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. 2013 Apr 25;12(10):1598–1604. doi: 10.4161/cc.24741

Deletion of p21/Cdkn1a confers protective effect against prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate model

Anil K Jain 1,†, Komal Raina 1,†, Rajesh Agarwal 1,2,*
PMCID: PMC3680539  PMID: 23624841

Abstract

Cyclin-dependent kinase inhibitors (CDKIs) p21Cip1/Waf1 (p21) and p27Kip1 (p27) play a determining role in cell cycle progression by regulating CDK activity; however, p21 role in prostate cancer (PCa) is controversial. Whereas p21 upregulation by anticancer agents causes cell cycle arrest in various PCa cell lines, elevated p21 levels have been associated with higher Gleason score, poor survival and increased PCa recurrence. These conflicting findings suggest that more studies are needed to examine p21 role in PCa. Herein, employing genetic approach, transgenic mice harboring p21/Cdkn1a homozygous deletion (p21−/−) were crossed with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice to characterize in vivo consequences of p21 deletion on prostate tumorigenesis. Lower urogenital tract weight of p21−/−/TRAMP mice was significantly lower than those of p21+/−/TRAMP and TRAMP mice. Histopathology further supported these observations, showing less aggressiveness in prostates of p21−/−/TRAMP. Furthermore, a significantly higher incidence of low-grade prostatic intraepithelial lesions (PIN) with a concomitant reduction in adenocarcinoma incidence was observed in p21−/−/TRAMP mice compared with TRAMP mice. In addition, whereas TRAMP mice showed the presence of poorly differentiated adenocarcinoma lesions, no such lesions were observed in p21/TRAMP transgenic mice. Specifically, there was a significant reduction in the severity of lesions in both p21−/−/TRAMP and p21+/−/TRAMP mice compared with TRAMP mice. Together, our data showed that p21 deletion reduces prostate tumorigenesis by slowing-down progression of PIN (pre-malignant) to adenocarcinoma (malignant), suggesting that intact p21 expression is associated with PCa aggressiveness, while its decreased levels may in fact confer protection against prostate tumorigenesis.

Keywords: TRAMP, prostate cancer, cell cycle, p21 knockdown

Introduction

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men in the United States, and is second only to lung cancer in deaths.1 Progression of PCa is a sequential multistage process involving the onset as a small neoplastic lesion of low histological grade progressing slowly to the lesions of advanced grade.2-5 With time, PCa progresses to a more aggressive hormone refractory stage, rendering anti-androgen therapy ineffective.2-6 An aberrant regulation of cell cycle has been an underlying cause of this uncontrolled cell proliferation and survival, leading to the neoplastic transformation and cancer progression to advanced stages in PCa.6,7 Cell cycle progression is regulated by the activity of cyclin-dependent kinases (CDK) in association with their regulatory subunits, cyclins and CDK inhibitors (CDKI).7-9 p21Cip1/Waf1 (hereafter referred as p21) and p27Kip1 are the members of the Cip/Kip family of CDKI that determine the progression through the G1 and S phases of cell cycle by regulating CDK activity.9 While lower protein p27Kip1 expression is often associated with poor prognosis in PCa, the prognostic significance of p21 in PCa is still controversial.10,11 Whereas upregulation of p21 by anticancer and chemopreventive agents has been shown to induce growth arrest in several androgen-dependent and -independent PCa cell lines,6,12,13 in many cases, the elevated p21 levels in human PCa tissues have been correlated with higher Gleason score, poor survival and increased recurrence rate of the disease.14-17 Interestingly, some reports have also associated high p21 levels with metastasis-free survival in PCa patients.18 Together, these mixed and contradictory observations warrant extensive studies to examine the roles of p21 molecule in PCa progression. In this regard, we recently reported the effect of knockdown of p21/Cdkn1a and p27/Cdkn1b alone or together on the growth characteristics, tumorigenicity and angiogenic potential of advanced human PCa DU145 cells.11 The results indicated that the diminished expression of either p21 or p27 Kip1 alone does not significantly alter these malignancy-associated characteristics of this PCa cell line, both in vitro and in vivo in nude mouse xenograft, and that simultaneous ablation of both of these molecules is required to enhance the aggressive PCa phenotype.11 The limitation of that study in relation to the practical and translational aspect is that it is difficult to extrapolate the data to the real clinical scenario, where PCa is initiated in its own tumor microenvironment. An effective alternative, so as to provide more relevant scientific insight into the role of p21 in PCa growth and progression would be to study the implication of p21 knockdown in a prostate tumorigenesis pre-clinical model that closely mimics the clinical scenario.

