Introduction

The seven signal transducer and activator of transcription (STAT) molecules are effectors of hormonal or cytokine stimulation through receptors. In unstimulated cells, STAT proteins exist largely in the cytoplasm as latent transcription factors. In response to exogenous factors, specific STATs undergo tyrosine phosphorylation, nuclear translocation, and DNA binding resulting in the transcriptional activation of distant target genes. There are many genes regulated by the STAT pathway that are required for cell growth, survival, differentiation, and motility [7].

STAT 5a, also called mammary growth factor, was isolated from prolactin-stimulated mammary cells, and is activated through the binding of prolactin to its receptor [17]. STAT 5a has been shown to be involved in integrin-mediated c-fos transcription and integrin-mediated adhesion affects control of cell cycle entry and inhibition of apoptosis [5]. In knockout mice that lack STAT 5a, the mammary gland does not develop properly [1, 14]. STAT 5a-deficient mice develop normally, but lobuloalveolar growth during pregnancy is aberrant [15].

It has been suggested that STAT 5a contributes to normal proliferation, and it has been shown that STAT 5a is essential for mammary gland differentiation in mice [15]. Furthermore, known defects in the Janus Kinase (JAK)/STAT pathway have led to the inhibition of growth restraint [19]. STAT 5a is also involved in adipocyte differentiation, its expression correlating with lipid accumulation, and it can be activated in the stroma by growth hormone and the epidermal growth factor [9, 24]. Recently, STAT 5a activation in breast cancer has been associated with a favorable prognosis [18].

Using a monoclonal antibody, we immunohistochemically tested different examples of breast disease for the expression of STAT 5a in comparison to normal breast ductal and lobular epithelial cells.

Materials and methods

For this study, 100 formalin-fixed, paraffin-embedded tissues representing various lesions of the breast were obtained from the files of the Armed Forces Institute of Pathology. The sections were reviewed for the presence of normal breast ductal and lobular epithelium, low-risk ductal intraepithelial neoplasia (DIN) (usual ductal hyperplasia), DIN 1 (atypical ductal hyperplasia), lobular intraepithelial neoplasia (LIN) (atypical lobular hyperplasia or lobular carcinoma in situ), DIN 1–3 (ductal carcinoma in situ), and invasive carcinoma [27]. Specimens were sectioned at 6 μm and then assayed with an antibody to STAT 5a (clone ST5a-2H2, Zymed Laboratories, South San Francisco, CA). Immunohistochemical analysis was performed as previously described [4]. Briefly, formalin-fixed, paraffin-embedded sections were heated at 70°C for 30 min and placed into a prewarmed pressure cooker in a solution of Reveal (Biocare Medical, Concord, CA) in which deparaffinization and antigen retrieval were performed simultaneously at 20 lb/in.2 for 3 min. Following depressurization, sections were placed into a prewarmed fresh solution of Reveal for 30 min before being rinsed in warm water. Endogenous peroxide and oxidative compounds were quenched by incubation in 5% H2O2 in methanol. Sections were rehydrated in Tris-buffered saline (TBS Plus, Biocare Medical) with 0.1% Tween 20 (DakoCytomation, Carpinteria, CA) added, prior to antibody application. Primary antibody incubations were performed for 60 min, with the ST5a-2H2 antibody diluted in TBS–Tween at 1:400. Detection reagents (ABC Universal Elite, Vector Laboratories Inc., Burlingame, CA) were applied for 45 min each, and all dilutions and intervening rinses were with TBS–Tween. Diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) (0.08%) with 0.025% H2O2 added in a solution of TBS–Tween (DAB) served as the chromogenic substrate. Sections were allowed to develop in DAB for 12 min, counterstained with hematoxylin, dehydrated, and coverslipped with Permount (Fisher Chemical Co., Pittsburgh, PA) in xylene.

Assayed sections were examined for accurate interpretation of positive signal on a qualitative intensity scale. Tissues were regarded as positively expressing STAT 5a when qualitative intensity was at least 2 on a 4-point scale, and more than 20% of the cells were reactive. The cell type along with reaction product distribution was noted.

Results

The results are listed in Table 1. In normal mammary gland epithelium, STAT 5a was found to exist in the cytoplasm and the nucleus of about 80% of the luminal breast epithelial cells in the lobules and larger ducts (Fig. 1). Myoepithelial cells failed to demonstrate any STAT 5a, and stromal cell fibroblasts were also nonreactive. STAT 5a was, as expected, found in endothelial cells, adipocytes, and leukocytes [2, 12, 29].

