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

Introduction

The ubiquitin E3 ligases catalyze the transfer of ubiquitins from E2 enzymes to substrate proteins for their proteasomal degradation. The stability of many oncoproteins and tumor suppressors is controlled by E3 ligases [¹–³], which are divided into the following three major subtypes according to their molecular structure and mechanisms of action: the homologous to E6-associated protein carboxyl terminus (HECT), really interesting new gene (RING), and RING-in-between-RING (RBR) [⁴]. E3 ligases exhibit higher substrate specificity than E1 and E2; thus, targeting oncogenic E3 ligases is a possible strategy for cancer treatment [⁵,⁶].

The E3 ligase RING finger and WD repeat domain 3 (RFWD3 or RNF201/FLJ10520) was identified as a substrate protein of the Ataxia Telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) in 2007 [⁷]. RFWD3 can stabilize p53 in response to DNA damage [⁸]. RFWD3 also plays a role in controlling replication checkpoints and responses to DNA damage in cancer cells despite E3 ligase activity [⁹,¹⁰]. Biallelic mutations have been found in RFWD3 in patients with Fanconi anemia, and these mutations cause this disease [¹¹]. A genome-wide association study demonstrated that RFWD3 is a susceptibility locus for malignant neoplasms, including multiple myeloma [¹²] and testicular germ cell tumor [¹³]. However, the significance of RFWD3 in cancers, including lung cancer, remains to be elucidated.

To unveil critical ubiquitin pathway genes (UPGs) for non-small cell lung cancer (NSCLC), the 696 UPGs found in human genome were silenced by small interfering RNA (siRNA) in NSCLC cells, and 31 UPGs including RFWD3 were identified [¹⁴]. Here, the significance of RFWD3 in NSCLC was investigated.

Materials and methods

Patients’ samples and immunohistochemistry analysis

This study was approved by the research ethics committees of Institute of Zoology, Chinese Academy of Sciences and the Third Affiliated Hospital of Kunming Medical University (Yunnan Tumor Hospital). All lung cancer samples were collected with informed consent. Immunohistochemistry (IHC) assay was performed using an anti-RFWD3 antibody (Proteintech, Chicago, USA) and the formalin-fixed, paraffin-embedded lung cancer tissue specimens (5 µm). Immunoreactivity score (IRS) was calculated as IRS (0–12) = RP (0–4) × SI (0–3), where RP is the percentage of staining-positive cells and SI is the staining intensity.

Cell culture and siRNA library

NSCLC lines A549 and H1975 were obtained from the American Tissue Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. The Human siGENOME SMARTpool siRNA Libraries containing 696 UPGs were purchased from the Thermo Scientific Dharmacon (Lafayette, CO, USA). siRNAs were transfected into the cells according to the manufacturer’s instructions, and cell viability was assessed using a CellTiter-Glo Reagent (Promega, Fitchburg, WI, USA) following the manufacturer’s instructions. Z-scores were calculated as follows: z= (x−m)/s, where x is the raw score to be standardized, m is the mean of the plate, and s is the standard deviation of the plate [¹⁵,¹⁶]. The Z-score of a gene reflects its requirement for cell proliferation, and Z-score≤-2 indicates that the gene is required for cell proliferation and that the silencing of the gene significantly inhibits cell proliferation. Z-score≥2 indicates that the gene inhibits lung cancer cell proliferation and that inhibition of the gene significantly promotes cell proliferation [¹⁵].

Cell proliferation and colony-forming activity assessment

A549 and H1975 cells were transfected with siRFWD3 (5′-GUUAAGAUGUUGAGUACUG-3′) for 24 h, and cell proliferation was assayed by a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instruction. Cell viability was estimated by trypan blue dye exclusion. Foci formation was facilitated by seeding A549 or H1975 cells transfected with negative control or RFWD3-specific siRNA onto 35 mm plates (300 cells per plate) in triplicate. The cells were stained with Giemsa after 14 days of culturing, and clones containing more than 50 cells were counted.

