Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug 18;2(84):ra44.
doi: 10.1126/scisignal.2000053.

Differential p53-independent outcomes of p19(Arf) loss in oncogenesis

Zhenbang Chen  1 , Arkaitz Carracedo, Hui-Kuan Lin, Jason A Koutcher, Nille Behrendt, Ainara Egia, Andrea Alimonti, Brett S Carver, William Gerald, Julie Teruya-Feldstein, Massimo Loda, Pier Paolo Pandolfi
Affiliations

Differential p53-independent outcomes of p19(Arf) loss in oncogenesis

Zhenbang Chen et al. Sci Signal. .

Abstract

One reported function of the tumor suppressor p19(Arf) is to stabilize p53, providing a critical checkpoint in the response to oncogenic insults. Acute loss of Pten leads to an increase in the abundance of p19(Arf), p53, and p21 proteins as part of a fail-safe senescence response. Here, we report that loss of p19(Arf) in prostate epithelium does not accelerate-but rather partially inhibits-the prostate cancer phenotype of Pten-deficient mice. Moreover, cellular senescence and a further decrease in the number of pre-neoplastic glands were observed in prostates of the Pten-p19(Arf) double-mutant mice. In both prostate epithelium and primary mouse embryo fibroblasts (MEFs), the increase in p53 protein abundance found upon loss of Pten was unaffected by the simultaneous loss of p19(Arf). However, in contrast to that in the prostate epithelium, p19(Arf) deficiency in MEFs lacking Pten abolished cell senescence and promoted hyperproliferation and transformation despite the unabated increase in p53 abundance. Consistent with the effect of p19(Arf) loss in Pten-deficient mouse prostate, we found that in human prostate cancers, loss of PTEN was not associated with loss of p14(ARF) (the human equivalent of mouse p19(Arf)). Collectively, these data reveal differential consequences of p19(Arf) inactivation in prostate cancer and MEFs upon Pten loss that are independent of the p53 pathway.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Loss of p19Arf does not accelerate prostate tumorigenesis
(A) Cumulative survival analysis (Kaplan-Meier plot) for p19Arf−/−(blue line), Ptenpc−/− (black line) and p19Arf−/−; Ptenpc−/−(red line). (B) Actual sizes of representative tumors from anterior prostates (AP) of Wt, p19Arf−/−, Ptenpc−/−, and p19Arf−/−; Ptenpc−/− double-mutant mice at 6 months of age. (C) Comparison of AP tumor masses from Ptenpc−/− and p19Arf−/−; Ptenpc−/− mice at 6 months of age indicates that p19Arf–deficiency does not accelerate the tumorigenesis in Pten–deficient mice (N=10 for each group, P = 0.91 > 0.05, t-test).
Fig. 2
Fig. 2. Loss of p19Arf constrains prostate cancer progression
(A) Histopathology analysis (haematoxylin/eosin staining) of ventral prostates in Wt, p19Arf−/−, Ptenpc−/−, and p19Arf−/−; Ptenpc−/− double mutant mice at 11–16 weeks of age. (B) Quantification of high-grade prostatic intraepithelial neoplasia (HG-PIN) in Wt, p19Arf−/−, Ptenpc−/− and p19Arf−/−; Ptenpc−/− double-mutant mice at 11–16 weeks of age (N=4 for each group). (C) Immunohistochemistry staining of pAkt (indicated by arrows) in adjacent sections from ventral prostates of (A). Insets indicate the lower magnification. Error bars in (B) represent S.D. for a representative experiment performed in triplicate. (D) KI-67 in adjacent sections from ventral prostates of (A), and an inset showing the KI-67 positivity in PIN lesions in p19Arf−/−; Ptenpc−/− double mutant mice prostate.
Fig. 3
Fig. 3. In vivo p19Arf p53 uncoupling and cellular senescence contribute to cancer suppression in compound mutant mice
(A) Histopathology and senescence analysis of 11-week-old prostates, stained as indicated for H&E, and senescence associated-β-galactosidase (β-gal) in anterior prostates (AP). (B) Quantification (percentage of cells) of the β-gal staining seen on (A) sections from 11-week-old mice (19.6 ± 5.3% for p19Arf−/−; Ptenpc−/− double mutant mice compared with 17.8 ± 4.3% for Ptenpc−/− mice; P = 0.33>0.05, t-test) . (C) p53 staining on AP sections from 11-week-old mice, arrows denote the positive staining of p53 in nucleus of epithelial cells. (D) Quantification of the p53 staining seen on (C) sections from 11-week-old mice. Senescence and histopathology analysis of 11-week-old prostates. Representative sections from three mice were counted for each genotype. Error bars in (B) and (D) represent S.D. for a representative experiment performed in triplicate.