Table 1 STAT 5a reactivity in various examples of breast epithelium
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Fig. 1
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a A normal breast lobule showing positive decoration for the presence of STAT 5a by immunohistochemistry (200×). b Normal breast lobule, immunohistochemical demonstration of STAT 5a in the cytoplasm and nucleus (400×)

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STAT 5a was detected in the cytoplasm and nucleus of epithelial cells in cases of usual ductal hyperplasia and in benign neoplasms such as fibroadenoma. However, in atypical proliferations, ductal carcinoma in situ (DIN 1–3) and in invasive carcinomas, the STAT 5a protein (with rare exceptions) was either not expressed or greatly reduced (Figs. 2, 3 and 4). It was absent in all cells with apocrine differentiation but was readily detected in cells undergoing secretory or lactational change. The reactivity in the secretory cells was often more intense than that seen in the surrounding normal cells, perhaps signaling a higher concentration of antigen in these cells (Fig. 5). Although most examples of LIN did not express STAT 5a, a few examples demonstrated some reduced reactivity. Also, two tubular carcinomas and three invasive carcinomas with squamoid features and inflammation exhibited focal reactivity (data not shown). This may be secondary to increased background STAT 5a in these cases, since it occurred only when the adjacent normal epithelial and inflammatory cells also showed increased STAT 5a expression as compared to STAT 5a-negative carcinomas. All control sections assayed with a normal mouse IgG in substitution for the STAT 5a antibody were nonreactive.

Fig. 2
figure 2

a Hematoxylin and eosin (H&E) stain of a focus of low-risk DIN (usual ductal hyperplasia) (200×). b Immunohistochemical demonstration of STAT 5a in low-risk DIN (usual ductal hyperplasia) (200×). c H&E stain of a focus of DIN 1 (atypical ductal hyperplasia) (200×). d Immunohistochemical demonstration of the absence of STAT 5a in DIN 1 (atypical ductal hyperplasia) (200×). Note: scattered residual STAT 5a-positive normal epithelial cells

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Fig. 3
figure 3

a H&E stain of a focus of DIN 3 (ductal carcinoma in situ, grade 3) (200×). b Immunohistochemical demonstration of the absence of STAT 5a in the carcinoma (200×). Note: STAT 5a-positive inflammatory cells. c,d Immunohistochemical demonstration of the absence of STAT 5a in DIN 1 (ductal carcinoma in situ, grade 1) (400×). Note: residual STAT 5a-positive normal epithelial cells

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Fig. 4
figure 4

a H&E stain of invasive ductal carcinoma (200×). b Immunohistochemical demonstration of the absence of STAT 5a in the carcinoma (200×). Note: adjacent normal STAT 5a-positive epithelial cells. c H&E stain of invasive ductal carcinoma (400×). d Immunohistochemical demonstration of the absence of STAT 5a in the carcinoma (400×). Note: entrapped normal STAT 5a-positive epithelial cells

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Fig. 5
figure 5

a H&E stain of a focus of epithelial cell secretory change (200×). b Immunohistochemical demonstration of STAT 5a in the cells with secretory changes (200×). Note: intensity greater than that seen in neighboring epithelial cells

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Discussion

A reduction of STAT 5a was observed, associated with abnormal breast epithelial cells. The absence of STAT 5a in the abnormal breast epithelial cells may indicate a defect contributory to the abnormal state. Since STAT 5a is required for the development of breast lobules, repressors of its activation or inappropriate signaling through other mechanisms might alter the function and phenotype of these cells. Because the demonstrated expression was seen specifically in cells known from other studies to contain STAT 5a, it was reasoned that the reaction was specific for STAT 5a or a STAT 5a-like protein. In all of the sections, normal ductal and lobular epithelium reacted strongly in the cytoplasm and nucleus with the antibody to STAT 5a, serving as an internal control for cells with weak or absent staining.

STAT 5a expression may be affected by an abnormal genotype. Certain genomic deletions may affect the STAT 5a gene locus, found on chromosome 17q11, incidentally. However, it is also possible that any number of biochemical events involving STAT 5a may act to provide an atmosphere in which cellular transformation can occur. The absence of STAT 5a could result in aberrant signaling or suboptimal transcription of developmental, regulatory, or growth-restraining proteins contributing to altered (metaplastic or neoplastic) phenotypic differentiation.

Numerous reports implicate the suppression of STAT 5a in normal and disease processes [20, 22, 24]. Suppressors of cytokine signaling (SOCS) and protein inhibitors of activated STATs (PIAS) can suppress signal transduction by inhibiting the STAT pathway [10, 26, 28]. Activated SOCS can also stimulate cell proliferation and increase cell survival through the Ras signaling pathway [6]. Therefore, proliferation can occur through at least one mechanism that would inactivate STAT 5a in the process and, in fact, a recent study demonstrated increased SOCS gene expression in breast cancer [21].

