SEARCH

SEARCH BY CITATION

Keywords:

  • breast;
  • breast neoplasms;
  • androgen;
  • androgen receptor;
  • phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND

Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser-213 (AR-Ser[P]-213) and Ser-650 (AR-Ser[P]-650), in breast cancer and evaluated their association with clinicopathological parameters.

METHODS

Immunohistochemistry was performed on primary and distant metastatic breast cancers and benign breast tissue using antibodies against AR, AR-Ser(P)-213, and AR-Ser(P)-650. The levels of cytoplasmic and nuclear expression were scored semiquantitatively using a histoscore.

RESULTS

Nuclear staining of AR was observed in all benign breast tissue and 67% of cancer cases. Nuclear and cytoplasmic AR-Ser(P)-213 was increased in breast cancers 2-fold (P = .0014) and 1.7-fold (P = .05), respectively, compared with benign controls, whereas nuclear and cytoplasmic AR-Ser(P)-650 expression was decreased in tumors by 1.9-fold and 1.7-fold (both P < .0001), respectively. Increased expression of nuclear or cytoplasmic AR-Ser(P)-213 was observed in metastatic breast cancers (1.3-fold, P = .05), ER-negative (2.6-fold, P = .001), and invasive ductal carcinoma (6.8-fold, P = .04). AR-Ser(P)-650 expression was downregulated in lymph node-positive breast cancers (1.4-fold, P = .02) but was upregulated in invasive ductal carcinomas (3.2-fold, P < .0001) and metastases (1.5-fold, P = .003). Moreover, in ER-negative breast cancers, nuclear AR-Ser(P)-650 was decreased (1.4-fold, P = .005), and cytoplasmic AR-Ser(P)-650 was increased (1.4-fold, P = .003).

CONCLUSIONS

AR and its phosphorylation at serines 213 and 650 are differentially expressed in breast cancer tumorigenesis and progression. Phosphorylation of AR at serines 213 and 650 is increased in ER-negative breast cancers, ductal carcinomas, and metastases and may have predictive value in breast cancer prognosis. Cancer 2013;119:2532–2540. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

The study of steroid receptor (SR) function in breast cancer biology has been largely focused on estrogen and progesterone receptors. However, recent studies have revealed the presence and importance of the androgen receptor (AR) in the biology of breast cancer (reviewed in references[1] and [2]). In fact, it may be possible to target the AR as a therapeutic strategy in estrogen receptor–negative breast cancer.[3, 4] Thus, in addition to the estrogen receptor, it is predicted that modulating the activity of ARs will provide novel prevention and treatment approaches for breast cancer patients.

Androgens act on target cells by binding to the androgen receptor, a ligand-dependent transcription factor, and are important in the development of male reproductive organs and prostate cancer. ARs have also been shown to have an inhibitory effect on estrogen receptor (ER)-α activity and to be a critical player in the growth and malignancy of breast cancer cells.[5] AR expression has been examined in several subtypes of breast cancer from patients with clinical follow-up. A study by Agoff et al showed high levels of AR expression in 49% of ER-negative and 89% of ER-positive cases.[6] AR expression is associated with a good prognosis in ER/progesterone receptor (PR)-negative cancers. Conversely, loss of ARs is associated with a poor prognosis in lymph node–positive ER/PR/HER2-negative breast cancers.[7] These findings are consistent with in vitro studies, in which AR activation with the agonist 5-α-dihydrotestosterone[8] or dehydroepiandrosterone sulfate (DHEAS)[9] inhibited cell growth in AR-positive breast cancer cell lines, suggesting that AR initiates a growth inhibitory effect in breast cancer. In evaluating the metastatic potential of breast cancer cells, ligand-activated ARs have also been reported to induce cell motility by changing both cellular morphology and E-cadherin expression.[10] Immunohistochemical studies on large cohorts of breast tumor samples have shown the potential for AR to be an effective biomarker for breast cancer survival.[11] Studies looking at different subsets and classes of breast cancer tumors based on immunohistochemical staining and gene expression microarray analysis have determined that AR plays an inherent role in cell signaling in breast cancer and that targeting AR could be a potential therapeutic strategy for treatment of some ER-negative cancers.[3, 4]

The expression and activity of steroid receptors, including ERs, PRs, and ARs, can be regulated by posttranslational modifications including phosphorylation, ubiquitination, acetylation, and sumoylation.[12] A large number of phosphorylation sites have been identified in these receptors, with the majority within the N-terminus. Site-specific phosphorylation of SRs has been shown to be modulated by a wide variety of kinases depending on the cellular context, affecting hormone sensitivity, receptor stability and localization, DNA binding, and cofactor interaction.[12] The expression and function of phosphorylated forms of AR have been reported in prostate cancer, but the biological implications of phosphorylated AR, particularly in breast cancer, remain largely unknown.

