A prostatic intraepithelial neoplasia-dependent p27 Kip1 checkpoint induces senescence and inhibits cell proliferation and cancer progression - PubMed (original) (raw)

. 2008 Aug 12;14(2):146-55.

doi: 10.1016/j.ccr.2008.06.002.

Chiara Grisanzio, Fionnuala O'Connell, Marc Barry, Joseph M Brito, Qing Xu, Isil Guney, Raanan Berger, Paula Herman, Rachel Bikoff, Giuseppe Fedele, Won-Ki Baek, Shunyou Wang, Katharine Ellwood-Yen, Hong Wu, Charles L Sawyers, Sabina Signoretti, William C Hahn, Massimo Loda, William R Sellers

Affiliations

A prostatic intraepithelial neoplasia-dependent p27 Kip1 checkpoint induces senescence and inhibits cell proliferation and cancer progression

Pradip K Majumder et al. Cancer Cell. 2008.

Abstract

Transgenic expression of activated AKT1 in the murine prostate induces prostatic intraepithelial neoplasia (PIN) that does not progress to invasive prostate cancer (CaP). In luminal epithelial cells of Akt-driven PIN, we show the concomitant induction of p27(Kip1) and senescence. Genetic ablation of p27(Kip1) led to downregulation of senescence markers and progression to cancer. In humans, p27(Kip1) and senescence markers were elevated in PIN not associated with CaP but were decreased or absent, respectively, in cancer-associated PIN and in CaP. Importantly, p27(Kip1) upregulation in mouse and human in situ lesions did not depend upon mTOR or Akt activation but was instead specifically associated with alterations in cell polarity, architecture, and adhesion molecules. These data suggest that a p27(Kip1)-driven checkpoint limits progression of PIN to CaP.

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Figures

Figure 1

Figure 1. Induction of p27Kip1 and senescence in prostatic intraepithelial neoplasia of _AKT1_-Tg and _Pten_L/L;Cre+ mice

(A) VPs from wild-type (WT) and _AKT1_-Tg mice were stained by immunohistochemisty using antibodies directed against phospho-Akt (S473) (upper panel) or against p27Kip1 (lower panel). Scale bar, 50 μM (B) The number of p27kip1 positively staining cells was determined in the VPs of wild type (WT) and _AKT1_-Tg mice of the indicated ages. Data are presented as mean ± SEM. (C) Wild type (PtenL/L;Cre−) and Pten conditional knock out (prostate specific) (PtenL/L;Cre+) VPs were stained with phospho-Akt (S473) (upper panel) or anti-p27Kip1 (lower panel). Scale bar, 50 μM; insert 100μM (D) Western blot analysis of p27Kip1 and Tubulin in whole cell lysates from ventral prostates of Wild type (WT), _AKT1_-Tg and Pten conditional knock out (PtenL/L;Cre+) mice of 6 weeks old (E) VPs from wild type and _AKT1_-Tg mice were stained with antibody against HP1α. Scale bar,50μM. (F) Area of HP1α staining were measured both in wild type and _AKT1_-Tg prostates. Data are presented as mean ± SD.

Figure 2

Figure 2. Genetic inactivation of Cdkn1b rescues cell from senescence and increases proliferation in _AKT1_-Tg mice

(A–L) Twelve hours after BrdU administration, mice were sacrificed and the VPs of wild-type (A, E, I), _AKT1_-Tg (B, F, J), Cdkn1b+/− (C, G, E) and _AKT1_-Tg, Cdkn1b+/− (D, H, L) mice were stained with either β-Gal (A– D), H&E (E–H) or with anti-BrdU antibody (I–L). Scale bar, 50 μM; insert 100μM. (M) The numbers of BrdU stained cells per 100 ducts was determined by manual counting of BrdU positive cells in all lobes of VP. Data are presented as mean ± SD.

Figure 3

Figure 3. Genetic inactivation of Cdkn1b in _AKT1_-Tg mice results in the development of invasive prostate cancer

(A) Prostatic intraepithelial neoplasia in a representative section from the VP of _AKT1_-Tg mouse (~52 weeks old). Scale bar, 100 μM (B) Invasive prostate cancer in representative sections from _AKT1_-Tg/Cdkn1b+/−. Sections were stained with H&E. Scale bar, 100 μM (C) VP from _AKT1_-Tg mice were stained with antibodies directed against p63. Scale bar, 100 μM (D) VP from _AKT1_-Tg/Cdkn1b+/− mice were stained with antibodies directed against p63. Scale bar, 100 μM (E) Summary of tumor incidence by age and genotype.

