nrm3758.
Larue L, Bellacosa A. Epithelial–mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/AKT pathways. Oncogene. 2005;24(50):7443–54 http://www.nature.com/articles/1209091.
Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial–mesenchymal transition: new insights in signaling, development, and disease. J. Cell Biol. 2006;172(7):973–81 https://rupress.org/jcb/article/172/7/973/44183/The-epithelialmesenchymal-transition-new-insights.
Li H-J, Yu P-N, Huang K-Y, Su H-Y, Hsiao T-H, Chang C-P, et al. NKX6.1 functions as a metastatic suppressor through epigenetic regulation of the epithelial–mesenchymal transition. Oncogene. 2016;35(17):2266–78 http://www.nature.com/articles/onc2015289.
Lleras RA, Adrien LR, Smith RV, Brown B, Jivraj N, Keller C, et al. Hypermethylation of a cluster of Krüppel-type zinc finger protein genes on chromosome 19q13 in oropharyngeal squamous cell carcinoma. Am. J. Pathol. 2011;178(5):1965–74 https://linkinghub.elsevier.com/retrieve/pii/S0002944011001659.
Lupo A, Cesaro E, Montano G, Zurlo D, Izzo P, Costanzo P. KRAB-zinc finger proteins: a repressor family displaying multiple biological functions. Curr. Genomics. 2013;14(4):268–78 http://www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2029&volume=14&issue=4&spage=268.
Martin TC, Yet I, Tsai P-C, Bell JT. coMET: visualisation of regional epigenome-wide association scan results and DNA co-methylation patterns. BMC Bioinformatics. 2015;16(1):131. https://doi.org/10.1186/s12859-015-0568-2.
Marttila S, Kananen L, Häyrynen S, Jylhävä J, Nevalainen T, Hervonen A, et al. Ageing-associated changes in the human DNA methylome: genomic locations and effects on gene expression. BMC Genomics. 2015 Dec 14;16(1):179. https://doi.org/10.1186/s12864-015-1381-z.
Mirantes C, Espinosa I, Ferrer I, Dolcet X, Prat J, Matias-Guiu X. Epithelial-to-mesenchymal transition and stem cells in endometrial cancer. Hum Pathol. 2013;44(10):1973–81 https://linkinghub.elsevier.com/retrieve/pii/S0046817713001780.
Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, et al. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer. 2014;14(1):54. https://doi.org/10.1186/1471-2407-14-54.
Stephen JK, Chen KM, Havard S, Harris G, Worsham MJ. Promoter methylation in head and neck tumorigenesis. 2012:187–206. https://doi.org/10.1007/978-1-61779-612-8_11.
Sun R, Xiang T, Tang J, Peng W, Luo J, Li L, et al. 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer. Theranostics. 2020;10(5):2243–59 http://www.thno.org/v10p2243.htm.
Tao C, Luo J, Tang J, Zhou D, Feng S, Qiu Z, et al. The tumor suppressor Zinc finger protein 471 suppresses breast cancer growth and metastasis through inhibiting AKT and Wnt/β-catenin signaling. Clin. Epigenetics. 2020;12(1):173. https://doi.org/10.1186/s13148-020-00959-6.
Teschendorff AE, Jones A, Fiegl H, Sargent A, Zhuang JJ, Kitchener HC, et al. Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation. Genome Med. 2012;4(3):24. https://doi.org/10.1186/gm323.
Vidal E, Sayols S, Moran S, Guillaumet-Adkins A, Schroeder MP, Royo R, et al. A DNA methylation map of human cancer at single base-pair resolution. Oncogene. 2017;36(40):5648–57 http://www.nature.com/articles/onc2017176.
Wang S, Cheng Y, Du W, Lu L, Zhou L, Wang H, et al. Zinc-finger protein 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA transcription in gastric cancer. Gut. 2013;62(6):833–41. https://doi.org/10.1136/gutjnl-2011-301776.
Wentzensen N, Sherman ME, Schiffman M, Wang SS. Utility of methylation markers in cervical cancer early detection: appraisal of the state-of-the-science. Gynecol. Oncol. 2009;112(2):293–9 https://linkinghub.elsevier.com/retrieve/pii/S0090825808008822.
Wheeler CM. Advances in primary and secondary interventions for cervical cancer: human papillomavirus prophylactic vaccines and testing. Nat. Clin. Pract. Oncol. 2007;4(4):224–35 http://www.nature.com/articles/ncponc0770.
Wiggins H, Rappoport J. An agarose spot assay for chemotactic invasion. Biotechniques. 2010;48(2):121–4. https://doi.org/10.2144/000113353.
Xiao Y, Xiang T, Luo X, Li C, Li Q, Peng W, et al. Zinc-finger protein 545 inhibits cell proliferation as a tumor suppressor through inducing apoptosis and is disrupted by promoter methylation in breast cancer. Das GM, editor. PLoS One. 2014;9(10):e110990. https://doi.org/10.1371/journal.pone.0110990.
Xu N, Zhang L, Meisgen F, Harada M, Heilborn J, Homey B, et al. MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion. J. Biol. Chem. 2012;287(35):29899–908. https://doi.org/10.1074/jbc.M112.391243.
Yates C. Prostate tumor cell plasticity: a consequence of the microenvironment. 2011:81–90. https://doi.org/10.1007/978-1-4614-0254-1_7.
Yu J, Liang QY, Wang J, Cheng Y, Wang S, Poon TCW, et al. Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer. Oncogene. 2013 Jan 27;32(3):307–17 http://www.nature.com/articles/onc201254.
Zhang J, Huang K. Pan-cancer analysis of frequent DNA co-methylation patterns reveals consistent epigenetic landscape changes in multiple cancers. BMC Genomics. 2017;18(S1):1045. https://doi.org/10.1186/s12864-016-3259-0.
Zhuang J, Jones A, Lee S-H, Ng E, Fiegl H, Zikan M, et al. The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women’s cancer. Absher D, editor. PLoS Genet. 2012;8(2):e1002517. https://doi.org/10.1371/journal.pgen.1002517.