The androgen receptor can be phosphorylated at multiple serine residues. AR phosphorylation is increased in response to androgen at serines 16, 81, 256, 308, 424, and 650. In addition, Ser-94 is constitutively phosphorylated.[13, 14] Studies in prostate cancer cell lines show AR phosphorylation at Ser-650 (AR-Ser[P]-650) is enhanced by treatment with forskolin, epidermal growth factor, and phorbol-12-myristate-13-acetate, suggesting that AR phosphorylation may be intricately linked to signal transduction processes regulating tumor promotion and cell growth.[13] AR-Ser(P)-650 phosphorylation also plays an important role in nuclear export of AR in response to stress kinase signaling through p38 and JNK kinases.[15] In the prostate, expression of AR phosphorylated at Ser-213 (AR-Ser[P]-213) has been shown to be restricted to epithelial cells and specific developmental and cellular contexts (high levels of androgens and differentiated luminal cells).[16] It has been reported that Akt phosphorylates AR at serines 213 and 791 in vitro,[17, 18] and we have recently observed that serine 213 can also be phosphorylated by the PIM1 kinase. In addition, detection of AR-Ser(P)-213 was more prevalent in recurring compared with nonrecurring prostate cancers.[19] Interestingly, a recent study found that PIM1 kinase expression was higher in breast cancers compared with benign breast tissues and was associated with increased invasiveness, higher tumor grade, ER/PR/HER2-negative status, and poorer survival rates.[20]

In this study, immunohistochemistry was performed using antibodies against the androgen receptor (recognizing phosphorylated and nonphosphorylated [total] AR) and AR phosphorylated at serine residues 213 and 650. We characterized the expression of AR, AR-Ser(P)-213, and AR-Ser(P)-650 in breast cancer epithelial cells on a tissue microarray (TMA) and correlated AR expression and phosphorylation with clinicopathological parameters.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Breast Cancer Specimens

Tissue microarrays (TMAs) of 156 formalin-fixed, paraffin-embedded breast cancer cases were obtained from therapeutic or diagnostic procedures performed as part of routine clinical management between 1999 and 2004 at the Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York. The TMA includes primary (n = 104) and distant metastatic (n = 52) breast cancers with 3 tissue cores representing each case. The predominant site of disease and metastatic deposits was used for the TMA. The primary cases were not matched to the distant metastases cases. The clinicopathological parameters included age, tumor size and type, hormone receptor status, clinical stage, grade, lymph node status, and clinical follow-up. Tumors were graded according to the modified Scarff-Bloom-Richardson system. The H&E stain of the TMA was reviewed by 2 pathologists at MSKCC to confirm the tumor histology, grade, and stage based on the most recent American Joint Commission on Cancer (AJCC) cancer staging criteria.[21] ER, PR, and HER2 statuses were obtained from the original pathology reports (≥10% immunohistochemical staining for ER or PR was considered positive). Lymph node positivity was defined as nodes with macrometastases (>2.0 mm). The benign controls (n = 34) included 17 cases from mammoplasty and 17 cases from the benign breast tissue of cancer cases from therapeutic or diagnostic procedures performed as part of routine clinical management between 2009 and 2010 at New York University Langone Medical Center, New York, New York. Tissues were acquired according to each institution's institutional review board's policies.

Immunohistochemistry

Immunohistochemistry was performed using affinity-purified antibodies against AR, AR-Ser(P)-213, and AR-Ser(P)-650. Paraffin-embedded tissue sections were dewaxed in xylene, rehydrated, and washed in phosphate-buffered saline (pH 7.4). For antigen retrieval, paraffin sections were heated in a microwave oven (900 watts) in 10 mM citrate buffer followed by treatment with 3% H2O2 and blocked with 20% normal goat serum. Sections were then incubated with antibody against AR (1:250 dilution; N-20; Santa Cruz Biotechnology, Santa Cruz, CA), AR-Ser(P)-213 (1:100),[16] and AR-Ser(P)-650 (1:250) (unpublished results, S. Logan), followed by incubation with a biotinylated rabbit secondary (1:1000; Vector Labs, Burlingame, CA). An avidin-biotin complex was formed and developed using diaminobenzidine chromagen, followed by a counterstain with hematoxylin. Microscopy was performed on a Zeiss Axio Imager A2 microscope, and representative images were acquired using QCapture Pro Software.

