Comparative studies of the estrogen receptors beta and alpha and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma - PubMed (original) (raw)

Comparative Study

Comparative studies of the estrogen receptors beta and alpha and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma

I Leav et al. Am J Pathol. 2001 Jul.

Abstract

An antibody, GC-17, thoroughly characterized for its specificity for estrogen receptor-beta (ER-beta), was used to immunolocalize the receptor in histologically normal prostate, prostatic intraepithelial neoplasia, primary carcinomas, and in metastases to lymph nodes and bone. Comparisons were made between ER-beta, estrogen receptor-alpha (ER-alpha), and androgen receptor (AR) immunostaining in these tissues. Concurrently, transcript expression of the three steroid hormone receptors was studied by reverse transcriptase-polymerase chain reaction analysis on laser capture-microdissected samples of normal prostatic acini, dysplasias, and carcinomas. In Western blot analyses, GC-17 selectively identified a 63-kd protein expressed in normal and malignant prostatic epithelial cells as well as in normal testicular and prostatic tissues. This protein likely represents a posttranslationally modified form of the long-form ER-beta, which has a predicted size of 59 kd based on polypeptide length. In normal prostate, ER-beta immunostaining was predominately localized in the nuclei of basal cells and to a lesser extent stromal cells. ER-alpha staining was only present in stromal cell nuclei. AR immunostaining was variable in basal cells but strongly expressed in nuclei of secretory and stromal cells. Overall, prostatic carcinogenesis was characterized by a loss of ER-beta expression at the protein and transcript levels in high-grade dysplasias, its reappearance in grade 3 cancers, and its diminution/absence in grade 4/5 neoplasms. In contrast, AR was strongly expressed in all grades of dysplasia and carcinoma. Because ER-beta is thought to function as an inhibitor of prostatic growth, androgen action, presumably mediated by functional AR and unopposed by the beta receptor, may have provided a strong stimulus for aberrant cell growth. With the exception of a small subset of dysplasias in the central zone and a few carcinomas, ER-alpha-stained cells were not found in these lesions. The majority of bone and lymph node metastases contained cells that were immunostained for ER-beta. Expression of ER-beta in metastases may have been influenced by the local microenvironment in these tissues. In contrast, ER-alpha-stained cells were absent in bone metastases and rare in lymph nodes metastases. Irrespective of the site, AR-positive cells were found in all metastases. Based on our recent finding of anti-estrogen/ER-beta-mediated growth inhibition of prostate cancer cells in vitro, the presence of ER-beta in metastatic cells may have important implications for the treatment of late-stage disease.

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Figures

Figure 1.

Figure 1.

Competitive ELISA. This graphic representation illustrates the concentration-dependent competition of the immunizing peptide (ERβC) with the GC-17 antibody. In addition, preincubation with 4 mg/ml of the control N-terminus peptide (ERβN) and GC-17 failed to compete out the binding to the recombinant ER-β peptide, demonstrating the specificity of the antibody. The GC-17 antibodies, bound to recombinant ER-β protein on ELISA plates, were recognized by alkaline phosphatase-conjugated anti-rabbit IgG antibodies. The whole complexes were visualized by incubation with _p_-nitrophenyl phosphate. The optical density at 405 nmol/L was measured. The entire assay was done four times. The column represents the mean value of optical density at 405 nmol/L of four measurements and the bar represents the SD. w/o, GC-17 preincubated without peptide antigen.

Figure 2.

Figure 2.

Competitive ER-β immunostaining DU145 cells (A and B) and normal prostate (C and D). A: DU145 cells immunostained in the absence of competing peptide are shown. Note the strong nuclear staining in the majority of cells (original magnification, ×265). B: After incubation of GC-17 with 40 μg of the immunizing peptide, there was almost a total absence of cells with positively stained nuclei (compare with A) [original magnifications: ×115 (left), ×350 (right)]. C: Tissue section of normal prostate. In the absence of competing peptide strong immunostaining of cells in the basal layer of glands is seen in this section of prostate (see also Figure 4, A and B ▶ ) [original magnifications: ×115 (left), ×230 (right)]. D: A replicate section of the prostate illustrated in C after preincubation of GC-17 with 40 μg of immunizing peptide. Note the absence of immunostaining in these sections [original magnifications: ×90 (left), ×280 (right)]. For details of the procedures used in these studies see Materials and Methods. All sections were counterstained with 10% Harris hematoxylin.

Figure 3.

Figure 3.

Western blot analysis. These autoradiographs illustrate the binding ability and specificity of GC-17 to ER-β protein and its lack of cross-reactivity with ER-α protein. A and B: Shown from left to right are the ER-α recombinant protein, short form of ER-β recombinant protein, and tissue lysates from human testis and normal prostate (1 to 4). C: From left to right are cell lysates of PrEC and DU145 cells, tissue lysates from human normal prostate (1 and 2), and long form of ER-β recombinant protein. The recombinant proteins and cell or tissue lysates were separated with SDS-PAGE gel and the separated proteins were transferred onto PolyScreen polyvinylidene difluoride transfer membrane. The blot was incubated with the ER-α monoclonal antibody (A) or GC-17 ER-β polyclonal antibody (B and C) and the complexes were visualized by the chemiluminescence ECL detection system followed by autoradiography. Note that the antibody detects only the ER-α protein using ER-α monoclonal antibody and GC-17 ER-β polyclonal antibody does not detect the ER-α protein but clearly identifies single bands for both forms of ER-β recombinant proteins and for the cell or tissue lysates. These bands correspond to the reported size of the short and long forms of the receptor (see Results).

Figure 4.

Figure 4.

