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Protein Expression in Basal Cells Occurs in the Early Stage of Human Prostate Cancer Development
1 Department of Epidemiology, School of Public Health; Departments of 2 Urology and 3 Pathology, School of Medicine and 4 Jonsson Comprehensive Cancer Center, University of California-Los Angeles, Los Angeles, CA; and Departments of 5 Pathology, 6 Surgery, and 7 Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY
Requests for reprints: Jianyu Rao, M.D., Department of Pathology and Lab Med, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095. Phone: (310) 794-1567; Fax: (310) 825-7795. E-mail: jrao{at}mednet.ucla.edu
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Abstract |
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(RXR) null mouse suggests that RXR may protect against neoplasia. The purpose of this study was to characterize RXR protein expression in human prostate to determine if RXR is altered in early stages of tumor progression. Immunohistochemistry with anti-RXR antibody was performed on 138 fresh frozen prostate specimens collected from 27 noncarcinomatous prostates and 111 radical prostatectomy samples of prostate adenocarcinoma (CA). The RXR signal intensity was scored using a scale of 0–3. In normal glands, RXR was expressed strongly in basal cells and only weakly in secretory epithelial cells. This finding was confirmed by double immunofluorescence labeling of RXR and Keratin-903, a basal cell marker, followed by confocal microscopic examination. In basal cells, a gradual decrease of RXR expression was noted from normal glands of noncarcinomatous prostate (3.0 ± 0) to "normal" glands distant to CA (2.13 ± 0.44) to "normal" glands adjacent to CA (1.25 ± 0.53) and high-grade PIN (0.56 ± 0.58). While nearly all "normal" glands from 138 specimens were positive for RXR in basal cells, only 48% (13 of 27) of the high-grade PIN glands appeared positive. Moreover, basal cell expression of RXR in "normal" tissue was less in specimens with poorly differentiated tumor (Gleason score 
8; 1.83 ± 0.36) compared with well-differentiated tumor (Gleason score < 6; 2.35 ± 0.34; P = 0.04). Thus, a decrease of RXR in the basal cells may serve as a marker for prostate CA-associated field change, which may represent an early event in the prostate carcinogenic process. These findings suggest that chemoprevention strategies with retinoids may be most effective if applied during the early stages of transformation. |
Introduction |
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Most actions of retinoids are thought to be mediated by its nuclear receptors, which function as ligand-activated transcription factors (reviewed in 16–18). There are two types of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The RARs and RXRs each have three subtypes: , ß, and 
. Each subtype has distinct and conserved sequences, a specific pattern of expression during embryonal development, and a different distribution in adult tissue. The subtypes are thought to regulate the expression of specific sets of genes (19, 20). Heterodimers of the RARs and RXRs bind to a specific DNA promoter sequence, termed the retinoic acid response element, and regulate gene transcription (21, 22). RXRs can also form homodimers and activate the retinoid X response element or form heterodimers with other members of the steroid receptor superfamily, thus serving in multiple regulatory functions in different signaling pathways (23).
Among the retinoid receptor subtypes, RXR appears to have a critical functional role in the development of prostatic preneoplastic lesions. In the transgenic mouse model with the conditional disruption of the RXR gene in the prostate epithelium, an age-dependent development of multifocal prostatic intraepithelial neoplasias (PIN) occurred, with high-grade PIN developing in some animals beginning at 10 months of age (24). The heterozygous RXR mutant mice developed PIN, but in a temporally delayed manner, suggesting a dose dependence on RXR in the epithelium (24). In human prostate cancer cell lines, RXR protein was decreased relative to noncancer prostate cell lines, and a reduction of cell growth or increased susceptibility to apoptosis was demonstrated with increases in the level of RXR in RXR-transduced prostate cancer cells (25).
In light of the evidence of a functional role for RXR in prostate tumor progression, we undertook an immunohistochemical (IHC) examination of a relatively large number of fresh frozen human prostate specimens and report our analysis of the distribution and semiquantitative expression levels of RXR protein in noncarcinomatous prostate, PIN, adenocarcinoma (CA), and histopathologically "normal" glands distant and adjacent to CA. Our study provides a detailed characterization of RXR protein expression in prostate cancer, which may elucidate its role in prostate neoplasia.
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Materials and Methods |
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Tissue Preparation
Immediately after surgical removal, prostates were separated into anterior, posterior, right, and left lobes. Between six and eight blocks containing portions of the lobes were embedded in OCT medium (Fisher Scientific, Pittsburgh, PA), freshly frozen, and stored at -70°C. The blocks of frozen tissues were shipped with dry ice to the Molecular Epidemiology Laboratory of the University of California-Los Angeles (UCLA) Jonsson Comprehensive Cancer Center, and the study was approved by the UCLA Institutional Review Board for Human Subjects. One to three blocks from each prostate were selected for histological analyses. Frozen tissues were cut with a cryostat into 7-µm thick sections. The first and last sections for each block of tissue were stained with H&E for histological diagnosis and the adjacent middle sections were used for IHC analysis. Two independent IHC analyses were conducted for each prostate specimen.