Transgenic adenocarcinoma of the mouse prostate (TRAMP) model is one such mouse model that mimics the human type of PCa progression in a stochastic fashion19-23 and has been used extensively by us and others to evaluate the molecular mechanisms of prostate tumorigenesis as well as the efficacy of various anticancer agents.24-27 TRAMP was developed in C57BL/6 mice in which minimal rat probasin promoter (PB) drives the expression of SV-40 early genes (T/t; Tag) specifically in prostatic epithelium.20,23 The transgene is under hormonal regulation, expressed at sexual maturity, which then induces spontaneous neoplastic epithelial transformation in the prostate.20,23 The function of p53 and Rb is abrogated by SV-40 large T antigen; as a result, TRAMP male mice develop spontaneous progressive stages of PCa with time from early lesions of prostatic intraepithelial neoplasia (PIN) to late stage adenocarcinoma.20,23 Furthermore, this PCa model has been also crossed with other genetically manipulated mice so as to generate bigenic mice,28-31 which are employed to study the role of specific molecules in PCa progression. The changing patterns of CDKs and cyclins have been also well characterized during the progression of PCa in TRAMP model;32 notably, we have reported the increased expression of p21 protein in TRAMP prostate with advanced stage of the disease.25

Taken together, the objective of the present study was to cross the knockout mice harboring p21/Cdkn1a homozygous deletion (p21−/−)33-35 with the TRAMP mice so as to characterize the in vivo consequences of p21 deletion (both heterozygous and homologous deletion) on prostate tumor initiation and progression.

Results

p21 deletion reduces LUT weight in p21+/−/TRAMP and p21−/−/TRAMP mice

At the time of necropsy, LUT weight of heterozygous TRAMP males (hereafter referred to as TRAMP mice), TRAMP mice with heterozygous deletion of p21/Cdkn1a (hereafter referred to as p21+/−/TRAMP), TRAMP mice with homozygous deletion of p21 (hereafter referred to as p21−/−/TRAMP mice), mice with heterozygous deletion of p21 (p21+/−) and mice with homozygous deletion of p21 (p21−/−) were compared with each other. There was no significant difference in LUT weight (normalized to body weight) (Fig. 1A) between non-transgenic mice, p21+/− mice or p21−/− mice. The LUT weight of TRAMP mice and p21−/−/TRAMP mice was significantly different; the latter was 72% (p < 0.001) less compared with TRAMP mice (Fig. 1A). The LUT weight of p21+/−/TRAMP mice, though lesser compared with TRAMP mice, was not statistically significant. Importantly, there was no considerable difference in body weight gain profiles between the mice in these three different groups (data not shown).

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Figure 1. Effect of p21 deletion on prostate tumorigenesis in p21+/−/TRAMP and p21−/−/TRAMP mice. (A) Effect of p21 deletion on the weight of the lower urogenital tract (LUT) organs. At the time of necropsy, 24 wk of age, each mouse was weighed, and the LUT including the bladder, seminal vesicles and prostate was removed en bloc and weighed. The dorsolateral prostates were histopathologically analyzed for the different stages of the neoplastic progression. (B) Effect of p21 deletion on the incidence of PIN lesions (C) Effect of p21 deletion on the incidence of adenocarcinoma of prostate. Prostatic intraepithelial neoplasia (PIN), well differentiated (WD), moderately differentiated (MD) and poorly differentiated (PD) adenocarcinoma characteristics. NC, negative control (non-transgenic mice); NS, no significant difference.

