A molecular mechanism for the "digital" response of p53 to stress.

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

Constitutive low expression of antiviral effectors sensitizes melanoma cells to a novel oncolytic virus.

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

Regulation of stability and inhibitory activity of the tumor suppressor SEF through casein-kinase II-mediated phosphorylation.

Inflammation and cancer are intimately linked. A key mediator of inflammation is the transcription-factor NF-κB/RelA:p50. SEF (also known as IL-17RD) is a feedback antagonist of NF-κB/RelA:p50 that is emerging as an important link between inflammation and cancer. SEF acts as a buffer to prevent excessive NF-κB activity by sequestering NF-κB/RelA:p50 in the cytoplasm of unstimulated cells, and consequently attenuating the NF-κB response upon pro-inflammatory cytokine stimulation. SEF contributes to cancer progression also via modulating other signaling pathways, including those triggered by growth-factors. Despite its important role in human physiology and pathology, mechanisms that regulate SEF biochemical properties and inhibitory activity are unknown. Here we show that human SEF is an intrinsically labile protein that is stabilized via CK2-mediated phosphorylation, and identified the residues whom phosphorylation by CK2 stabilizes hSEF. Unlike endogenous SEF, ectopic SEF was rapidly degraded when overexpressed but was stabilized in the presence of excess CK2, suggesting a mechanism for limiting SEF levels depending upon CK2 processivity. Additionally, phosphorylation by CK2 potentiated hSef interaction with NF-κB in cell-free binding assays. Most importantly, we identified a CK2 phosphorylation site that was indispensable for SEF inhibition of pro-inflammatory cytokine signaling but was not required for SEF inhibition of growth-factor signaling. To our knowledge, this is the first demonstration of post-translational modifications that regulate SEF at multiple levels to optimize its inhibitory activity in a specific signaling context. These findings may facilitate the design of SEF variants for treating cytokine-dependent pathologies, including cancer and chronic inflammation.