Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis

Abstract

Cellular senescence has been theorized to oppose neoplastic transformation triggered by activation of oncogenic pathways in vitro, but the relevance of senescence in vivo has not been established. The PTEN and p53 tumour suppressors are among the most commonly inactivated or mutated genes in human cancer including prostate cancer. Although they are functionally distinct, reciprocal cooperation has been proposed, as PTEN is thought to regulate p53 stability, and p53 to enhance PTEN transcription. Here we show that conditional inactivation of Trp53 in the mouse prostate fails to produce a tumour phenotype, whereas complete Pten inactivation in the prostate triggers non-lethal invasive prostate cancer after long latency. Strikingly, combined inactivation of Pten and Trp53 elicits invasive prostate cancer as early as 2 weeks after puberty and is invariably lethal by 7 months of age. Importantly, acute Pten inactivation induces growth arrest through the p53-dependent cellular senescence pathway both in vitro and in vivo, which can be fully rescued by combined loss of Trp53. Furthermore, we detected evidence of cellular senescence in specimens from early-stage human prostate cancer. Our results demonstrate the relevance of cellular senescence in restricting tumorigenesis in vivo and support a model for cooperative tumour suppression in which p53 is an essential failsafe protein of Pten-deficient tumours.

Figure 1. Loss of Trp53 does not initiate prostate tumours but renders Pten-deficient carcinomas lethal

a, Histopathological analysis (haematoxylin/eosin staining) of anterior prostates (AP) in WT, Pten and Trp53 single and double mutants at 9 weeks of age reveals normal glands in WT and Trp53^pc−/− mice but PIN lesions in Pten^pc−/− mice and invasion (arrow) in Pten^pc−/−;Trp53^pc−/− mice. b, Disease-free survival curve (Kaplan–Meier plot) for prostate cancer. Adenocarcinoma was found only in the Pten^pc−/−;Trp53^pc−/− cohort (P < 0.05). The arrow indicates puberty. c, Cumulative survival analysis. A statistically significant decrease in lifespan (P < 0.0001) compared with the Pten^pc−/− cohort was found for the Pten^pc−/−;Trp53^pc−/− cohort (asterisk) and for the Pten^pc−/−;Trp53^pc+/− cohort (double asterisk). d–f, MRI of AP tumours (dashed circles) at 23–31 weeks (d) and their actual sizes (e) and weights (f) after biopsy. Error bars in f indicate s.d. of AP weight for the numbers of mice indicated above the bars.

Figure 2. Acute loss of Pten triggers the p53-dependent senescence pathway in primary mouse embryonic fibroblasts (MEFs)

a, Top: growth curves of primary MEFs, infected with retroviral Cre (with selection) and followed over a 6-day period. Bottom: cellular senescence assay of cells from a. b, Top: growth curves of primary MEFs infected with Pten shRNA (with selection) and followed over a 6-day period. Bottom: cellular senescence assay of cells from b. c, Western blots of MEF lysates. Numbers indicate densitometrically determined protein levels relative to β-actin. d, Growth curves (top) and senescence staining (bottom) of Pten^{Δ /Δ} ;Trp53^{Δ /Δ} double-null MEFs (black squares), Pten^{Δ /Δ} ;Trp53^{Δ /+} MEFs (red triangles) and Pten^{Δ /Δ} MEFs (blue circles). Error bars indicate s.d. L/L indicates Pten^loxP/loxP. e, Transformation assay of all combinations of Pten and Trp53 inactivation. H-Ras-infected NIH 3T3 cells served as positive control (grey bar). Error bars indicate s.d. for a representative experiment performed in triplicate.

Figure 3. Acute loss of Pten results in ARF upregulation and p53/p21 stabilization in primary MEFs

a, Left: inhibition of protein synthesis by cycloheximide (CHX) and western blotting at the indicated times (minutes) shows that the half-life of p53 and p21 proteins is prolonged on acute loss of Pten in MEFs. Right: quantification of p53 and p21 half-life from the western blots, normalized to β-actin. Blue circles, Pten^+/+-Cre; red squares, Pten^{Δ /Δ} -Cre. b, Western blotting demonstrates upregulation of p19^Arf protein after acute Pten loss in MEFs. Pten^+/+-Cre, Pten^{Δ /+}-Cre, Pten^loxP/loxP (L/L, Ctrl) and Pten^loxP/loxP-MSCV vector (Pten^L/L-vec) serve as controls. c, Expression of myristoylated Akt (mAkt) in MEFs induces growth arrest (top) and cellular senescence (bottom). Black squares, control; red triangles, pBabe; blue circles, pBabe-mAkt. Error bars represent s.d. for a representative experiment performed in triplicate.

Figure 4. The p53-dependent cellular senescence pathway restricts tumorigenesis in Pten-deficient prostates

a, Senescence and histopathological analysis of 11-week-old prostates, stained as indicated. H&E, haematoxylin/eosin. Scale bars, 50μm (β-Gal and haematoxylin/eosin stain) and 10μm (p53, p19^Arf and p21). b, Quantification of the β-Gal staining seen on AP sections at 8 weeks (open bars) and 11 weeks (filled bars). Representative sections from three mice were counted for each genotype. c, Quantification of TUNEL assay for apoptosis in the AP at 11 weeks (of more than two mice per genotype). d, Quantification of Ki-67 staining of 11-week-old AP done as in b. Error bars in b–d represent s.d. for a representative experiment performed in triplicate. Asterisk indicates statistical significance between Pten^pc−/− and Pten^pc−/−;Trp53^pc−/− double mutants (P < 0.05). e, Western blot analysis of AP tissue from each genotype at 11 weeks shows Akt activation and p53 induction in Pten^pc−/− mice. f, Senescence (β-Gal) and histopathological (haematoxylin/eosin stain) analysis of cryosections from human radical prostatectomy samples, showing strong (+++) β-Gal staining from a hyperproliferative (preneoplastic) prostate gland (left) or weak (+) β-Gal staining from a neoplastic gland from a different patient (right). Scale bars, 25μm. g, A model for Pten-deficient tumorigenesis, p53 cooperativity and its therapeutic implications. Pten-deficient tumours may benefit from treatment with drugs that restore the activity of mutated p53 (for example, PRIMA28) or that stabilize WT p53 through the inactivation of MDM2 (for example, Nutlin29). h, A model for prostate tumour initiation, development and progression synergistically contributed by Pten, Trp53 and other genes. Loss of Trp53 accelerates cancer progression by a senescence escape mechanism in Pten-deficient tumours.