p27(Kip1) deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process

Abstract

Background: p27 is a cell cycle suppressor gene, whose protein is a negative regulator of cyclin/cdk complexes. p27 is also a potential target of retinoids in cancer prevention studies. In benign prostate hyperplasia (BPH), and in most carcinomas, p27(Kip1) is down-regulated, suggesting its potential resistance to retinoids. To test this hypothesis, we examined the efficacy of 9-cis retinoic acid (9cRA) to suppress prostate cell proliferation (PECP) and carcinogenesis in p27(Kip1) deficient mice.

Methods: p27(Kip1) deficient (-/-), heterozygous (+/-) and homozygous (+/+) mice were treated for 7 days with testosterone, 9cRA, or with both, and cell proliferation in dorsolateral prostate (DLP) was determined by BrdU labeling. Prostate carcinogenesis was induced by N-Methyl-N-Nitrosourea (MNU) and hormone stimulation.

Results: PECP in DLP of two-month-old mice of all genotypes was similar but significantly increased in old p27-/- mice only. Testosterone treatment increased PECP in all three p27 genotypes with the highest values in p27-/- mice. p27(Kip1) deficiency did not affect the response of PEC to 9cRA and to 9cRA+testosterone. The decrease of p27(Kip1) in p27+/- and p27-/- mice progressively increased the incidence and frequency of PIN and tumors. 9cRA suppressed PIN in all three p27 genotypes and this was associated with decreased PECP and increased cellular senescence.

Conclusions: This data indicates that p27(Kip1) deficiency promotes prostate cell proliferation and carcinogenesis but does not affect 9cRA's potential to suppress prostate carcinogenesis, suggesting that patients with PIN and carcinomas lacking or having a low level of p27(Kip1) expression may also benefit from clinical trials with retinoids.

Figure 1

Morphological changes in DLP of p27 deficient mice in the course of carcinogenesis. 1A: Dorso-lateral prostate of 6-month-old p27-/- mice. Note the glandular structures are covered by a single layer of prostate epithelial cells (PEC). Glands are surrounded by a thick layer of myoepithelial cells, stroma cells and collagen (arrows). H&E staining × 200. 1B: High grade PIN in a p27-/- mice treated with MNU and sacrificed at the age of 9 months. Hyperplastic and dysplastic PEC cover entire glands and form papillary and alveolar structures (arrows). The surrounding connective tissue stroma is very abundant (arrow had). H&E × 200. 1C: Ductal carcinoma of papillary type in DLP of a p27-/- animal treated with MNU and sacrificed at the age of 10 months. Note, tumor cells occupy the entire glands lumen. H&E × 200. 1D: Infiltrative prostate carcinoma in a p27-/- animal sacrificed at the age of 12 months. Tumor cells infiltrate the surrounding stroma (arrows). H&E × 200. 1E: Pituitary tumor in a p27-/- mice sacrificed at the age of 10 months. H&E × 200. 1F: Intestinal carcinoma in a p27-/- mice sacrificed at the age of 9 months. Note tumor cell infiltration in the surrounding stroma (arrow). H&E × 200. 1G: RARα expression in DLP of a 6-month-old p27+/- mice. RARα was detected by anti-RARα antibody (SC-251) and ABC kit. The receptor is expressed in papillary structures (arrow) and in the basal layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 1H: RXRα expression in PIN of p27-/- 9-month-old mice. Note that most epithelial cells express RXRα receptor (arrow). The slide is counterstained by hematoxylin × 200.

Figure 2

Effects of 9cRA on cell proliferation and senescence in DLP of p27 deficient mice. Fig. 2A: Cell proliferation, as determined by Ki-67 antibody in DLP of 9-month-old p27+/+ mice. Note several Ki-67-positive cells (brawn-stained, arrows) in glandular structures which are covered by a single layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 2B: Ki-67 positive cells (arrows) in anterior (large left) and DLP (two right glands) of 9-month-old p27-/- mice. Note the thick connective tissue surrounding glandular structures. The slide is counterstained by hematoxylin × 200. 2C: Ki-67 positive cells in PIN of MNU-treated mice. The number of Ki-67 positive cells is higher (arrow), as compared to those in Fig. 2B. The slide is counterstained by hematoxylin × 200. 2D: Treatment of p27-/- mice with 9cRA for 6 months reduced the number of Ki-67 positive cells in PIN (arrow). Note the inflammation associated cells in PINs' lumen. The slide is counterstained by hematoxylin × 200. 2E: Senescent cells (blue stained) determined by SA-β-Gal staining in glandular and ductal structures of DLP of 9-month-old p27+/- mice. Senescent cells are also detectable among myoepithelial and stroma cells (arrows). The slide is counterstained by nuclear fast red × 200. 2F: Treatment of animals with 9cRA increased senescent cells (blue stained) in DLP and AP of a p27+/- mice. The slide is counterstained by nuclear fast red × 200. 2G: 9cRA increased senescent cells in low grade PIN (arrows) as compared to non-PIN areas of a 10-month-old p27+/- animal. The slide is counterstained by nuclear fast red × 200. 2H: 9cRA also increased senescent cells in high grade PIN as shown in the right glandular structure. The slide is counterstained by nuclear fast red × 200.