IFI16 is a member of the PYRIN superfamily that has been implicated in BRCA1-mediated apoptosis and inflammation signaling pathways. Here we report that most breast cancer cell lines examined expressed decreased mRNA and protein levels of IFI16, although IFI16 is expressed in human primary normal mammary epithelial cells. Significantly, immunohistochemical analysis of tissues from 25 breast cancer patients demonstrated that carcinoma cells showed negative or weaker staining of IFI16 compared with positive nuclear staining in normal mammary duct epithelium. si-RNA-mediated reduction of IFI16 resulted in perturbation of p53 activation when treated with ionizing radiation (IR). Expression of IFI16 enhanced p53 transcriptional activity in cells exposed to IR. Adenovirus expression of IFI16 in IFI16-deficient MCF7 induced apoptosis, which was enhanced by radiomimetic neocarcinostatin treatment. Tetracycline-regulated IFI16 also induced apoptosis when coexpressed with p53 in p53-deficient EJ cells subjected to IR, suggesting that IFI16 is involved in p53-mediated transmission of apoptosis signaling. Consistent with these results, expression of IFI16 enhanced activation of the known p53 target genes, including p21, Hdm2, and bax in MCF7 cells. These results suggest that loss of IFI16 results in deregulation of p53-mediated apoptosis, leading to cancer development.

We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502-802) interacted with the IFI16 Pyrin domain (aa 1-130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNAdamaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.

The branching of complex N-glycans attached to growth factor receptors promotes tumor progression by prolonging growth factor signaling. The addition of the bisecting GlcNAc to complex N-glycans by Mgat3 has varying effects on cell adhesion, cell migration and hepatoma formation. Here we show that Chinese hamster ovary (CHO) cells expressing Mgat3 and the Polyoma Middle T (PyMT) antigen have reduced cell proliferation and growth factor signaling dependent on a galectin lattice. The Mgat3 gene is not expressed in virgin mammary gland but is upregulated during lactation and is expressed in MMTV/PyMT tumors. Mice lacking Mgat3 that cannot transfer the bisecting GlcNAc to N-glycans acquire PyMT-induced mammary tumors more rapidly, have an increased tumor burden, increased migration of tumor cells, and increased early metastasis to lung. Tumors and tumor-derived cells lacking Mgat3 exhibit enhanced signaling through the Ras pathway, and reduced amounts of functionally-glycosylated α-dystroglycan. Constitutive overexpression of an MMTV/Mgat3 transgene inhibits early mammary tumor development and tumor cell migration. Thus the addition of the bisecting GlcNAc to complex Nglycans of mammary tumor cell glycoprotein receptors is a cell-autonomous mechanism serving to retard tumor progression by reducing growth factor signaling.

. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.Breast cancer is the most common cancer and the second leading cause of cancer mortality in women, with approximately one of nine women being affected in her lifetime (1). Inheritance in breast cancer families follows the classic mendelian pattern of autosomal dominant transmission, with 50% of carriers' children inheriting BRCA1 2 mutations. This inheritance pattern, as well as loss-of-heterozygosity studies in tumors from affected members of BRCA1-linked families, supports the hypothesis that BRCA1 fits the model of a classic tumor suppressor gene, with loss of the normal allele in the tumors of all informative cases (2). Female mutation carriers are estimated to have an 85% lifetime risk of breast cancer (3) and a 40 -50% risk of ovarian cancer (4). BRCA1, first identified as a breast cancer susceptibility gene, encodes a 1863-amino acid protein with an N-terminal RING finger domain and a C-terminal acidic domain termed the BRCT domain (5). Mutations in both alleles of BRCA1 greatly increase the risk of breast and ovarian cancers, identifying this gene as a tumor suppressor. Gene disruption experiments in mice result in early embryonic lethality and have therefore provided no information regarding BRCA1 function in adult animals (6 -8). Mice resulting from conditional knockout show immature mammary development and form tumors after long latency with p53 mutation (9). The BRCA1 protein may act at a number of points in nuclear function and growth control (10 -12). For example, the immunofluorescence pattern of BRCA1 dramatically changes from discrete nuclear dots to a dispersed pattern when cells are treated with chemical compounds or IR, implying that BRCA1 is involved in a replication checkpoint after DNA damage (10, 13-15). More recently, we found that BRCA1 plays a crucial role in activating caspase-3 upon UV damage (16).An appropriate response to DNA damage is crucial for maintenance of genome stability. Several cellular proteins have been implicated in such processes, such as ATM/ATR protein Ser/Thr kinases, the MRN complex, Fanconi anemia proteins, and the BRCA1 breast cancer tumor-susceptible protein (17)(18)(19)(20). In human and murine cells, ATM is required for early response to agents such as IR that induce DSBs. Despite considerable overlap between the processes regulated by ATM and its relative ATR, cells that lack ATM are extremely sensitive to IR. Thus, ATM plays a unique and essential role in determining survival following IR. In its unstimulated state, ATM is proposed to exist as a homodimer in which the kinase domain of one subunit faces the autophosphorylation of another (21). Upon stimulation, the intermolecular phosphorylation site of the subunits promotes dissociation, and the monomers are free to phosphorylate other substrates (21-23). Use of the phosphorylation site-specific antibod...

Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects, leukemia, and squamous cell carcinoma of the head and neck (HNSCC) and cervix. The FA/BRCA pathway includes 8 members of a core complex and 5 downstream gene products closely linked with BRCA1 or BRCA2. Precancerous lesions are believed to trigger the DNA damage response (DDR), and we focused on the DDR in FA and its putative role as a checkpoint barrier to cancer. In primary fibroblasts with mutations in the core complex FANCA protein, we discovered that basal expression and phosphorylation of ATM (ataxia telangiectasia mutated) and p53 induced by irradiation (IR) or mitomycin C (MMC) were upregulated. This heightened response appeared to be due to increased basal levels of ATM in cultured FANCA-mutant cells, highlighting the new observation that ATM can be regulated at the transcriptional level in addition to its well-established activation by autophosphorylation. Functional analysis of this response using γ-H2AX foci as markers of DNA double-stranded breaks (DSBs) demonstrated abnormal persistence of only MMC-and not IR-induced foci. Thus, we describe a processing defect that leads to general DDR upregulation but specific persistence of DNA crosslinker-induced damage response foci. Underscoring the significance of these findings, we found resistance to DNA crosslinker-induced cell cycle arrest and apoptosis in a TP53-mutant, patient-derived HNSCC cell line, whereas a lymphoblastoid cell line derived from this same individual was not mutated at TP53 and retained DNA crosslinker sensitivity. Our results suggest that cancer in FA may arise from selection for cells that escape from a chronically activated DDR checkpoint.

Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway.

Supplementary Methods, Figures 1-5, Table 1 from The Bisecting GlcNAc on <i>N</i>-Glycans Inhibits Growth Factor Signaling and Retards Mammary Tumor Progression

Supplementary Methods, Figures 1-5, Table 1 from The Bisecting GlcNAc on <i>N</i>-Glycans Inhibits Growth Factor Signaling and Retards Mammary Tumor Progression