Inactivation of PTEN is associated with a low p27Kip1 protein expression in breast carcinoma

Authors


Abstract

BACKGROUND

It was previously demonstrated that PTEN protein expression is reduced in 67 of 236 (28%) breast carcinomas. Recent experimental studies suggested that the cell cycle inhibitor p27Kip1 (p27) is a downstream mediator through which PTEN negatively regulates cell cycle progression.

METHODS

The immunohistochemic expression of p27 and PTEN protein expression was evaluated in a series of 228 invasive ductal carcinomas of the breast.

RESULTS

PTEN protein expression was found to have decreased in 65 of 228 (29%) cases, while the nuclear accumulation of p27 protein was low in 99 of 228 (43%) cases. A reduced PTEN protein expression correlated significantly (P = 0.0214) with a low p27 protein expression. Univariate analysis indicated that the patients demonstrating a combined decrease in PTEN and p27 protein expression have a significantly (P = 0.0044) worse disease-free survival (DFS) than those with other combinations of these two protein expression patterns, while multivariate analysis indicated that the lymph node status, MIB-1 counts, and the combination of PTEN/p27 protein expression (P = 0.0452) are independently significant prognostic factors for DFS.

CONCLUSIONS

A reduced PTEN protein expression correlated significantly with a low p27 protein expression in breast carcinoma. The finding that the patients with a combined decrease in both protein expressions had a poor prognosis thus suggests that a combined loss of PTEN and p27 function is associated with an aggressive phenotype in breast carcinoma. Cancer 2005. © 2005 American Cancer Society.

The PTEN gene, a tumor-suppressor gene, encodes a dual-specificity phosphatase with lipid and protein phosphatase activities.1, 2 PTEN antagonizes phosphoinositide (PI) 3(phosphotidylinositol 3)-kinase by dephosphorylating PIP3 (phosphotidylinositol-3,4,5-triphosphate) and induces G1 cell cycle arrest by negatively regulating the PI 3-kinase/Akt signaling pathway, while the inactivation of PTEN function results in increased Akt activity.3–5 The inactivation of PTEN has been demonstrated in various cell lines and tumor specimens.1, 2, 6 Although the PTEN gene mutation was thought to be frequent in the initial study,6 PTEN gene mutations have not been recognized in a majority of breast carcinomas.7 The inactivation of PTEN function is thought to be due not only to gene mutation and deletion but also to epigenetic mechanisms, such as promoter methylation.8, 9 We previously demonstrated a reduced expression of PTEN protein in 67 of 236 (28%) breast carcinomas, while the patients with reduced PTEN protein expression had a worse prognosis than those with a normal PTEN protein expression.1 Conversely, p27Kip1 (p27), a member of the Cip/Kip family of CDK inhibitors, was found to play a role in regulating the progression from the G1 to the S phase.10, 11 An up-regulation of p27 induced by the exogenous PTEN gene transfer was demonstrated in different cell lines12, 13 and recent experimental studies have suggested p27 to be a downstream mediator through which PTEN negatively regulates cell cycle progression.14–19

There have been several studies which evaluated the relation between PTEN and p27 protein expression in surgical specimens of thyroid,17 prostate,20–22 endometrial,23 and ovarian24 carcinomas and glioma.25 To the best of our knowledge, however, no study has yet been conducted regarding the relation between the PTEN and p27 protein expression in surgical specimens for breast carcinoma. In the present study, the immunohistochemic expression of p27 protein was evaluated in a series of 228 invasive ductal carcinomas of the breast in which PTEN protein expression alone had been determined previously.1 The aim of this study was to investigate the relation between PTEN and p27 protein expression in invasive ductal carcinoma of the breast. The prognostic value of the combination of PTEN and p27 protein expression for breast carcinoma was also evaluated.

MATERIALS AND METHODS

Patients

This study comprised 228 women with breast carcinoma who underwent surgery for breast carcinoma between 1985 and 1998 at the Beppu Medical Center Hospital, Japan, without any evidence of distant metastasis at the time of surgery. This series was described in our previous study.1 The histological type of breast carcinoma in all patients was invasive ductal carcinoma; any histological types other than invasive ductal carcinoma were excluded in this study. The patients' ages ranged from 23 to 85 years, with a mean age of 58.1 years. The patients were treated either by mastectomy (193 patients) or by breast conservation treatment (35 patients). Lymph node dissection was performed in 226 patients. Adjuvant postoperative hormone therapy was given to 201 patients and 197 patients received adjuvant postoperative chemotherapy; 52 patients received postoperative radiotherapy. The median follow-up duration was 6.7 years.