Pathological changes in the prostate of p21+/−/TRAMP and p21−/−/TRAMP mice

Using the modified classification by Shappell et al.36 as described by us earlier,24 the TRAMP mouse prostate pathology was classified as: (1) low-grade PIN (LGPIN), which has intact gland profiles with foci of two or multilayers of atypical cells and elongated hyper chromatic nuclei; (2) high-grade PIN (HGPIN), which has enlarged diameter of glands, distorted duct profiles, having increased epithelial stratification and cribriform architecture, a larger number of hyperchromatic and pleomorphic nuclei, and lumen is almost completely filled with cells; (3) well differentiated (WD) adenocarcinoma, which has increased quantity of small glands; the basement membranes of glands display signs of invasion, and the spaces between the ducts are considerably reduced; (4) moderately differentiated (MD) adenocarcinoma, which is characterized by merged glands; the spaces between the ducts are completely lost; and (5) poorly differentiated (PD) adenocarcinoma, which displays a continuous sheet of undifferentiated cells.

Importantly, as reported earlier for the TRAMP prostate,19,24 prostate tumorigenesis in terms of its pathological manifestation was more apparent in dorsolateral lobe of prostate than in other lobes (anterior and ventral) in p21/TRAMP mice, thus detailed histopathological analysis was done on the dorsolateral lobe only (Figs. 1–3). Analysis of H&E-stained sections showed that the tumor incidence was markedly different between TRAMP and p21/TRAMP male mice (Fig. 1B and C). It was further inferred from histopathological data that the prostates of p21−/−/TRAMP mice had less signs of aggressive prostatic disease compared with TRAMP mice. As shown in Figure 1B, p21−/−/TRAMP mice showed a significant increase in the incidence of lesions with PIN characteristics and, in parallel, also showed a reduction in the incidence of lesions with adenocarcinoma characteristics compared with TRAMP mice (Fig. 1C). Also, in p21−/−/TRAMP mice, there was an increase in the incidence of lesions of LGPIN type together with a decrease in the incidence of HGPIN lesions (Fig. 1B). On the other hand, in p21+/−/TRAMP mice, the incidence of HGPIN was higher, while the incidence of LGPIN was lesser; there was, however, no incidence of PIN lesions in TRAMP mice (Fig. 1B). In the p21−/−/TRAMP group, there was also no incidence of moderately or poorly differentiated adenocarcinoma (Fig. 1C); however, there was an 8% incidence of MD but no incidence of PD adenocarcinoma in p21+/−/TRAMP mice. Importantly, TRAMP mice showed the presence of WD (36%), MD (43%) and PD (21%) adenocarcinoma lesions (Fig. 1C). Furthermore, in TRAMP-negative mice, while no histopathological lesions were observed in the prostate (Fig. 1), in the p21+/− and p21−/− mice there was 29% and 20% incidence of LGPIN lesions, respectively. The photomicrographs, representative of different pathological lesions observed in the dorsolateral and anterior prostates of TRAMP, p21+/−/TRAMP and p21−/−/TRAMP mice, are shown in Figure 2.

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Figure 3. The p21+/−/TRAMP and p21−/−/TRAMP mice show less severe prostatic lesions (tumor grade) of dorsolateral prostate. (A) Different stages of prostate tissues were graded, and the maximum histological score for the prostate lobe was used to calculate a mean for the group. Data are presented as mean peak histological score and ± SEM (error bars) of each group. (B) Effect of p21 deletion on the percentage of area of dorsolateral prostate having histological grade, low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade prostatic intraepithelial neoplasia (LGPIN), well differentiated (WD), moderately differentiated (MD) and poorly differentiated (PD) adenocarcinoma characteristics. Columns indicate mean ± SEM. NC, negative control (non-transgenic mice); NS, no significant difference.

