dna stain br Results br Discussion CSL serves as the central

Results
Discussion CSL serves as the central node in canonical Notch signaling by transmitting signaling from all Notch receptors upon ligand activation. In this work, we report that genetic ablation of CSL in breast tumor dna stain leads to enhanced tumor growth after transplantation into mammary fat pads in mice, an unexpected finding given that blocking of Notch at the receptor level reduces and activation of Notch promotes tumor growth (Bolós et al., 2013; Suman et al., 2013). The genome-wide transcriptome data support the view that CSL does not merely mediate Notch signaling, as the set of genes upregulated by CSL ablation was considerably larger than the Notch signature in the MDA-MB-231 cells. This conclusion differs from a recent report, which used small hairpin RNA to knock down CSL expression in MDA-MB-231 cells (Kulic et al., 2014). While Kulic et al. (2014), like us, showed that reduced CSL expression in MDA-MB-231 cells promotes tumor growth, they argue that the observed phenotype was linked to derepression of Notch-activated genes. This notion, however, was based on gene-expression analysis of a set of only 170 genes that were on theoretical grounds considered to be Notch responsive (Kulic et al., 2014). Only five of the 170 genes, however, were among the 1,768 genes upregulated in the CSL−/− cells and only two genes were common to the 139 genes in our Notch signature (Figures S4B and S4C). The notion that CSL has Notch-independent functions is in line with the fact that CSL interacts with a number of proteins that are not linked to Notch signaling, such as CTCF, EBNA3c, interferon regulatory factor 4, and RITA (Collins et al., 2014). One hypothesis to explain the large Notch-independent gene set posits that CSL binds to a larger number of genomic sites, only a subset of which can bind Notch ICD, and loss of CSL would thus lead to derepression also of Notch-independent genes. Recent data, however, support a view whereby CSL is dynamically recruited by Notch ICD and not statically bound as a repressor (Castel et al., 2013; Krejcí and Bray, 2007). An alternative hypothesis is that CSL may not directly repress all genes whose expression is altered, but rather control expression of a smaller set of chromatin modifiers or transcriptional regulators, which in turn reset the chromatin landscape and/or alter gene expression on a broader scale. In support of this view, we noted that expression of a number of such factors, such as Serpin, was upregulated in the CSL-deficient cells. That loss of CSL enhances tumor development is further underlined by a recent study reporting that combined silencing of CSL and p53 in cancer-associated fibroblasts leads to stromal and cancer cell expansion (Procopio et al., 2015). An unexpected consequence of removing CSL was the unlocking of a hypoxic response during normoxia, manifested by a post-transcriptional elevation of HIF1α protein levels and the upregulation of hypoxia-regulated genes such as VEGF-A, STC2, and KLF8. This is in line with previous reports on normoxic HIF1α protein stabilization (Palmer et al., 2000; Ranasinghe et al., 2015; for review see Kuschel et al., 2012), and adds to the emerging view of a multifaceted interplay between Notch and the cellular hypoxic response (Andersson and Lendahl, 2014; Lendahl et al., 2009). Notch1 ICD interacted with HIF1α, and it is noteworthy that blocking Notch ICD generation reduced the amount of HIF1α, raising the intriguing possibility that Notch ICD in some way plays a role in the observed HIF1α stabilization. Furthermore, the HIF1α protein levels in the CSL-deficient cells were reduced by DTT treatment, suggesting a role for redox potential, possibly linked to nitrosylation of HIF1α (Palmer et al., 2000) or destabilization of the ODD domain. The unleashing of a hypoxic response during normoxia may also be linked to the acquisition of aberrant cell morphology, a phenotype strongly reminiscent of the recently described polyploid giant cancer cell (PGCC) phenotype (Zhang et al., 2014). Interestingly, PGCC cells, which are endowed with cancer stem cell-like properties, were recently identified in a number of tumor contexts in response to hypoxia or chemical induction of the hypoxic response by CoCl2 (Zhang et al., 2014), suggesting that loss of CSL may lead to a PGCC-like state via upregulation of HIF1α. As PGCC cells are endowed with reduced proliferative rate in vitro combined with accelerated tumor growth capacity (Zhang et al., 2014), the induction of HIF1α protein levels in the CSL-deficient cells may underlie their enhanced growth rates, invasive capacity, and accelerated tumor growth.