Immunohistochemistry

The immunohistochemic method to determine the PTEN protein has been described previously.1 Briefly, 3-μm sections were heated with a microwave for antigen retrieval and endogenous peroxidase in sections was inactivated in 0.1% pepsin in 0.01 N HCl buffer, pH 2.5, followed by incubation in 0.05% saponin for 30 minutes at room temperature. The sections were blocked in 1.5% normal horse serum in phosphate-buffered saline PBS and then were incubated at 4 °C overnight with monoclonal anti-PTEN antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 in PBS. The sections were subsequently stained according to the labeled streptavidin biotin (LSAB) method using a Dako LSAB + kit (Dako, Glostrup, Denmark) and then were observed using diaminobenzidine, followed by counterstaining with hematoxylin. A normal mammary gland was used as an internal control for PTEN protein expression. The immunohistochemic expression of breast carcinoma cells was judged to be either a normal or reduced expression compared with the PTEN protein expression of a normal mammary gland in the same slide.

For the immunohistochemic analysis of p27 protein, 3-μm sections were dewaxed and rehydrated and antigen retrieval was performed by microwave heating for 15 minutes in a 10-mM citrate buffer at pH 6.0. Next the sections were reacted with mouse monoclonal antibody for p27 (Novocastra Laboratories, Newcastle, UK) diluted at 1:40 for 60 minutes at room temperature, then subsequently stained by the universal immunoperoxidase polymer method using a Histofine Simple Stain MAX PO(M) kit (Nichirei, Tokyo, Japan) according to the protocol provided by the manufacturer. Positive reactions were observed with diaminobenzidine, followed by counterstaining with hematoxylin. The positivity of p27 protein expression was defined as high when more than 50% of the tumor cells displayed distinct nuclear staining and as low when less than 50% of the tumor cells displayed nuclear staining. A cutoff value of 50% was also used in our previous study for gastric carcinoma26 and this level has also been frequently used to determine the p27 protein expression levels in various tumor specimens.27, 28

Both protein expressions were determined independently by three of the authors (S.T., K.Y., and S.E.), of whom two did not know any clinicopathologic information for each patient. The methods and results of assessing p53 protein expression and the cell proliferation index (percentage of MIB-1-immunostained tumor cells) have been described previously.29

Statistical Analysis

The chi-square test was used to investigate any categorical variables. The disease-free survival (DFS) was estimated using the Kaplan–Meier method, and any differences in the survival curves were compared by the log-rank test. A multivariate analysis was performed by the Cox proportional hazards model. A P-value of < 0.05 was regarded as statistically significant. All statistical analyses were performed by using the StatView 5.0 software (SAS Institute, Cary, NC).

RESULTS

Immunohistochemic Expression of PTEN and p27 Protein

PTEN protein expression was found to have decreased in 65 of 228 (29%) cases, while the nuclear accumulation of p27 protein was low in 99 of 228 (43%) cases. The relation between PTEN and p27 protein expression is shown in Table 1. A significant (P = 0.0214) correlation was observed between PTEN and p27 protein expression. A reduced PTEN protein expression correlated significantly with a low p27 protein expression.

Table 1. The Relation between the PTEN and p27 Protein Expressions
 No. of patientsp27 protein (%)P value
High n = 129Low n = 99
PTEN protein   0.0214
 Normal163100 (61)63 (39) 
 Reduced6529 (45)36 (55) 

Relation Between the Combination of the PTEN/p27 Protein Expression and Clinicopathologic Factors

Table 2 shows the relation between the combination of the PTEN/p27 protein expression patterns and the clinicopathologic factors of breast carcinoma. The combination of PTEN/p27 protein expression correlated significantly with the nuclear grade, estrogen receptor status, p53 protein expression, and MIB-1 counts, while the combination of a normal PTEN/low p27 protein expression as well as the combination of a reduced PTEN/low p27 protein expression were associated with nuclear grade III, a negative estrogen receptor status, a positive p53 protein expression, and positive MIB-1 counts.