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Figure 2. The photomicrographs (100× magnification), depicting different pathological lesions observed in H&E staining of the dorsolateral, anterior and ventral prostates of TRAMP, p21+/−/TRAMP and p21−/−/TRAMP mice are shown.

p21 deletion reduces prostate tumor grade in p21+/−/TRAMP and p21−/−/TRAMP mice

Dorsolateral prostate tissues were further pathologically classified and graded as described earlier.24,37 The grades ascribed were: (1) normal epithelium with no pathological lesions, grade 1.0; (2) LGPIN, grade 2.0; (3) HGPIN, grade 3.0; (4) WD adenocarcinoma, grade 4.0; (5) MD adenocarcinoma, grade 5.0; and (6) PD adenocarcinoma -grade 6.0. The lesions in p21−/−/TRAMP and p21+/−/TRAMP mice were significantly less severe (p < 0.001) compared with TRAMP mice as indicated by the decrease in tumor grades in these bigenic mice; a better protective effect due to homozygous p21 deletion was observed in p21−/−/TRAMP mice (Fig. 3A). Specifically, p21−/−/TRAMP mice (mean peak score, 2.7) demonstrated only a slightly lower tumor grade than p21+/−/TRAMP mice (mean peak score, 3.2) but showed a significantly lower tumor grade than the TRAMP mice (mean peak score, 4.9). These results together indicate that p21 deletion reduces both adenocarcinoma incidence and tumor grade in the prostate of TRAMP mice.

p21 deletion reduces the area covered by adenocarcinoma lesions in the prostate of p21+/−/TRAMP and p21−/−/TRAMP mice

As shown in Figure 3B, in p21+/−/TRAMP and p21−/−/TRAMP mice, about 7–11 ± 2% area of dorsolateral prostate also displayed normal histology, which was not the case in TRAMP mice. Furthermore, the distribution of PIN lesions also indicated that compared with TRAMP mice, the prostatic area showing PIN lesions in p21/TRAMP mice was significantly higher (Fig. 3B). Specifically, there was a significantly increased area (59 ± 11%) covered by LGPIN lesions in p21−/−/TRAMP mice, compared with 28 ± 9% and 7 ± 2.6% in p21+/−/TRAMP and TRAMP mice, respectively (Fig. 3B). Also, prostate area showing characteristics of more aggressive forms of the disease was significantly different between p21/TRAMP mice and TRAMP group; p21−/−/TRAMP mice displayed 87% (p < 0.05) lesser area of adenocarcinoma in comparison to the TRAMP mice (Fig. 3B). While LGPIN lesions were observed in p21+/− and p21−/− mice, the p21−/− mice had significantly less area covered by these PIN lesions compared with that in p21+/−/mice.

Discussion

Mutations associated with genes encoding for tumor suppressors and oncogenes, and constitutively active mitogenic signaling are known to cause deregulation of the essential steps involved in cell cycle, leading to unrestrained proliferation of cancer cells associated with tumorigenesis.6-8 Progression through cell cycle is governed by CDKs and associated cyclins (subunits which positively regulate cell cycle) as well as CDK inhibitors (CDKI) (subunits which negatively regulate cell cycle).7,9,38 The different cell cycle phases are associated with the specific activation of different CDKs; for example, progression through the early and middle-phases of G1 are controlled by CDK4/CDK6 in association with cyclin D, late G1 and G1-S transition is regulated by CDK2-cyclin E complex, S-phase progression is controlled by CDK2-cyclin A association, and G2-M phase progression is associated with CDK1-cyclin B1 complex.7-9 Briefly, the molecular mechanisms governing various cell cycle steps involve the association of specific CDK/cyclin complexes, which are in turn regulated by the synthesis and degradation of cyclins,7,9 phosphorylation of CDK and the binding to CDKI.7,9 In the G1 and S phases, CDKI members, belonging to the Cip/Kip family, display negative effects on the cell cycle-associated activity of the complexes between CDK4/6/2 and cyclins D, E and A,7,9 while the CDKI members, belonging to the Ink4 family, specifically inhibit the activity of complexes between CDK4/6 and cyclin D, thus playing a role in the regulation of G1 phase only.7,9 Furthermore, one of the substrates of the CDK4/6-cyclin D complexes, retinoblastoma (Rb), plays a key role in cell cycle progression; importantly, its mutation is associated with various cancers.7,39 The phosphorylation status of Rb, controlled by CDK/cyclin complexes and its association/disassociation with E2F, play an essential role in cell cycle progression through various cell check points involved in G1 and G1-S transition.7,39 On the withdrawal of mitogenic signaling, synthesis of cyclin D is stalled, and Cip/Kip proteins block the catalytic activity of the cyclin E/A-CDK2 complex, resulting in cell cycle arrest.7,39 Furthermore, at this point in cell cycle phase, an insult causing DNA damage leads to a p53-dependent transcriptional activation (enhances) of CDKI (Cip1/p21), which negatively affects CDK/cyclin complex activity, thereby inhibiting phosphorylation of Rb and inhibiting the replication of DNA, thus stopping the cells from S phase transition.7,39 Apart from its induction by p53, p21 induction pathways independent of p53 have also been reported.40,41

