Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Abstract

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. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. 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. 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.

Figures

Figure 1. Reactivation of p53 results in liver tumour regression

a, Embryonic liver progenitor cells were transduced with a tetracycline-regulatable p53 shRNA (TRE.shp53), tTA and H-rasV12. After onset of liver tumours, p53 expression could be restored by doxycycline (Dox) treatment. b, Reactivation of p53 leads to rapid tumour regression. Tumour-bearing mice were treated with Dox starting at day 0 and imaged at the indicated time points (n = 9). c, Subcutaneous tumours derived from ras-transformed liver progenitor cells with tet-off shRNA (TRE.shp53) or a non-regulatable shRNA (MLS.shp53) were grown in nude mice. Values represent mean ± s.d. (n = 4). d, p53 reactivation is reversed by Dox withdrawal. Protein lysates from liver progenitor cells pulse-treated with Dox for 4 days were immunoblotted for p53. e, Representative mice (n = 6) as in b were pulse-treated with Dox for 4 days and imaged at the indicated time.

Figure 2. The primary response to p53 reactivation is not apoptosis

a, Haematoxylin and eosin (H&E) immunohistochemical staining for apoptotic cells (TUNEL and Caspase 3 staining) and proliferating cells (Ki67 staining) of liver tumours before (p53 off) and after Dox treatment (p53 on). Tumours showed histopathology of human hepatocellular and cholangiocellular carcinoma. Inset (P) denotes positive controls (irradiated thymus, 20Gy (20 Gray)). ‘p53 on’ shows representative tumour on day 6. Scale bar, 100 μm. b, Quantification of a. Values represent mean ± s.d. (n = 4; **P<0.002).

Figure 3. p53 reactivation induces cellular senescence

a, SA-β-Gal staining of representative tumour-bearing livers untreated (p53 off) or treated with Dox (p53 on, day 6) (n= 3). b, SA-β-Gal staining of tumour sections (n = 3). Tumours were either untreated (p53 off), constantly treated with Dox for 8 days (p53 on 8 days) or briefly treated for 4 days and left untreated for 8 days (p53 on 4 days/off 8 days). Scale bar, 25 μm. c, Immunoblotting for senescence markers in normal liver or liver tumours treated with Dox for 0, 4 and 6 days. d, Liver progenitor cells harbouring ras and tet-off shp53 were cultured in Dox-containing medium for 6 days (p53 on) and stained for SA-β-Gal. Values represent mean±s.d. (n = 3; **P < 0.0001). e, Representative pictures from d. f, Cells as in d were cultured with (p53 on, red line) or without Dox (p53 off, black line) and cell numbers were counted. Values represent mean±s.d. (n=4).

Figure 4. Clearance of liver tumours by an innate immune response

a–f, Progressive immune infiltration in regressing tumours following p53 reactivation (H&E; n = 4). Arrows denote peri-tumoral polymorphonuclear (PMN) leukocytes. Arrowheads denote intra-tumoral polymorphonuclear leukocytes. Scale bar, 100 μm. e, High magnification view of d. g, p53 reactivation leads to increased expression of chemokines and adhesion molecules in senescent tumours (in vivo) and cultured liver progenitor cells (in vitro). Values represent mean± s.d. (duplicate samples with triplicate qPCR). All the D4 and D8 values are statistically significant compared with D0 (P<0.05). h, p53 reactivation is accompanied by increased immune cell transcripts in senescent tumours (in vivo) but not in cultured cells (in vitro). Values represent the average of duplicate samples from microarrays. i, Subcutaneous hepatocarcinomas were treated with Dox to induce tumour regression. The macrophage toxin GdCl (red line), an anti-neutrophil antibody (purple) or an anti-natural killer-cell antibody (blue) was administered. Saline (solid black line) or an isotype control antibody (dashed black line) served as controls. Values represent mean ± s.d. (n = 4; *P < 0.02; **P < 0.002 for day 16). j, Blockade of innate immune cells does not prevent p53-induced senescence. Frozen sections from control animals or immune antagonist treated animals were stained for SA-β-Gal activity (n = 3). Scale bar, 50 μm.