Immunohistochemistry
A RXR affinity-purified rabbit polyclonal antibody that specifically recognizes human RXR (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:100 concentration. A Keratin-903 (K903) antibody (Enzo Diagnostics, Inc., Farmingdale, NY) mouse monoclonal antibody that is specific for high molecular weight cytokeratins 1, 5, 10, and 14 was used to identify epithelial basal cells (26). The K903 antibody was used at 1:500 concentration. Manual IHC was performed as follows. The frozen prostate sections were warmed and air-dried at room temperature for 30 min. The slides were immersed in acetone at 4°C for 10 min to fix the sections. Slides were rinsed in PBS, incubated in PBS-hydrogen peroxide (0.3% hydrogen peroxide) for 30 min, and then equilibrated in PBS-Tween (0.25% Tween). Sections were first blocked with 5% normal donkey serum (Sigma Chemical Co., St. Louis, MO) for 45 min at room temperature and then incubated at room temperature with primary antibody diluted in PBS-Tween to the specified concentration for 14–16 h. Secondary antibody [donkey anti-rabbit for RXR or donkey anti-mouse for cytokeratin K903 (CK903); Sigma Chemical] at a 1:200 concentration was applied to the slides for 90 min at room temperature. Vecastain (Vector Laboratories, Inc., Burlingame, CA) was used as the conjugate and 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical) was used as the chromagen for detection. The slides were counterstained with hematoxylin. The specificity of the IHC procedure was confirmed by using negative controls; tissues were incubated in either preimmune serum or by using the RXR antibody preabsorbed with an excess of purified RXR antigen (Santa Cruz Biotechnology).
The staining intensity was scored using a scale of 0–3 as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. RXR staining in secretory epithelial cells was judged as either present or absent.
Double-Label Immunofluorescence and Confocal Examination
The frozen prostate sections were fixed and blocked as described above. The slides were simultaneously incubated with RXR antibody (1:50) and CK903 antibody (1:150) for 14–16 h at room temperature. Slides were rinsed with PBS and then incubated with secondary antibodies consisting of donkey anti-rabbit labeled with FITC for RXR (Jackson ImmunoResearch Laboratories, Wesgrove, PA) and donkey anti-mouse labeled with rhodamine (Jackson ImmunoResearch Laboratories) for CK903 for 3 h at room temperature. The signal was observed with confocal microscopy (Olympus AX 70, Melville, NY) and photographed using Kodak (Rochester, NY)Elite Chrome 100 film.
Statistical Analyses
t test analyses were used to compare the mean expression levels of RXR among the histologically distinct tissues and to compare mean expression levels of RXR in tissues with Gleason scores 2–5 to tissues with higher Gleason scores. Two-sided t tests were used and P values were reported.
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Results |
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expression. In all of the prostate specimens, RXR protein was detectable by IHC in stromal cells and included endothelial cells, fibroblasts, and vascular smooth muscles. Positive signal for RXR in these stromal cells was uniformly strong, localized to the nucleus, and served as internal positive controls for RXR IHC. No staining was observed in the negative controls, which included incubation of tissues in preimmune serum or preabsorption of RXR antibody with an excess of purified RXR antigen.
Noncarcinomatous Prostate (Normal/Normal)
Representative images of H&E and IHC staining for RXR are presented in Fig. 1, A and B, respectively. RXR signal was uniformly strong in basal cells and weak in secretory epithelial cells. The intensity was much stronger in the nucleus versus in the cytoplasm. The expression of RXR in basal cells was confirmed by colocalization of the RXR signal with the basal cell marker K903 by double-label immunofluorescence under confocal microscopy as shown in Fig. 1, C and D. Table 1 shows the relative RXR protein expression levels in basal cells of the histologically distinct areas (normal/normal, "normal" tissue distant or adjacent to CA, and PIN). Noncarcinomatous prostates had strong positive RXR signal in the basal cells (expression level 3.0 ± 0). Weak expression of RXR signal in the secretory epithelial cells as clearly shown in Fig. 1D was found in all (27 of 27) of the noncarcinomatous specimens, which is graphically presented in Fig. 2.