Considering these steps involved in cell cycle progression, it can be speculated that overexpression of p21 would be associated with significant growth arrest, having a negative impact on cell cycle progression causing a strong tumor growth inhibition. However, clinically, the relation of overexpression of p21 to prognosis as well as to disease endpoint in different malignancies is variable.11 For example, increased expression of p21 is associated with favorable clinical results in various cancers, including that of pancreas and bladder, whereas p21 expression marks poor clinical end results in cancers of the breast and liver.42-45 Regarding PCa, whereas in vitro studies delineating the mechanisms involved in the efficacy of anticancer agents indicate the possible involvement of overexpressed p21 in causing cell arrest in various PCa cell lines,12,13 clinically in many cases, the elevated p21 levels in human PCa tissues have been reported to be associated with prostatic disease graded with high Gleason scores, reduced survival outcomes and increase in the recurrence of this malignancy.14-17

On similar lines, evaluation of human PCa samples from androgen-dependent/-independent tumors and xenograft studies employing androgen-responsive cell lines have shown that increased expression of p21 is associated with high proliferative index and progression to androgen-independent PCa.46 Given the fact that p21 is a growth inhibitory molecule,7-9 the findings indicating toward an association of overexpression of p21 with poor outcome in PCa patients raises questions about the inhibitory role of this molecule in PCa growth and progression. One possible justification explaining this anomaly is the recent finding that p21-induced cells may have a paracrine growth stimulatory effect; specifically, conditioned cell culture media from cells induced with p21 has been shown to cause anti-apoptotic and pro-mitogenic effect on non-induced cells, although p21 induces cell cycle arrest under in vitro conditions.47 Furthermore, evaluation of prostatectomy tissues from patients with PCa has shown that the ethnic background of patients is an important variable while correlating expression of p21 levels with disease survival.48 While expression of p21 predicts that patients with Caucasian background will be cancer-free; in the case of patients with African-American background, the p21 expression does not indicate a better prospective for the patients.48 This phenomenon associated with the in vivo expression of p21 in tumors cannot be related to genetic alteration of the p21 gene, as polymorphism and mutations of p21 gene are reported to be rare in human malignancies, including PCa.49 In addition, some reports have also associated high p21 levels with favorable prognosis and no incidence of metastasis in future in patients diagnosed with PCa and having undergone treatment by radiation therapy followed by salvage prostatectomy.18

Accordingly, the present study was an effort to characterize for the first time the in vivo consequences of p21 deletion on initiation and progression of prostate tumorigenesis employing TRAMP mice. We generated a TRAMP mice model with a p21-null genetic background and provided compelling genetic evidence suggesting that the p21 homologous deletion provides a significant protective effect against prostate tumorigenesis. At 24 wk of age, necropsy was performed across the groups, where LUT weights of the p21/TRAMP mice were found to be significantly lower than the LUT weights of TRAMP mice. Detailed histopathological analysis and its comparison across different mice groups indicated significant differences in the incidence of prostatic tumor lesions between p21/TRAMP and TRAMP male mice. Overall, in this study, p21 deletion reduced the incidence and severity of prostate adenocarcinoma by retarding the progression of PIN lesions (pre-malignant stage) to adenocarcinoma stages, suggesting the possibility that intact p21 expression is associated with PCa aggressiveness, while its decreased levels may in fact confer protection against prostate tumorigenesis.

Materials and Methods

Animals, treatment and necropsy

Heterozygous TRAMP females (Jackson Laboratory), developed on a pure C57BL/6 background,19 were cross-bred with non-transgenic C57BL/6 breeder males to generate heterozygous TRAMP males (TRAMP mice). To generate male/female TRAMP mice with heterozygous deletion of p21/Cdkn1a (p21+/−/TRAMP), transgenic male mice harboring homozygous deletion of p21 (p21−/−) in C57BL/6 background35 [kindly obtained from Dr Michael A. O’Reilly,35 University of Rochester Medical Center, on authorized permission from Dr Philip Leder33 (Harvard Medical School), the innovator scientist] were crossed with heterozygous TRAMP females. While the male p21+/−/TRAMP mice were used in the study, the female p21+/−/TRAMP mice were crossed with male p21−/− mice to generate TRAMP mice with homozygous deletion of p21 (p21−/−/TRAMP mice). As an overall control, male p21−/− and p21+/−/mice were routinely obtained by intercrossing p21−/− mice with each other, or crossing p21−/− mice with C57BL/6 mice, respectively. Tail DNA was subjected to PCR-based screening assay for PB-Tag, Cdkn1a (wild type, heterozygous and homozygous deletion) and routinely obtained 4-wk-old TRAMP male mice from the specific genetics were used in the study. There were 15, 13 and 14 mice in TRAMP, p21+/−/TRAMP and p21−/−/TRAMP groups, respectively. While in the controls: p21+/− and p21−/− groups, there were six mice each, and in the TRAMP-negative (C57BL/6 wild type) group there were nine mice.

During the study, animals had free access to drinking water and food. Food consumption and animal body weight were recorded on weekly basis, and animals were monitored daily for their general health. All animal care was in accordance with institutional guidelines with approved protocol. At the time of sacrifice (24-wk-old mice), the animals were euthanized by carbon dioxide asphyxiation followed by exsanguination. At the time of necropsy, mice were individually weighed and examined for gross pathology, and lower urogenital tract (LUT) including bladder, seminal vesicles and prostate, was removed en bloc. LUT wet weight was then recorded, and prostate glands were harvested and microdissected; if a tumor obscured the boundaries of lobes it was taken as such. Collected tissues were fixed overnight in 10% (v/v) phosphate-buffered formalin, processed conventionally and paraffin embedded. Sections (5 μm) were stained with hematoxylin and eosin (H&E) for histopathological assessment. Given the fact that the p21-deficient mice are known to develop spontaneous tumors of hematopoietic, endothelial and epithelial origin at an average of 16 mo,33,34 and that the TRAMP mice in C57BL/6 background do not show significant metastatic lesions,19,21 we did not study the incidence of non-prostatic lesions in both bigenic p21/TRAMP mice and the TRAMP mice.

Statistical and microscopic analyses

All statistical analyses were done with Sigma Stat software version 2.03 (Jandel Scientific). Fishers’ exact test was employed to compare incidence of PIN and adenocarcinoma between groups. For other analyses, the statistical significance of difference between groups was analyzed by one-way ANOVA followed by Bonferroni t-test for pair wise multiple comparisons. Two-sided p values < 0.05 were considered significant. All microscopic analyses employed Zeiss Axioscope 2 microscope (Carl Zeiss, Inc.) and photomicrographs were taken with Carl Zeiss AxioCam MrC5 camera.

Acknowledgments

This work was supported by NCI R01 grants CA102514 and CA116636. The two mating pairs of p21-knockout mice on C57BL/6 background were kindly provided by Dr Michael A. O’Reilly, University of Rochester Medical Center, on authorized permission from Dr Philip Leder (Harvard Medical School).

Glossary

Abbreviations: PCa

prostate cancer

TRAMP

transgenic adenocarcinoma of the mouse prostate

LUT

lower urogenital tract

LGPIN

low grade prostatic intraepithelial neoplasia

HGPIN

high grade prostatic intraepithelial neoplasia

CDK

cyclin-dependent kinase

CDKI

cyclin-dependent kinase inhibitors

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

References

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