Evaluation of Immunostaining

Immunohistochemical staining was examined and scored independently, in a blinded manner, by 2 observers using a semiquantitative weighted histoscore. The weighted histoscore represents staining intensity (negative, 0; weak, 1; moderate, 2; and strong, 3) and the percentage of positive cells within each intensity category, providing a score of 0-300.[22] Nuclear staining and cytoplasmic staining were scored separately. The final histoscore was determined for each case by taking the mean histoscore of all the present cores and was used for statistical analysis.

Statistical Analysis

The cases were divided into different groups according to clinicopathological parameters. Statistical analyses of the histoscores were performed by the unpaired Student t-test with Prism 4 software (GraphPad Software, Inc., La Jolla, CA). The standard deviations were calculated to estimate the degree of variation in each group. All testing was 2-tailed, with .05 as the level of significance. Cases with missing data were excluded from analyses. Based on the results of the univariate analysis, variables with significant P values were selected and evaluated with multivariate linear regression analysis to estimate their potential as independent predictors for clinical parameters. The multivariate linear regression analysis was performed with SPSS software package (IBM Inc., Armonk, NY).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Patient and Tumor Characteristics

The breast cancer patients ranged in age from 30 to 87 years old (median, 54 years). Of the 156 cancer cases, 104 cases were localized cancers (67%), and 52 cases were distant metastatic breast cancers (33%). The sites of metastatic breast cancer included bone (n = 17), lung (n = 9), brain (n = 8), ovary (n = 8), liver (n = 5), soft tissue (n = 3), and chest wall (n = 2). The majority of tumors were ductal carcinomas (n = 84), with a few lobular carcinomas (n = 15). Fifty-nine percent of tumors (n = 92) were high stage (T ≥ 2). Seventy-eight cases (50%) were ER-positive cancers, whereas 65 were ER-negative (42%). Eighty-three cases (53%) were recurrent cancer, either locally (n =31) or at a distant metastatic site (n =52). Immunohistochemical staining was performed, and the mean histoscores for AR, AR-Ser(P)-213, and AR-Ser(P)-650 are presented in Table 1.

Table 1. AR, AR-Ser(P)-213, and AR-Ser(P)-650 Expression in Clinical Subgroups
Clinical SubgroupAR-NAR-Ser(P)-213-NAR-Ser(P)-213-CAR-Ser(P)-650-NAR-Ser(P)-650-C
  1. Abbreviations: N, nuclear staining; C, cytoplasmic staining.

  2. Mean histoscores, standard deviations, and P values are shown. Bold indicates statistical significance using univariate analysis. The level of significance is P ≤ .05.

