Identification of an E3 ligase-encoding gene RFWD3 in non-small cell lung cancer

Abstract

In order to unveil ubiquitin pathway genes (UPGs) that are essential for non-small cell lung cancer (NSCLC) cell proliferation, we recently conducted a siRNA screening experiment to knockdown the expression of 696 UPGs found in the human genome in A549 and H1975 NSCLC cells. We found that silencing of one of the candidates, RFWD3 that encodes an E3 ubiquitin ligase essential for the repair of DNA interstrand cross-links in response to DNA damage, led to dramatic inhibition of NSCLC cell proliferation with significant Z-scores. Knockdown of RFWD3 suppressed colony forming activity of NSCLC cells. We further evaluated the significance of RFWD3 in NSCLCs and found that this gene was more elevated in tumor samples than in paired normal lung tissues and was inversely associated with the clinical outcome of patients with NSCLC. Moreover, RFWD3 expression was significantly higher in smokers than in non-smokers. These results show for the first time that RFWD3 is required for NSCLC cell proliferation and may have an important role in lung carcinogenesis.

Keywords

RFWD3 / NSCLC / prognosis / tobacco smoke

References

[1] Wang D, Ma L, Wang B, Liu J, Wei W. E3 ubiquitin ligases in cancer and implications for therapies. Cancer Metastasis Rev 2017; 36(4): 683–702

[2] Kitagawa K, Kitagawa M. The SCF-type E3 ubiquitin ligases as cancer targets. Curr Cancer Drug Targets 2016; 16(2): 119–129

[3] Uchida C, Kitagawa M. RING-, HECT-, and RBR-type E3 ubiquitin ligases: involvement in human cancer. Curr Cancer Drug Targets 2016; 16(2): 157–174

[4] Zheng N, Shabek N. Ubiquitin ligases: structure, function, and regulation. Annu Rev Biochem 2017; 86(1): 129–157

[5] Liu J, Shaik S, Dai X, Wu Q, Zhou X, Wang Z, Wei W. Targeting the ubiquitin pathway for cancer treatment. Biochim Biophys Acta 2015; 1855(1): 50–60

[6] Gabrielsen M, Buetow L, Nakasone MA, Ahmed SF, Sibbet GJ, Smith BO, Zhang W, Sidhu SS, Huang DT. A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants. Mol Cell 2017; 68(2): 456–470.e10

[7] Mu JJ, Wang Y, Luo H, Leng M, Zhang J, Yang T, Besusso D, Jung SY, Qin J. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints. J Biol Chem 2007; 282(24): 17330–17334

[8] Fu X, Yucer N, Liu S, Li M, Yi P, Mu JJ, Yang T, Chu J, Jung SY, O’Malley BW, Gu W, Qin J, Wang Y. RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage. Proc Natl Acad Sci USA 2010; 107(10): 4579–4584

[9] Gong Z, Chen J. E3 ligase RFWD3 participates in replication checkpoint control. J Biol Chem 2011; 286(25): 22308–22313

[10] Liu S, Chu J, Yucer N, Leng M, Wang SY, Chen BPC, Hittelman WN, Wang Y. RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response. J Biol Chem 2011; 286(25): 22314–22322

[11] Knies K, Inano S, Ramírez MJ, Ishiai M, Surrallés J, Takata M, Schindler D. Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia. J Clin Invest 2017; 127(8): 3013–3027

[12] Mitchell JS, Li N, Weinhold N, Försti A, Ali M, van Duin M, Thorleifsson G, Johnson DC, Chen B, Halvarsson BM, Gudbjartsson DF, Kuiper R, Stephens OW, Bertsch U, Broderick P, Campo C, Einsele H, Gregory WA, Gullberg U, Henrion M, Hillengass J, Hoffmann P, Jackson GH, Johnsson E, Jöud M, Kristinsson SY, Lenhoff S, Lenive O, Mellqvist UH, Migliorini G, Nahi H, Nelander S, Nickel J, Nöthen MM, Rafnar T, Ross FM, da Silva Filho MI, Swaminathan B, Thomsen H, Turesson I, Vangsted A, Vogel U, Waage A, Walker BA, Wihlborg AK, Broyl A, Davies FE, Thorsteinsdottir U, Langer C, Hansson M, Kaiser M, Sonneveld P, Stefansson K, Morgan GJ, Goldschmidt H, Hemminki K, Nilsson B, Houlston RS. Genome-wide association study identifies multiple susceptibility loci for multiple myeloma. Nat Commun 2016; 7: 12050

