Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic “hit” contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

[1]  A. Knudson.  Mutation and cancer: statistical study of retinoblastoma. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[2]  B. Hill.  Cancer chemotherapy. The relevance of certain concepts of cell cycle kinetics. , 1978, Biochimica et biophysica acta.

[3]  D. Easton,et al.  Allele losses in the region 17q12–21 in familial breast and ovarian cancer involve the wild–type chromosome , 1992, Nature Genetics.

[4]  Steven E. Bayer,et al.  A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. , 1994, Science.

[5]  D. Easton,et al.  Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. , 1995, American journal of human genetics.

[6]  C. Liu,et al.  Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development. , 1996, Genes & development.

[7]  J. Rossant,et al.  The Tumor Suppressor Gene Brca1 Is Required for Embryonic Cellular Proliferation in the Mouse , 1996, Cell.

[8]  Ralph Scully,et al.  Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage , 1997, Cell.

[9]  T. Mak,et al.  Partial rescue of Brca15–6 early embryonic lethality by p53 or p21 null mutation , 1997, Nature Genetics.

[10]  T. Ludwig,et al.  Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. , 1997, Genes & development.

[11]  J. Peto,et al.  p53 mutation with frequent novel codons but not a mutator phenotype in BRCA1- and BRCA2-associated breast tumours , 1998, Oncogene.

[12]  M. Jasin,et al.  Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. , 1998, Genes & development.

[13]  K. Kinzler,et al.  Genetic instabilities in human cancers , 1998, Nature.

[14]  X. Wang,et al.  Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. , 1999, Molecular cell.

[15]  M. Perricaudet,et al.  Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2 , 1999, Oncogene.

[16]  B. Koller,et al.  Brca1 controls homology-directed DNA repair. , 1999, Molecular cell.

[17]  D. Livingston,et al.  In search of the tumour-suppressor functions of BRCA1 and BRCA2 , 2000, Nature.

[18]  W. Vogel,et al.  Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families. , 2000, Cancer research.

[19]  M. King,et al.  BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. , 2001, Human molecular genetics.

[20]  D. Evans,et al.  Correspondence re: A. Rothfuss et al., Induced micronucleus frequencies in peripheral blood lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families. Cancer Res., 60: 390-394, 2000. , 2001, Cancer research.

[21]  M. Moynahan,et al.  The cancer connection: BRCA1 and BRCA2 tumor suppression in mice and humans , 2002, Oncogene.

[22]  G. Mills,et al.  Evidence of haplotype insufficiency in human cells containing a germline mutation in BRCA1 or BRCA2 , 2002, International journal of cancer.

[23]  M. Loda,et al.  Growth factor requirements and basal phenotype of an immortalized mammary epithelial cell line. , 2002, Cancer research.

[24]  H. Kampinga,et al.  BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA damage , 2002, International journal of radiation biology.

[25]  S. Elledge,et al.  The BRCA1 suppressor hypothesis: an explanation for the tissue-specific tumor development in BRCA1 patients. , 2002, Cancer cell.

[26]  D. Easton,et al.  Cancer incidence in BRCA1 mutation carriers , 2003 .

[27]  R. Walker,et al.  Breast cell invasive potential relates to the myoepithelial phenotype , 2003, International journal of cancer.

[28]  H Thierens,et al.  Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers , 2004, International journal of radiation biology.

[29]  D. Papadopoulo,et al.  Fidelity of DNA double-strand break repair in heterozygous cell lines harbouring BRCA1 missense mutations , 2004, Oncogene.

[30]  W. Foulkes,et al.  BRCA1 and BRCA2: 1994 and beyond , 2004, Nature Reviews Cancer.

[31]  C. Perou,et al.  MYC Is Amplified in BRCA1-Associated Breast Cancers , 2004, Clinical Cancer Research.

[32]  Jeremy M. Stark,et al.  Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences , 2004, Molecular and Cellular Biology.

[33]  K. Offit,et al.  Increased Progesterone Receptor Expression in Benign Epithelium of BRCA1-Related Breast Cancers , 2004, Cancer Research.

[34]  G. Chenevix-Trench,et al.  Germ‐line mutations in BRCA1 or BRCA2 in the normal breast are associated with altered expression of estrogen‐responsive proteins and the predominance of progesterone receptor A , 2004, Genes, chromosomes & cancer.

[35]  A. Ashworth,et al.  Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way. , 2004, Biochimica et biophysica acta.

[36]  L. Cupples,et al.  Allele imbalance, or loss of heterozygosity, in normal breast epithelium of sporadic breast cancer cases and BRCA1 gene mutation carriers is increased compared with reduction mammoplasty tissues. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[37]  J. Jonkers,et al.  Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects , 2006, Oncogene.

[38]  R. Eeles,et al.  Increased level of chromosomal damage after irradiation of lymphocytes from BRCA1 mutation carriers , 2005, British Journal of Cancer.

[39]  A. Spurdle,et al.  Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability , 2007, Breast Cancer Research and Treatment.

[40]  L. Norton,et al.  Heterogenic Loss of the Wild-Type BRCA Allele in Human Breast Tumorigenesis , 2007, Annals of Surgical Oncology.

[41]  J. Bartek,et al.  DNA Damage Response as an Anti-Cancer Barrier: Damage Threshold and the Concept of 'Conditional Haploinsufficiency' , 2007, Cell cycle.

[42]  B. Park,et al.  Knock-in of mutant K-ras in nontumorigenic human epithelial cells as a new model for studying K-ras mediated transformation. , 2007, Cancer research.

[43]  B. Park,et al.  A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting , 2007, Nature Protocols.

[44]  Y. Jeng,et al.  Brca1 heterozygous mice have shortened life span and are prone to ovarian tumorigenesis with haploinsufficiency upon ionizing irradiation , 2007, Oncogene.

[45]  J. Jonkers,et al.  Mouse models for BRCA1 associated tumorigenesis: From fundamental insights to preclinical utility , 2008, Cell cycle.

[46]  Alan Ashworth,et al.  A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[47]  P. Argani,et al.  Tamoxifen-stimulated growth of breast cancer due to p21 loss , 2008, Proceedings of the National Academy of Sciences.

[48]  Bernard T. Lee,et al.  Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers. , 2009, Cancer research.

[49]  Karthik Devarajan,et al.  Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations , 2010, Cancer Prevention Research.

[50]  H. Cunliffe,et al.  Genomic alterations in histopathologically normal breast tissue from BRCA1 mutation carriers may be caused by BRCA1 haploinsufficiency , 2010, Genes, chromosomes & cancer.