Mutant p53 (mtp53) promotes chemotherapy resistance through multiple mechanisms, including disabling proapoptotic proteins and regulating gene expression. Comparison of genome wide analysis of mtp53 binding revealed that the ETS-binding site motif (EBS) is prevalent within predicted mtp53-binding sites. We demonstrate that mtp53 regulates gene expression through EBS in promoters and that ETS2 mediates the interaction with this motif. Importantly, we identified TDP2, a 59-tyrosyl DNA phosphodiesterase involved in the repair of DNA damage caused by etoposide, as a transcriptional target of mtp53. We demonstrate that suppression of TDP2 sensitizes mtp53-expressing cells to etoposide and that mtp53 and TDP2 are frequently overexpressed in human lung cancer; thus, our analysis identifies a potentially ''druggable'' component of mtp53's gain-of-function activity.[Keywords: TDP2; cancer; p53] Supplemental material is available for this article. One of the definitive characteristics of the mutant p53 (mtp53) protein is that it can alter the cellular phenotype, resulting in the acquisition of gain-of-function activities such as abnormal cell growth, suppression of apoptosis, chemotherapy resistance, increased angiogenesis, and metastasis ( For example, mtp53 can interact with its family members, p63 and p73, and disable their ability to induce apoptosis (Di Como et al. 1999;Marin et al. 2000;Strano et al. 2000Strano et al. , 2002Gaiddon et al. 2001;Bergamaschi et al. 2003;Irwin et al. 2003;Lang et al. 2004). mtp53 can also interact with other transcription factors (such as NF-Y, E2F1, VDR, and p63) and thereby can be recruited to target genes that have consensus binding sites for these transcription factors (Di Agostino et al. 2006;Adorno et al. 2009;Fontemaggi et al. 2009;Stambolsky et al. 2010). Notably, some of these interactions help explain how the mtp53 protein can deregulate gene expression and promote abnormal cell growth, angiogenesis, and metastasis (Di Agostino et al. 2006;Adorno et al. 2009;Fontemaggi et al. 2009;Muller et al. 2009Muller et al. , 2011. However, thus far, none of these transcription factors have been shown to play a fundamental role in regulating the expression of genes that can confer chemotherapy resistance by modulating the response to DNA damage. The main goal of this study was to identify a transcriptional regulatory mechanism through which mtp53 can promote chemotherapy resistance. Results Identification of mtp53 target genesTo identify transcriptional targets of mtp53, we employed two different approaches: chromatin immunoprecipitation (ChIP)-on-chip and ChIP combined with deep sequencing (ChIP-seq). The ChIP-on-chip was performed with Nimblegen arrays that have oligonucleotide probes for all of the promoters in the human genome (Nimblegen Promoter Arrays). The ChIP-seq analysis was performed using the Illumina platform. We conducted these analyses in the Li-Fraumeni cell line MDAH087, which expresses only the R248W mtp53 protein (Bischoff et al. 1990). The ChIP-on-chip analysis identif...

SUMMARY Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP dependent protein (GTPase) activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53’s control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Posttranslational modifications play a crucial role in the proper control of c-Myc protein stability and activity. c-Myc can be modified by small ubiquitin-like modifier (SUMO). However, how SUMOylation regulates c-Myc stability and activity remains to be elucidated. The deSUMOylation enzyme, SENP1, has recently been shown to have a prooncogenic role in cancer; however, mechanistic understanding of this is limited. Here we show that SENP1 is a c-Myc deSUMOylating enzyme. SENP1 interacts with and deSUMOylates c-Myc in cells and in vitro. Overexpression of wild-type SENP1, but not its catalytically inactive C603S mutant, markedly stabilizes c-Myc and increases its levels and activity. Knockdown of SENP1 reduces c-Myc levels, induces cell cycle arrest, and drastically suppresses cell proliferation. We further show that c-Myc can be comodified by both ubiquitination and SUMOylation. SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination, suggesting that SUMOylation promotes c-Myc degradation through the proteasome system. Interestingly, SENP1-mediated deSUMOylation promotes the accumulation of monoubiquitinated c-Myc and its phosphorylation at serine 62 and threonine 58. SENP1 is frequently overexpressed, correlating with the high expression of c-Myc, in breast cancer tissues. Together, these results reveal that SENP1 is a crucial c-Myc deSUMOylating enzyme that positively regulates c-Myc’s stability and activity.

Glucose metabolism is biochemically intertwined between energy metabolism and building block biosynthesis in living cells. However, it has not been investigated yet how its metabolic network is orchestrated to govern glucose flux in space and time. Since we reported that human enzymes in glucose metabolism are spatially organized into metabolically active membraneless compartments (i.e., glucosomes), we have employed lattice light sheet microscopic imaging and other biophysical and biochemical techniques to understand their functional significance in cellular metabolism. Now, we demonstrated that glucosome assemblies behave like liquid droplets in human cells and thus reversibly respond to environmental changes. In addition, we characterized a molecular architecture of the glucosome, which appears to be constructed from higher-ordered oligomeric structures of its scaffolder enzyme along with transient enzyme-enzyme interactions. Importantly, we found that enzymatic compositions of glucosomes are altered when they are spatially in proximity to mitochondria to functionally couple glycolysis with mitochondrial metabolism in human cells. Collectively, we envision that the subcellular localization-function relationship between glucosomes and mitochondria may represent one of fundamental principles by which 4-dimensional metabolic networks are not only dynamically but also efficiently regulated in living human cells.One Sentence SummaryInvestigation of a 4D functional network of glucose metabolism uncovers a fundamental principal of subcellular metabolic regulation in human cells.

The stability and activity of the p53 tumor suppressor protein are tightly regulated by various posttranslational modifications, including SUMOylation. p53 can be modified by both SUMO1 and SUMO2, although how SUMOylation regulates p53 activity is still obscure. Whether p53 activity is directly regulated by deSUMOylation is also unclear. Here, we show that SENP1, a SUMOspecific protease implicated in pro-oncogenic roles, is a p53 deSUMOylating enzyme. SENP1 interacts with p53 and deSUMOylates p53 in cells and in vitro. Knockdown of SENP1 markedly induced p53 transactivation activity. We further show that SENP1 depletion synergizes with DNA damage-inducing agent etoposide to induce p53 activation and the expression of p21, leading to synergistic growth inhibition of cancer cells. Our results reveal that SENP1 is a critical p53 deSUMOylating enzyme and a promising therapeutic target in wild-type p53 containing cancer cells.