Reduced Nuclear Expression of Transcription Factor AP-2 Associates with Aggressive Breast Cancer

Patients.

The present prospective study consisted of 520 breast cancer patients of the Kuopio Breast Cancer Project (30, 31, 32, 33) . All of the women with a susceptible breast lump in the project catchment area between April 1990 and December 1995 were invited to Kuopio University Hospital for further examinations and final diagnosis. Women willing to participate in the project were interviewed and examined by a trained nurse before any diagnostic procedures. The participation rate of the patients with diagnosed breast cancer was 98%. A healthy population control matched by age (±5 years) was drawn for each patient from the Finnish National Population Register covering the same catchment area. Altogether, 479 invasive and 41 noninvasive carcinomas were included in the project. After surgical treatment, the patients were offered adjuvant chemotherapy and/or hormonal therapy and radiotherapy depending on the mode of the surgery, the patient’s menopausal status, and the stage of the disease, according to the national guidelines (34) . The stage was assessed using UICC classification (35) . Patients with noninvasive carcinomas, earlier breast cancer, metastatic disease, or insufficient tumor material were excluded from the present study. Thus, 420 patients with sufficient primary tumors and complete clinical histories were included in the study. The mean age of the 420 patients was 59.1 years and the median 56.7 years (range, 23.3–91.6 years). The mean follow-up time was 55.0 months and the median, 57.3 months (range, 1.2–115.1 months). During the first 5 years of follow-up, 76 patients (18%) had a recurrence, 50 patients (12%) died of breast cancer and 37 patients (9%) died of other causes. The overall 5-year survival rate was 76%. The 5-year RFS rate of the patients was 79% and the BCRS, 85%. The 5-year survival of excluded stage IV patients (n = 17) was 24%. The clinicopathological data of the patients are summarized in Table 1⇓ .

Histology.

The tumor samples were fixed in 10% buffered formalin and embedded in paraffin. The histological diagnosis was confirmed by reviewing one to four original sections of the primary tumor. All of the tumors were simultaneously re-evaluated for histological type and grade by two senior pathologists, who were unaware of the clinical data. The most representative blocks were selected to be cut into new 5-μm thick sections for immunohistochemical analyses.

Immunohistochemistry.

The sections were deparaffinized in xylene, rehydrated in ethanol, and washed twice with distilled water. For better antigen retrieval, the samples were boiled in a microwave oven for three times for 5 min in a citrate buffer (pH 6.0). Endogenous peroxidases were blocked by 5% hydrogen peroxidase treatment for 5 min. The samples were washed with PBS (pH 7.2) and incubated in 1.5% normal goat serum for 25 min to prevent nonspecific antigen binding. The samples were incubated with the primary antibody, a rabbit polyclonal antibody for human AP-2α (C-18, specific for AP-2α, AP-2β, and AP-2γ; Santa Cruz Biotechnology 2002, Santa Cruz, CA) used at a working dilution of 1:2000 overnight at 4°C. Before applying the secondary antibody, the samples were washed twice with PBS. The slides were incubated for 35 min with the biotinylated secondary antibody, followed by a wash and 45-min incubation in an avidin-biotinylated peroxidase complex-reagent (Vectastain Rabbit ABC Elite kit; Vector Laboratories, Burlingame, CA). AP-2 expression was visualized with a 5-min diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) treatment. The slides were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with DePex (BDH Ltd, Poole, United Kingdom). Each staining series had positive and negative control slides (Fig. 1⇓ , F1 and F2), which stained as expected. In addition, strongly positive inflammatory cells in the tumors served as internal controls.

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Fig. 1.

A, ductal carcinoma, grade I. Nuclear staining (arrow). B, strong nuclear (arrows) AP-2 staining in ductal carcinoma, grade II. C, ductal carcinoma, grade III. Nuclear (arrow) and cytoplasmic (asterisk) expressions. D, cytoplasmic expression (asterisk) with a few positive nuclei in ductal carcinoma, grade III. E1–E2, lobular carcinoma, nuclear staining. F1–F2, negative control slides of samples B and C, respectively. A–D, E2–F2: bar, 40 μm; E1: bar, 80 μm.

Western Blot.

