Conditional deletion of the Pten gene in the mouse prostate induces prostatic intraepithelial neoplasms at early ages but a slow progression to prostate tumors

Abstract

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten(LoxP):Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten(LoxP/LoxP):Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten(LoxP/LoxP):Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten(LoxP/+):Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten(LoxP/LoxP):Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten(LoxP/LoxP):Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.

Figure 1. Generating and analyzing Pten Osr1-Cre conditional knockout mice.

A. Diagram for Pten knockout strategy. In Pten^loxP/loxP mice, LoxP recognition sequences were inserted into the endogenous Pten locus flanking exon 5 as previously reported . Two different Cre transgenic lines carrying Osr1-Cre or PB-Cre4 transgene were crossed to Pten^loxP/loxP mice for the generation of Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP;PB-Cre4 mice. B and C. Genomic PCR analyses for Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP:PB-Cre4 mice. PCR was performed using genomic DNA samples harvested from various organs in Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP:PB-Cre4 mice. Primers in the Pten locus were used to detect either the loxP allele or the Cre-recombined allele. The 0.65 kb fragment of the Pten loxP allele (solid arrows) was observed in all 15 organs from both Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP:PB-Cre4 mice. The 0.3kb Cre-recombined fragment (open arrows) was detected only in the prostate of Pten^loxP/loxP:PB-Cre4 mice and in the prostate, urinary bladder, brain, lung, kidney, stomach, and ovary of Pten^loxP/loxP:Osr1-Cre mice. D. Western blotting to detect Pten expression in Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP:PB-Cre4 mice. Whole cell lysates isolated from prostate lobes were analyzed by Western blot with the PTEN antibody. b-actin was used as a loading control. AP, anterior; DLP, dorsolateral and VP, ventral prostate lobes.

Figure 2. Development of PIN lesion in Pten^loxP/loxP:Osr1-Cre mice.

A–C. H&E staining for 2 to 4 week old Pten^loxP/loxP Osr1-Cre mice. Representative images of mouse PIN lesions are shown from different prostate lobes of 2 week old Pten^loxP/loxP:Osr1-Cre mice; AP, anterior; D/LP, dorso/lateral, and VP, ventral prostate lobes. Immunohistochemical analyses of these samples show the PIN lesions with concurrent absence of Pten expression (please see Figure S1). The typical high-grade PIN lesions are mainly cribriform or stratified in nature, and often distended the prostate glandular unit profile and/or completely filled the prostate glandular unit lumen. Mitoses were rare. The fibromuscular stroma remained intact, with overall glandular unit profiles being recognizable. D–G. Histological analyses of the prostate tissues isolated from different age groups of Pten^loxP/loxP:Osr1-Cre mice. However, the nature and extent of the high grad PIN in Pten^loxP/loxP:Osr1-Cre mice did not appear to change with advancing age, as evidence by the similarities of lesions in the 4-week old mice as compared to their 2-, 7-, and 12-month of cohorts.

Figure 3. Deletion of Pten increases expression and activity of Akt in Pten^loxp/loxp:Osr1-Cre mice.

A. Analysis of Akt and pAkt expression in prostate tissues of 3 and 12-month old Pten^loxP/loxP:Osr1-Cre and Pten^loxP/loxP mice. Westernblotting was performed to examine both Akt and phospho-Akt expression. β-tubulin was used as a loading control. B–I. Immunohistochemical analysis of Akt and phospho-Akt in prostate sections. Akt expression levels are increased in the epithelial cells within high-grade PIN regions of Pten^loxP/loxP:Osr1-Cre prostates (F, G), compared with Pten^loxP/loxP mice (B, C). Activation of Akt was assessed with a phospho-Akt antibody. High levels of phospho-Akt immunoreactivity was observed in the high grade PIN regions of Pten^loxP/loxP:Osr1-Cre prostates (H, I) compared with the low signal observed in the prostate epithelial cells of Pten^loxP/loxP:prostates (D, E).

Figure 4. Deletion of Pten induces cell proliferative advantage in prostate tissues.

A–I. Cell proliferation was examined by immunostaining for Ki-67. Prostate sections isolated from 4-week-old Pten^loxP/loxP, Pten^loxP/+:Osr1-Cre, and Pten^loxP/loxP Osr1-Cre mice were stained for Ki-67. A total of 1000 epithelial cells in each lesion from three different lesions from three mice of each genotype were evaluated for Ki-67 immunoreactivity. J. The positive immunoreactive cells for Ki-67 in Pten^loxP/loxP:Osr1-Cre+ mice are significantly greater than both Pten^loxP/loxP control mice and Pten^loxP/wt:Osr1-Cre mice, P<0.01.

Figure 5. Immunohistochemistry analyses of the prostate tissues isolated from Pten^loxP/loxP:Osr1-Cre.

H&E staining and immunohistochemistry were performed on prostate tissues of 4-week-old Pten^loxP/loxP:Osr1-Cre mice. Adjacent prostate tissue sections from two Pten^loxP/loxP:Osr1-Cre mice were analyzed with different antibodies as marked, including the AR (C, D), CK8 (G, H), E-cadherin (E, F), synaptophysin (M,N), p63 (I, J) and CK5 (K, L). A pair of adjacent slides from the prostate tissues of either Pten^loxP/loxP:Osr1-Cre or Pten^loxP/loxP:PB-Cre4 mice were subjected to immunohistochemistry using the Pten and p63 antibodies. Representative images focusing on p63 positive cells (arrows) are shown (Q–T).

Figure 6. Development of malignant prostate tumors in older Pten^loxP/loxP:Osr1-Cre mice.

H&E staining was performed to analyze prostate tissues isolated from greater than 12-month-old Pten^loxP/loxP:Osr1-Cre mice. Intra-glandular solid prostatic carcinomas were observed in 5 mice and three were shown here (A to C), each of which appear wholly contained within enlarged prostatic glandular units that otherwise contain abundant proliferative changes consistent with high grade PIN. In addition to increased cellular and nuclear atypia, an observable increase in mitotic activity is noted in the solid carcinoma areas. No evidence for invasion beyond the basement membrane or fibromuscular stroma was noted. In another single mouse, invasion of a cribriform high grade PIN lesion beyond the basement membrane and fibromuscular stroma of the glandular unit was observed (D), which meets the criteria for a diagnosis of well-differentiated prostatic adenocarcinoma.

Figure 7. Examining AKT and AR signaling pathways in castrated Pten-null tumors.

Immunohistochemical characterization was carried out to assess Akt and pAkt expression in Pten null prostate tumors isolated from intact (A, B) and castrated (C, D) 12-month-old Pten^loxP/loxP:Osr1-Cre mice. The same tissue samples were also analyzed with the antibodies against, the AR, pS6, Ki67, and Pten immunohistochemcially (E to L).