Table 2. Relation Between the Combination of PTEN/p27 Protein Expressions and Clinicopathologic Factors
 No. of patients (%)Combination of PTEN/p27 protein expressions (%)P value
Normal/high n = 100Normal/low n = 63Reduced/high n = 29Reduced/low n = 36
  • a

    Lymph node dissection was performed in 226 patients.

Lymph node metastasisa     0.1611
 Absent127 (56)63 (63)35 (56)14 (48)15 (43) 
 Present99 (44)37 (37)27 (44)15 (52)20 (57) 
Nuclear grade     < 0.0001
 I or II152 (67)81 (81)31 (49)23 (79)17 (47) 
 III76 (33)19 (19)32 (51)6 (21)19 (53) 
Estrogen receptor     0.0002
 Positive100 (44)55 (55)18 (29)18 (62)9 (25) 
 Negative128 (56)45 (45)45 (71)11 (38)27 (75) 
p53 protein expression     0.0031
 Negative168 (74)83 (83)37 (59)24 (83)24 (67) 
 Positive60 (26)17 (17)26 (41)5 (17)12 (33) 
MIB-1 counts     0.0098
 Negative134 (59)64 (64)30 (48)23 (79)17 (47) 
 Positive94 (41)36 (36)33 (52)6 (21)19 (53) 

Univariate and Multivariate Analyses of Disease-Free Survival

Univariate analyses of DFS indicated that patients with a low p27 protein expression have a significantly (P = 0.0095) worse DFS than those with a high p27 protein expression. Figure 1 shows the survival curves stratified according to the combination of PTEN/p27 protein expression. Univariate analyses indicated that patients with the combination of reduced PTEN/low p27 protein expression have a significantly (P = 0.0044) worse DFS than those with other combinations of the two protein expressions. Multivariate analyses of DFS indicated that lymph node status, MIB-1 counts, and the combination of the PTEN/p27 protein expression are independently significant prognostic factors for DFS (Table 3).

Figure 1.

The disease-free survival curves stratified according to the combination of PTEN and p27 protein expression.

Table 3. Multivariate Analyses of the DFS
VariablesaP valueRelative risk95% CI
  • a

    A multivariate analysis was performed on 12 variables consisting of age, tumor size, surgical operation, adjuvant hormone therapy, adjuvant chemotherapy, postoperative radiotherapy, and the 6 variables demonstrated in the table. The P value for the 6 variables other than the 6 variables demonstrated in the table was not statistically significant.

Lymph node metastasis (present vs. absent)< 0.00017.363.56–15.2
Nuclear grade (III vs. I or II)0.12531.600.88–2.90
Estrogen receptor (negative vs. positive)0.50831.230.67–2.27
p53 protein expression (positive vs. negative)0.62841.170.62–2.24
MIB-1 counts (positive vs. negative)0.00102.861.53–5.36
The combination of the PTEN/p27 protein expressions   
 Reduced PTEN/low p27 (vs. others)0.04521.871.01–3.44

DISCUSSION

The present study demonstrated a significant correlation between PTEN and p27 protein expression in invasive ductal carcinoma of the breast. A reduced PTEN protein expression correlated with a low p27 protein expression. To the present, seven studies have evaluated the relation between PTEN and p27 protein expression in various tumor specimens.17, 20–25 However, no such study has yet been conducted on the surgical specimens of breast carcinoma. A significant (P < 0.05) correlation between PTEN and p27 protein expression has been demonstrated in thyroid,17 prostate,20–22 and endometrial carcinomas.23 Conversely, no significant correlation was found between PTEN and p27 protein expression in ovarian carcinoma24 or glioma.25 The sample size of the latter two studies, however, was relatively small, while statistical analyses were performed on 49 ovarian carcinomas20 and 25 gliomas.25 We also demonstrated here a strong association between the combined PTEN/p27 protein expressions and nuclear grade, estrogen receptor status, p53 protein expression, and MIB-1 counts. Our previous study demonstrated that PTEN protein expression alone was not associated with nuclear grade, estrogen receptor status, p53 protein expression, or MIB-1 counts.1 In the present study, a low p27 protein expression, combined either with or without a reduced PTEN protein expression, was associated with a nuclear grade III, a negative estrogen receptor status, a positive p53 protein expression, and positive MIB-1 counts. The role of p27, negatively regulating G1 progression and S phase entry, accounts for a close correlation between the low expression of p27 protein and the increased MIB-1 (Ki-67 antigen) counts,10, 11 while a close correlation had been demonstrated between nuclear grade, estrogen receptor status, p53 protein expression, and MIB-1 counts in our previous study.29

