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FANCF methylation contributes to chemoselectivity in ovarian cancer

A new model of ovarian cancer tumor progression implicates aberrant FANCF promoter methylation that is associated with silencing and disruption of the Fanconi-anemia-BRCA pathway. Disruption of the pathway occurs de novo in ovarian and may contribute to selective sensitivity to platinum salts.

Ovarian cancer cells accumulate genetic pathways, including BRCA1, RAD51, complex. BRCA2/FANCD1 binds to changes that allow them to evade ATM, and NBS1 (D’Andrea and Grompe, RAD51 and to DNA, promoting a DNA chemotherapeutic drugs and become 2003). Five of the FANC gene products repair response. The ubiquinated increasingly dangerous. In view of the (FANCA, FANCC, FANCE, FANCF, and FANCD2 also colocalizes with NBS- high mortality rates associated with ovar- FANCG) are subunits of a nuclear com- MRE11-RAD50 complex in DNA dam- ian cancer, a better understanding of the plex (FA complex) that is required for the age nuclear foci. Germline of molecular mechanisms underlying tumor monoubiquitination of the downstream several genes in the pathway result in progression in the disease could reveal FANCD2 (Figure 1A). The sev- impaired response to DNA damage and novel pathways of high clinical rele- enth gene, FANCD1, was recently shown increased cancer susceptibility. vance. A key feature of ovarian cancer is to be identical to BRCA2 (Howlett et al., FANCD2 exists as two isoforms in its sensitivity to platinum salts such as 2002). Defects in the Fanconi-anemia- normal cells, nonubiquitinated FANCD2- Cisplatin (CDDP) and Carboplatin, two BRCA (FA-BRCA) pathway are associat- S and monoubiquitinated FANCD2-L. drugs that have been the mainstay of ed with genomic instability and Inducible expression of monoubiquitinat- therapy for decades. Unfortunately, ovar- increased sensitivity to DNA-damaging ed FANCD2 in response to DNA damage ian cancer cells, with their unstable agents such as ionizing radiation (IR), requires an intact FA-BRCA pathway. genomes, are initially sensitive to this mitomycin C (MMC), and CDDP. In Taniguchi et al.(2003) screened 25 ovar- class of drugs, but the cells invariably response to ionizing radiation-mediated ian cancer cell lines with varying sensi- become resistant. double-strand breaks, ATM phosphory- tivities to CDDP and found two cell lines, In a recent study, Taniguchi et al. lates the NBS1 protein. Phosphorylation 2008 and TOV-21G, without the (2003) describe a model for ovarian of NBS1 is required for FANCD2 phos- FANCD2-L isoform. Compared to other tumor progression in which the initial phorylation at serine 222, leading to acti- ovarian cancer cell lines, both 2008 and methylation of FANCF, a gene associat- vation of an S phase checkpoint. In TOV-21G are hypersensitive to CDDP ed with , is followed by response to DNA damage, the FA com- with half maximal inhibitory concentra- FANCF demethylation and CDDP resis- plex mediates ubiquitination of FANCD2 tion (IC50) less than 1.0 µm of CDDP. tance. FANCF is one of seven recently at lysine 561. Activated FANCD2 is TOV-21G cells were retrovirally trans- cloned Fanconi anemia genes whose translocated to chromatin and DNA duced with various FANC cDNAs protein products were found to interact repair foci, which contain the BRCA1 (FANCA, FANCC, FANCE, FANCF, with involved in DNA repair protein and BRCA2/FANCD1 protein FANCG) in an attempt to correct any

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Figure 1. The Fanconi anemia/BRCA pathway and inactivation of BRCA1 in breast/ovarian cancer A: FANCD2 protein functions at the intersection of two signaling pathways. In response to ionizing radiation-mediated double-strand breaks (DSB), ATM phosphorylates the NBS1 protein. Phosphorylation of NBS1 is required for FANCD2 phosphorylation at serine 222 (S222), leading to acti- vation of an S phase checkpoint. In response to DNA damage, the FA complex mediates the Ub of FANCD2 at lysine 561. Activated FANCD2 is translocated to chromatin and DNA repair foci, which contain the BRCA1 protein and BRCA2/FANCD1 protein complex. BRCA2/FANCD1 binds to RAD51 and to DNA, promoting a DNA repair response. The Ub-FANCD2 also colocalizes with NBS-MRE11-RAD50 complex in DNA damage nuclear foci. B: The mechanism of inactivation of BRCA1 in breast/ovarian cancer. Hypermethylation of the BRCA1 promoter was detected in sporadic breast and ovarian cancer samples with proportions ranging from 11% to 31% and from 5% to 15%, respectively. This model shows that promoter methy- lation can serve as a “first hit” in the BRCA1 and FANCF genes just as inherited mutation can serve as a “first hit” in the BRCA1 and BRCA2 genes. In cases where the first copy is methylated, the second copy may be inactivated by LOH or methylation. abnormalities in proteins upstream of ascites of a woman previously exposed methylation. Thus, their study suggests FANCD2 in the FA-BRCA pathway. Only to CDDP, were also methylated. When that disruption of the FANC-BRCA path- FANCF was able to correct the defect in C13* was treated with 5-aza-2’-deoxycy- way occurs de novo in ovarian cancers FANCD2 monoubiquitination in the tidine, a demethylating agent, FANCF and may contribute to selective sensitivi- transfected cells. The FANCF-corrected mRNA expression and monoubiquina- ty to platinum salts, which has significant TOV-21G cells became resistant to MMC tion of FANCD2 were restored.The treat- clinical implication. and CDDP with IC50 > 1 µm CDDP. No ed C13* cells then became less sensitive Fanconi anemia patients are predis- FANCF gene were found in to CDDP. The authors established a posed to many types of cancer, including TOV-21G, but its promoter was densely more general role for FANCF promoter acute myeloid leukemia, squamous cell methylated. Two additional cell lines, methylation in ovarian cancer with the carcinoma of the head and neck, gyne- C13*, a CDDP-resistant derivative of observation that 4 of 19 (21%) primary cological cancers, and esophageal can- 2008 with low levels of FANCF protein ovarian cancers, not previously exposed cer. About 10% of women diagnosed and OAW42, a cell line derived from the to CDDP, also demonstrated FANCF with breast or ovarian cancer each year

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Figure 2. The heterogeneity of DNA methylaiton and In cancer, the dynamics of genetically and epigenetically mediated loss of gene function are very different. The results of the progressive and heterogeneous methylation are an increasing degree of transcriptional loss and a variable decrease in protein production in individual cells of tumor. are estimated to carry highly penetrant, 11, band p15 (11p15), a region contain- defects in structure, cell germline mutations in the BRCA1 or ing a number of imprinted genes associ- division, and viability, so that a BRCA1- BRCA2 gene (Antoniou et al., 2003). ated with cancer, which is also frequently deficient cell must acquire additional Somatic mutations of BRCA1 or BRCA2 lost in ovarian cancer and other tumor alterations that overcome these prob- genes are rare, yet epigenetic changes types (Lu et al., 1997). In a preliminary lems and presumably force tumor evolu- in the form of promoter methylation study of 75 primary breast tumors, we tion down a limited set of pathways resulting in transcriptional silencing of have observed 13 tumors with FANCF (Venkitaraman, 2002). While it is not the BRCA1 gene have been demonstrat- promoter methylation. Further explo- clear that methylation of FANCF will have ed in about 5%–15% of nonfamilial ovar- ration of the epigenetic silencing of similar effects as BRCA1, a model of car- ian cancer cases and 11%–31% of FANCF gene will likely provide insight cinogenesis involving the FA-BRCA nonfamilial breast cancers (Catteau and into the etiology of nonfamilial cancers pathway can be proposed in which pro- Morris, 2002). Interestingly, BRCA2 is as has been demonstrated for the moter methylation can serve as a “first generally hypomethylated and overex- BRCA1 gene. hit” in the BRCA1 or FANCF genes just pressed in breast and ovarian cancers DNA microarray analyses indicate as inherited mutation can serve as a (Collins et al., 1997). Though the num- that breast cancers arising in the setting “first hit” in the BRCA1 and BRCA2 bers are small, this study suggests that of germline BRCA1 mutations have genes (Figure 1B). In cases where the 8% of established ovarian cancer cell unique gene expression profiles, which first copy is methylated, the second copy lines and about 20% of primary ovarian are identical to sporadic tumors with may be inactivated by LOH or methyla- cancers have hypermethylation at the methylated BRCA1 and distinct from tion. FANCF gene promoter. It is not clear other types of breast cancer (van’t Veer Taniguchi et al. (2003) also provide whether methylation was bi-allelic or et al., 2002). The observed similarities further evidence that, as is the case with associated with gross chromosomal between BRCA1-mutated and some deleterious point mutations and gross deletion or loss of heterozygosity (LOH) BRCA1-methylated sporadic tumors chromosomal deletions, aberrant pro- in the primary tumors. FANCF gene is support a tumor progression model in moter methylation is associated with a located on the short arm of chromosome which early loss of BRCA1 causes loss of gene function that can provide a

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selective advantage to transformed cells. the highest degree of methylation, leav- Selected reading However, in contrast to the clonal evolu- ing an outgrowth of a population of cells tion associated with genetic mutations, that are nonmethylated. Antoniou, A., Pharoah, P.D., Narod, S., Risch, promoter CpG island hypermethylation is This study raises important ques- H.A., Eyfjord, J.E., Hopper, J.L., Loman, N., Olsson, H., Johannsson, O., Borg, A., et al. a more gradual and progressive process tions regarding the use of demethylating (2003). Am. J. Hum. Genet. 72, 1117–1130. (Jones and Baylin, 2002). The process agents in the treatment of ovarian can- Catteau, A., and Morris, J.R. (2002). Semin. can vary between individual DNA cer. We do not know nearly enough Cancer Biol. 12, 359–371. strands and between cells, creating about the different pathways that are methylation heterogeneity even in long subject to epigenetic silencing, hence Collins, N., Wooster, R., and Stratton, M.R. (1997). Br. J. Cancer 76, 1150–1156. established cell culture. Because the clinical trials using these agents should degree of transcriptional silencing is usu- proceed cautiously. Nonetheless, we D’Andrea, A.D., and Grompe, M. (2003). Nat. Rev. Cancer 3, 23–34. ally dependent on the density of methy- now have sophisticated techniques to lated CpG sites in the island, this detect de novo methylation, and these Howlett, N.G., Taniguchi, T., Olson, S., Cox, B., methylation heterogeneity can lead to tools should prove useful as we develop Waisfisz, Q., De Die-Smulders, C., Persky, N., Grompe, M., Joenje, H., Pals, G., et al. (2002). heterogeneous populations of cells with novel strategies for the early detection, Science 297, 606–609. varying levels of gene expression and prevention, and treatment of ovarian Jones, P.A., and Baylin, S.B. (2002). Nat. Rev. different properties (Figure 2). This phe- cancer. Genet. 3, 415–428. nomenon could, in part, explain why OAW42 cell line that has 54% of its CpG Lu, K.H., Weitzel, J.N., Kodali, S., Welch, W.R., Olufunmilayo I. Olopade* Berkowitz, R.S., and Mok, S.C. (1997). Cancer sites methylated is partially CDDP sensi- Res. 57, 387–390. tive while 2008 cell line that is 95% and Minjie Wei Taniguchi, T., Tischkowitz, M., Ameziane, N., methylated is CDDP hypersensitive. Center for Clinical Cancer Genetics Hodgson, S.V., Mathew, C.G., Joenje, H., Mok, Likewise, while initial methylation of Department of Medicine S.C., and D’Andrea, A.D. (2003). Nature Med., in FANCF followed by FANCF demethyla- University of Chicago Medical Center press. tion could be one explanation for the Chicago, Illinois 60637 van ‘t Veer, L.J., Dai, H., van de Vijver, M.J., He, observed CDDP resistance in C13* cell *E-mail: Y.D., Hart, A.A., Mao, M., Peterse, H.L., van der line, methylation heterogeneity might [email protected] Kooy, K., Marton, M.J., Witteveen, A.T., et al. (2002). Nature 415, 530–536. also explain the phenomenon. It is con- ceivable that CDDP eradicated cells with Venkitaraman, A.R. (2002). Cell 108, 171–182.

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