Stanford BIOC 230 - Senescence and tumour clearance is triggered

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LETTERSSenescence and tumour clearance is triggered by p53restoration in murine liver carcinomasWen Xue1*, Lars Zender1*, Cornelius Miething1, Ross A. Dickins1,2, Eva Hernando3, Valery Krizhanovsky1,Carlos Cordon-Cardo3& Scott W. Lowe1,2Although cancer arises from a combination of mutations in onco-genes and tumour suppressor genes, the extent to which tumoursuppressor gene loss is required for maintaining establishedtumours is poorly understood. p53 is an important tumour sup-pressor that acts to restrict proliferation in response to DNAdamage or deregulation of mitogenic oncogenes, by leading tothe induction of various cell cycle checkpoints, apoptosis or cel-lular senescence1,2. Consequently, p53 mutations increase cell pro-liferation and survival, and in some settings promote genomicinstability and resistance to certain chemotherapies3. To deter-mine the consequences of reactivating the p53 pathway intumours, we used RNA interference (RNAi) to conditionally regu-late endogenous p53 expression in a mosaic mouse model of livercarcinoma4,5. We show that even brief reactivation of endogenousp53 in p53-deficient tumours can produce complete tumourregressions. The primary response to p53 was not apoptosis, butinstead involved the induction of a cellular senescence programthat was associated with differentiation and the upregulation ofinflammatory cytokines. This program, although producing onlycell cycle arrest in vitro, also triggered an innate immune responsethat targeted the tumour cells in vivo , thereby contributing totumour clearance. Our study indicates that p53 loss can berequired for the maintenance of aggressive carcinomas, and illus-trates how the cellular senescence program can act together withthe innate immune system to potently limit tumour growth.p53 mutations are common in human liver cancer6, which is typ-ically highly aggressive and resistant to non-surgical therapies. Todetermine the requirement for p53 loss in the maintenance of suchcarcinomas, we used reversible RNAi7to control p53 in a chimaericliver cancer mouse model (Fig. 1a)4,5. Purified embryonic liver pro-genitor cells (hepatoblasts) were transduced with retroviruses expres-sing oncogenic ras (HrasV12), the tetracycline transactivator protei ntTA (‘tet-off’) and a tet-responsive p53 miR30 design short hairpinRNA (shRNA; Supplementary Fig. 1a)7,8, and seeded into the livers ofathymic nude mice following intrasplenic injection4,5. To facilitate invivo imaging, the oncogenic ras retrovirus co-expressed green fluor-escent protein (GFP) and, in some experiments, hepatoblasts werealso co-transduced with a luciferase reporter.p53 expression was efficiently suppressed in the absence ofdoxycycline (Dox) and rapidly restored following Dox addition(Supplementary Fig. 1b, c). On transplantation into the livers ofrecipient mice, hepatoblast populations co-expressing Ras and theconditional p53 shRNA rapidly produced invasive hepatocarcinomasin the absence of Dox (Fig. 1b), whereas cells expressing each vectoralone did not (data not shown). These tumours were GFP-positiveand, if expressing luciferase, could be visualized externally by bio-luminescence imaging (Fig. 1b).Animals with advanced tumours were treated with Dox to re-establish p53 expression (Fig. 1b). Shortly after Dox administration,the p53 microRNA (miRNA) was shut off and p53 expressionincreased (Supplementary Fig. 1). Although tumours in untreatedmice rapidly progressed, those in Dox-treated animals began toinvolute and became nearly undetectable within 12 days (Fig. 1b).Similar results were observed in a subcutaneous setting, wheretumours could be accurately monitored using calliper measurements(Fig. 1c, left panel). Importantly, ras-induced liver carcinomas pro-duced using a constitutive p53 shRNA grew similarly irrespective ofDox treatment (Fig. 1c, right), indicating that tumour regression wasnot due to Dox toxicity. Such regressions also occurred when p53 wasreactivated in tumours co-expressing a constitutively activated Aktor an endogenous oncogenic K-ras allele and the conditional p53shRNA (W.X., L.Z. and S.W.L., unpublished data).To determine whether transient p53 reactivation could also causetumour regression, we treated transformed cells in culture ortumour-bearing mice with Dox for 4 days and then removed thedrug. Immunoblotting revealed that p53 could be transientlyinduced following Dox addition and withdrawal (Fig. 1d). In cul-tured cells, even two days of Dox treatment reduced colony forma-tion to levels observed following continuous Dox treatment(Supplementary Fig. 2a). Furthermore, both in situ and subcutan-eous liver carcinomas showed complete regressions after only fourdays of Dox treatment (Fig. 1e; Supplementary Fig. 2b). Thus, p53can induce tumour involution through a process that, once activated,seems irreversible. These observations are analogous to results seenin murine tumours conditionally expressing various oncogenes,where silencing of the initiating oncogene often causes tumourregression9,10.The rapid involution of hepatocarcinomas re-expressing p53 isconsistent with p53’s well-characterized ability to promote apopto-sis. We therefore examined apoptosis and proliferation in tumoursbefore and after p53 restoration (Fig. 2a, b). Surprisingly, weobserved few cells that were TUNEL-positive or contained activatedcaspase 3 following p53 reactivation, suggesting that the primaryresponse to p53 was not apoptosis. Similarly, substantial necrosiswas not observed in the regressing tumours. Instead, these tumoursshowed a marked decrease in proliferation (Ki67) that was associatedwith signs of cellular differentiation (Supplementary Fig. 3).p53 can also promote cellular senescence, an apparently irrevers-ible form of cell cycle arrest that is a potent barrier to tumori-genesis11–14and can be triggered by hyperactive Ras or PI3Ksignalling12,15. Interestingly, hepatocarcinomas expressing eitheroncogenic ras or Akt showed clear signs of senescence following p53reactivation in vivo (Fig. 3a–c; data not shown for Akt), including theaccumulation of senescence-associated-b-galactosidase (SA-b-Gal)*These authors contributed equally to this work.1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.2Howard Hughes Medical Institute, Cold Spring Harbor, New York 11724, USA.3Division of MolecularPathology, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.Vol 445|8 February 2007|doi:10.1038/nature05529656Nature


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Stanford BIOC 230 - Senescence and tumour clearance is triggered

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