Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence 1,2 . Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies 3 . To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma 4,5 . We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.p53 mutations are common in human liver cancer 6 , which is typically highly aggressive and resistant to non-surgical therapies. To determine the requirement for p53 loss in the maintenance of such carcinomas, we used reversible RNAi 7 to control p53 in a chimaeric liver cancer mouse model (Fig. 1a) 4,5 . Purified embryonic liver progenitor cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), the tetracycline transactivator protein tTA ('tet-off') and a tet-responsive p53 miR30 design short hairpin RNA (shRNA; Supplementary Fig. 1a) 7,8 , and seeded into the livers of athymic nude mice following intrasplenic injection 4,5 . To facilitate in vivo imaging, the oncogenic ras retrovirus co-expressed green fluorescent protein (GFP) and, in some experiments, hepatoblasts were also co-transduced with a luciferase reporter.p53 expression was efficiently suppressed in the absence of doxycycline (Dox) and rapidly restored following Dox addition ( Supplementary Fig. 1b, c). On transplantation into the livers of recipient mice, hepatoblast populations co-expressing Ras and the conditional p53 shRNA rapidly produced invasive hepatocarcinomas in the absence of Dox (Fig. 1b), whereas cells expressing each vector alone did not (data not shown). These tumours were GFP-positive and, if expressing luciferase, could be visualized externally by bioluminescence imaging (Fig. 1b).

Summary Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to non-cancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene.

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