Tumor/benign     
Tumor (n = 152)67.7 ± 5.788.9 ± 5.329.6 ± 4.291.2 ± 5.274.5 ± 4.5
Benign (n = 34)12.4 ± 9.644.9 ± 11.817.1 ± 4.7170.5 ± 11.3125.4 ± 8.7
P< .0001.0014.05< .0001< .0001
Histologic subtype     
Ductal (n = 84)63.9 ± 7.885.0 ± 6.534.7 ± 6.077.9 ± 6.682.1 ± 5.3
Lobular (n = 15)100.6 ± 19.374.6 ± 11.35.1 ± 4.194.6 ± 13.525.8 ± 9.3
P.0757.5197.0413.3147< .0001
ER status     
Negative (n = 65)42.2 ± 7.186.7 ± 8.245.7 ± 7.674.5 ± 7.491.0 ± 7.0
Positive (n = 78)90.7 ± 8.391.8 ± 7.217.5 ± 4.5104.1 ± 7.363.1 ± 5.7
P< .0001.6459.0012.0054.0025
PR status     
Negative (n = 85)53.0 ± 6.893.0 ± 7.541.0 ± 6.783.0 ± 6.785.0 ± 6.0
Positive (n = 56)96.0 ± 10.087.0 ± 7.615.0 ± 3.9110.0 ± 8.663.0 ± 7.1
P.0005.5947.0012.0461.02
HER2 status     
Negative (n = 107)69.0 ± 7.187.8 ± 6.425.9 ± 4.786.6 ± 6.069.7 ± 5.3
Positive (n = 13)73.5 ± 20.1101.9 ± 18.052.2 ± 16.2113.8 ± 18.197.3 ± 10.4
P.8369.4731.1415.1759.029
Tumor stage     
≤1 (n = 13)76.4 ± 19.4115.9 ± 16.236.0 ± 12.894.2 ± 12.3103.5 ± 17.7
≥2 (n = 89)97.4 ± 22.884.4 ± 5.929.0 ± 5.877.8 ± 6.767.4 ± 5.3
P.4936.0627.6603.3546.0187
Lymph nodes involved     
0 (n = 25)56.7 ± 14.8100.4 ± 12.242.2 ± 13.385.7 ± 12.295.9 ± 10.0
≥1 (n = 81)75.7 ± 8.181.8 ± 6.524.6 ± 4.982.5 ± 6.966.6 ± 5.9
P.2511.1632.1282.8241.0162
Distant metastases     
Yes (n = 52)75.6 ± 9.8102.6 ± 10.829.9 ± 7.0117.5 ± 9.376.9 ± 8.2
No (n = 101)65.6 ± 7.081.2 ± 5.528.9 ± 5.278.0 ± 5.973.2 ± 5.3
P.4091.05.9077.0003.7007
Recurrence     
No (n = 64)65.4 ± 9.189.5 ± 7.431.0 ± 6.985.5 ± 7.777.7 ± 7.1
Yes (n = 33)70.7 ± 12.6667.3 ± 8.328.2 ± 8.865.0 ± 8.562.5 ± 7.9
P.7378.0686.8081.0972.1838
Disease-specific survival     
NED/AWD (n = 77)70.1 ± 8.686.9 ± 6.51927.77 ± 6.084.7 ± 7.273.6 ± 6.5
DOD (n = 38)58.5 ± 9.588.0 ± 10.832.9 ± 8.185.4 ± 9.477.3 ± 7.5
P.4038.923.6174.9516.9428

Androgen Receptor, AR-Ser(P)-213, and AR-Ser(P)-650 Expression in Benign and Malignant Breast Cells

We first examined the expression profiles of nuclear and cytoplasmic ARs in benign and malignant breast epithelial cells, including primary and metastatic cancers. We observed nuclear staining of ARs in all the benign breast tissue (100% cases), with 105 of 156 cancers (67%) showing nuclear AR staining (Fig. 1A). The mean expression of nuclear AR was decreased in breast cancers by 1.8-fold compared with benign tissues (P < .0001). Cytoplasmic detection of total AR under the staining conditions of the tissue microarray was negative or weak and therefore not scored.

image

Figure 1. AR and phosphorylated AR expression in benign breast tissue. Immunohistochemical staining showing the representative expression patterns of AR (A), AR-Ser(P)-213 (B), and AR-Ser(P)-650 (C) in benign breast tissue. Images are at 20× magnification, and inserts are at 40× magnification.

Download figure to PowerPoint

Nuclear and cytoplasmic expression of AR-Ser(P)-213 and AR-Ser(P)-650 was observed in both malignant and benign breast tissue (Fig. 1B,C). However, the patterns of phosphorylated AR expression were distinct. Nuclear staining and cytoplasmic staining of AR-Ser(P)-213 were both increased in breast cancers compared with benign controls by 2-fold (P = .0014) and 1.7-fold (P = .05), respectively. Conversely, nuclear AR-Ser(P)-650 expression and cytoplasmic AR-Ser(P)-650 expression were both significantly decreased in tumors compared with benign tissue by 1.9-fold and 1.7-fold (P < .0001), respectively (Table 1).