Fig. 4
Fig. 4. p19Arf–p53 uncoupling in primary mouse embryonic fibroblasts (MEFs)
(A) Western Blot of lysates of primary MEFs with indicated antibodies in Wt, PtenΔ/Δ, and p19Arf−/−–PtenΔ/Δ double-null cells. β-actin is used as a loading control. (B) Quantification of protein abundance in primary MEFs for p53, p16 and pRb from (A). (C) Western blotting of cellular lysates of primary MEFs with indicated antibodies in p19Arf−/−, p19Arf−/−–PtenΔ/+ and p19Arf−/−–PtenΔ/Δ double-null cells. (D) Inhibition of protein synthesis by cycloheximide (CHX) combined with Western blot at the indicated times (minutes) shows that the half-life of p53 in p19Arf−/−–PtenΔ/Δ double-null MEFs is comparable to that in Wt MEFs. Right panel: quantification of p53 half-life from the Western blots in left panel normalized to β-actin. Blue circles, Wt-Cre; red squares, p19Arf−/−–PtenΔ/Δ Cre. (E) Cellular senescence assay of Wt, p19Arf−/−, PtenΔ/Δ, p19Arf−/−–PtenΔ/Δ double-null cells. (F) Growth curves of primary MEFs, infected with retroviral Cre (under puromycin selection) followed over a 6 day period: p19Arf−/−–PtenΔ/Δ double-null (red squares), PtenΔ/Δ (black squares), p19Arf−/− (black circles) and Wt cells (blue triangles). (G) Transformation assay as determined by colony formation in soft agar from (E), and PtenΔ/Δ; p53Δ/Δ double-null cells served as positive control. Error bars for (B, E, F and G) indicate S.D. for a representative experiment performed in triplicate.
Fig. 5
Fig. 5. Loss of p19Arf in Pten null prostate mutant mice results in decreased E2F-1 and PCNA upregulation
Imunohistochemical (IHC) staining for E2F-1 (A) and PCNA (B) of ventral prostates in Wt, p19Arf−/−, Ptenpc−/− and p19Arf−/−; Ptenpc−/− double mutant mice at 11 weeks of age. PIN lesions in p19Arf−/−; Ptenpc−/− double mutant mice prostate in (B) are represented in an inset.
Fig. 6
Fig. 6. Overexpression of p14ARF in human prostate cancer specimens correlate with PTEN-loss
(A) p14ARF protein abundance in prostate cancer biopsies. Right: Representative images for p14ARF staining in specimens at low and high severity grades. Left: graph showing the correlation of disease aggressiveness and p14ARF abundance; 0= tumor cells negative for p14ARF; 1= tumor cells focally positive for p14ARF; 2= tumor cells diffusely positive for p14ARF. (B) Correlation of PTEN and p14ARF protein abundance in human prostate specimens by immunohistochemistry (IHC). Representative images showing p14ARF staining in specimens with normal PTEN abundance (1) and PTEN loss (0). Yellow indicates abundance 2, orange abundance 1 and red abundance 0. (C) Schematic representation of the findings highlighted in this study.
See this image and copyright information in PMC

References

    1. Kamijo T, Bodner S, van de Kamp E, Randle DH, Sherr CJ. Tumor spectrum in ARF-deficient mice. Cancer Res. 1999;59:2217–2222. - PubMed
    1. Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Pandolfi PP. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005;436:725–730. - PMC - PubMed
    1. Groth A, Weber JD, Willumsen BM, Sherr CJ, Roussel MF. Oncogenic Ras induces p19ARF and growth arrest in mouse embryo fibroblasts lacking p21Cip1 and p27Kip1 without activating cyclin D-dependent kinases. J Biol Chem. 2000;275:27473–27480. - PubMed
    1. Cleveland JL, Sherr CJ. Antagonism of Myc functions by Arf. Cancer Cell. 2004;6:309–11. - PubMed
    1. Weber JD, Taylor LJ, Roussel MF, Sherr CJ, Bar-Sagi D. Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol. 1999;1:20–26. - PubMed

Publication types

MeSH terms

Substances