Figure 4

Figure 4. Induction of p27Kip1 and increases of senescence markers do not require mTOR activation and is correlated with the PIN phenotype

(A–O) _AKT1_-Tg mice were treated with RAD001 at 10mg/kg/QD/PO for 0, 2, and 14 days (P–R) _AKT1_-Tg mice were treated with placebo for 0, 2, and 14 days. (A–C) Tissue sections from the VPs were stained with H&E, (D–F) Tissue sections were stained with antibody directed against phospho-Akt (S473) (G–I) Tissue sections were satained with antibody directed against phospho-S6RP (J–L) Immunohistochemical analysis was done in tissue sections with antibody against HP1α Scale bar, 200μM (M–R) Tissue sections were stained with antibody directed against p27Kip1. Shown are sections representative of the results obtained in at least 12 animals evaluated after each specific treatment period. Scale bar, 100 μM (A–I and M–R),

Figure 5

Figure 5. Stabilization of p27Kip1 in human prostate intraepithelial neoplasia

(A) Tissue sections from normal human prostate were stained with H&E (B) Normal prostate tissue sections were stained with antibody directed against p27Kip1. (C) Prostatic intraepithelial neoplasia (PIN) tissue sections were stained with H&E (D) Tissue sections from human PIN were stained with antibody directed against p27Kip1 (E) Tissue sections from human prostate cancer (CaP) were stained with H&E (F) Human prostate cancer (CaP) tissue sections were stained with antibody directed against p27Kip1. Scale bar, 50 μM (A–F). (G) The number of p27kip1 positive cells was determined in the normal, PIN, PIN adjacent to invasive cancer and CaP. Data are presented as mean ± SEM.

Figure 6

Figure 6. Markers of cellular senescence are elevated in human PIN

(A) Frozen sections of normal human prostate were subjected to β-Gal and hematoxylin/eosin staining. The blue staining indicates the β-Gal activity, haematoxylin and eosin was used as a counter stain (B) Frozen sections of humn Prostate Intraepithelial Neoplasia (PIN) were subjected to β-Gal and hematoxylin/eosin staining. The blue staining indicates the β-Gal activity, haematoxylin and eosin was used as a counter stain to visualize the PIN lesion (C) Tissue sections from paraffin embedded normal human prostate and PIN lesions were stained with antibodies against HP1α (D) Tissue sections from paraffin embedded normal human prostate and PIN lesions were also stained with antibodies against HP1γ. Scale bar, 50μM (A–D); insert 100 μM. (E) Average area of HP1α positive stain was measured in both normal and PIN lesion Data are presented as mean ± SD. (F) Average area of HP1γ positive stain was measured in both normal and PIN lesion Data are presented as mean ± SD.

Figure 7

Figure 7. Disruption of cellular polarization and adhesion is associated with upregulation of p27Kip1 level

(A) Ventral prostates of both wild type (WT) and _AKT1_-Tg mice stained with Z0-1 antibody and imaged by confocal microscope. Scale bar, 50μM.. (B) Rat embryonic fibroblast cells stably transfected with either vector or Myr-AKT1 were cultured under adherent and suspension condition. Protein lysates were prepared and immunoblotted with antibodies directed against p27Kip1 (upper panel), phospho-Akt (S473), phosho-GSK3 (α and β) (middle panel) and Tubulin (lower panel). (C) Protein lysates were made from primary human epithelial cells (LEAR and LEKAR) cultured under suspension (Susp) or adherent (Ad) conditions. Immunoblot analysis using antibodies directed against p27Kip1(upper panel), phospho-Akt (S473) (middle panel), and Tubulin (lower panel) (D) Primary human epithelial cells (LEAR) were transduced with three different shRNAs against E-Cadherin and shGFP as control. Cell were harvested 2 days after post selection and total protein lysates were prepared. E-Cadherin (upper panel), p27Kip1 (middle panel) and Actin (lower panel) western blot analysis was performed (E) LEAR cells with E-Cadherin ShRNAs and control (ShGFP) were grown in plate and pictures were taken 2 days after post selection. Scale bar, 100 μM.

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