The absence of STAT 5a in most abnormal breast epithelial cells including carcinomas is not something that has been widely noted, however. Because STAT 5a is a transcription factor, one might imagine that an over-expression or constitutive expression of STAT 5a would be seen in the uncontrolled growth of breast carcinomas like the reports of over-expression of STAT 3 in these tumors [3]. This has been reported in one study where 76% of the breast cancers examined showed phosphorylated STAT 5a to be present in the nucleus [8]. It has also been shown that in 65% of induced rat mammary gland tumors, STAT 5a was detected in the nucleus using phosphorylation-specific antibodies [23].

These data may seem, initially, to contradict the present findings. Antibody variability, however, can certainly make a difference in detection. Nevalainen et al. saw a decrease in activated STAT 5a in more aggressive breast cancers using a phosphor-specific antibody, while detection of the STAT 5a protein using another anti-STAT 5a was maintained [18]. In contrast, the report by Shan et al. [23] stated that high STAT 5a nuclear expression closely correlated with higher-grade carcinomas and identified a cytoplasmic location for STAT 5a in the normal rat mammary gland. Because the STAT 5a expression in this present study was seen in the nucleus, it was reasoned that at least some of the identified STAT 5a might be of an activated form. The ST5a-2H2 clone used for this study, while not described as an activation specific clone, demonstrated a lack of detection of STAT 5a in higher-grade lesions corroborating the results of Nevalainen et al. [18].

The ST5a-2H2 antibody is directed against the molecule's carboxy-terminus near where the active phosphorylation site is located. Some functional STAT 5a variants may exist that are not detectable with this antibody, and some naturally occurring splice site variants of STAT 5a have been shown to have the ability to suppress the transcriptional activity of the wild-type STAT 5a [30]. Also, a few early studies have pointed to distinct carboxy-terminal truncated forms of STAT 5a [13, 16]. If truncated forms were present in the tumor cells, they would not be visible with this antibody. Tantalizingly, a report examining the expression of various forms of STAT 5a in transgenic mice demonstrated that a carboxy-terminal truncated form of STAT 5a produced the most undifferentiated carcinomas in the mammary glands of these mice [11].

It was of interest to note that a relative over-expression of STAT 5a was observed in cells undergoing secretory change. This was considered unusual because cells undergoing apocrine differentiation (a secretory cell) were uniformly nonreactive. Perhaps the secretory nature of breast epithelium is dependent on viable STAT 5a, and the functional ability of the cell is lost when defects occur in this pathway leading to abnormal cell behavior. Also, the rare lesions showing some expression were either well-differentiated tubular carcinomas or tumors surrounded by a large amount of STAT 5a-positive inflammatory cells. Examples of LIN (merely an indicator of generalized risk) demonstrated the expression nearest to that of normal or hyperplastic samples, and in the study by Cotarla et al., the STAT 5a-positive tumors correlated with increased levels of differentiation [8]. This may indicate that STAT 5a function is maintained in lesions that are differentiated enough to allow normal processes to occur. These findings support STAT 5a as a marker of good prognosis, and the percentage of positive carcinomas reported by Nevalainen et al. is also similar to the number reported here [18]. In addition, a recent report described STAT 5a as a suppressor of invasion due to its loss associated with metastatic lesions and a relationship between STAT 5a and adhesion [25]. STAT 5a, then, has been alternately reported to be an active factor associated with breast carcinoma or acting almost like a tumor suppressor gene in the breast. The correlation of function and tumor grade with respect to STAT 5a expression as determined by the individual antibody employed will have to be investigated further.

Whether or not STAT 5a has an effect on the maintenance of the normal phenotype as it has an effect on the establishment of the normal phenotype is subject to conjecture. In most instances, abnormal cell growth can be identified by an absence of STAT 5a reactivity (Fig. 6). The St5a-2H2 antibody, therefore, might also have a possible clinical utility in distinguishing atypical ductal hyperplasia or grade one ductal carcinoma in situ (DIN 1) from usual ductal hyperplasia (low-risk DIN). Since STAT 5a is required for the normal development of breast lobules, repressors of its activation or inappropriate signaling through other mechanisms might alter the function and phenotype of breast epithelial cells.

Fig. 6
figure 6

a H&E stain showing small foci of DIN 1 Flat type (flat type epithelial atypia) and DIN 1 (atypical ductal hyperplasia) (400×). b Immunohistochemical detection of STAT 5a demonstrating its absence in these abnormal foci (400×)

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