Bladder cancer is the fourth most common cancer and the ninth leading cause of cancer death in the United States.1 Most of bladder cancers are transitional cell carcinomas (TCC). Other types include squamous cell carcinoma and adenocarcinoma. In most patients with bladder cancer, death results from metastatic disease and high recurrence. In bladder cancer treatment, surgery, chemotherapy, radiation therapy, immunotherapy and vaccine therapy are all critical elements. Among chemotherapies, cisplatin or mitomycin C (MMC) DNA cross-linking agent is one of most commonly used intravesical drugs for treating non-invasive bladder cancer. About 50–68% of patients with superficial bladder cancer (non-invasive) have a very good response to intravesical therapy. This type of chemotherapeutic agents is also considered in the treatment of advanced bladder cancer (invasive) that has extended beyond the bladder wall. Therefore, DNA cross-link agents appear to be very effective in killing bladder cancer cells, suggesting the cellular machinery in processing DNA cross-link damage is somehow insufficient in bladder cancer cells, eventually dying of irreparable DNA damage. Bladder cancer development/progression attributed to many defective cellular signaling pathways, including HRAS,2 p53 and RB,2-4 MMP-9,5 IL-8,6 VEGF,7 EGFR8,9 and many others.10-12 The cellular response specifically to DNA cross-link damage is the Fanconi Anemia (FA) /FA-BRCA pathway.13 However, it is largely unclear whether the FA/FA-BRCA pathway is insufficient in bladder cancer cells, and the insufficiency then contributes to the development of bladder cancer.
FA is a rare autosomal recessive or X-linked disease characterized by severe bone marrow failure, developmental abnormalities and an extremely high incidence of both hematological and non-hematological malignancies.14-19 FA cells display chromosome abnormalities and hypersensitivity to DNA cross-linking agents,14,20,21 which can be recapitulated in many types of non-FA cells by disrupting the function of FA genes in our studies22-25 and those of others.26,27 To date, there are 14 or 15 classified FA groups FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P,14,15,18-21,28-37 each of which can be accounted for by mutations in a given gene unique to that group. The sensitivity of FA cells from all complementation groups to DNA cross-linking agents and their similar clinical phenotypes suggest that all FA proteins function in a common DNA damage response pathway. Eight of the known FA proteins (FANCA, FANCB, FANCC, FANCG, FANCE, FANCF, FANCL and FANCM)15,30,32,38 and other known39,40 and unknown proteins function together by forming a multi-protein complex to serve as an E3 ubiquitin ligase to monoubiquitinate FANCD2 at lysine 561 (K561)26,41-48 and its paralog FANCI at K523.49 This pathway is activated during DNA replication as well as following DNA damage triggered especially by DNA cross-linking agents, such as mitomycin C (MMC), diepoxybutane (DEB) or cisplatin,50,51 and the monoubiquitinated FANCD2 and FANCI along with FANCD1/BRCA2,42 FANCN/PALB2,30,32,38,52 FANCJ/BRIP153,54 and others work in concert to repair DNA damage. FANCD2 thus is the central player of the pathway,28 and monoubiquitinated FANCD2 can be used as a measure for activation of this signaling pathway. Of the eight FA complex proteins, FANCL was identified as a catalytic subunit of the complex E3 ubiquitin ligase, because it contains a C-terminal PHD finger domain that exhibits E3 ubiquitin ligase activity in vitro.26
In FA patients, cancer incidence is about 5-fold higher than that of the general population and up to several hundred-fold higher for particular malignancies.55 This cancer-prone phenomenon indicated that FA genes play crucial roles in tumor suppression. Thus, the signaling pathway constituted by FA proteins has been termed the FA tumor suppressor pathway. This pathway is also called the FA-BRCA tumor suppressor pathway, because three FA proteins, FANCD1, FANCJ and FANCN, are BRCA or BCRA-related proteins, which are BRCA2, BRIP1 (BRCA1 interaction protein 1) and PALB2 (partner and localizer of BRCA2), respectively. Gene therapy was found to improve the clinical symptoms of FA in the hematopoietic system;56 however, patients still eventually develop cancer57 and die, with a mean age of 16 y.58 The FA tumor suppressor pathway has long been suggested,59 but the implications of this disordered signaling pathway directly in non-FA human cancer just began to be appreciated in our recent work.22 Despite many studies aimed at investigating various aspects of this cancer-prone genetic disease using a variety of methods, including those of genetics, biochemistry and the clinical sciences,21,26,46,60-65 much remains to be learned. For example, how the FA pathway influences specific types of human cancer development is unknown. In this study, we report for the first time that FAVL impairment of the FA pathway has a substantial effect on the development of human bladder cancer, providing novel insights into the development of additional effective tools to fight bladder cancer.
