Familial Breast Cancer Screening Reveals an Alteration in the RAP80 UIM Domain That Impairs DNA Damage Response Function
Total Page：16
File Type：pdf, Size：1020Kb
Oncogene (2009) 28, 1843–1852 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Familial breast cancer screening reveals an alteration in the RAP80 UIM domain that impairs DNA damage response function J Nikkila¨ 1,4, KA Coleman2,4, D Morrissey2,4, K Pylka¨ s1, H Erkko1, TE Messick2, S-M Karppinen1, A Amelina1, RWinqvist 1 and RA Greenberg2,3 1Laboratory of Cancer Genetics, Department of Clinical Genetics and Biocenter Oulu, Oulu University Hospital, University of Oulu, Oulu, Finland; 2Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA and 3Department of Pathology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Germline mutations in two major susceptibility genes, Introduction BRCA1 and BRCA2, account for nearly 20% of familial breast cancers. A majority ofthe remaining genetic Approximately 5–10% of all breast cancers stem from factors involved in heritable breast cancer susceptibility hereditary predisposition to the disease. Mutations in are, however, unknown. Recently, a new BRCA1-inter- the two main susceptibility genes BRCA1 and BRCA2 acting protein, receptor associated protein 80 (RAP80), (also called FANCD1), together with mutations in a was identified. RAP80 plays an important role in BRCA1- number of other high-penetrance genes such as TP53 mediated DNA damage responses (DDRs) by recruiting and PTEN, account for 20% of familial breast cancer BRCA1 to DNA double-strand breaks (DSBs). A cases. For the remaining 80%, the genetic factors are comprehensive screening of DNA from affected index largely unknown and are likely to involve mutations in cases of112 BRCA1/BRCA2 mutation-negative Finnish moderate- and low-penetrance susceptibility genes, breast cancer families revealed altogether 10 alterations in plausibly acting together with certain environmental or RAP80, one ofwhich, c.241-243delGAA, resulted in a other hereditary factors (Rahman and Stratton, 1998; single glutamic acid deletion at residue 81 in a highly Schwab et al., 2002; Wooster and Weber, 2003; Stratton conserved region ofubiquitin interaction motif1. The and Rahman, 2008). However, the involvement of rare resultant delE81 protein product displayed significantly constitutional mutations in additional unknown high- reduced ubiquitin binding and DSB localization. Expres- penetrance gene(s) cannot currently be ruled out. sion ofthe RAP80 delE81 allele impaired both BRCA1 BRCA1 and BRCA2 are linked to various processes and ABRA1 DSB recruitment, thus compromising involved in the DNA damage response (DDR), includ- BRCA1-mediated DDR signaling. Compared with wild- ing both repair of DNA double-strand breaks (DSBs) type RAP80, expression ofthe delE81 allele was and checkpoint control. Furthermore, BRCA1 and associated with a significant increase in cytogenetically BRCA2 reside in part as members of a biochemically detectable chromosomal aberrations, particularly chro- active multiprotein complex and colocalize at sites of matid breaks. Although evidently quite rare, these results DNA damage. This suggests that functions carried out suggest that critical constitutional mutations in RAP80 by BRCA1/BRCA2-containing complexes in the DDR abrogate DDR function and may be involved in genetic are necessary for both genome integrity and the predisposition to cancer. suppression of breast and ovarian epithelial malig- Oncogene (2009) 28, 1843–1852; doi:10.1038/onc.2009.33; nancies (Chen et al., 1998; Greenberg et al., 2006). published online 23 March 2009 Consequently, the genes encoding proteins interacting with BRCA1 or BRCA2 could represent important Keywords: RAP80; breast cancer; BRCA1; ubiquitin candidates for harboring new cancer susceptibility alleles. Consistent with this notion, breast cancer suscept- ibility alleles in BRIP1 (also called FANCJ or BACH1) and PALB2 (alias FANCN) have been detected because Correspondence: Dr RA Greenberg, Department of Cancer Biology, of earlier identification of each of these proteins as Abramson Family Cancer Research Institute, University of Pennsyl- important mediators of BRCA1 and BRCA2 DNA vania School of Medicine, 421 Curie Blvd, BRB II/III Rm 513, repair activities. Moreover, BRCA1 is extensively Philadelphia, PA 19104-6160, USA. E-mail: [email protected] or phosphorylated by the checkpoint kinases and breast Dr RWinqvist, Laboratory of Cancer Genetics, Department of cancer suppressors ATM and CHK2, and such phos- Clinical Genetics and Biocenter Oulu, University of Oulu, Oulu phorylation events are necessary for DNA damage University Hospital, P.O. Box 5000, Oulu FIN-90014, Finland. inducible formation of supercomplexes