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Collaboration of Brca1 and Chk2 in Tumorigenesis

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Collaboration of Brca1 and Chk2 in Tumorigenesis Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Collaboration of Brca1 and Chk2 in tumorigenesis John Peter McPherson,1,2,5,6 Bénédicte Lemmers,1,2,6 Atsushi Hirao,3 Anne Hakem,1,2 Jacinth Abraham,1,2 Eva Migon,1,2 Elzbieta Matysiak-Zablocki,1,2 Laura Tamblyn,1,2 Otto Sanchez-Sweatman,2 Rama Khokha,2 Jeremy Squire,2 M. Prakash Hande,4 Tak W. Mak,1,2 and Razqallah Hakem1,2,7 1Advanced Medical Discovery Institute, Ontario Cancer Institute, Toronto, Ontario M5G 2C1, Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; 3The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan; 4Department of Physiology, Faculty of Medicine, National University of Singapore, 117597 Singapore Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. However the activation of p53-dependent checkpoint/apoptotic signaling in the absence of Brca1 is poorly understood. Here, we show that Chk2 inactivation is partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic integrity. Brca1 deficiency was found to result in Chk2 phosphorylation and the Chk2-dependent accumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 was cooperative in breast cancer. Our findings identify a critical role for Chk2 as a component of the DNA damage-signaling pathway activated in response to Brca1 deficiency. [Keywords: Breast cancer; T cell; genomic instability; apoptosis; cell cycle; proliferation] Supplemental material is available at http://www.genesdev.org. Received February 10, 2004; revised version accepted April 2, 2004. BRCA1 germline mutations predispose women to early et al. 1997; Shen et al. 1998), radiosensitivity (Shen et al. onset, familial breast and ovarian cancer (Scully and Liv- 1998; Welcsh et al. 2000), chromosomal abnormalities ingston 2000; Welcsh et al. 2000; Venkitaraman 2002). (Xu et al. 1999; Mak et al. 2000; Welcsh et al. 2000), p53 Despite its role in maintenance of genome integrity activation (Hakem et al. 1996), G2/M checkpoint loss (Kinzler and Vogelstein 1997), transcriptional regulation (Larson et al. 1997; Xu et al. 1999), and impaired homolo- (Scully et al. 1997a; Somasundaram et al. 1997), and gous recombination repair (Moynahan et al. 1999). Ac- chromatin remodeling (Yarden and Brody 1999; Bochar cordingly, the pleiotropic effects of BRCA1 mutation et al. 2000; Pao et al. 2000; Wang et al. 2000; Welcsh et could be attributed to its involvement in DNA repair and al. 2000), the exact mechanism of tumor suppression by transcriptional regulation. BRCA1 remains to be defined. BRCA1 associates with Brca1-targeted disruption in mice results in embry- proteins that function in DNA replication and repair onic lethality (Gowen et al. 1996; Hakem et al. 1996; Liu (Scully et al. 1997b; Wang et al. 2000), transcriptional et al. 1996; Ludwig et al. 1997). To circumvent this limi- activation (Chapman and Verma 1996; Monteiro et al. tation, conditional targeting strategies have been em- 1996; Scully et al. 1997a; Li et al. 2000), and the DNA ployed that facilitate the study of the consequences of damage response (Scully et al. 1997b; Zhong et al. 1999; Brca1 disruption in vivo and ex vivo (Xu et al. 1999; Mak Li et al. 2000; Wang et al. 2000; Welcsh et al. 2000). Cells et al. 2000). Mice that are tBrca1−/− (Mak et al. 2000) with mutant Brca1 display defects in survival and pro- carry a targeted null mutation of Brca1 restricted to the liferation (Gowen et al. 1996; Hakem et al. 1996; Ludwig T-cell lineage, thus bypassing the associated lethality of Brca1 germline disruption. Brca1 disruption in the T-cell compartment results in a drastic depletion of thymo- cytes and peripheral T cells, the accumulation of chro- 5Present address: Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. mosomal abnormalities, and activation of p53. Thymo- −/− 6These authors contributed equally to this work. cyte development of tBrca1 mice is restored in the 7Corresponding author. absence of p53 or the presence of overexpressed anti- E-MAIL [email protected]; FAX (416) 204-2277. Article published online ahead of print. Article and publication date are apoptotic protein Bcl2 that antagonizes the p53 pathway. at http://www.genesdev.org/cgi/doi/10.1101/gad.1192704. These findings suggest that genomic instability and p53 1144 GENES & DEVELOPMENT 18:1144–1153 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Brca1 and Chk2 collaborate in tumor suppression activation due to the ablation of Brca1 contribute to the component of these genome surveillance pathways, observed defects in T-cell development, survival, and Chk2 (also known as Cds1, for review, see Bartek et al. proliferation. 