Online data availability

The cancer microarray database Oncomine [¹⁷], including the databases of Hou Lung [¹⁸], Selamat Lung [¹⁹], Landi Lung [²⁰], Okayama Lung [²¹], Wachi Lung [²²], and Su Lung [²³], were explored. The transcriptome data and clinical data of 518 lung adenocarcinomas (LUADs) and 500 lung squamous cell carcinomas (LUSCs) were downloaded from the Cancer Genomics Hub. The Cancer Genome Atlas (TCGA) database was assessed using accession code phs000178. The expression of RFWD3 in Asian population, including Chinese [²⁴,²⁵] and Japanese [²⁶,²⁷], NSCLCs, was assessed in the Gene Expression Omnibus (GEO) datasets under the accession codes GSE19804, GSE29250, GSE31210, and GSE66759. The Online Survival Analysis Software containing the microarray data of 1928 LUADs and LUSCs [²⁸] was utilized. The correlation of RFWD3 expression with overall survival (OS) was analyzed by entering RFWD3 into the database and setting different clinical parameters. The Kaplan–Meier survival plots and log rank P values were obtained from the webpage. The baseline demographic characteristics of the patients are available in the reference [²⁸].

Statistical analyses

Statistical analyses were conducted using SPSS 16.0 for Windows (Chicago, IL, USA). Differences between data groups were evaluated for significance using Student’s t-test of unpaired data, and all statistical tests were two-sided. The survival curve was estimated with the Kaplan–Meier method and log-rank test using the Online Survival Analysis Software [²⁸]. P values less than 0.05 were deemed statistically significant in all cases.

Results

Unveiling RFWD3 in NSCLCs

We conducted a systematic knockdown of UPGs in NSCLC cell lines A549 and H1975 (Fig. 1A) by using the siRNA human siGENOME SMARTpool library obtained from Dharmacon [¹⁴,¹⁶]. The robust Z-scores of the SMARTpools were calculated from duplicate experiments [¹⁵]. A total of 31 candidates were identified, and RFWD3 was required for the proliferation of the two NSCLC cell lines with significant Z-scores (Fig. 1B). We verified the results by transfecting siRFWD3 in A549 and H1975 NSCLC cells. siRFWD3 significantly inhibited cell proliferation (Fig. 1C) and suppressed the colony-forming activity of the cells (Fig. 1D). These data suggest that RFWD3 may have important roles in NSCLC.

RFWD3 expression in NSCLCs

The expression level of RFWD3 in NSCLCs was evaluated by performing IHC assays in tumor samples of 22 NSCLCs (Table 1). RFWD3 staining among 14 (64%) patients was strong, whereas RFWD3 staining was weak among 8 (36%) cases (Fig. 2A and 2B). RFWD3 was mainly localized in the cytoplasm of the lung cancer cells (Fig. 2A).

The observations were confirmed by exploring the Oncomine cancer microarray database [¹⁷] (www.oncomine.org). In the databases of Hou Lung [¹⁸], Selamat Lung [¹⁹], Landi Lung [²⁰], Okayama Lung [²¹], and Wachi Lung [²²], the RFWD3 RNA levels were more elevated in the tumor tissues than in normal lung tissues (Fig. 3A–3E). In the Su Lung database [²³], the RFWD3 expression was slightly higher in the tumor samples than in normal control lung tissues, although the difference was not statistically significant (Fig. 3F, P = 0.1).

We explored RFWD3 RNA levels in the TCGA transcriptome database containing 1018 NSCLC tumor samples and 109 normal lung specimens. A significantly elevated expression of RFWD3 was found in the NSCLC tumor samples relative to normal lung tissue samples (Fig. 3G). The expression level of RFWD3 was significantly higher in the tumor tissues than in normal lung samples (Fig. 3H–3I; P<0.0001) in LUADs (n = 518) and LUSCs (n = 500). In the paired tumor-adjacent samples of TCGA datasets, RFWD3 was more significantly expressed in the tumor tissues than in their adjacent lung samples (Fig. 3J and 3K). The expression of RFWD3 in Asian NSCLCs was assessed in GEO datasets, and the results showed that the Chinese and Japanese NSCLCs exhibited higher RFWD3 in tumor tissues than in normal lung tissues (Fig. 4A and 4B).