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that was positive in all (111 of 111) of the specimens. Similar to noncarcinomatous normal/normal prostates, RXR protein signal was more intense in the basal cells than in the secretory epithelial cells. However, the mean expression level of basal cells in the distant "normal" tissue was 2.13 ± 0.44 as shown in Table 1 and was notably less than in normal/normal prostate (P < 0.001). Weak RXR expression was noted in the secretory epithelium in 100% (111 of 111) of the "normal" cancerous specimens (Fig. 2).
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expression in the basal cells was noted as compared with the basal cells of the noncarcinomatous and "normal" distant glands (Fig. 3F). The mean RXR expression level of the adjacent tissue was 1.25 ± 0.53, which is significantly lower (P < 0.001) than either normal/normal or "normal" distant tissue (Table 1). One of the CA adjacent specimens lacked visible secretory cell RXR signal (Fig. 2). Thus, histologically "normal" adjacent tissues display reduced RXR protein, which may indicate molecular field changes in the neoplastic process.
Prostatic Intraepithelial neoplasia
Focal regions of PIN were identified by H&E as shown in Fig. 4A. Figure 4B shows K903 signal that was positive in all of the PIN specimens, indicating the presence of basal cells. However, RXR signal was absent in the basal cells in 52% (14 of 27) of the PIN specimens (Fig. 4C; Table 1). Double-label immunofluorescence under confocal microscopy clearly demonstrates K903 expression in basal cells as shown in Fig. 4D compared with the absence of RXR in the basal cells shown in Fig. 4E. Among the PIN specimens that did have RXR signal, most were positive in <20% of the basal cells. The mean basal cell expression of RXR was significantly lower in PIN (0.56 ± 0.58) as compared with noncarcinomatous tissue or "normal" tissue from carcinomatous prostates (P < 0.001; Table 1). Weak RXR expression in secretory epithelium was present in 48% of PIN samples (Fig. 2).
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signal in both CA and PIN lesions. Signal for both RXR and K903 was typically negative in CA as shown by double-label immunofluorescence under confocal microscopy (Fig. 4, D and E). However, 2 of 26 CA specimens (8%) showed weak expression of RXR protein in the tumor cells (Fig. 2).
Correlation of RXR Protein Expression in Basal Cells of Distant Benign Glands with Gleason Score
The mean signal levels of RXR in the basal cells distant from CA were determined for specimens categorized into three groups of Gleason scores: 2–5, 6–7, and >7 as shown in Table 2. Tissues with Gleason scores of 2–5 corresponding to well-differentiated tumor had an average RXR expression level of 2.35 ± 0.34 in distant "normal" glands. A lower RXR expression level, 2.10 ± 0.44, was found in distant "normal" glands with moderately differentiated tumors with Gleason scores of 6 or 7. Poorly differentiated glands corresponding to Gleason scores of 8 or 9 had a significantly lower expression (1.83 ± 0.36) of RXR in distant "normal" glands (P = 0.04) as compared with Gleason scores 2–5 tumor.
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Discussion |
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protein expression in human prostate carcinogenesis. Our major finding is that RXR protein expression decreases in the basal cells in the field as neoplasia progresses from noncancerous normal (normal/normal) to "normal" cells in cancerous prostate and then to PIN. While RXR signal was weakly present in non-neoplastic secretory epithelial cells, the expression is lost in epithelial cells of most PIN and CA areas. Furthermore, the level of RXR protein varies among what was pathologically considered to be normal tissue (i.e., RXR signal was significantly different among normal/normal, "normal" adjacent to CA, and "normal" distant to CA tissues). Moreover, we found a correlation between the decrease in RXR protein expression in distant "normal" tissue from carcinomatous prostates and increasing Gleason score. That "normal" glands undergo a reduction in RXR protein preceding histopathological changes indicates that control of RXR may be an early event that extends to a field of affected tissue. These findings support the hypothesis that RXR protein loss begins at an early stage of carcinogenesis and may represent a field disease marker for prostate malignancy.
Descriptions of the expression of RXR in human prostate have been limited to a few published studies. A highly heterogeneous pattern of RXR protein expression, with some areas of low or no staining, was reported in 13 CA specimens by Zhong et al. (25). Alfaro et al. (27) and Kikugawa et al. (28) have detected RXR protein in samples from normal and carcinomatous prostates. However, artifactual antibody staining due to formalin fixation may have occurred in some studies because RXR positivity in inflammatory cells was observed as well (29). Our study not only compared the expression in normal prostates and cancer but also carefully examined the expression in the distant and adjacent fields including PIN. We found that RXR protein expression was absent in basal and luminal secretory epithelial cells in 52% of PIN specimens, a finding highly consistent with the results obtained using prostate-specific RXR null mouse model (24). In addition, a careful examination of expression of RXR in basal versus epithelial cells was performed by side-by-side IHC labeling with K903 (29), a basal cell marker, as well as double-immunofluorescence labeling of K903 and RXR followed by confocal microscopic examination.
