Zoë Weaver

Centrosome Amplification and a Defective G2–M Cell Cycle Checkpoint Induce Genetic Instability in BRCA1 Exon 11 Isoform–Deficient Cells

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.

Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation.

Cre-mediated excision of exon 11 of the breast-tumour suppressor gene Brca1 in mouse mammary epithelial cells causes increased apoptosis and abnormal ductal development. Mammary tumour formation occurs after long latency and is associated with genetic instability characterized by aneuploidy, chromosomal rearrangements or alteration of Trp53 (encoding p53) transcription. To directly test the role of p53 in Brca1-associated tumorigenesis, we introduced a Trp53-null allele into mice with mammary epithelium-specific inactivation of Brca1. The loss of p53 accelerated the formation of mammary tumours in these females. Our results demonstrate that disruption of Brca1 causes genetic instability and triggers further alterations, including the inactivation of p53, that lead to tumour formation.

A targeted disruption of the murine Brca1 gene causes γ-irradiation hypersensitivity and genetic instability

Germline mutations of the Brca1 gene are responsible for most cases of familial breast and ovarian cancers, but somatic mutations are rarely detected in sporadic events. Moreover, mouse embryos deficient for Brca1 have been shown to die during early embryogenesis due to a proliferation defect. These findings seem incompatible with the tumor suppress function assigned to this gene and raise questions about the mechanism by which Brca1 mutations cause tumorigenesis. We now directly demonstrate that BRCA1 is responsible for the integrity of the genome. Murine embryos carrying a Brca1 null mutation are developmentally retarded and hypersensitive to γ-irradiation, suggesting a failure in DNA damage repair. This notion is supported by spectral karyotyping (SKY) of metaphase chromosomes, which display numerical and structural aberrations. However, massive chromosomal abnormalities are only observed when a p53−/− background is introduced. Thus, a p53 dependent cell cycle checkpoint arrests the mutant embryos and prevents the accumulation of damaged DNA. Brca1−/− fibroblasts are not viable, nor are Brca1−/− : p53−/− fibroblasts. However, proliferative foci arise from Brca1−/− : p53−/− cells, probably due to additional mutations that are a consequence of the accumulating DNA damage. We believe that the increased incidence of such additional mutations accounts for the mechanism of tumorigenesis associated with Brca1 mutations in humans.

Mammary tumors in mice conditionally mutant for Brca1 exhibit gross genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer.

BRCA1 mutation carriers have an increased susceptibility to breast and ovarian cancer. Excision of exon 11 of Brca1 in the mouse, using a conditional knockout (Cre-loxP) approach, results in mammary tumor formation after long latency. To characterize the genomic instability observed in these tumors, to establish a comparative map of chromosomal imbalances and to contribute to the validation of this mouse model of breast cancer, we have characterized chromosomal imbalances and aberrations using comparative genomic hybridization (CGH), and spectral karyotyping (SKY). We found that all tumors exhibit chromosome instability as evidenced by structural chromosomal aberrations and aneuploidy, yet they display a pattern of chromosomal gain and loss that is similar to the pattern in human breast carcinomas. Of note, nine of 15 tumors exhibited a gain of distal chromosome 11, a region that is orthologous to human chromosome 17q11-qter, the mapping position of Erbb2. However, our analysis suggests that genes distal to Erbb2 are the main targets of amplification. Four of the tumors also exhibited a copy number loss of proximal chromosome 11 (11A-B), a region orthologous to human 17p. In eight of the tumors we observed whole or partial gain of chromosome 15 centering on 15D2-D3 (orthologous to human chromosome 8q24), the map location of the c-Myc gene, and six of the tumors exhibited copy number loss of whole or partial chromosome 14, including 14D3, the map location of Rb1. We conclude that despite the tremendous shuffling of chromosomes during the course of mammalian evolution, the pattern of genomic imbalances is conserved between BRCA1-associated mammary gland tumors in mice and humans. Western blot analysis showed that while p53 is absent or mutated in some tumors, at least two tumors revealed wild-type protein, suggesting that other genetic events may lead to tumorigenesis. Similar to BRCA1-deficient mouse embryonic fibroblasts, the tumor cells contained supernumerary functional centrosomes with intact centrioles whose presence results in multipolar mitoses and aneuploidy.

Myc/p53 interactions in transgenic mouse mammary development, tumorigenesis and chromosomal instability

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV–LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-deficient strain as a means of examining the effect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Specifically, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53−/−), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53+/+); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53+/−) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53+/− mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53+/+ mice. Also, as previously observed (), lymphomas arose with extremely short latency in MMTV-myc/ p53−/− mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53+/+ and MMTV-myc/p53+/− mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-myc-overexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV‐cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/− tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31–p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses. Genes Chromosomes Cancer 25:251–260, 1999. Published 1999 Wiley‐Liss, Inc.

RAG-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice

ABSTRACT Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcrα/δ locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm−/− mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2−/− Atm−/− mice contained multiple chromosomal abnormalities, but none of these involved the Tcrα/δ locus. These findings indicated that tumorigenesis in Atm−/− mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1−/− Atm−/− mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1−/− Atm−/− mice in parallel to our previous Rag2−/− Atm−/− study. We found that thymic lymphomas occur at high frequency in Rag1−/− Atm−/− mice and resemble those that occur in Rag2−/− Atm−/− mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcrα/δ locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.

Comparative Genomic Hybridization (CGH)—Detection of Unbalanced Genetic Aberrations Using Conventional and Micro‐Array Techniques

This unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor samples. Specific emphasis is placed on recent applications to the analysis of murine model systems for human cancer. CGH is an invaluable tool for identifying the characteristic genetic rearrangements in these models. The authors discuss an exciting new method currently being developed, array CGH, which results in a tremendous increase in resolution. Oncogene amplifications and deletions of tumor‐suppressor genes are detected on a single‐gene level. Detailed protocols are supplied for CGH analysis of both human and mouse chromosomes. Keywords: comparative genomic hybridization; tumor genetics; unbalanced chromosomal aberrations; DNA copy number changes; gene amplification; oncogenes; tumor suppressor genes; mouse models of human cancer; array technology; array‐CGH This unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor

Abstract 1556: Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors.

Optical imaging of labeled cells is the most prevailing method for cell tracking in mouse models. However, the immunogenicity of xenobiotic reporter genes, such as the commonly used firefly luciferase (ffLuc) and enhanced green fluorescence protein (eGFP), results in inconsistency in cell labeling and tumor progression, preventing their use in host mice with normal immunity. To resolve this issue, we have generated a reporter gene-tolerized transgenic mouse in which an ffLuc-eGFP fusion gene was targeted to the anterior pituitary gland using a rat Growth Hormone promoter (dubbed the “glowing-head” mouse). When ffLuc-eGFP-labeled Lewis Lung Carcinoma (LLC) tissue was subcutaneously transplanted into syngeneic glowing-head mice (GH-c-brd), their non-transgenic litter-mates (WT-c-brd), and immunocompromised Nod-Scid (balb/c) mice, anti-GFP antibody was induced only in the circulation of WT-c-brd. Labeled LLC also exhibited fewer CD4+ T cells but more macrophages in GH-c-brd than in WT-c-brd, suggesting it induced adaptive immunity in the latter. Moreover, GH-c-brd exhibited faster subcutaneous tumor growth and post-resection metastatic progression, and maintained more consistent tumor labeling, as compared to WT-c-brd and even Nod-Scid mice. Interestingly, in an adjuvant chemotherapeutic setting, the immunogenicity of the labeled tumor was able to deter metastatic progression in a manner similar to that obtained by drug treatment. We conclude that the immunogenicity of xenobiotic labeling markers can significantly influence disease progression and therapeutic responses thereby compromising immunocompetent preclinical cancer models, and that the glowing-head mouse can be used to circumvent such problems and promote consistent disease monitoring. Citation Format: Chi-Ping Day, Zoe Weaver, John Carter, Carrie Bonomi, Terry Van Dyke, Melinda Hollingshead, Glenn Merlino. Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2013-1556

Abstract A33: Integrated analysis of molecular and metabolomic data to identify drug resistance mechanisms in lung cancers

Consistent bioinformatics analysis approaches of chemically perturbed biological systems are needed in order to achieve meaningful integration of results from multiple experiments in support of cancer systems biology research. Our initial efforts have focused on identifying and contrasting molecular mechanisms that reveal biochemical events affecting non-small cell lung cancers in the context of combination drug therapies as a model for defining these approaches. While the link between growth factor receptor mutations, target inhibitor drugs and non-small cell lung cancer is well established, the basis of variable response in different patient cohorts is still poorly understood and is the subject of ongoing investigations. Data for our efforts were provided by the NCI9s Center for Advanced Preclinical Research. Mice genetically engineered to develop erlotinib-sensitive lung adenocarcinomas were subjected to drug compounds that are currently in clinical trials, as well as erlotinib, individually and in combination, to study early response and development of late resistance. Metabolite data, gene expression data, and histological data were collected and used in defining the initial analysis approach. While evaluation of analysis approaches continues, preliminary results already indicate these data can be effective in assessing drug efficacy on reducing tumor burden. Further efforts are underway to develop analysis techniques to explore the underlying dynamic processes. Citation Format: Anand S. Merchant, Natalie Fedorova Abrams, Eric A. Stahlberg, Zoe Weaver. Integrated analysis of molecular and metabolomic data to identify drug resistance mechanisms in lung cancers [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A33.