Top

Radiation Oncology

Published in:

01-12-2020 | Cervical Cancer | Research

Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription

Authors: Jing-Jie Zheng, Yue He, Yang Liu, Feng-Shuang Li, Zhen Cui, Xiao-Meng Du, Chun-Peng Wang, Yu-Mei Wu

Published in: Radiation Oncology | Issue 1/2020

Abstract

Background

Radiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes “induced” during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy.

Methods

PAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9.

Results

We confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers.

Conclusions

PAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.

Available only for authorised users

Pimple S, Mishra G. Global strategies for cervical cancer prevention and screening. Minerva Ginecol. 2019;71(4):313–20. https://doi.org/10.23736/S0026-4784.19.04397-1.CrossRefPubMed

Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66(2):115–32. https://doi.org/10.3322/caac.21338.CrossRefPubMed

Ye C, Sun NX, Ma Y, et al. MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells. FEBS Lett. 2015;589(6):702–9. https://doi.org/10.1016/j.febslet.2015.01.037.CrossRefPubMed

Lamarca A, Barriuso J, McNamara MG, et al. Biliary tract Cancer: state of the art and potential role of DNA damage repair. Cancer Treat Rev. 2018;70:168–77. https://doi.org/10.1016/j.ctrv.2018.09.002.CrossRefPubMed

Goel S, DeCristo MJ, McAllister SS, et al. CDK4/6 inhibition in Cancer: beyond cell cycle arrest. Trends Cell Biol. 2018;28(11):911–25. https://doi.org/10.1016/j.tcb.2018.07.002.CrossRefPubMedPubMedCentral

Oh SJ, Cho H, Kim S, et al. Targeting Cyclin D-CDK4/6 sensitizes immune-refractory Cancer by blocking the SCP3-NANOG Axis. Cancer Res. 2018;78(10):2638–53. https://doi.org/10.1158/0008-5472.CAN-17-2325.CrossRefPubMedPubMedCentral

Liu HM, Wu Q, Cao JQ, et al. A phenanthroline derivative enhances radiosensitivity of hepatocellular carcinoma cells by inducing mitochondria-dependent apoptosis. Eur J Pharmacol. 2019;843:285–91. https://doi.org/10.1016/j.ejphar.2018.10.031.CrossRefPubMed

Assani G, Zhou Y. Effect of modulation of epithelial-mesenchymal transition regulators Snail1 and Snail2 on cancer cell radiosensitivity by targeting of the cell cycle, cell apoptosis and cell migration/invasion. Oncol Lett. 2019;17(1):23–30. https://doi.org/10.3892/ol.2018.9636.CrossRefPubMed

Gursoy-Yuzugullu O, Carman C, Serafim RB, et al. Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity. Oncotarget. 2017;8(15):24518–32. https://doi.org/10.18632/oncotarget.15543.CrossRefPubMedPubMedCentral

10.

Koo T, Cho BJ, Kim DH, et al. MicroRNA-200c increases radiosensitivity of human cancer cells with activated EGFR-associated signaling. Oncotarget. 2017;8(39):65457–68. https://doi.org/10.18632/oncotarget.18924.CrossRefPubMedPubMedCentral

11.

Liu B, Han D, Zhang T, et al. Hypoxia-induced autophagy promotes EGFR loss in specific cell contexts, which leads to cell death and enhanced radiosensitivity. Int J Biochem Cell Biol. 2019;111:12–8. https://doi.org/10.1016/j.biocel.2018.09.013.CrossRefPubMed

12.

Liu Y, Tian M, Zhao H, et al. IER5 as a promising predictive marker promotes irradiation-induced apoptosis in cervical cancer tissues from patients undergoing chemoradiotherapy. Oncotarget. 2017;8(22):36438–48. https://doi.org/10.18632/oncotarget.16857.CrossRefPubMedPubMedCentral

13.

Tian M, Yu XP, Zhou PK, et al. Immediate early response gene 5 promotes irradiation combined with cisplatin-induced apoptosis in HeLa cells. Int J Clin Exp Pathol. 2018;11(1):262–8 eCollection 2018. (no doi).PubMedPubMedCentral

14.

Li XN, Ji C, Zhou PK, et al. Establishments of IER5 silence and overexpression cervical cancer SiHa cell lines and analysis of radiosensitivity. Int J Clin Exp Pathol. 2016;9(7):6672–82 (no doi).

15.

Yang C, Yin L, Zhou P, et al. Transcriptional regulation of IER5 in response to radiation in HepG2. Cancer Gene Ther. 2016;23(2–3):61–5. https://doi.org/10.1038/cgt.2016.1.CrossRefPubMed

16.

Asano Y, Kawase T, Okabe A, et al. IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis. Sci Rep. 2016;6:19174. https://doi.org/10.1038/srep19174.CrossRefPubMedPubMedCentral

17.

Kim J, Guermah M, Roeder RG. The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS. Cell. 2010;140(4):491–503. https://doi.org/10.1016/j.cell.2009.12.050.CrossRefPubMedPubMedCentral

18.

Karmakar S, Dey P, Vaz AP, et al. PD2/PAF1 at the crossroads of the Cancer network. Cancer Res. 2018;78(2):313–9. https://doi.org/10.1158/0008-5472.CAN-17-2175.CrossRefPubMedPubMedCentral

19.

Vaz AP, Deb S, Rachagani S, et al. Overexpression of PD2 leads to increased tumorigenicity and metastasis in pancreatic ductal adenocarcinoma. Oncotarget. 2016;7(3):3317–31. https://doi.org/10.18632/oncotarget.6580.CrossRefPubMed

20.

Zhi X, Giroux-Leprieur E, Wislez M, et al. Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer. Biochem Biophys Res Commun. 2015;465(4):685–90. https://doi.org/10.1016/j.bbrc.2015.08.017.CrossRefPubMed

21.

Nimmakayala RK, Seshacharyulu P, Lakshmanan I, et al. Cigarette smoke induces stem cell features of pancreatic Cancer cells via PAF1. Gastroenterology. 2018;155(3):892–908 e896. https://doi.org/10.1053/j.gastro.2018.05.041.CrossRefPubMed

22.

Vaz AP, Ponnusamy MP, Rachagani S, et al. Novel role of pancreatic differentiation 2 in facilitating self-renewal and drug resistance of pancreatic cancer stem cells. Br J Cancer. 2014;111(3):486–96. https://doi.org/10.1038/bjc.2014.152.CrossRefPubMedPubMedCentral

23.

Chen FX, Xie P, Collings CK, et al. PAF1 regulation of promoter-proximal pause release via enhancer activation. Science. 2017;357(6357):1294–8. https://doi.org/10.1126/science.aan3269.CrossRefPubMedPubMedCentral

24.

Leroy B, Girard L, Hollestelle A, et al. Analysis of TP53 mutation status in human cancer cell lines: a reassessment. Hum Mutat. 2014;35(6):756–65. https://doi.org/10.1002/humu.22556.CrossRefPubMedPubMedCentral

25.

Yu XP, Wu YM, Liu Y, et al. IER5 is involved in DNA double-Strand breaks repair in association with PAPR1 in Hela cells. Int J Med Sci. 2017;14(12):1292–300. https://doi.org/10.7150/ijms.21510.CrossRefPubMedPubMedCentral

26.

Lopez J, Poitevin A, Mendoza-Martinez V, et al. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance. BMC Cancer. 2012;12:48. https://doi.org/10.1186/1471-2407-12-48.CrossRefPubMedPubMedCentral

27.

Li X, Matilainen O, Jin C, et al. Specific SKN-1/Nrf stress responses to perturbations in translation elongation and proteasome activity. PLoS Genet. 2011;7(6):e1002119. https://doi.org/10.1371/journal.pgen.1002119.CrossRefPubMedPubMedCentral

28.

Haring M, Offermann S, Danker T, et al. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods. 2007;3:11. https://doi.org/10.1186/1746-4811-3-11.CrossRefPubMedPubMedCentral

29.

Yang C, Wang Y, Hao C, et al. IER5 promotes irradiation- and cisplatin-induced apoptosis in human hepatocellular carcinoma cells. Am J Transl Res. 2016;8(4):1789–98 eCollection 2016. (no doi).PubMedPubMedCentral

30.

Hou L, Wang Y, Liu Y, et al. Paf1C regulates RNA polymerase II progression by modulating elongation rate. Proc Natl Acad Sci U S A. 2019;116(29):14583–92. https://doi.org/10.1073/pnas.1904324116.CrossRefPubMedPubMedCentral

31.

Moniaux N, Nemos C, Schmied BM, et al. The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis. Oncogene. 2006;25(23):3247–57. https://doi.org/10.1038/sj.onc.1209353.CrossRefPubMed

32.

Van Oss SB, Cucinotta CE, Arndt KM. Emerging insights into the roles of the Paf1 complex in gene regulation. Trends Biochem Sci. 2017;42(10):788–98. https://doi.org/10.1016/j.tibs.2017.08.003.CrossRefPubMedPubMedCentral

33.

Peng Y, Zhang Y. Enhancer and super-enhancer: positive regulators in gene transcription. Animal Model Exp Med. 2018;1(3):169–79. https://doi.org/10.1002/ame2.12032.CrossRefPubMedPubMedCentral

34.

Chen FX, Woodfin AR, Gardini A, et al. PAF1, a molecular regulator of promoter-proximal pausing by RNA polymerase II. Cell. 2015;162(5):1003–15. https://doi.org/10.1016/j.cell.2015.07.042.CrossRefPubMedPubMedCentral

Title: Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription
Authors: Jing-Jie Zheng
Yue He
Yang Liu
Feng-Shuang Li
Zhen Cui
Xiao-Meng Du
Chun-Peng Wang
Yu-Mei Wu
Publication date: 01-12-2020
Publisher: BioMed Central
Keywords: Cervical Cancer
Cervical Cancer
Published in: Radiation Oncology / Issue 1/2020
Electronic ISSN: 1748-717X
DOI: https://doi.org/10.1186/s13014-020-01580-w

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2020

125I seeds irradiation inhibits tumor growth and induces apoptosis by Ki-67, P21, survivin, livin and caspase-9 expression in lung carcinoma xenografts

Feasibility study: spot-scanning proton arc therapy (SPArc) for left-sided whole breast radiotherapy

A monocentric, open-label randomized standard-of-care controlled study of XONRID®, a medical device for the prevention and treatment of radiation-induced dermatitis in breast and head and neck cancer patients

p63+Krt5+ basal cells are increased in the squamous metaplastic epithelium of patients with radiation-induced chronic Rhinosinusitis

Deep learning vs. atlas-based models for fast auto-segmentation of the masticatory muscles on head and neck CT images

Early circulating tumour DNA kinetics measured by ultra-deep next-generation sequencing during radical radiotherapy for non-small cell lung cancer: a feasibility study