Our analysis showed that the basal cell expression of RXR in PIN was significantly reduced compared with expression in basal cells of normal glands. That the loss of RXR signal in PIN was a result of reduced RXR expression in basal cells and not due to loss of basal cells was confirmed by positive K903 signal in PIN. The reduced RXR signal in PIN basal cells supports the hypothesis that PIN is a precursor to prostate cancer and that loss of RXR protein may indicate a premalignant alteration to basal cells along the pathway to neoplasia. It is generally accepted that the basal cells act as reserve cells for the production of secretory cells (30). Prostate CA, by definition, should not contain basal cells (31). Because most expression of RXR appeared to be in the basal cells and only weakly in the epithelial cells, one should be cautious in the interpretation of findings in the cancer area. Although we also observed a decreased expression of RXR in secretory epithelial cells from normal to PIN to cancer and 92% (24 of 26) of the cancer areas showed a total absence of the staining, a more sensitive and quantitative type of analysis will be needed to confirm the observation.
In recent years, several genes in the retinoid signaling pathway have been identified as critical factors during prostate cancer initiation and progression (32). The mRNA expression levels of RARß and RXRß have been shown to be significantly less in patients with prostate cancer compared with normal patients, and notably, RARß mRNA expression was lost in adjacent, benign prostatic glands (33). There is strong evidence that the critical RXR in prostate biology appears to be RXR. Mice lacking both RXRß and RXR are normal in terms of prostate morphology (34), whereas prostate epithelium in mice with the conditional disruption of the RXR gene displays increased proliferation and induction of PIN, which is considered to be a preneoplastic lesion of CA (24). The underlying mechanism responsible for decreased RXR in prostate epithelium is not clear and is not the subject of this study. Aberrant promoter methylation has been identified as an epigenetic mechanism for transcriptional silencing (35); however, Lotan et al. (33) observed RXR transcript signal in prostate cancer. Although RXR transcript is present in prostate cancer, the levels of RXR protein may be regulated. For example, RXR protein is degraded by ubiquitin-mediated proteolysis that occurs on binding of RXR agonist ligands or when heterodimeric partners (i.e., RAR) become activated (36). Decreased RXR would result in deficient RAR/RXR heterodimers and RXR/RXR homodimers. Thus, in the model in which RXR functions as a transcriptionally active partner (37), this could result in functional cellular retinoid deficiency. Moreover, to mediate multiple signaling pathways in the prostate, RXR may partner with other nuclear receptors, such as the peroxisomal proliferator-activated receptor- and vitamin D receptor, the ligands of which have been shown to inhibit prostatic cell growth (17, 38).
The importance of the RXRs is underscored by the effectiveness of RXR-specific ligands to suppress tumorigenesis. RXR-specific ligands have been considered as promising agents for the prevention of prostate cancer and are generally less toxic than RAR-selective retinoids (39). The RXR-selective retinoid, SR11246, was able to inhibit the clonal growth of prostate cancer cells (11). In the LNCaP human prostate cancer cell line, 13-cis-retinoic acid inhibited the tumorigenic potential and expression of prostate-specific antigen, and there was also a significant increase in the expression of RXR mRNA after 13-cis-retinoic acid treatment as compared with untreated cells (10). To assess the potential for retinoids as a therapeutic modality, the RAR/RXR content of the tumors is important, and specific determination of the relative levels of RXR is a prerequisite for the use of RXR-selective ligands. IHC using biopsy specimens from tumors and preneoplastic lesions is a simple and reliable procedure to identify patients with RXR as well as those patients who are most likely to benefit from retinoid treatments.
In conclusion, our results show that suppressed RXR protein expression in basal cells occurs early in prostate carcinogenesis and that RXR undergoes a statistically significant decline during the progression from normal tissue to PIN and on to primary CA, supporting its role as a field disease marker. Further investigation into its potential as an intermediate end point marker is warranted. If the loss of RXR protein is proven to be a premalignant feature, it may be an important marker for use in retinoid-based chemoprevention trials to evaluate core biopsy material to determine who should take retinoid therapies. Alternatively, it may be potentially informative to evaluate changes in receptor protein expression in conjunction with clinical outcomes in those who take retinoid-based therapies but fail to show response to retinoids.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6/ 9/03; revised 10/ 9/03; accepted 11/25/03.
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J. L. Stebbins, D. Jung, M. Leone, X.-k. Zhang, and M. Pellecchia A Structure-based Approach to Retinoid X Receptor-{alpha} Inhibition J. Biol. Chem., June 16, 2006; 281(24): 16643 - 16648. [Abstract] [Full Text] [PDF]
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