RFWD3 is inversely associated with the prognosis of NSCLCs

The Online Survival Analysis Software [²⁸] was utilized to analyze the association between RFWD3 expression and the lifespan of patients. The OS of NSCLCs with high RFWD3 expression was significantly shorter than that of NSCLCs with low RFWD3 expression (Fig. 5A, P = 1.9 × 10⁻⁷). NSCLCs with high RFWD3 exhibited a shorter survival time than those with low levels of RFWD3 in LUADs and LUSCs (Fig. 5A). In the TCGA datasets, the NSCLC patients with high RFWD3 had slightly worse prognosis than those with low RFWD3, although the difference was not statistically significant (Fig. 5B). Thus, RFWD3 might play a role in the pathogenesis of NSCLCs.

Association between RFWD3 expression and tobacco smoke

The association between RFWD3 expression levels and the tobacco smoking status of patients was explored in the Oncomine and TCGA databases. Data showed that RFWD3 expression was significantly higher in smoker NSCLCs than in non-smoker NSCLCs in the databases of Lee Lung [²⁹] (Fig. 6A; P<0.0001) and TCGA (Fig. 6B; P = 0.03). In the GEO Sweden dataset (GSE29016) [³⁰], smoker LUADs had higher RFWD3 than their non-smoking counterparts. Thus, tobacco smoke and related carcinogens may upregulate RFWD3 and facilitate lung carcinogenesis.

Discussion

Pulmonary homeostasis facilitates normal respiration and is maintained by coping with various circumstances, including airborne particles, pathogens, and other noxious stimuli. The accumulation of misfolded or dysfunctional proteins from critical proteins may cause cell abnormality during division, differentiation, senescence, and death. E3 ligases mediate poly-ubiquitination and the subsequent degradation of proteins in normal and abnormal cells. Thus, alterations in UPGs may lead to the hyper-activation of oncoproteins and loss of function of tumor suppressors, thereby facilitating cancer initiation and progression [³¹,³²]. Abnormalities in UPGs, such as UBE1L [³³], UbcH10 [³⁴], UBE2C [³⁵], Hrad6B [³⁶], c-Cbl [³⁷], SINA [³⁸], Ataxin-3 [³⁹], USP8 [⁴⁰], USP17 [⁴¹], USP37 [⁴²], and Skp1-Cul1-F-box protein complex essential component Skp1 [⁴³], have been reported in lung cancer. Herein, we showed for the first time that RFWD3 is required for NSCLC cell proliferation because the knockdown of this E3 ligase led to the dramatic inhibition of NSCLC cell proliferation (Fig. 1). We analyzed the expression level of RFWD3 in NSCLCs in our setting and datasets and found that RFWD3 was elevated in the tumor samples and inversely associated with the clinical outcome of the patients (Figs. 2–4). These results indicate that RFWD3 may play an important role in NSCLC, and further research should be performed to unveil how this E3 ligase is involved in lung carcinogenesis.

RFWD3 stabilizes p53 [⁸] and participates in replication checkpoint [⁹]. The stabilization of RFWD3 by PCNA at the replication fork is essential for DNA replication [⁴⁴]. RFWD3-mediated ubiquitination promotes the timely removal of RPA and RAD51 from DNA damage sites to facilitate homologous recombination [⁴⁵], and the RPA-mediated recruitment of RFWD3 is vital for interstrand crosslink repair and human health [⁴⁶]. Tobacco smoke and haze (smohaze), which are responsible for>90% of lung cancer deaths [⁴⁷–⁴⁹], result in DNA damage and cause somatic mutations in the genome [⁵⁰–⁵²]. We found that RFWD3 was associated with tobacco smoke and that RFWD3 was more significantly expressed in smokers than in non-smokers (Fig. 6). Thus, RFWD3 may play a role in lung carcinogenesis by perturbing DNA damage repair and/or modifying the expression of oncoproteins/tumor suppressors, thereby affecting the critical pathways involving p53 and other crucial oncoproteins/tumor suppressors and the immune system. These possibilities and the potential of RFWD3 as a therapeutic target for lung cancer should be further investigated in the future.

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. 2018. Accessed November 22, 2018

[48] World Health Organization. Household air pollution and health. 2018. Accessed December 24, 2018

[49] World Health Organization. Tobacco fact sheet. 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

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature