We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6 ؊/؊ mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IB kinase . In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor. Intracellular signaling initiated by latent membrane protein 1 (LMP1), the principal oncoprotein of the human gammaherpesvirus Epstein-Barr virus (EBV), is of interest not only for viral oncogenesis but also for the reason that LMP1 shares early steps in pathways used by CD40, interleukin-1 (IL-1) receptor-Toll-like receptor (TLR), and tumor necrosis factor receptor (TNFR) for activation of NFB (reviewed in references 16 and 35). As a member of the TNFR superfamily, LMP1 recruits TNFR-associated death domain protein, TNFRinteracting protein (RIP), and several TNFR-associated factors (TRAFs). Unlike CD40 and receptor activator of NF-B (RANK), which contain TRAF6-binding sites, LMP1 does not have a consensus TRAF6-binding sequence but associates with TRAF6 indirectly (reviewed in references 8, 16, 35, 51, and 61).Ubiquitination through K48-polyubiquitin linkage is well known as a process whereby proteins are targeted for proteasomal degradation. Recently, proteasome-independent functions for ubiquitination through K63 polyubiquitin or monoubiquitin linkages have been identified, and the importance of these ubiquitin...

Background: Pancreatic Ductal Adenocarcinoma (PDA) ranks fourth among cancer-related deaths in the United States. The poor prognosis of PDA is due to resistance to current therapies. Contributing to this resistance is the tumor architecture comprising dense desmoplastic stroma, extracellular matrix (ECM), tumor cells and tumor hypoxia. We hypothesized that destabilizing the compact tumor architecture by weakening the interactions between tumor cells alone or tumor cells along with the ECM may result in more effective therapies for PDA. Methods and Results: Using the pancreatic cell line Panc1, we developed a three dimensional (3D) spheroid model system, that exhibits oxygen gradients and hypoxia that mimics the tumor microenvironment. We used the pancreatic cancer cell line PANC1-HRE that stably expresses HRE-luciferase and detects HIF-1α activation, and a platform comprising a non-matrix nano-structured scaffold, to generate compact spheroids with hypoxic inner cores and HIF-1α activation. A high throughput siRNA screen using the kinome siRNA library and our 3D spheroid modal system identified kinases whose silencing reduced both the level of hypoxia and activity of HIF-1α. The positive control, siRNA against HIF-1α, reduced activity of HIF-1α with no effect on the levels of hypoxia within the spheroids. We identified the kinases Toll-like receptor 4 (TLR4) and its downstream target spleen tyrosine kinase (SYK) as our lead candidates following target validation. Image analysis of spheroids transfected with siRNA against both TLR4 and SYK resulted in the loss of compactness of the spheroids. In addition, these spheroids also demonstrated a significant reduction in the levels of hypoxia determined by the hypoxia probe Lox-1. These findings suggest that the alteration of the spheroid architecture resulted in the reduction in hypoxia. Previous studies have shown that SYK is a direct target of TLR4. Therefore to determine the mechanism through which TLR4 dependent signaling regulates the integrity of the spheroidal architecture we are generating stable cell lines in Panc1 of shRNA targeting TLR4 under an inducible system. We will use our inducible shRNA targeting TLR4 and the two 3D model systems, the hanging drop system (analogous to a primary tumor) and the nanoculture plate system (analogous to the progression of a metastatic loci) to further confirm our findings. Conclusion: TLR4 mediated signaling regulates the integrity of spheroid architecture. Inhibiting this pathway destabilized the tumor architecture altering the hypoxic state of the spheroid. Our long-term goals are to use known pharmacological inhibitors of TLR4 to study the mechanisms by which this kinase regulates tumor architecture, determine the pre-clinical significance of targeting TLR4 utilizing autochtonous mouse models for PDA and develop TLR4-targeted therapy for PDA. Citation Format: Geoffrey A. Bartholomeusz, Alex Campos, Geoffrey Grandjean, Garth Powis. A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2013-3836