During the surgery a fresh breast cancer specimen was obtained from some of the patients. Immediately after resection, the samples were covered with OCT, cooled in liquid isopentane, cooled in liquid nitrogen, and re-stored at −70°C until analysis. Four tumor samples with high AP-2 expression in immunohistochemical staining were selected for Western blot analyses. The same antibody as in immunohistochemistry was used to detect AP-2.

The frozen tissue samples were homogenized, incubated on ice for 30 min, and centrifuged at 13,000 rpm for 15 min at 4°C. Homogenization was performed using lysis homogenization buffer [0.02 m Tris-HCl, 0.002 m EDTA, 0.1 m NaCl (pH 8.0)] with protease inhibitors (1 mmol VO₄, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmol phenylmethylsulfonyl fluoride). After centrifugation, the samples were either refrigerated and stored at −70°C until analysis or prepared immediately for gel transfer. In the latter case, samples were heated at 100°C for 5 min with equal amount of SDS sample buffer [0.125 m Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol (pH 6.8)]. Next, the centrifuged samples were separated on an SDS-polyacrylamide gel (100 V for 3 h at room temperature) and transferred onto a nitrocellulose membrane (Hybond ECL nitrocellulose membrane; Amersham Pharmacia Biotech United Kingdom Limited, Little Chalfant, Buckinghamshire, United Kingdom) by electrophoresis (100 V for 2 h at 4°C). Filters were blocked with 0.1% soy buffer solution [0.137 m NaCl, 1.47 mm KH₂PO₄, 8.1 mm Na₂HPO₄, 2.7 mm KCl, 0.1% Tween, and 0.1% soy (pH 7.4)] and then incubated with the primary antibody diluted 1:5000 for 30 min. After a wash with PBS-Tween buffer, the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was applied onto the filter diluted 1:12,000 and was incubated for 45min. After a wash, the signals were visualized using a commercial substrate kit (Supersignal West Pico Trial kit; Pierce, Perbio Science Deutschland GmbH, Bonn, Germany). A prestained marker (Bio-Rad Laboratories, Hercules, CA) and Trial Mix marker (Novagen, CN Bioscience, Ltd., Beeston, Nottingham, United Kingdom) were run in parallel to the samples. A benign breast tissue sample served as a positive control.

Scoring of Immunoreactivity.

The specimens were analyzed by three observers (J. P., K. R., V-M. K.) unaware of the patients’ clinical outcome. Discrepancies between the observers were found in less than 10% of the slides examined, and consensus was reached on a further review. All of the tumor cells with detectable nuclear staining were considered as positive. For statistical analyses, the percentage distribution of stained tumor cell nuclei in the sample was divided into low (<80%) or high (≥80%) expression groups using the median as a cutoff value. Other cutoff values were also tested (e.g., mean, and values between the 40th and 50th percentiles), but the median value was chosen because it does not introduce a bias that could be caused by the use of a minimum P approach (36) . Cytoplasmic expression was considered positive if >10% of cells in the tumor area were stained.

Statistical Analyses.

The statistical analyses were carried out by using the SPSS for Windows 9.0 program (SPSS Inc., Chicago, IL). Differences between benign and malignant tissues were examined with a nonparametric Mann-Whitney U test, in which the variables were considered as continuous. The associations between AP-2 immunohistochemistry and clinicopathological parameters were tested with contingency tables and a χ² test. The univariate survival analyses were performed using the Kaplan Meier’s log-rank analysis, and the independent prognostic value of variables was further examined with Cox’s regression analysis. Ps ≤ 0.05 were considered as significant in the analyses. In the Cox’s multivariate analysis, the Enter-method was used with an additional removal limit of P < 0.10. Both BCRS and RFS were examined. The recurrence-free time was defined as a time between the diagnosis and the date of the first local recurrence or a distant metastasis, whichever appeared first. Patients who remained healthy or died without breast cancer during the follow-up were censored at the time of the last control examination or death.

AP-2 Expression.

To compare the expression of AP-2 in malignant tumors and in benign ductal breast epithelium, an additional set of 24 samples containing benign epithelium were stained for AP-2. The nuclear expression was significantly reduced in carcinomas (P < 0.001) compared with that in benign breast epithelium. The staining pattern for AP-2 in carcinomas was mainly nuclear (Fig. 1, A and B)⇓ although cytoplasmic expression was detectable in 47% of the cases (Fig. 1, C and D)⇓ . Lobular carcinomas expressed more often AP-2-positive nuclear staining (63% of the cases; Fig. 1, E1 and E2⇓ ) than ductal (48% of the cases; Fig. 1, A–C⇓ ) or other types of (45% of the cases) carcinomas (P = 0.048; Table 2⇓ ).