Recent experimental studies have demonstrated a close association between the PTEN and p27 functions.12–19 Di Cristofano et al.14 reported that a combined loss of PTEN and p27 function was found to strongly increase the development of prostate carcinoma in double mutant mice. An accelerated G1 to S transition accompanied by a down-regulation of p27 was found in the cell line lacking both PTEN alleles.15 The restoration of the wild-type PTEN gene resulted in G1 cell cycle arrest with an up-regulation of p27 in breast carcinoma cell lines,16 while the increased p27 level by the PTEN gene transfer was also demonstrated in glioblastoma cell lines.12, 13 The PTEN gene was also demonstrated to induce G1 arrest by up-regulating p27 via its lipid phosphatase activity in MCF-7 breast carcinoma cells.19 PTEN expression blocked the G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, with a significant accumulation of p27.12 Conversely, the inhibition of p27 using antisense oligonucleotides abrogated the PTEN-induced transcriptional growth arrest, thus suggesting that p27 was required for PTEN-induced G1 arrest.17, 18

The role of PTEN in negatively regulating the PI 3-kinase signaling pathway has been established.3–5 Because PTEN inhibits cell cycle progression and induces G1 cell cycle arrest through negatively regulating the PI 3-kinase/Akt signaling pathway, the inactivation of PTEN function thus leads to an increased Akt activity.3–5 Furthermore, the increased Akt activity was demonstrated to decrease p27 expression30 while also blocking p27 entry into the nucleus.31 Conversely, the Forkhead transcription factor family has been demonstrated to mediate the G1 cell cycle regulation that depends on p27,32 while the transcription factors were also demonstrated to be controlled by Akt phosphorylation.33 The Forkhead transcription factors FKHRL1 and FKHR could not activate the transcription in PTEN-deficient cells.34 The above-mentioned experimental studies12–19, 30–34 have all demonstrated that PTEN regulates the G1 cell cycle progression by negatively regulating the PI 3-kinase/Akt signaling pathway, while also suggesting that p27 is a critical target of the signaling process.

We demonstrated that patients with a reduced PTEN protein expression have a worse prognosis than those with a normal PTEN protein expression in our previous study.1 Conversely, according to the role of p27 in negatively regulating the cell cycle and proliferation, a decreased expression of p27 protein has been shown to be associated with a poor clinical outcome in various carcinomas.11, 26–28 In the present study, the patients with a combined decrease in PTEN and p27 protein expression had a poor prognosis in breast carcinoma. In the seven studies mentioned above where the relation between PTEN and p27 protein expression was evaluated using surgical specimens,17, 20–25 only one study on prostate carcinoma, by Halvorsen et al.,20 evaluated the prognostic value of the combination of PTEN and p27 protein expression. They demonstrated that the concomitant loss of PTEN and p27 protein expression was found in 18 of 103 (17.5%) prostate carcinomas and they determined this to be an independent prognostic factor based on a multivariate analysis.20 These findings were consistent with the results of the present study on breast carcinoma. These findings therefore suggest that a combined loss of PTEN and p27 function was associated with an aggressive phenotype in breast and prostate carcinomas.

In conclusion, a significant correlation was observed between PTEN and p27 protein expression in invasive ductal carcinoma of the breast. A reduced PTEN protein expression correlated with a low p27 protein expression. The finding that patients with a combined decrease in both protein expressions had a poor prognosis thus suggests a combined loss of PTEN and p27 function to be associated with an aggressive phenotype in breast carcinoma.

Acknowledgements

The authors thank Yuji Ogino and Yuji Shimoda of Sumikin Bioscience for expert technical assistance and Brian Quinn for review of the article.

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