Decreased BRCA1 expression in the absence of genetic mutation is observed frequently in sporadic cancers of the breast and other sites, although little is known regarding the mechanisms by which the expression of this gene can be repressed. Here, we show that activating and repressive E2Fs simultaneously bind the BRCA1 promoter at two adjacent E2F sites in vivo, and that hypoxia induces a dynamic redistribution of promoter occupancy by these factors resulting in the transcriptional repression of BRCA1 expression. Functionally, we show that hypoxia is associated with impaired homologous recombination, whereas the nonhomologous end-joining (NHEJ) repair pathway is unaffected under these conditions. Repression of BRCA1 expression by hypoxia represents an intriguing mechanism of functional BRCA1 inactivation in the absence of genetic mutation. We propose that hypoxiainduced decreases in BRCA1 expression and consequent suppression of homologous recombination may lead to genetic instability by shifting the balance between the highfidelity homologous recombination pathway and the errorprone NHEJ pathway of DNA repair. Furthermore, these findings provide a novel link between E2Fs and the transcriptional response to hypoxia and provide insight into the mechanisms by which the tumor microenvironment can contribute to genetic instability in cancer. (Cancer Res 2005; 65(24): 11597-604)

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptors that organize signaling complexes downstream of activated cell surface receptors. Here, we show that IRS-1 and IRS-2, despite significant homology, play critical yet distinct functions in breast cancer, and we identify specific signaling pathways that are influenced by IRS-1 using the polyoma virus middle-T (PyV-MT) transgenic mouse model of mammary carcinoma and Irs-1 null (Irs1 ؊/؊ ) mice. The absence of Irs-1 expression enhanced metastatic spread significantly without a significant effect on primary tumor growth. Orthotopic transplant studies revealed that the increased metastatic potential of Irs1-deficient tumor cells is cell autonomous. Mammary tumors that developed in PyV-MT::Irs1 ؊/؊ mice exhibited elevated Irs-2 function and enhanced phosphatidylinositol 3-kinase/ Akt/mTor activity, suggesting that one mechanism by which Irs-1 impedes metastasis is to suppress Irs-2-dependent signaling. In support of this mechanism, reduction of Irs-2 expression in Irs1 ؊/؊ tumor cells restored mTor signaling to wild-type levels. PyV-MT::Irs1 ؊/؊ tumors also exhibited a significant increase in vascular endothelial growth factor expression and microvessel density, which could facilitate their dissemination. The significance of our findings for human breast cancer is heightened by our observation that Irs-1 is inactivated in wild-type, metastatic mammary tumors by serine phosphorylation. Collectively, our findings reveal that inactivation of IRS-1 enhances breast cancer metastasis and support the novel hypothesis that IRS-1 has metastasis suppressor functions for breast cancer.The insulin receptor substrate (IRS) proteins are cytoplasmic docking proteins that function as essential signaling intermediates downstream of activated cell surface receptors, including the insulin, insulin-like growth factor 1 (IGF-1), prolactin, growth hormone (GH), and vascular endothelial growth factor (VEGF) receptors, members of the integrin receptor family, and select cytokine receptors (38,68,70,78,81,85). The IRS proteins are recruited to receptors through pleckstrin homology and phosphotyrosine-binding domains in their N termini and mediate their functions by organizing signaling complexes at sites of receptor activation (81). Upon binding they are phosphorylated on tyrosine residues in their C termini, creating multiple phosphotyrosine binding motifs that recruit downstream effectors, including phosphatidylinositol 3-kinase (PI3K), Grb-2, Fyn, and Shp-2 to initiate intracellular signaling cascades (81). The IRS proteins were originally identified as substrates of the insulin receptor, and they have been predominantly studied for their role in metabolic signaling (81). Although the IRS proteins are highly homologous, there is evidence for unique functions for each of the four IRS family members. Irs-1 null mice are born small and remain runted throughout life, and these mice develop insulin resistance. However, Irs-1 null mice do not develop diabetes, because they mai...

No abstract

Chk2 is a serine/threonine kinase that signals to cell cycle arrest, DNA repair, and apoptotic pathways following DNA damage. It is activated by phosphorylation in response to ionizing radiation, UV light, stalled replication forks, and other types of DNA damage. Hypoxia is a common feature of solid tumors and has been shown to affect the regulation of many genes, including several DNA repair factors. We show here that Chk2 is phosphorylated on Thr68 and thereby activated in cells in response to hypoxia, and that this phosphorylation is dependent on the damage response kinase ataxia telangiectasia mutated (ATM) but not on the related kinase ATM and Rad3-related. Moreover, phosphorylation of Chk2 under hypoxia was attenuated in cells deficient in the repair factors MLH1 or NBS1. Finally, Chk2 serves to protect cells from apoptosis under hypoxic growth conditions. These results identify hypoxia as a new stimulus for Chk2 activation in an ATM-, MLH1-, and NBS1-dependent manner, and they suggest a novel pathway by which tumor hypoxia may influence cell survival and DNA repair.

No abstract