Among all types of cancer, cisplatin is a relatively most effective chemotherapeutic drug in the treatment of bladder cancer, suggesting cellular signaling pathways responsible for repairing cross-link damage harbored in bladder cancer cells are most likely defective, leading cells to be sensitive to the treatment. Indeed, the FA-BRCA signaling pathway, responsible especially for repairing cross-link DNA damage, was found to be impaired in more than 35% of bladder carcinoma cases, resulting from an elevated level of FAVL expression, which is comparable to the one in cells found to have an impaired FA pathway (Fig. 1). Similar to FA cells that are sensitive to cross-linking agents, bladder cancer cells displayed hypersensitivity to cross-linking agents when ectopically expressing FAVL (Fig. 5). More importantly, elevated FAVL promotes not only the growth of bladder cancer cells in vitro and in vivo, but also their invasiveness (Figs. 2 and 3), which confers the development of advanced bladder cancer. Therefore, FAVL appears to be a tumor promotion factor that may function through all phases of bladder tumor development. For bladder cancer, tumor development undergoes specific phase-transitions, such as, Ta, non-invasive papillary carcinoma; Tis, carcinoma in situ, “flat tumor”; T1, tumor invades subepithelial connective tissue; T2, tumor invades muscularis propria; T2a, tumor invades inner half muscle; T2b, tumor invades outer half muscle; T3, tumor invades perivesical tissue and T4, tumor invades other organs and pelvic wall. How FAVL affects the development of particular phases needs to be further defined in the future.
Research advances in the field of molecular biology have provided a new understanding of the genetic background of human bladder cancer. For example, it is known that bladder tumorigenesis results from numerous defective cellular signaling pathways, including HRAS,2 p53 and RB,2,3 MMP-9,5 IL-8,6 VEGF7 and EGFR8 pathways. However, bladder cancer remains to be one of leading causes of cancer death, indicating studies on improving our understanding of pathogenesis of human bladder cancer are much needed. We here for the first time found that FAVL impairment of the FA pathway contributes to bladder tumorigenesis. Knowledge learned herein, like genetic abnormalities and biologic aberrations known to have roles in tumorigenesis,2,3 will be the basis for investigations on the development of specific methods for bladder cancer prevention, diagnosis and/or treatment.
Increasing studies provide evidence linking the compromised FA-BRCA signaling cascade to sporadic human cancers. In these cancers, the FA-BRCA pathway is impaired by epigenetic silencing and somatic or inherited mutations of one or several FA genes, supporting the long proposed concept that the FA-BRCA pathway is a tumor suppressor pathway. We previously found a novel alternative splice variant of FANCL, named FAVL, which was increased in nearly 50% of cancer tissue samples detected. More importantly, the study on FAVL demonstrated, for the first time, the essential role of an intact FA-BRCA signaling in suppressing the development of non-FA human cancer (11). Here, we aimed to address how the FA-BRCA tumor suppressor pathway affects specific types of human cancer. The results presented in this study are among the first to indicate that the FA-BRCA tumor suppressor pathway may emerge to be an important guardian pathway responsible for protecting human cells, particularly bladder cells, from going awry and becoming neoplasm.
George B, Datar RH, Wu L, Cai J, Patten N, Beil SJ, et al.
p53 gene and protein status: the role of p53 alterations in predicting outcome in patients with bladder cancer
J Clin Oncol 2007;
25:5352-8; PMID: 18048815
; DOI: 10.1200/JCO.2006.10.4125
Jee HJ, Kim AJ, Song N, Kim HJ, Kim M, Koh H, et al.
Nek6 overexpression antagonizes p53-induced senescence in human cancer cells
Cell Cycle 2010;
9:4703-10; PMID: 21099361
; DOI: 10.4161/cc.9.23.14059
Izawa JI, Slaton JW, Kedar D, Karashima T, Perrotte P, Czerniak B, et al.
Differential expression of progression-related genes in the evolution of superficial to invasive transitional cell carcinoma of the bladder
Oncol Rep 2001;
8:9-15; PMID: 11115562
Crew JP, O’Brien T, Bradburn M, Fuggle S, Bicknell R, Cranston D, et al.
Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer
Cancer Res 1997;
57:5281-5; PMID: 9393750
Chow NH, Liu HS, Yang HB, Chan SH, Su IJ.
Expression patterns of erbB receptor family in normal urothelium and transitional cell carcinoma. An immunohistochemical study
Virchows Arch 1997;
430:461-6; PMID: 9230911
; DOI: 10.1007/s004280050056
Sayan AE, Stanford R, Vickery R, Grigorenko E, Diesch J, Kulbicki K, et al.
Fra-1 controls motility of bladder cancer cells via transcriptional upregulation of the receptor tyrosine kinase AXL
31:1493-503; PMID: 21822309
; DOI: 10.1038/onc.2011.336
Sugano G, Bernard-Pierrot I, Laé M, Battail C, Allory Y, Stransky N, et al.
Milk fat globule--epidermal growth factor--factor VIII (MFGE8)/lactadherin promotes bladder tumor development
30:642-53; PMID: 20956946
; DOI: 10.1038/onc.2010.446
Ahmad I, Morton JP, Singh LB, Radulescu SM, Ridgway RA, Patel S, et al.
β-Catenin activation synergizes with PTEN loss to cause bladder cancer formation
30:178-89; PMID: 20818428
; DOI: 10.1038/onc.2010.399
Askham JM, Platt F, Chambers PA, Snowden H, Taylor CF, Knowles MA.
AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K
29:150-5; PMID: 19802009
; DOI: 10.1038/onc.2009.315
Sengerová B, Wang AT, McHugh PJ.
Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair
Cell Cycle 2011;
10:3999-4008; PMID: 22101340
; DOI: 10.4161/cc.10.23.18385
Huang TT, D’Andrea AD.
Regulation of DNA repair by ubiquitylation
Nat Rev Mol Cell Biol 2006;
7:323-34; PMID: 16633336
; DOI: 10.1038/nrm1908
Fei P, Yin J, Wang W.
New advances in the DNA damage response network of Fanconi anemia and BRCA proteins. FAAP95 replaces BRCA2 as the true FANCB protein
Cell Cycle 2005;
4:80-6; PMID: 15611632
; DOI: 10.4161/cc.4.1.1358
Huang TT, Nijman SM, Mirchandani KD, Galardy PJ, Cohn MA, Haas W, et al.
Regulation of monoubiquitinated PCNA by DUB autocleavage
Nat Cell Biol 2006;
8:339-47; PMID: 16531995
; DOI: 10.1038/ncb1378
Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins
Nat Rev Genet 2007;
8:735-48; PMID: 17768402
; DOI: 10.1038/nrg2159
D’Andrea AD, Grompe M.
The Fanconi anaemia/BRCA pathway
Nat Rev Cancer 2003;
3:23-34; PMID: 12509764
; DOI: 10.1038/nrc970
Zhang J, Zhao D, Park HK, Wang H, Dyer RB, Liu W, et al.
FAVL elevation in human tumors disrupts Fanconi anemia pathway signaling and promotes genomic instability and tumor growth
J Clin Invest 2010;
120:1524-34; PMID: 20407210
; DOI: 10.1172/JCI40908
Zhang J, Wang X, Lin CJ, Couch FJ, Fei P.
Altered expression of FANCL confers mitomycin C sensitivity in Calu-6 lung cancer cells
Cancer Biol Ther 2006;
5:1632-6; PMID: 17106252
; DOI: 10.4161/cbt.5.12.3351
Zhang J, Zhao D, Wang H, Lin CJ, Fei P.
FANCD2 monoubiquitination provides a link between the HHR6 and FA-BRCA pathways
Cell Cycle 2008;
7:407-13; PMID: 18277096
; DOI: 10.4161/cc.7.3.5156
Park HK, Wang H, Zhang J, Datta S, Fei P.
Convergence of Rad6/Rad18 and Fanconi anemia tumor suppressor pathways upon DNA damage
PLoS ONE 2010;
5:e13313; PMID: 20967207
; DOI: 10.1371/journal.pone.0013313
Meetei AR, de Winter JP, Medhurst AL, Wallisch M, Waisfisz Q, van de Vrugt HJ, et al.
A novel ubiquitin ligase is deficient in Fanconi anemia
Nat Genet 2003;
35:165-70; PMID: 12973351
; DOI: 10.1038/ng1241
Taniguchi T, Tischkowitz M, Ameziane N, Hodgson SV, Mathew CG, Joenje H, et al.
Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors
Nat Med 2003;
9:568-74; PMID: 12692539
; DOI: 10.1038/nm852
Hahn SA, Greenhalf B, Ellis I, Sina-Frey M, Rieder H, Korte B, et al.
BRCA2 germline mutations in familial pancreatic carcinoma
J Natl Cancer Inst 2003;
95:214-21; PMID: 12569143
; DOI: 10.1093/jnci/95.3.214
Meetei AR, Medhurst AL, Ling C, Xue Y, Singh TR, Bier P, et al.
A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M
Nat Genet 2005;
37:958-63; PMID: 16116422
; DOI: 10.1038/ng1626
Reid SSD, Hanenberg H, Barker K, Hanks S, Kalb R, Neveling K, et al.
Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer. Nat Genet 2007; 39:162-4
; PMID: 17200671
Roest HP, Baarends WM, de Wit J, van Klaveren JW, Wassenaar E, Hoogerbrugge JW, et al.
The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice
Mol Cell Biol 2004;
24:5485-95; PMID: 15169909
; DOI: 10.1128/MCB.24.12.5485-5495.2004
Xia BDJ, Ameziane N, de Vries Y, Rooimans MA, Sheng Q, Pals G, et al.
Fanconi anemia is associated with a defect in the BRCA2 partner PALB2
Nat Genet 2007; 39:159-61 ;
; PMID: 17200672
Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A.
Mutations of the SLX4 gene in Fanconi anemia
Nat Genet 2011;
43:142-6; PMID: 21240275
; DOI: 10.1038/ng.750
Fanconi anemia and breast cancer susceptibility meet again
Nat Genet 2010;
42:368-9; PMID: 20428093
; DOI: 10.1038/ng0510-368
Meindl A, Hellebrand H, Wiek C, Erven V, Wappenschmidt B, Niederacher D, et al.
Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene
Nat Genet 2010;
42:410-4; PMID: 20400964
; DOI: 10.1038/ng.569
Stoepker C, Hain K, Schuster B, Hilhorst-Hofstee Y, Rooimans MA, Steltenpool J, et al.
SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype
Nat Genet 2011;
43:138-41; PMID: 21240277
; DOI: 10.1038/ng.751
Vaz F, Hanenberg H, Schuster B, Barker K, Wiek C, Erven V, et al.
Mutation of the RAD51C gene in a Fanconi anemia-like disorder
Nat Genet 2010;
42:406-9; PMID: 20400963
; DOI: 10.1038/ng.570
Rahman NSS, Thompson D, Kelly P, Renwick A, Elliott A, Reid S, et al.
Breast Cancer Susceptibility Collaboration (UK). PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat Genet 2007; 39:165-7
; PMID: 17200668
Ling C, Ishiai M, Ali AM, Medhurst AL, Neveling K, Kalb R, et al.
FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway
EMBO J 2007;
26:2104-14; PMID: 17396147
; DOI: 10.1038/sj.emboj.7601666
Ciccia A, Ling C, Coulthard R, Yan Z, Xue Y, Meetei AR, et al.
Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM
Mol Cell 2007;
25:331-43; PMID: 17289582
; DOI: 10.1016/j.molcel.2007.01.003
Morgan NV, Tipping AJ, Joenje H, Mathew CG.
High frequency of large intragenic deletions in the Fanconi anemia group A gene
Am J Hum Genet 1999;
65:1330-41; PMID: 10521298
; DOI: 10.1086/302627
Howlett NG, Taniguchi T, Olson S, Cox B, Waisfisz Q, De Die-Smulders C, et al.
Biallelic inactivation of BRCA2 in Fanconi anemia
297:606-9; PMID: 12065746
; DOI: 10.1126/science.1073834
Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins L, Alon N, Wijker M, Parker L, et al.
Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA
Nat Genet 1996;
14:320-3; PMID: 8896563
; DOI: 10.1038/ng1196-320
Strathdee CA, Gavish H, Shannon WR, Buchwald M.
Cloning of cDNAs for Fanconi’s anaemia by functional complementation
356:763-7; PMID: 1574115
; DOI: 10.1038/356763a0
de Winter JP, Rooimans MA, van Der Weel L, van Berkel CG, Alon N, Bosnoyan-Collins L, et al.
The Fanconi anaemia gene FANCF encodes a novel protein with homology to ROM
Nat Genet 2000;
24:15-6; PMID: 10615118
; DOI: 10.1038/71626
Timmers C, Taniguchi T, Hejna J, Reifsteck C, Lucas L, Bruun D, et al.
