and its role in FA and other Cancers FANCD2 and its role in FA and other Cancers Christina Miller

Fanconi Anemia is a congenital cancer predisposition syndrome that causes chromosomal instability Bone marrow failure Sensitivity to cross-linking agents http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2375970/ Congenital Malformations: short stature, small gonads, microphthalmia (small eye), skeletal defects Caused by mutations in any of 13 known Fanc genes- all participate in BRCA1/ FA pathway 8 complementation groups Autosomal Recessive FANCD2 is worse- I will explain later Mutations are hypomorphic meaning they only cause partial loss of function not null function

FANCD2 is expressed in tissues where proliferation occurs The four on the right are fetal tissues Northern Blot for FANCD2 FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). http://dx.doi.org.libproxy.lib.unc.edu/10.1016/S1097-2765(01)00172-1

FANCD2 is regulated by post Translational Modification Monoubiquitinated on Lys-561 Necessary for binding to chromatin Phosphorylated by ATM or ATR on Ser-222 Monoubiquination only during s phase, phosphorylation occurs after genotoxic stress FANCA, C, E, F, G, and L- complex ATM phosphorylates after IR ATR phosphorylates in response to replication stalling agents (ICLs) FANCL in complex with E2 ligases UBE2T or UBE2W These modifications are independent Don’t know why D2 and I form heterodimer or what regulates it- new results from 2012 study show that G2 and I have separate functions No known functional domains. 44 exons unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila http://www.ncbi.nlm.nih.gov/pmc/articles/PMC480901/ http://www.uniprot.org/uniprot/Q9BXW9 Image: http://www.scills.ac.uk/arno-alpi-research.shtml http://dx.doi.org/10.1016/j.mrfmmm.2005.05.010 http://www.scills.ac.uk/arno-alpi-research.shtml

Frontiers in Bioscience 9, 421-437, January 1, 2004 Interstrand Cross-links are covalent bonds that link DNA strands and block replication ICL agents such as mitomycin c and cisplatin are used as chemotherapeutic agents Also found in plants and cosmetics Agent must be taken into nucleus This inhibits replication, transcription 40 icl are enough to kill a mammalian cell that can’t repair itself Frontiers in Bioscience 9, 421-437, January 1, 2004

FANCD2 is necessary for replication-dependent ICL repair FANCI and FANCD2 are both monoubiquinated and are bound together Repair occurs when two replications forks run into the ICL The DNA is then cut to form a double stranded break and then repaired incisions is repaired via Rad51-dependent homologous recombination with the other sister Co-localizes with rad-51 and BRCA1 and BRCA2 https://walter.hms.harvard.edu/node/11 Nature Reviews Cancer 3, 23-34 (January 2003)

FANCD2 is also active in S Phase arrest pathway This is the reason why patients with mutation in FANCD2 have a worse prognosis- FANCD2 is the only one of the known FA proteins that functions in this pathway FANCD2 is phosphorylated by ATM and S phase arrest is activated There are other proteins phosphorylated by ATM that do the same thing through other pathways Nature Reviews Cancer 3, 23-34 (January 2003) http://dx.doi.org/10.1016/j.dnarep.2004.04.005

FANCD2-/- mice show phenotypes similar to those of FA patients Wild type and FANCD2-/- mutant one day after birth Wild type eye one day after birth Representative histology of eye from a Fancd2–/– pup (postnatal day 1) shows a disorganized retina with lack of a distinct chamber or lens development. There is some prenatal mortality suggesting fancd2 is important for embryo development Fancd2-/- mutant mice have higher incidence of tumors than other fanc muntants such as fancc-/- FANCd2 is conserved in eukaryotes- drosophila, c. elegans- unlike other fanc proteins D2 mutants show HSC loss in early development, loss of quiescence in hsc, compromised hsc function FANCD2-/- mutant eye one day after birth Genes Dev. 2003 August 15; 17(16): 2021–2035.

Reduced FANCD2 expression is correlated with decreased survival of breast cancer patients FANCD2 and BRCA participate in the same pathway so it makes sense that FANCD2 would play a role in breast cancer and possibly other cancers. staining for the four metastasis-inducing proteins AGR2, OPN, S100A4, and S100P are the most significantly associated with negative staining for cytoplasmic FANCD2 The cytoplasm (a) +++, strong staining (——– ; 100% = 5 patients); (b) ++, moderate staining (------; 100% = 36 patients); (c) +, intermediate staining (........ ; 100% = 55 patients); (d) +/−, borderline staining (– – –); 100% = 96 patients) and (e) −, negative staining (- — - —, 100% = 118 patients) for FANCD2. ic FANCD2 levels are reduced which may indicate that there is another function other than DNA repair Am J Pathol. 2010 June; 176(6): 2935–2947.

References Dronkert MLG, Kanaar R. Repair of DNA interstrand cross-links. Mutat Res /DNA Repair. 2001 9/4;486(4):217-47. Grompe M, D'Andrea A. Fanconi anemia and DNA repair. Hum Mol Genet. 2001;10:2253. Hölzel M, van Diest PJ, Bier P, Wallisch M, Hoatlin ME, Joenje H, et al. FANCD2 protein is expressed in proliferating cells of human tissues that are cancer‐prone in fanconi anaemia. The Journal of Pathology. 2003;201(2):198-203. Houghtaling S, Timmers C, Noll M, Finegold MJ, Jones SN, Meyn MS, et al. Epithelial cancer in fanconi anemia complementation group D2 (Fancd2) knockout mice. Genes & development. 2003;17(16):2021-35. Rudland PS, Platt-Higgins AM, Davies LM, et al. Significance of the Fanconi anemia FANCD2 protein in sporadic and metastatic human breast cancer. American Journal of Pathology.2010;176(6):2935–2947. Shukla P, Ghosh K, Vundinti BR. Current and emerging therapeutic strategies for fanconi anemia. The HUGO Journal. 2012;6(1):1-8. Venkitaraman AR. Tracing the network connecting BRCA and fanconi anaemia proteins. Nature reviews. Cancer. 2004;4(4):266-76. Wang X, D’Andrea AD. The interplay of fanconi anemia proteins in the DNA damage response. DNA Repair. 2004 0;3(8–9):1063-9.