Western Blot Analysis.

In Western blotting, the primary antibody recognized a protein M_r of about 50,000, which has been reported to be the size of AP-2α. The results of Western blotting are shown in Fig. 2⇓ .

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Fig. 2.

Western blot analysis for AP-2. Four representative AP-2-positive tumor samples (T) and one benign AP-2 positive breast sample (N) were included in the analysis. All of the samples showed a band M_r about 50,000, which is the same as given for AP-2α. Trial Mix Protein marker (M) was used as a size marker. kDa, M_r in thousands.

Relationship of AP-2 Expression to the Clinicopathological Data.

Low nuclear AP-2 expression was significantly associated with advanced stage (P = 0.002), axillary lymph node positivity (P = 0.012), poor differentiation of the tumor (P = 0.001), and recurrences (P = 0.003). Nuclear AP-2 expression was not associated with ER and PR receptor status. The results are summarized in Table 2⇓ . Cytoplasmic positivity, instead, was more often present in ER- (P = 0.005) and PR- (P = 0.043) negative tumors and was, in addition, associated with ductal carcinoma type (P = 0.001) and poor differentiation (P = 0.012).

Univariate Survival Analysis.

In univariate analyses, low nuclear AP-2 expression was a significant predictor of shorter RFS (Fig. 3A)⇓ . The 5-year RFS in patients with high nuclear AP-2 expression was 85% compared with 73% in the low expressing group (P = 0.0028). Similarly, in patients with axillary lymph node metastases, low nuclear AP-2 expression was related to shorter RFS (P = 0.0108; Fig. 3B⇓ ). A nonsignificant trend was observed between reduced nuclear AP-2 expression and poor BCRS (P = 0.0964). The other prognostic factors of shorter RFS and BCRS were advanced stage (P < 0.0001 for both), lymph node positivity (P < 0.0001 for both), and poor differentiation (P = 0.0314 for BCRS and P = 0.0498 for RFS; Table 3⇓ ). ER (P < 0.0001) and PR (P = 0.0012) negativity were also related to poor BCRS (Table 3)⇓ .

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Fig. 3.

The P of RFS according to nuclear AP-2 expression (median as a cutoff value). A, in all of the patients; B, in the lymph node-positive patients.

For further evaluation, nuclear AP-2 staining was combined with cytoplasmic expression of the protein. Interestingly, not only low nuclear expression of AP-2 but also cytoplasmic involvement was associated with decreased survival when compared with high, strictly nuclear AP-2 expression. High nuclear expression without cytoplasmic involvement predicted longer RFS (P = 0.0050; Fig. 4A⇓ ) and BCRS (P = 0.0314) in the whole cohort and in the lymph node-positive patients [P = 0.0030 for RFS (Fig. 4B)⇓ , and P = 0.0411 for BCRS]. Cytoplasmic expression alone did not have prognostic value for BCRS or RFS.

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Fig. 4.

Probability of RFS according to combined (nuclear/cytoplasmic) expression of AP-2. The group “nuclear” contains those patients with high nuclear AP-2 expression without cytoplasmic involvement. The group titled as “others” contains all of the patients with low nuclear AP-2 expression and those with positive cytoplasmic staining. A, all of the patients; B, the lymph node-positive patients.

Multivariate Survival Analysis.

Complete data were available for 409 patients for recurrence-free and 403 for BCRS analyses. The Cox’s multivariate analyses included statistically significant variables (stage, histological grade, and AP-2 expression in RFS analysis; stage, histological grade, and ER- and PR-status in BCRS analysis) derived from the univariate analyses. The independent predictors of short RFS were low nuclear AP-2 expression (P = 0.0292) and advanced stage (P = 0.0001). The BCRS was independently predicted by stage (P < 0.0001) and ER status (P = 0.0321). Similarly, in the lymph node-positive patients (n = 166), RFS was independently predicted by stage (P = 0.0110) and nuclear AP-2 status (P = 0.0151), which were, in addition, the only significant variables derived from univariate survival analysis in this group. The results are presented in Table 4⇓ . In the lymph node-negative patients (n = 244), the number of events was too low (27 recurrences and 14 breast cancer deaths) to have statistical power in the survival analysis.