Positional cloning of a novel Fanconi anemia gene, FANCD2
Mol Cell 2001;
7:241-8; PMID: 11239453
; DOI: 10.1016/S1097-2765(01)00172-1
Levitus M, Rooimans MA, Steltenpool J, Cool NF, Oostra AB, Mathew CG, et al.
Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes
103:2498-503; PMID: 14630800
; DOI: 10.1182/blood-2003-08-2915
Taniguchi T, Garcia-Higuera I, Xu B, Andreassen PR, Gregory RC, Kim ST, et al.
Convergence of the fanconi anemia and ataxia telangiectasia signaling pathways
109:459-72; PMID: 12086603
; DOI: 10.1016/S0092-8674(02)00747-X
Smogorzewska A, Matsuoka S, Vinciguerra P, McDonald ER, Hurov KE, Luo J, et al.
Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair
129:289-301; PMID: 17412408
; DOI: 10.1016/j.cell.2007.03.009
Sala-Trepat M, Rouillard D, Escarceller M, Laquerbe A, Moustacchi E, Papadopoulo D.
Arrest of S-phase progression is impaired in Fanconi anemia cells
Exp Cell Res 2000;
260:208-15; PMID: 11035915
; DOI: 10.1006/excr.2000.4994
Xia B, Sheng Q, Nakanishi K, Ohashi A, Wu J, Christ N, et al.
Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2
Mol Cell 2006;
22:719-29; PMID: 16793542
; DOI: 10.1016/j.molcel.2006.05.022
Levitus M, Waisfisz Q, Godthelp BC, de Vries Y, Hussain S, Wiegant WW, et al.
The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J
Nat Genet 2005;
37:934-5; PMID: 16116423
; DOI: 10.1038/ng1625
Litman R, Peng M, Jin Z, Zhang F, Zhang J, Powell S, et al.
BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ
Cancer Cell 2005;
8:255-65; PMID: 16153896
; DOI: 10.1016/j.ccr.2005.08.004
Williams DA, Croop J, Kelly P.
Gene therapy in the treatment of Fanconi anemia, a progressive bone marrow failure syndrome
Curr Opin Mol Ther 2005;
7:461-6; PMID: 16248281
The anaemia of cancer: death by a thousand cuts
Nat Rev Cancer 2005;
5:543-55; PMID: 15965494
; DOI: 10.1038/nrc1648
Kennedy RD, D’Andrea AD.
The Fanconi Anemia/BRCA pathway: new faces in the crowd
Genes Dev 2005;
19:2925-40; PMID: 16357213
; DOI: 10.1101/gad.1370505
Fanconi’s anaemia in the genetics of neoplasia
230:370-3; PMID: 4927726
; DOI: 10.1038/230370a0
Auerbach AD, Wolman SR.
Susceptibility of Fanconi’s anaemia fibroblasts to chromosome damage by carcinogens
261:494-6; PMID: 934283
; DOI: 10.1038/261494a0
Aplastic Anemia, Pediatric Aspects
1:361-6; PMID: 10388017
Meetei AR, Yan Z, Wang W.
FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination
Cell Cycle 2004;
3:179-81; PMID: 14712086
; DOI: 10.4161/cc.3.2.656
Rego MA, Harney JA, Mauro M, Shen M, Howlett NG.
Regulation of the activation of the Fanconi anemia pathway by the p21 cyclin-dependent kinase inhibitor
31:366-75; PMID: 21685936
; DOI: 10.1038/onc.2011.237
Wilson JB, Yamamoto K, Marriott AS, Hussain S, Sung P, Hoatlin ME, et al.
FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3
27:3641-52; PMID: 18212739
; DOI: 10.1038/sj.onc.1211034
Hoskins EE, Gunawardena RW, Habash KB, Wise-Draper TM, Jansen M, Knudsen ES, et al.
Coordinate regulation of Fanconi anemia gene expression occurs through the Rb/E2F pathway
27:4798-808; PMID: 18438432
; DOI: 10.1038/onc.2008.121
Park HK, Panneerselvam J, Dudimah FD, Dong G, Sebastian S, Zhang J, et al.
Wip1 contributes to cell homeostasis maintained by the steady-state level of Wtp53
Cell Cycle 2011;
10:2574-82; PMID: 21734451
; DOI: 10.4161/cc.10.15.15923