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. 2013 Jul 31;8(7):e69484.
doi: 10.1371/journal.pone.0069484. Print 2013.

Loss of Survivin in the Prostate Epithelium Impedes Carcinogenesis in a Mouse Model of Prostate Adenocarcinoma

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Free PMC article

Loss of Survivin in the Prostate Epithelium Impedes Carcinogenesis in a Mouse Model of Prostate Adenocarcinoma

Helty Adisetiyo et al. PLoS One. .
Free PMC article

Abstract

The inhibitor of apoptosis protein survivin is expressed in most cancers. Using the conditional PTEN deletion mouse model, we previously reported that survivin levels increase with prostate tumor growth. Here we evaluated the functional role of survivin in prostate tumor growth. First, we demonstrated that mice lacking the survivin gene in prostate epithelium were fertile and had normal prostate growth and development. We then serially, from about 10-56 weeks of age, evaluated histopathologic changes in the prostate of mice with PTEN deletion combined with survivin mono- or bi-allelic gene deletion. While within this time period most of the animals with wild-type or monoallelic survivin deletion developed adenocarcinomas, the most severe lesions in the biallelic survivin deleted mice were high-grade prostatic intra-epithelial neoplasia with distinct histopathology. Many atypical cells contained large hypertrophic cytoplasm and desmoplastic reaction in the prostatic intra-epithelial neoplasia lesions of this group was minimal until the late ages. A reduced proliferation index as well as apoptotic and senescent cells were detected in the lesions of mice with compound PTEN/survivin deficiency throughout the time points examined. Survivin deletion was also associated with reduced tumor expression of another inhibitor of apoptosis member, the X-linked inhibitor of apoptosis. Our findings suggest that survivin participates in the progression of prostatic intraepithelial neoplasia to adenocarcinoma, and that survivin interference at the prostatic intraepithelial neoplasia stages may be a potential therapeutic strategy to halt or delay further progression.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Survivin deletion has no effect on normal prostate development.
(A) Prostate epithelium-specific Survivin deletion was produced by homologous recombination via ARR2PB promoter-driven Cre expression and Lox P sites flanking all four exons of Survivin. (B) Illustration of PCR analysis for ascertaining genotypes, in this case, using tissues from 20-week old mice. Tail DNA extracts from cPten-/- and Pten f/f mice were used as positive (+) and negative (-) controls for Cre. H2O, water; AP, anterior prostate; VP, ventral prostate; DLP, dorsolateral prostate. (C) Ventral view of prostate showing generally normal gross morphology whether with either single or biallelic inactivation of Survivin. (D) Examples of H&E or immunostaining for androgen receptor (AR), luminal epithelial cell marker cytokeratin 8 (CK8), and proliferation marker Ki67, using ventral prostate lobes collected from 20-week old mice. Positive expression is indicated by the brown staining in the cytoplasm (CK8) or nucleus (AR and Ki67). The results illustrate retention of normal tissue morphology, protein marker expression pattern, and proliferation rate in prostates with heterozygous or homozygous deletion of Survivin. Bar, 50 µm.
Figure 2
Figure 2. Generation of double conditional knockout mice lacking alleles of Pten and Survivin.
(A) Mouse genotype of interest was obtained by crossing mice carrying Cre transgene and floxed phosphatase region of Pten (exon V) with floxed Survivin mice. (B) Genotypes of mice (shown: 10 weeks old) were determined by PCR analysis of the tissue samples. Tail DNA extracts from cPten-/- and Pten f/f mice were used as positive (+) and negative (-) controls for Cre, H2O, water. (C) Status of Survivin deletion was also confirmed at the protein level. This is illustrated by a representative Western blot from the ventral prostate of mice from the 10-, 21-, and 37-week groups, with β-actin serving as loading control. Prostate tissue lysates from Pten f/f S f/f mice were used as normal control (ctrl).
Figure 3
Figure 3. Loss of Survivin in conditional Pten deletion mouse model delays prostate tumor progression.
Representative ventral view of prostate of various genotypes at different time points of aging, (A), (B), (C), (D) indicating animals from the 10-, 21-, 37-, 56-week groups, respectively. Deletion of both Survivin alleles yielded a significantly smaller prostate relative to the sizes seen in the other groups. AP, VP, DLP as in Figure 1; SV, seminal vesicles. (E) Representative depiction of histological analysis of H&E staining of paraffin-embedded prostate tissue sections of mice from 10, 21, 37, and 56 week-old groups: cPten -/- S +/+ mice appeared to develop high grade PINs [–30] in the majority of glands as early as 9 weeks of age. At later time points invasive adenocarcinomas (arrow, 56 weeks) could be detected. cPten -/- S +/- mice of the 10-week group displayed PINs 1-2, followed by detection of PINs 3-4 in the 21-week age group. Prostate epithelium of cPten -/- S -/- mice encompasses single atypical cells containing large hyperchromatic nuclei (arrow, 20 weeks) and large cytoplasm between 8 and 20 weeks of age. PINs 2-3 were observed in some of the glands from the 37- and 56-week age groups. Note that desmoplastic reaction was absent or minimal in lesions of cPten -/- S -/- mice. Dorsolateral lobes. Bar, 50 µm.
Figure 4
Figure 4. Representative pattern of expression of cellular markers in prostates of conditional Pten knockout mice with heterozygous and homozygous deletion of Survivin.
(A) Immunostaining of dorsolateral prostate lobes of animals from the 56-week old group of different genotypes using antibodies against androgen receptor (AR), cytokeratin 8 (CK8), basal epithelial marker p63, PTEN, phosphorylated Akt (P-Akt), and Ki67. All images were taken at 400x magnification. Bar, 50 µm. (B) Comparison of proliferation index as assessed by Ki67 staining of dorsolateral lobes of the 56-week old group. * P < 0.05.
Figure 5
Figure 5. Effects of Survivin deletion on other molecular and cellular parameters.
(A) Representative Western blot analysis of dorsolateral prostates from the 37-week old group showed that XIAP level was down-regulated in prostate tissues lacking both alleles of Survivin, while Livin was relatively unaffected. A similar pattern of XIAP expression was also observed when samples were obtained from in 10 and 21 week- old groups. (B) Detection of higher levels of cleaved-caspase 3 expression, indicated by arrows, in the PIN lesions from the conditional Pten deleted prostate tumors lacking single or both alleles of Survivin compared to tumors with intact Survivin. * P < 0.05; · P <0.01. (C) Illustration of senescence-activated β-galactosidase staining results. An increase in senescence was indicated in the conditional Pten deleted prostate samples with complete inactivation of Survivin especially at the high-grade PIN stage compared to samples with intact and monoallelic deletion of Survivin. Note only low level of senescence in prostate adenocarcinoma tissues of conditional Pten knockout mice with either with intact and single deletion of Survivin. PINs are denoted as low-grade (LG) or high-grade (HG), and adenocarcinoma as AdCa. All images were taken at 400x magnification. Bar, 50 µm.
Figure 6
Figure 6. Assessment of γ-H2AX immunostaining in prostate tissue sections.
(A) Representative view of γ-H2AX expression, indicated by arrows, in the prostate samples at 10 weeks from the conditional Pten knockout mice with single or double deletion of Survivin as compared to corresponding samples with intact Survivin. Images were taken at 400x magnification. Bar, 50 µm. (B) (C), and (D) show the results of quantitation of γ-H2AX positive cells in samples at 10, 21 and 37 weeks, respectively. * P < 0.05; · P <0.01.

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