[13] Chung CC, Kanetsky PA, Wang Z, Hildebrandt MAT, Koster R, Skotheim RI, Kratz CP, Turnbull C, Cortessis VK, Bakken AC, Bishop DT, Cook MB, Erickson RL, Fosså SD, Jacobs KB, Korde LA, Kraggerud SM, Lothe RA, Loud JT, Rahman N, Skinner EC, Thomas DC, Wu X, Yeager M, Schumacher FR, Greene MH, Schwartz SM, McGlynn KA, Chanock SJ, Nathanson KL. Meta-analysis identifies four new loci associated with testicular germ cell tumor. Nat Genet 2013; 45(6): 680–685

[14] Zhang DL, Qu LW, Ma L, Zhou YC, Wang GZ, Zhao XC, Zhang C, Zhang YF, Wang M, Zhang MY, Yu H, Sun BB, Gao SH, Cheng X, Guo MZ, Huang YC, Zhou GB. Genome-wide identification of transcription factors that are critical to non-small cell lung cancer. Cancer Lett 2018; 434: 132–143

[15] Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R. Statistical practice in high-throughput screening data analysis. Nat Biotechnol 2006; 24(2): 167–175

[16] Zhao XC, Wang GZ, Zhou YC, Liu J, Ma L, Zhang C, Zhang DL, Gao SH, Qu LW, Zhang B, Wang CL, Huang YC, Chen L, Zhou GB. Genome-wide identification of CDC34 that stabilizes EGFR and promotes lung carcinogenesis. bioRxiv 2018

[17] Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Varambally R, Yu J, Briggs BB, Barrette TR, Anstet MJ, Kincead-Beal C, Kulkarni P, Varambally S, Ghosh D, Chinnaiyan AM. Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 2007; 9(2): 166–180

[18] Hou J, Aerts J, den Hamer B, van Ijcken W, den Bakker M, Riegman P, van der Leest C, van der Spek P, Foekens JA, Hoogsteden HC, Grosveld F, Philipsen S. Gene expression-based classification of non-small cell lung carcinomas and survival prediction. PLoS One 2010; 5(4): e10312

[19] Selamat SA, Chung BS, Girard L, Zhang W, Zhang Y, Campan M, Siegmund KD, Koss MN, Hagen JA, Lam WL, Lam S, Gazdar AF, Laird-Offringa IA. Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression. Genome Res 2012; 22(7): 1197–1211

[20] Landi MT, Dracheva T, Rotunno M, Figueroa JD, Liu H, Dasgupta A, Mann FE, Fukuoka J, Hames M, Bergen AW, Murphy SE, Yang P, Pesatori AC, Consonni D, Bertazzi PA, Wacholder S, Shih JH, Caporaso NE, Jen J. Gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival. PLoS One 2008; 3(2): e1651

[21] Okayama H, Kohno T, Ishii Y, Shimada Y, Shiraishi K, Iwakawa R, Furuta K, Tsuta K, Shibata T, Yamamoto S, Watanabe S, Sakamoto H, Kumamoto K, Takenoshita S, Gotoh N, Mizuno H, Sarai A, Kawano S, Yamaguchi R, Miyano S, Yokota J. Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas. Cancer Res 2012; 72(1): 100–111

[22] Wachi S, Yoneda K, Wu R. Interactome-transcriptome analysis reveals the high centrality of genes differentially expressed in lung cancer tissues. Bioinformatics 2005; 21(23): 4205–4208

[23] Stearman RS, Dwyer-Nield L, Zerbe L, Blaine SA, Chan Z, Bunn PA Jr, Johnson GL, Hirsch FR, Merrick DT, Franklin WA, Baron AE, Keith RL, Nemenoff RA, Malkinson AM, Geraci MW. Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model. Am J Pathol 2005; 167(6): 1763–1775

[24] Lu TP, Tsai MH, Lee JM, Hsu CP, Chen PC, Lin CW, Shih JY, Yang PC, Hsiao CK, Lai LC, Chuang EY. Identification of a novel biomarker, SEMA5A, for non-small cell lung carcinoma in nonsmoking women. Cancer Epidemiol Biomarkers Prev 2010; 19(10): 2590–2597