A and B: Immunolocalization of ER-β in the normal human prostate using the GC-17 antibody. Note the strong staining of cells in the basal layer and its virtual absence in the nuclei and cytoplasm of secretory cells (best seen in B). Some nuclear membrane staining was however evident in a few secretory cells (A and B). Nuclear staining is also evident in stromal cells. Original magnifications: ×100 (A), ×400 (B). C: ER-β immunostaining in low/moderate-grade dysplasia. Moderate to strong expression of the receptor is evident in dysplastic secretory cells. Note also the strongly stained cells in the basal layer (original magnification, ×400). D: ER-β-immunostained section of high-grade dysplasia. Nuclear staining is almost totally absent in this lesion. In some dysplastic cells light staining of nuclear membranes is evident. This area of the lesion is almost totally devoid of receptor-stained basal cells. A few residual-stained basal cells are however evident in the bottom left corner of the lesion. Note the presence of two positively stained basal cells in a portion of a normal gland on the right (original magnification, ×250). E: ER-α immunostaining in dysplasia of the central zone. The majority of dysplastic cells are immunostained for the receptor in this lesion. There was however great variation in the percentage of positive cells found in these central zone lesions. Intraluminal pseudo-gland formation seen here was common in dysplasias of the central zone (original magnification, ×200). All sections were counterstained with 10% Harris hematoxylin.

Figure 5.

Figure 5.

A: ER-β staining in grade 3 carcinoma. Strong nuclear immunostaining is evident in this grade 3 carcinoma. Light cytoplasmic staining of neoplastic cells is also present. Although positive immunostaining for the receptor was found in the majority of grade 3 cancers there was variation in the percentage of stained cells in any given lesion. This was especially the case in areas of transition from grade 3 to grade 4/5 carcinoma (see C) (original magnification, ×400). B: ER-β immunostaining of a clear cell carcinoma in the transition zone. Immunostaining for the receptor is absent in the majority of cells in this grade 3 clear-cell carcinoma. Scattered among the negatively stained cells are cells with small nuclei that are immunopositive for the receptor. The occurrence of these positive cells was uncommon in clear cell carcinomas. In most of these cancers all of the cells were unstained for the receptor. Interestingly, in replicate sections, these ER-β-positive cells were negative for AR immunostaining whereas the reverse was true for the majority of cells that were negative for the β receptor (original magnification, ×400). All sections were counterstained with 10% Harris hematoxylin. C: ER-β staining in an area of transition from grade 3 to grade 4/5 carcinoma. The majority of nuclei in this cancer are unstained. A few positively stained nuclei are however seen in two grade 4/5 glands (bottom left) and in a single cell in a grade 3 gland (bottom right). Light cytoplasmic staining is evident in most cells. Nuclear membrane staining is also present in many of these cells. Cytoplasmic staining was common in all grades of dysplasia and carcinoma. Nuclear membrane immunostaining was however only a feature of high-grade dysplasias and grade 4/5 carcinoma (original magnification, ×400).

Figure 6.

Figure 6.

A: ER-β immunostaining in a prostatic carcinoma metastatic to bone. Note the strong nuclear immunostaining for the β receptor in this metastatic lesion. The neoplastic cells are localized between spicules of bone. Strong to moderate staining was present in the majority of metastases to bone (original magnification, ×400). B: ER-β immunostaining in a prostatic carcinoma metastatic to an internal iliac lymph node. Strong nuclear immunostaining is evident in this metastatic lesion. Strong PSA immunostaining of these cells were found (not illustrated). Note that the nuclei of several stromal cells in this lymph node are also positively stained for the receptor (original magnification, ×400). C: ER-β immunostaining of a prostatic carcinoma metastatic to a lymph node. In this example the metastatic cells were unstained for the receptor. Cells in this same cancer were however immunostained for ER-α and AR (see D and E). As was the case for the metastasis illustrated in B these cells were strongly PSA-positive. Note that lymphocytes are strongly stained for the receptor, which was a consistent finding and provided an internal positive control for immunostaining with the β antibody (original magnification, ×100). D: ER-α immunostaining of the same lesion illustrated in C. Immunopositive cells are seen scattered throughout this metastatic lesion. In one other case of lymph node metastasis a very few positive cells (<10%) were present. ER-β was however strongly expressed in that metastatic lesion. Note the absence of ER-α immunostaining of lymphocytes, a consistent finding that was in contrast to ER-β staining in these cells (original magnification, ×100). E: Representative section of AR immunostaining in metastatic lymph node lesions. Strong nuclear staining was uniformly found in the majority of cells that comprised these metastatic lesions. Cytoplasmic staining occurred only in metastases from the patient who had received anti-androgenic therapy. Lymphocytes were lightly stained for AR (original magnification, ×100). All sections were counterstained with 10% Harris hematoxylin.

Figure 7.

Figure 7.

A and B: LCM dissection of a grade 3 carcinoma. A: The grade 3 neoplastic gland that was microdissected is seen in the center of this microscopic field (original magnification, ×325). B: The microdissected lesion is seen on the transfer cap (original magnification, ×325). Both sections were lightly stained with 10% Harris hematoxylin. C: Results of RT-PCR analysis of ER-β mRNA on microdissected human normal prostate acini (N-1 and N-2) and grade 3 (G3–1 and G3–2) and 4/5 (G4–1 to G4–4) carcinomas. Total RNAs were extracted from the microdissected tissues on transfer cap and reverse-transcribed. The resultant cDNAs were subjected to PCR analyses. The amplified products were run into 2% agarose gel with ethidium bromide. Representative fluorographs for ER-β and GAPDH RT-PCR analyses are shown.

Comment in

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