AR, AR-Ser(P)-213, and AR-Ser(P)-650 Expression in ER-Positive Versus ER-Negative Breast Cancers

The breast cancer cases were grouped according to the available clinicopathological parameters, and detection of AR, AR-Ser(P)-213, and AR-Ser(P)-650 was analyzed. Among the ER-negative cancers, AR nuclear staining was detected in 34 cases (52%), compared with 64 cases (82%) of ER-positive cancers. ER-negative cancers, which in general have a more aggressive clinical course, showed increased expression in cytoplasmic AR-Ser(P)-213 (2.6-fold, P = .001) and AR-Ser(P)-650 (1.4-fold, P = .003) compared with the ER-positive breast cancers (Fig. 2). The ER-negative breast cancers also showed decreased expression of nuclear AR (2.1-fold, P < .0001) and nuclear AR-Ser(P)-650 (1.4-fold, P = .005). There was no significant difference in the nuclear expression of AR-Ser(P)-213 between ER-negative and -positive cancers. These data indicate that the phosphorylation of AR at Ser-213 and Ser-650 may play a role in aggressive ER-negative tumors. We also observed a similar expression pattern in PR-negative breast cancers (Table 1).

image

Figure 2. AR and phosphorylated AR expression in ER-positive versus ER-negative breast cancer. Immunohistochemical staining showing the expression patterns of AR (A and D), AR-Ser(P)-213 (B and E), and AR-Ser(P)-650 (C and F) in ER-positive and ER-negative breast cancers. AR-Ser(P)-213 and AR-Ser(P)-650 cytoplasmic staining is increased in ER-negative compared with ER-positive cancers. Images are at 20× magnification, and inserts are at 40× magnification.

Download figure to PowerPoint

AR-Ser(P)-213 and AR-Ser(P)-650 Expression in Histologic Subtypes of Breast Cancer

Compared with invasive lobular carcinoma (n = 15), invasive ductal carcinoma (n = 84) exhibited increased expression of cytoplasmic AR-Ser(P)-213 (6.8-fold, P = .04) and AR-Ser(P)-650 (3.2-fold, P < .0001; Fig. 3B, C, E, F). These data are consistent with the findings from ER-negative breast cancers, indicating that cytoplasmic AR phosphorylation may correlate with more aggressive breast cancers. There were no significant differences in the expression of nuclear AR, AR-Ser(P)-213 or AR-Ser(P)-650 between ductal and lobular-type cancers (Table 1).

image

Figure 3. AR and phosphorylated AR expression in invasive lobular carcinoma, invasive ductal carcinoma, and distant metastasis. Immunohistochemical staining showing the staining patterns of AR (A, D, and G), AR-Ser(P)-213 (B, E, and H), and AR-Ser(P)-650 (C, F, and I) in invasive lobular carcinoma, invasive ductal carcinoma, and distant metastasis. There is increased cytoplasmic AR-Ser(P)-213 and AR-Ser(P)-650 staining in ductal carcinomas, and there is also increased nuclear AR-Ser(P)-213 and AR-Ser(P)-650 in breast metastases. Images are at 20× magnification, and inserts are at 40× magnification.

Download figure to PowerPoint

AR-Ser(P)-213 and AR-Ser(P)-650 Expression in High-Stage Tumors

According to the AJCC Cancer Staging Manual (7th ed.) criteria, 92 cases (59%) were high stage (T ≥ 2). We observed decreased cytoplasmic AR-Ser(P)-650 expression in T ≥ 2 cancers by 1.7-fold (P = .02) compared with T ≤ 1. The nuclear expression of AR-Ser(P)-213 was decreased in high-stage breast cancers (1.4-fold), but the difference was not statistically significant (P = .06). The expression of nuclear AR, nuclear AR-Ser(P)-650, and cytoplasmic AR-Ser(P)-213 did not reach statistical significance comparing T ≤ 1 and T ≥ 2 (Table 1).

AR-Ser(P)-213 and AR-Ser(P)-650 Expression in Tumor Progression

A total of 81 breast cancer cases (52%) had metastasized to the lymph nodes at the time of diagnosis. We observed decreased cytoplasmic AR-Ser(P)-650 expression (1.4-fold, P = .02) in breast cancers with positive metastatic lymph nodes compared with cancers with negative lymph node status. There was no significant difference in the expression of nuclear AR, nuclear AR-Ser(P)-650, and nuclear and cytoplasmic AR-Ser(P)-213 between lymph node–positive versus –negative breast cancers.

Distant metastases to other organs were represented in 52 breast cancer cases (33%). Breast cancers with distant metastasis had higher expression of nuclear AR-Ser(P)-213 (1.3-fold, P = .05) and of nuclear AR-Ser(P)-650 (1.5-fold, P = .0003); see Figure 3H,I. There was no significant difference in the expression of nuclear AR, cytoplasmic AR-Ser(P)-213, and cytoplasmic AR-Ser(P)-650 between primary breast cancers and those with distant metastases. These data suggest that phosphorylation of AR (Ser-213 and Ser-650) in the nucleus may play a role in metastasis.

Ninety-seven of the 104 primary tumor cases with and without lymph node involvement were followed for disease recurrence. Ninety-six of the primary cancers and 23 metastatic cases were followed for tumor progression. The follow-up interval ranged from 1 to 171 months, with a mean of 43 months. During this time, 33 patients with primary tumor (34%) developed recurrent cancer, locally or at a distant site. As for tumor progression, 58 patients (49%) remained disease free (NED), 19 patients (16%) were alive with recurrent disease (AWD), and 22 patients (18%) were deceased from recurrent cancer (DOD). We did not observe any significant difference in the expression pattern of AR, AR-Ser(P)-213, or AR-Ser(P)-650 in primary versus recurrent breast cancers or in the DOD versus NED/AWD groups (Table 1).

Multivariate Analysis of AR Expression and Phosphorylation

Clinical parameters found to be statistically significant via univariate analysis (tumor histologic type, ER status, pathologic stage, lymph node status, and distant metastasis; see Table 1) were evaluated using multivariate linear regression analysis for AR and phosphorylated AR expression in the breast cancer cases (Table 2). The fold change in the mean histoscores of AR or phosphorylated AR expression within each clinical parameter was used to examine if ARs or phosphorylated ARs are independent predictors associated with the clinical parameter. Increased expression of AR-Ser(P)-213 correlated with ductal carcinoma, ER-negative status, and distant metastases using univariate analysis, but multivariate analysis of AR-Ser(P)-213 and these parameters did not maintain statistical significance. However, decreased nuclear AR-Ser(P)-650 expression was an independent predictor of ER status (P = .006), and increased expression was predictive of distant metastasis (P = .007). In addition, increased cytoplasmic AR-Ser(P)-650 expression was associated with histological subtype (ductal carcinoma, P = .007).

Table 2. Fold Change in Expression of AR, AR-Ser(P)-213, and AR-Ser(P)-650 in Clinical Subgroups
Subgroup RatioAR-NAR-Ser(P)-213-NAR-Ser(P)-213-CAR-Ser(P)-650-NAR-Ser(P)-650-C
  1. Abbreviations: N, nuclear staining; C, cytoplasmic staining.

  2. P values from univariate analysis (U) and multivariate analysis (M) are shown. Bold indicates statistical significance using multivariate analysis. P ≤ .05 was considered statistically significant.

Tumor/benign0.5 P < .0001 (U)1.9 P = .0025 (U)1.8 P = .05 (U)0.5 P < .0001(U)0.6 P < .0001(U)
ER−/ER+0.5 P < .0001 (U)0.9 P = .65 (U)2.61 P = .0012(U)/.71 (M)0.72 P = .0054(U)/.006(M)1.44 P = .0025 (U)/.295 (M)
Her2−/Her2+0.9 P = .84 (U)0.9 P = .47 (U)0.5 P = .14 (U)0.8 P = .18 (U)0.72 P = .029 (U)/.78 (M)
Ductal/lobular0.6 P = .076 (U)1.1 P = .52 (U)6.79 P = .0413 (U)/.338(M)0.8 P = .31 (U)3.18 P < .001 (U)/.007(M)
T stage 2-4/T stage 11.3 P = .49 (U)0.73 P = .0627 (U)/.267 (M)0.8 P = .66 (U)0.8 P = .35(U)0.65 P = .0187 (U)/.101 (M)
Lymph node+/lymph node−1.3 P = .25 (U)0.8 P = .16 (U)0.6 P = .13(U)1.0 P = .82(U)0.69 P = .0162 (U)/.076 (M)
Distant met+/Distant met−1.2 P = .41 (U)1.26 P = .05 (U)/.074 (M)1.0 P = .9 (U)1.51 P = .003 (U)/.007 (M)1.0 P = .7 (U)

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Steroid hormone receptors and their coactivators play an important role in breast cancer development and progression. Although the functions of ARs and androgens in breast cancer are still unclear, multiple studies in epidemiology and human breast cancer implicate androgens in breast carcinogenesis. High levels of androgen in postmenopausal women have been correlated with an increased incidence of breast cancer.[23, 24] A significant correlation between AR status and tumor grade has been reported.[25] Also, increased expression of ARs in ER-negative breast cancer correlated to increased age, postmenopausal status, tumor grade, tumor size, and HER-2/neu overexpression has been shown.[6] However, increased AR expression has also been reported in ER/PR-positive breast cancers with low histological grades.[26, 27] Our results are consistent with the studies by Secreto et al and Park et al: 64 of 77 of ER-positive breast cancers (83%) and 34 of 62 of ER-negative breast cancer cases (55%) expressed AR. The ER-positive cancers also showed significantly higher expression of AR than did the ER-negative cancers.

