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Fanconi Anemia D2 Protein Confers Chemoresistance in Response to the Anticancer Agent, Irofulven

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Fanconi Anemia D2 Protein Confers Chemoresistance in Response to the Anticancer Agent, Irofulven 3153 Fanconi anemia D2 protein confers chemoresistance in response to the anticancer agent, irofulven Yutian Wang,1 Timothy Wiltshire,2 Jamie Senft,1 Introduction 3 1,2 Sharon L. Wenger, Eddie Reed, Fanconi anemia is a genetic cancer-susceptibility syn- and Weixin Wang1,2 drome characterized by congenital abnormalities, bone marrow failure, and cellular sensitivity to DNA cross- 1 2 Mary Babb Randolph Cancer Center, Department of linking agents (1, 2). There are at least 12 Fanconi anemia Microbiology, Immunology, and Cell Biology, and 3Department of Pathology, West Virginia University School of Medicine, complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, Morgantown, West Virginia and M), and 10 Fanconi anemia genes (A, C, D1/BRCA2, D2, E, F, G, J, L, and M) have been cloned (1–8). All of the Fanconi anemia proteins function in a common path- Abstract way. Fanconi anemia proteins (A, B, C, E, F, G, L, and M) The Fanconi anemia-BRCA pathway of genes are fre- assemble in a nuclear complex that is required for mono- quently mutated or epigenetically repressed in human ubiquitination/activation of the downstream FANCD2 cancer. The proteins of this pathway play pivotal roles in protein (1, 2, 4, 6, 9–13). FANCL has been shown to DNA damage signaling and repair. Irofulven is one of a possess the E3 ubiquitin ligase activity to monoubiqui- new class of anticancer agents that are analogues of tinate FANCD2at Lys 561 (11). The deubiquitinating mushroom-derived illudin toxins. Preclinical studies and enzyme of FANCD2, USP1, has also been recently clinical trials have shown that irofulven is effective identified (14). FANCD1 was shown to be identical to against several tumor cell types. The exact nature of BRCA2(15). FANCD2colocalizes with BRCA1 and irofulven-induced DNA damage is not completely under- BRCA2, and BRCA1 is required for monoubiquitination stood. Previously, we have shown that irofulven acti- and foci formation of FANCD2in response to DNA vates ATM and its targets, NBS1, SMC1, CHK2, and damage(1,2,4,16–18).Therefore,BRCA1,BRCA2 p53. In this study, we hypothesize that irofulven induces (FANCD1), and Fanconi anemia proteins functionally DNA double-strand breaks and FANCD2 may play an merge in a common Fanconi anemia-BRCA pathway of important role in modulating cellular responses and DNA damage response (1, 2). chemosensitivity in response to irofulven treatment. By The Fanconi anemia-BRCA pathway of DNA damage using cells that are proficient or deficient for FANCD2, response is frequently impaired in cancers due to genetic ATR, or ATM, we showed that irofulven induces mutation or epigenetic repression (1, 2, 4, 9, 10, 19). BRCA1 FANCD2 monoubiquitination and nuclear foci formation. and BRCA2 (FANCD1) are frequently mutated in familial ATR is important in mediating irofulven-induced breast and ovarian cancers (>50%; refs. 1, 20, 21). Germ FANCD2 monoubiquitination. Furthermore, we showed line mutations in FANCD1 (BRCA2), FANCA, FANCC,or that FANCD2 plays a critical role in maintaining chromo- FANCG have been found in pancreatic cancer (>10%; refs. some integrity and modulating chemosensitivity in re- 22–27). The BRCA2 (FANCD1) interacting protein, EMSY, sponse to irofulven-induced DNA damage. Therefore, this which binds to BRCA2and represses its function, was study suggests that it might be clinically significant to found to be amplified in sporadic breast (13%) and higher- target irofulven therapy to cancers defective for proteins grade ovarian (17%) cancers (28). FANCF, a protein of the Fanconi anemia-BRCA pathway. [Mol Cancer Ther upstream of FANCD2and essential for its activation, is 2006;5(12):3153–61] silenced by promoter methylation in several types of sporadic cancers including ovarian (21%), breast (17%), head and neck (15%), non–small cell lung (14%), and cervical (30%) cancers (19, 29–31). The silencing of FANCF may be linked to acquired cisplatin resistance in a subset of Received 7/20/06; revised 9/28/06; accepted 11/1/06. ovarian cancers (31). Grant support: National Cancer Institute grant 5R03CA107979 (W. Wang). Irofulven (6-hydroxymethylacylfulvene, HMAF; MGI The costs of publication of this article were defrayed in part by the 114, NSC no. 683863) is a member of a new class of payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to anticancer agents that are analogues of mushroom-derived indicate this fact. illudin toxins. Preclinical studies and clinical trials have Note: Current address for E. Reed: Centers for Disease Control and shown that irofulven is effective against several tumor Prevention, Atlanta, Georgia. cell types (32–40). Thus far, the structure and nature Requests for reprints: Weixin Wang, Mary Babb Randolph Cancer Center, of DNA damage caused by irofulven have not yet been West Virginia University, 1835 Health Sciences South, P.O. Box 9300, Morgantown, WV 26506. Phone: 304-293-2243; Fax: 304-293-4667. fully characterized. Earlier studies suggested that the E-mail: [email protected] DNA damage caused by the illudin family of com- Copyright C 2006 American Association for Cancer Research. pounds might be repaired by the nucleotide excision doi:10.1158/1535-7163.MCT-06-0427 repair pathway (41, 42). A recent study indicated that Mol Cancer Ther 2006;5(12). December 2006 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. 3154 FANCD2 Confers Chemoresistance to Irofulven transcription-coupled nucleotide excision repair is the cells (American Type Culture Collection, Manassas, VA) exclusive pathway in repairing illudin S and irofulven- weregrowninDMEM. elicited DNA lesions (43). However, in these studies, the Clonogenic Survival Assay homologous recombination pathway for DNA double- To determine chemosensitivity and IC50 concentration, the strand break repair was not evaluated (41–43). Previously, clonogenic survival assay was done on 60 mm cell culture we have shown that irofulven activates ATM and its dishes as described previously (44). Cells were treated with targets, NBS1, SMC1, CHK2, and p53 (44). Recent reports different concentrations of irofulven for 1 h, followed by indicate that ATM and CHK2are specifically activated by drug-free incubation for f10 days. Colonies were stained drug (calicheamicin) or radiation-induced double-strand with crystal violet and counted if 50 or more cells were pre- breaks (45, 46), and FANCD2may play a role in homo- sent. The IC50 concentration was calculated as the irofulven logous recombination pathway of double-strand break concentration that kills 50% of colonies of untreated controls. repair (1, 2). Therefore, we hypothesize that irofulven Western Blotting induces DNA double-strand breaks, and that FANCD2 Western blotting was done as described previously may play an important role in maintaining chromosome (44). Total cellular extracts (50 Ag) were electrophoresed stability and modulating chemosensitivity in response to in SDS-PAGE gels. The primary antibodies used includ- irofulven. ed: mouse anti-human actin (1:10,000 dilution), mouse To explore whether certain genetic defects in human anti-FLAG (1:200 dilution; Sigma), goat anti-human ATR cancer can be exploited to achieve preferential therapeutic (1:500 dilution), and mouse anti-human FANCD2(1:500 outcomes by irofulven, we did this study to investigate the dilution; Santa Cruz Biotechnology, Santa Cruz, CA). roles that FANCD2might play in irofulven-induced DNA The secondary antibodies used were: sheep anti-mouse damage response. We showed that irofulven induces the IgG-HRP (1:2,000 dilution; GE Healthcare Bio-Sciences monoubiquitination and foci formation of FANCD2. Corp., Piscataway, NJ) and donkey anti-goat IgG-HRP Irofulven-induced FANCD2monoubiquitination is medi- (1:2,000 dilution; Santa Cruz Biotechnology). The relative ated by ATR. FANCD2is critical for maintaining chromo- amount of FANCD2protein bands was quantified by some integrity and modulating chemosensitivity in densitometry. response to irofulven. Immunofluorescent Staining Cells were plated on cover-slips and treated with irofulven for 1 h followed by 12h of drug-free incubation. Materials and Methods Cells were then fixed and stained with mouse anti-human Cell Culture FANCD2(1:200dilution; Santa Cruz Biotechnology). All cell lines were maintained in various media sup- After staining with Alexa-fluor 546-conjugated goat anti- plemented with 10% fetal bovine serum in a 37jCincu- mouse IgG secondary antibody (1:200 dilution; Invitro- bator with 5% CO2 atmosphere. Human ovarian cancer gen), slides were mounted with Vectashield mounting cell lines A2780, CAOV3, and OVCAR3 were cultured in medium (Vector Laboratories, Burlingame, CA) contain- RPMI 1640. Human ovarian cancer cell line SKOV3 was ing 5 ng/mL of 4¶,6-diamidino-2-phenylindole. Staining cultured in McCoy’s 5A medium. The vector and short images were captured with an Olympus Provis AX70 hairpin FANCD2(shFANCD2)stably transfected SKOV3 fluorescence microscope (Olympus, Melville, NY), and cells were cultured in McCoy’s 5A medium containing Spot digital camera and software (Diagnostic Instruments, 200 Ag/mL of G418 (Invitrogen, Carlsbad, CA). The Sterling Height, MI). SV40-transformed Fanconi anemia D2fibroblasts (PD20F) RNA Interference transfected with the vector, wild-type FANCD2, or To knock-down ATR, one pair of 65-nucleotide short K561R mutant FANCD2(generously provided by Dr. hairpin ATR oligos containing the target sequence of
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