2001; McGowan 2002) has been identified as a tumor Although several studies have linked the compro- suppressor, as mutations in Chk2 have been implicated mised viability and genomic instability of Brca1-defi- in familial cancer syndromes including Li-Fraumeni cient cells to the activation of p53 by genome surveil- (Bell et al. 1999) and low penetrance breast cancer sus- lance (Hakem et al. 1997; Shen et al. 1998; Mak et al. ceptibility (Meijers-Heijboer et al. 2002; Vahteristo et al. 2000; Xu et al. 2001; Cao et al. 2003), the molecular 2002). Several studies have elucidated an ATM-Chk2- components that comprise this response are unclear. Re- p53 DNA damage signaling cascade that is activated fol- cent studies of p53 have elucidated upstream signaling lowing DNA damage (Hirao et al. 2000; Bartek et al. cascades that modulate its half-life, intracellular local- 2001). As Chk2 functions in a DNA damage response ization, and functional activity (Giaccia and Kastan pathway that result in p53 activation, we sought to de- 1998; Caspari 2000; Vousden and Lu 2002). DNA damage termine the contribution of this kinase to phenotypes alters the phosphorylation status of p53 and other DNA associated with Brca1 deficiency. We demonstrate that damage response proteins by triggering molecular signal- deficiency in Chk2 partially mimics the loss of p53 and ing cascades that include ATM, ATR, DNA-PK, Chk1, rescues the defective development, growth, and cellular and Chk2 kinases (for review, see Rich et al. 2000; Zhou demise of Brca1-deficient T cells at the expense of ge- and Elledge 2000; Nyberg et al. 2002; Shiloh 2003). A nomic instability and increased tumorigenicity. We have Figure 1. Chk2 deficiency restores the cellularity and development of the T-cell lineage in tBrca1−/− mice. Thymocyte development was assessed by fluorescence-activated cell sorting (FACS) on thymocytes from wild-type, tBrca1−/−, Chk2−/−,andtBrca1−/−Chk2−/− mice. Thymocytes were stained with antibodies against CD4, CD8, and TCR␣␤. TCR␣␤ expression was evaluated on gated CD4+CD8+ populations. Single-cell suspensions from spleen and lymph nodes were stained with antibodies against the T-cell lineage-specific marker Thy 1 and the B-cell lineage marker B220. Depicted FACScans with quadrant percentages are representive of least three independent experiments. GENES & DEVELOPMENT 1145 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press McPherson et al. Table 1. Aneuploidy and chromosomal abnormalities in tBrca1−/− peripheral T-cells Total end- Metaphases Chromosomes Aneuploid to-end Tri-radial-like Chromosome Chromatid Genotype analyzed per metaphasea cells (%) fusionsb,c structuresb breaksb breaksb Fragmentsb,d p53−/− 47 39.2 ± 2.7 17.0 0.06/cell 0 0.02/cell 0.06/cell 0.15/cell (26–40) 6.38% of 2.13% of 4.26% of 12.77% of cells cells cells cells tBrcal−/− 46 38.1 ± 6.7 60.9 0.87/cell 0.57/cell 0.33/cell 0.44/cell 0.70/cell p53−/− (24–56) 67.4% of 41.3% of 21.7% of 34.8% of 45.7% of cells cells cells cells cells Chk2−/− 136 39.93 ± 0.4 4.0 0 0 0.02/cell 0.01/cell 0 (36–40) 2.03% of 1.35% of cells cells tBrcal−/− 148 39.47 ± 1.4 25.7 0.14/cell 0.17/cell 0.07/cell 0.09/cell 0.21/cell Chk2−/− (32–40) 11.03% of 13.97% of 6.62% of 8.09% of 13.97% of cells cells cells cells cells Results shown are pooled from analyses of metaphase spreads from activated T-cell populations of at least three different mice per genotype. aChromosomes per metaphase presented as mean ± S.D. and range of chromosome numbers presented in parentheses. bThe incidence per cell is indicated in the upper row, the percentage of cells with abnormalities is indicated in the lower row. cIncludes Robertsonian fusionlike configurations, telomere associations, dicentric chromosomes, and ringlike structures. dIncludes centric and acentric fragments. also identified an important role for Chk2 in suppressing CD3␧ + anti-CD28 was attenuated in the absence of Brca1-associated breast cancer. Chk2 (Fig. 2C,D). The G1 + G2/M-phase arrest in anti- CD3 + IL-2 activated tBrca1−/− cells was reversed in the Results absence of Chk2, suggesting that the reversal of the pro- liferation impediment was at least partially the result of To assess genetic interplay between Brca1 and Chk2 in attenuation of the cell cycle arrest (Fig. 2E). vivo, mice with a conditional disruption of Brca1 (Mak Chk2 is phosphorylated and activated in response to et al. 2000) in the T-cell lineage (LckCre Brca1fl/fl DNA damage, resulting in the stabilization and activa- “tBrca1−/−”) or mammary epithelium (WapCre Brca1fl/fl tion of p53 (Chehab et al.
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