A global decrease in microRNA (miRNA) levels is often observed in human cancers 1,2 , indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wildtype and p53-deficient cells. Here we describe a family of miRNAs, miR-34a-c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.The p53 tumour suppressor lies at a nexus of cellular pathways that sense DNA damage, cellular stress and improper mitogenic stimulation 3 . p53 integrates such signals and, in response, induces growth arrest, promotes apoptosis, blocks angiogenesis, or mediates DNA repair in a context-dependent manner 4 . The importance of p53 in preventing tumour formation is indicated by the presence of mutations in the p53 pathway in nearly all cancers 5 . Although p53 is most studied as a transcriptional activator, several reports have suggested that p53 represses the expression of specific genes 6 . Studies of p53-mediated Reprints and permissions information is available at www.nature.com/reprints.

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).

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumourigenic properties. Here, we present evidence that the SASP can also induce “paracrine senescence” in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGFβ family ligands, VEGF, CCL2 and CCL20. Amongst them, TGFβ ligands play a major role by regulating p15INK4b and p21CIP1. Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

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.