[25] Ma L, Huang Y, Zhu W, Zhou S, Zhou J, Zeng F, Liu X, Zhang Y, Yu J. An integrated analysis of miRNA and mRNA expressions in non-small cell lung cancers. PLoS One 2011; 6(10): e26502

[26] Okayama H, Kohno T, Ishii Y, Shimada Y, Shiraishi K, Iwakawa R, Furuta K, Tsuta K, Shibata T, Yamamoto S, Watanabe S, Sakamoto H, Kumamoto K, Takenoshita S, Gotoh N, Mizuno H, Sarai A, Kawano S, Yamaguchi R, Miyano S, Yokota J. Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas. Cancer Res 2012; 72(1): 100–111

[27] Tai MC, Kajino T, Nakatochi M, Arima C, Shimada Y, Suzuki M, Miyoshi H, Yatabe Y, Yanagisawa K, Takahashi T. miR-342-3p regulates MYC transcriptional activity via direct repression of E2F1 in human lung cancer. Carcinogenesis 2015; 36(12): 1464–1473

[28] 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

[29] Lee ES, Son DS, Kim SH, Lee J, Jo J, Han J, Kim H, Lee HJ, Choi HY, Jung Y, Park M, Lim YS, Kim K, Shim Y, Kim BC, Lee K, Huh N, Ko C, Park K, Lee JW, Choi YS, Kim J. Prediction of recurrence-free survival in postoperative non-small cell lung cancer patients by using an integrated model of clinical information and gene expression. Clin Cancer Res 2008; 14(22): 7397–7404

[30] Staaf J, Jönsson G, Jönsson M, Karlsson A, Isaksson S, Salomonsson A, Pettersson HM, Soller M, Ewers SB, Johansson L, Jönsson P, Planck M. Relation between smoking history and gene expression profiles in lung adenocarcinomas. BMC Med Genomics 2012; 5(1): 22

[31] Ravid T, Hochstrasser M. Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 2008; 9(9): 679–689

[32] Snoek BC, de Wilt LH, Jansen G, Peters GJ. Role of E3 ubiquitin ligases in lung cancer. World J Clin Oncol 2013; 4(3): 58–69

[33] Kok K, Hofstra R, Pilz A, van den Berg A, Terpstra P, Buys CH, Carritt B. A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme. Proc Natl Acad Sci USA 1993; 90(13): 6071–6075

[34] Pallante P, Malapelle U, Berlingieri MT, Bellevicine C, Sepe R, Federico A, Rocco D, Galgani M, Chiariotti L, Sanchez-Cespedes M, Fusco A, Troncone G. UbcH10 overexpression in human lung carcinomas and its correlation with EGFR and p53 mutational status. Eur J Cancer 2013; 49(5): 1117–1126

[35] Kadara H, Lacroix L, Behrens C, Solis L, Gu X, Lee JJ, Tahara E, Lotan D, Hong WK, Wistuba II, Lotan R. Identification of gene signatures and molecular markers for human lung cancer prognosis using an in vitro lung carcinogenesis system. Cancer Prev Res (Phila) 2009; 2(8): 702–711

[36] Sasaki H, Moriyama S, Nakashima Y, Yukiue H, Fukai I, Fujii Y. Decreased Hrad6B expression in lung cancer. Acta Oncol 2004; 43(6): 585–589

[37] Tan YHC, Krishnaswamy S, Nandi S, Kanteti R, Vora S, Onel K, Hasina R, Lo FY, El-Hashani E, Cervantes G, Robinson M, Hsu HS, Kales SC, Lipkowitz S, Karrison T, Sattler M, Vokes EE, Wang YC, Salgia R. CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases. PLoS One 2010; 5(1): e8972

[38] Ahmed AU, Schmidt RL, Park CH, Reed NR, Hesse SE, Thomas CF, Molina JR, Deschamps C, Yang P, Aubry MC, Tang AH. Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. J Natl Cancer Inst 2008; 100(22): 1606–1629

[39] Sacco JJ, Yau TY, Darling S, Patel V, Liu H, Urbé S, Clague MJ, Coulson JM. The deubiquitylase Ataxin-3 restricts PTEN transcription in lung cancer cells. Oncogene 2014; 33(33): 4265–4272

[40] Byun S, Lee SY, Lee J, Jeong CH, Farrand L, Lim S, Reddy K, Kim JY, Lee MH, Lee HJ, Bode AM, Won Lee K, Dong Z. USP8 is a novel target for overcoming gefitinib resistance in lung cancer. Clin Cancer Res 2013; 19(14): 3894–3904

[41] McFarlane C, McFarlane S, Paul I, Arthur K, Scheaff M, Kerr K, Stevenson M, Fennell DA, Johnston JA. The deubiquitinating enzyme USP17 is associated with non-small cell lung cancer (NSCLC) recurrence and metastasis. Oncotarget 2013; 4(10): 1836–1843

[42] Pan J, Deng Q, Jiang C, Wang X, Niu T, Li H, Chen T, Jin J, Pan W, Cai X, Yang X, Lu M, Xiao J, Wang P. USP37 directly deubiquitinates and stabilizes c-Myc in lung cancer. Oncogene 2015; 34(30): 3957–3967

[43] Liu YQ, Wang XL, Cheng X, Lu YZ, Wang GZ, Li XC, Zhang J, Wen ZS, Huang ZL, Gao QL, Yang LN, Cheng YX, Tao SC, Liu J, Zhou GB. Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors. Oncotarget 2015; 6(33): 34953–34967

[44] Lin YC, Wang Y, Hsu R, Giri S, Wopat S, Arif MK, Chakraborty A, Prasanth KV, Prasanth SG. PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication. Proc Natl Acad Sci USA 2018; 115(52): 13282–13287

[45] Inano S, Sato K, Katsuki Y, Kobayashi W, Tanaka H, Nakajima K, Nakada S, Miyoshi H, Knies K, Takaori-Kondo A, Schindler D, Ishiai M, Kurumizaka H, Takata M. RFWD3-mediated ubiquitination promotes timely removal of both RPA and RAD51 from DNA damage sites to facilitate homologous recombination. Mol Cell 2017; 66(5): 622–634.e8

[46] Feeney L, Muñoz IM, Lachaud C, Toth R, Appleton PL, Schindler D, Rouse J. RPA-mediated recruitment of the E3 ligase RFWD3 is vital for interstrand crosslink repair and human health. Mol Cell 2017; 66(5): 610–621.e4

[47] World Health Organization. Ambient (outdoor) air quality and health. http://www.who.int/mediacentre/factsheets/fs313/en/. 2018. Accessed November 22, 2018

[48] World Health Organization. Household air pollution and health. https://www.who.int/news-room/fact-sheets/detail/household-air-pollution-and-health. 2018. Accessed December 24, 2018

[49] World Health Organization. Tobacco fact sheet. http://www.who.int/mediacentre/factsheets/fs339/en/. 2019. Accessed December 24, 2018

[50] Alexandrov LB, Ju YS, Haase K, Van Loo P, Martincorena I, Nik-Zainal S, Totoki Y, Fujimoto A, Nakagawa H, Shibata T, Campbell PJ, Vineis P, Phillips DH, Stratton MR. Mutational signatures associated with tobacco smoking in human cancer. Science 2016; 354(6312): 618–622

[51] Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J, Chen K, Walker J, McDonald S, Bose R, Ornitz D, Xiong D, You M, Dooling DJ, Watson M, Mardis ER, Wilson RK. Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012; 150(6): 1121–1134

[52] Gibelin C, Couraud S. Somatic alterations in lung cancer: do environmental factors matter? Lung Cancer 2016; 100: 45–52

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2016YFC0905501), the National Natural Science Funds for Distinguished Young Scholar (No. 81425025), the Key Project of the National Natural Science Foundation of China (No. 81830093), the National Natural Science Foundation of China (Nos. 81672765 and 81802796), and the CAMS Innovation Fund for Medical Sciences (CIFMS; No. 2019-I2M-1-003).

Compliance with ethics guidelines

Yanfei Zhang, Xinchun Zhao, Yongchun Zhou, Min Wang, and Guangbiao Zhou declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975 as revised in 2000. Additional informed consent was obtained from all patients whose identifying information is included in this article.

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