with members E-mail: robert.winqvist@oulu.fi 4These authors contributed equally to work of the MRN complex (composed of MRE11, RAD50 Received 16 December 2008; revised 1 February 2009; accepted 12 and NBS1) and TOPBP1 (Topoisomerase II a binding February 2009; published online 23 March 2009 protein 1) (Greenberg et al., 2006). Mutations in the RAP80 germline mutation impairs DNA damage response J Nikkila¨ et al 1844 genes encoding these BRCA1-interacting proteins have a critical role in BRCA1-mediated DDR signaling also been associated with breast cancer susceptibility and harmful changes in this gene might lead to a (Heikkinen et al., 2006; Karppinen et al., 2006; Stratton disruption of genomic integrity, thus facilitating cancer and Rahman, 2008). development. Recently, a new BRCA1-associated complex has been To explore the possibility that constitutional muta- identified, consisting of a set of proteins that interacts tions in RAP80 are associated with an increased risk of with the BRCA1 C-terminal (BRCT) repeat domains. developing cancer, blood sample DNA of affected index RAP80 (receptor associated protein 80 alias UIMC1 cases from 112 Finnish BRCA1/BRCA2 mutation- ubiquitin-interacting motif containing 1) was identified negative breast cancer families were comprehensively as a BRCA1–BRCT-binding protein on the basis of its screened for possible mutations. One of the alterations co-immunoprecipitation with BRCA1 during purifica- observed, 243delGAA (delE81), mapped to a highly tion of the BRCA1–BARD1 complexes (Kim et al., conserved region in the functionally important UIM1 2007a; Sobhian et al., 2007; Wang et al., 2007; Yan domain and this change abrogated ubiquitin binding et al., 2007). The BRCA1–RAP80 association is and DDRfunction for RAP80and other members of mediated in a phosphorylation-dependent manner by the RAP80–BRCA1 protein complex. These results ABRA1 (Abraxas alias CCDC98), in which the BRCT suggest that the RAP80 delE81 defect is biologically repeats of BRCA1 form a direct biochemical interaction relevant and might be connected to hereditary predis- with phosphorylated ABRA1. This interaction bridges position to cancer. BRCA1 association with the RAP80 complex, which also includes BRCC36, a deubiquitinating enzyme with specificity for lysine 63-linked ubiquitin chains (K63- Ub) (Sobhian et al., 2007). RAP80 is required for the Results accumulation of BRCA1 and the other members of this complex to ionizing radiation-induced foci (IRIF). RAP80 mutation screening RAP80 migrates to IRIF and, through its tandem The screening for RAP80 mutations in affected index ubiquitin interaction motif (UIM) domains, targets cases from 112 BRCA1/BRCA2 mutation-negative BRCA1, ABRA1 and BRCC36 to K63-Ub at DSBs breast cancer families revealed altogether 10 germline (Kim et al., 2007b; Liu et al., 2007; Sobhian et al., 2007; alterations (Table 1). Eight of the detected changes were Wang and Elledge, 2007). The ubiquitin structures in exon regions and two were in intron regions. One of recognized by RAP80 are formed in response to a series the exonic alterations was novel and seven had been of signaling events emanating from MDC1 (mediator of reported earlier (Akbari et al., 2009; Osorio et al., 2009). DNA damage checkpoint protein 1) binding to phos- All detected variants were checked for potential splicing phorylated histone H2AX (gH2AX) at chromatin defects using NNsplice software (http://www.fruitfly. regions adjacent to DSBs (Kim et al., 2007a; Sobhian org/seq_tools/splice.html); however, nothing abnormal et al., 2007). This phosphorylation-dependent interac- was detected. We used ESEfinder 2.0 software (http:// tion targets the RNF8 E3 ligase in association with the rulai.cshl.edu/tools/ESE/) to identify exonic alterations K63-Ub-specific E2 enzyme UBC13 (alias UBE2N) to that fall within predicted exonic splicing enhancer DSB sites to synthesize K63-Ub on histones H2A and sequences that could possibly affect exonic splicing gH2AX, providing a strong DSB targeting signal for enhancer functions (summarized in Table 1). RAP80-containing complexes (Huen et al., 2007; Kolas Of the genetic alterations detected, one was located in et al., 2007; Mailand et al., 2007; Wang and Elledge, a functional domain, suggesting potential deleterious 2007; Zhao et al., 2007). As a consequence, RAP80 plays effects on RAP80 functions in the DDR (Figure 1a). Table 1 Observed sequence variants occurring in the RAP80 gene and possible effects assessed by using ESEfinder software Exon Variant ESE Carrier frequency (%) P-value (OR; 95% CI) Familial cases Controls 2 c.1–8A>Ga SF2/ASF, SRp40 1.8 (2/112) 1.8 (6/325) 1.00 (0.97; 0.19–4.86) 2 c.43C>T (R15Wa) — 4.5 (5/112) 2.2 (7/325) 0.20 (2.12; 0.66–6.83) 3 c.228T>C