Compared with other studies in which AR expression was dichotomized as positive or negative, we used a histoscore, which combines the intensity score with the percentage of positive staining, which we believe more accurately reflects the expression of AR and its phosphorylation status in breast cancer. We also used a collection of benign breast tissue as controls. Our analysis of the expression of AR and phosphorylated AR in breast cancer and benign controls suggests that AR plays an active role in breast cancer tumorigenesis and progression.

We observed decreased total AR expression in breast cancer compared with benign controls and in ER-negative cancers compared with ER-positive ones. Importantly, our data indicate that there are increased levels of phosphorylated AR in the cytoplasm compared with the nucleus in breast cancer, especially in ER-negative cancer and invasive ductal type carcinoma. Last, phosphorylated AR expression is increased, especially AR-Ser(P)-650, in nuclei of breast cancers with distant metastases. All these data support the role of the androgen receptor and its phosphorylation in the tumorigenesis and progression of breast cancer.

Our results show that both nuclear and cytoplasmic AR phosphorylation at Ser-213 is increased in breast cancer, suggesting that phosphorylation could alter gene expression in the nucleus and may also play a nongenomic role in the cytoplasm to promote breast cancer progression. This idea is consistent with the increased nuclear expression of AR-Ser(P)-213 observed in breast cancer with distant metastases. We also observe increased cytoplasmic expression of AR-Ser(P)-213 in the ER-negative cancers and invasive ductal carcinomas, which generally have a more aggressive clinical course. Invasive ductal carcinomas are traditionally considered more aggressive than invasive lobular carcinomas, but recent evidence showed that survival rates, stage for stage, may be similar.[28, 29] Altogether, our data suggest a role of AR phosphorylation at Ser213 in tumor progression. However, it should be noted that under multivariate analysis, these parameters did not maintain statistical significance, likely because of sample size and the relationship between the clinical parameters and aggressive breast cancers.

Our data show that both nuclear and cytoplasmic AR phosphorylation at Ser-650 is generally decreased in breast cancer cells, suggesting that regulation of the receptor plays a role in breast cancer progression. There were specific instances in which AR-Ser(P)-650 expression was increased. AR phosphorylation at Ser-650 was increased in the cytoplasm of ER-negative breast cancer cells and ductal carcinoma, although the nuclear AR phosphorylation at Ser-650 was decreased, supporting the idea that phosphorylation at Ser-650 may be important in nuclear export of AR in more aggressive breast cancers. In addition, nuclear expression of AR-Ser(P)-650 was increased in cancers with distant metastases, suggesting that phosphorylation in aggressive disease may direct the AR to activate transcription of genes that promote metastases. Of note, it is possible that results from the recurrences or metastases could be complicated by prior systemic and/or local therapy.

Recently, De Amicis et al reported the role of AR in tamoxifen-resistant breast cancers. Gene expression profiling showed that AR mRNA was increased, whereas ER-α mRNA was reduced in tamoxifen-resistant tumors, and overexpression of AR in the MCF-7 cell line (ERα-positive) caused them to become tamoxifen resistant, which could be reversed with an AR antagonist.[30] Although the expression of AR and ER was inversely correlated with histopathological grade, AR expression still remained significantly higher than ER expression. The prevalence of AR expression in breast cancer, particularly in triple-negative or hormone-resistant disease, makes it an attractive therapeutic target.[31]

In summary, we report that the androgen receptor and its phosphorylation at serines 213 and 650 may play a role in the development and progression of breast cancer. To our knowledge, this is the first study examining the phosphorylation status of the androgen receptor in breast cancers. Future studies will focus on the mechanism of AR phosphorylation in the tumorigenesis and progression of breast cancer. It may also be of great interest to study the pattern of other serine residues and their significance in breast versus prostate cancer progression.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

This study was supported by the NYU Clinical and Translational Science Institute (1UL1RR029893), a seed fund to Peng Lee, and NIH R01CA112226 (to Susan K. Logan).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES