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© 2000 American Society for Clinical Oncology Differential Expression of Nuclear Retinoid Receptors in Normal and Malignant ProstatesFrom the Scott Department of Urology and Department of Pathology, Baylor College of Medicine; and Departments of Clinical Cancer Prevention and Tumor Biology, University of Texas, M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Dov Kadmon, MD, Scott Department of Urology, Baylor College of Medicine, 6560 Fannin, Ste 2100, Houston, TX 77030; email dkadmon{at}bcm.tcm.edu
PURPOSE: To determine (1) whether nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are differentially expressed in normal and in cancerous human prostate tissues and (2) whether oral fenretinide therapy impacts the expression of these receptors in prostate cancer. PATIENTS AND METHODS: In situ hybridization with antisense riboprobes was used to probe for RAR and RXR transcripts in prostate tissues in a two-phased study: (1) expression of retinoid receptors in eight normal prostates was compared with their expression in 10 randomly picked radical prostatectomy specimens (group A); (2) expression of retinoid receptors was determined in 22 radical prostatectomy specimens from participants in a clinical study (group B). Twelve patients received oral fenretinide 200 mg/d, and 10 received placebo pills for 28 days before surgery.
RESULTS: RAR CONCLUSION: RARß and RXRß mRNAs are selectively lost in both prostate cancer and adjacent morphologically normal prostatic tissue, supporting the concept of a field of carcinogenesis. One month of oral fenretinide (200 mg/d) did not influence the expression of retinoid receptors in prostate cancer.
PROSTATE CANCER IS the most prevalent solid tumor in United States men and the second leading cause of cancer mortality in this population.1 Because of the presumed long latency period between initiation and full malignant transformation, because of the exceedingly long doubling time of early malignant disease, and because of its high prevalence in the population, prostate cancer is ideally suited for a chemoprevention approach. Retinoids (natural and synthetic derivatives of vitamin A) represent a premier class of chemopreventive agents. Preclinical and clinical studies document the efficacy of these agents in chemoprevention of oral leukoplakia, cervical cancer, and second primary tumors in head and neck neoplasia.2-12 Early experiments on mouse prostate explant cultures have shown that retinoic acid could both inhibit and reverse the proliferative effects of chemical carcinogens on prostatic epithelium.13,14 The ongoing interest in the ability of retinoids to inhibit prostate carcinogenesis led to several retrospective case control studies trying to link vitamin A intake and its serum level with the risk of developing prostate cancer.15,16 The results of these studies were conflicting, with one study even suggesting that vitamin A was associated with an increased risk of developing prostate cancer in men older than 70 years of age.17 The mechanism by which retinoids regulate the growth and differentiation of cells has not been completely elucidated. Retinoids are thought to act on nuclear-binding proteins, which act as transcription factors.18-24 There are two types of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs).23,25,26 Both belong to the superfamily of steroid hormone receptors.23 The  , ß, and  subtypes of the RARs and RXRs have distinct and conserved amino and carboxy terminal domains.23,25,26 Heterodimers of the RARs and RXRs bind to a specific DNA sequence known as the retinoic acid response element. Retinoids regulate this element, which is located in the promoter region of genes such as the RAR-b2 gene.27 Considering that each receptor subtype has a specific pattern of expression during embryonal development and a different distribution in adult tissues, the different subtypes are thought to regulate the expression of distinct sets of genes.25,28 The level of expression of the various subtypes may provide clues to the roles of these receptors in the development of cancer and to the biologic response to retinoid therapy. Decreases in RARß expression have been seen in oral premalignant and malignant lesions with upregulation of the receptor after treatment with isotretinoin.29 RARß expression is reportedly decreased in non–small-cell lung cancer as well.30
Our group identified the presence of RAR We report our detailed analysis of the expression of various RAR and RXR receptors in the prostate and in prostate cancer. Additionally, we describe the effect of 4 weeks of oral 4-HPR therapy (200 mg/d) on the expression of these receptors. We think this is the first report of in situ hybridization of retinoid receptors in clinical prostate cancer.
Altogether, 40 prostate specimens were analyzed. Eight specimens were from normal subjects (four from young organ donors and four from elderly patients with BPH), and 32 were from patients with localized prostate cancer who underwent a radical retropubic prostatectomy. The first 10 radical prostatectomy specimens were randomly picked from our surgical pathology department (group A). Group B patients (22 in all), participated in a clinical study and were randomly assigned to receive either 4-HPR (200 mg/d, orally) or placebo pills for 28 days before their radical prostatectomy. After a routine pathologic processing, formalin-fixed paraffin-embedded tissue sections 4-µm thick were selected from the benign, malignant, and adjacent nonmalignant prostate tissues. A nonradioactive mRNA in situ hybridization technique using digoxigenin-labeled antisense riboprobes for retinoid receptors was used, as previously described.34 The quality and the specificity of the digoxigenin-labeled probes were determined with northern blotting, and the specificity of the binding of antisense riboprobes was verified by using sense probes as controls.34 The stained sections were reviewed and scored using an Olympus Microscope (Olympus America Inc, Melville, NY). The observers were blinded as to whether the specimens came from patients who treated with either placebo or 4-HPR. For each prostate, an entire whole-mount section was cut off the block, hybridized, and analyzed at low magnification. Areas that appeared to be positive were verified by observation at higher magnification. Positive samples had retinoid receptor expression in at least 10% of cells. The negative samples were completely devoid of signal. Fisher’s exact test was used to determine statistical significance. This study was approved by the Baylor College of Medicine Institutional Review Board and is in accord with assurances filed with and approved by the Department of Health and Human Services. Informed consent was obtained from each subject.
Table 1 lists the expression of the various retinoid receptors in cancerous and noncancerous prostate tissues. RAR , RAR, RXR, and RXR mRNAs were detected in all the normal, as well as the cancerous, prostates. Interestingly, the intensity of the in situ hybridization signal for RXRß was much weaker than for the other receptor probes. An additional interesting observation is that RARß and RXRß expression was confined to the basal cell layer in benign glands, whereas the other retinoid receptors were expressed throughout the epithelium (Fig 1D).
RXRß mRNA was expressed in four of the eight normal prostates, but it was not detectable in any of the 10 malignant prostate specimens in group A. RARß mRNA expression was detected in seven of eight normal prostates as compared with only four of 10 group A cancerous glands. The only benign prostate that lacked RARß mRNA came from a BPH specimen. There were no statistically significant differences in expression of retinoid receptors between the young organ donors and the elderly patients with BPH. In group B, 12 patients were treated with 4-HPR, and 10 patients received placebo pills. Their clinical characteristics are listed in Table 2. The expressions of the various receptors in this group are listed in Table 1. In Table 3, the expressions of RARs and RXRs in malignant and in adjacent nonmalignant prostate tissues are listed side by side. Interestingly, in the cancerous prostates, retinoid receptor expression in the histologically normal-seeming tissue followed the same pattern as in the cancers.
RAR , RAR, RXR, and RXR mRNAs were detected in most cancerous and noncancerous prostate tissues. In the placebo group, RARß expression was detected in three of 10 specimens in the benign prostatic tissue and in one cancerous focus. RXRß was expressed in one of 10 specimens in the benign portion of the gland and in none of 10 specimens in the cancerous foci. The findings were similar in the group treated with 4-HPR. RARß expression was detected in the cancerous tissue in four of 12 specimens and in the adjacent, benign tissue in three of 12. RXRß was detected in the cancerous tissue in one of 12 specimens and in benign pericancerous areas in none of 12 specimens.
Human prostate cancer is a slow-growing tumor, with doubling times for local tumors estimated at 2 to 4 years. In addition, large populations of men are thought to be carriers of latent forms of prostate cancer. Consequently, an effective chemopreventive agent would be expected to have a significant impact on this disease. Retinoids are the most investigated class of chemopreventive drugs. They have a profound influence on the growth and differentiation of normal and malignant tissues and are involved in morphogenesis, apoptosis, extracellular matrix formation, and modulation of the immune system.19 Retinoids also have been demonstrated to inhibit carcinogenesis in epithelial cells in animal models and to reverse oral, skin, and cervical premalignancies.2-12,14,32 These data lead to the hypothesis that alterations in retinoid metabolism are involved in the carcinogenic process. A molecular lesion in the RAR gene was identified in human acute promyelocytic leukemia, and an altered expression of RARß was reported in lung cancer.35,36 The administration of retinoic acid corrected and upregulated the RARß expression in lung cancer and in premalignant oral lesions. The prostate was shown to contain endogenous retinol and its active metabolite, retinoic acid. The concentration of retinol in BPH tissues was double that in either normal prostate or in prostate cancer. Prostate cancer, on the other hand, contained five to eight times less retinoic acid than normal prostate or BPH.31 4-HPR, a synthetic retinoid, has shown long-term tolerability in breast cancer patients37 and has been studied as a possible chemopreventive agent for prostate cancer. Pollard et al32 showed that 4-HPR prevented the development of the primary tumor in methylnitrosourea-induced prostate malignancies. It also inhibited metastases of PA-III cells in Lobund-Wistar rats.32 Likewise, dietary 4-HPR decreased the tumor incidence and mass of ras+myc–induced carcinomas in the mouse prostate reconstitution model system.38
Several studies have attempted to explain the mechanism of 4-HPR’s actions. Igawa et al33 showed that at a concentration of 1 mmol, 4-HPR inhibits the growth of PC-3 cell lines and blocks the cell cycle transition from G1 to S phase. This was associated with a suppression of c-myc gene expression.33 Pienta et al39 demonstrated that 4-HPR may inhibit angiogenesis. In two studies, retinoids were found to inhibit 5- RARß and RXRß mRNA expression was found in fewer patients with prostate cancer compared with normal patients. RARß mRNA was expressed in seven of eight benign prostates, and its expression was significantly depressed in malignant prostates. RXRß expression was completely lost or markedly depressed in malignant prostates but was expressed in four of eight benign prostates. The mechanism that causes loss of RARß and RXRß mRNAs in prostate cancer is not understood. The finding of some expression of the RARß mRNA suggests that the changes are not because of structural damage to the genes but possibly because of changes in transcription. Suppression of the expression of RARß mRNA in head and neck and oral cancers did not involve changes in the structure of the RARß gene.44 The expression of RARß mRNA may depend on the level of retinoids in prostate tissue. Prostate cancer tissues were found to have five to eight times less retinoic acid than normal prostate or BPH.31 Could transforming growth factor beta (TGFß) be providing for a field effect as far as RARß expression is concerned? We have previously reported that TGFß expression is upregulated in prostate cancer,49 whereas Nugent et al50 demonstrated that TGFß might block the increase in RARß expression induced by retinoic acid. Finally, the observation that RARß is primarily expressed in basal cells could partially explain its loss in prostate cancer, which has a predominantly luminal cell phenotype. It does not explain, however, the loss of RARß expression in adjacent, benign prostatic glands that still have a morphologically normal appearing basal layer. Interestingly, a prospective study of vitamin A serum levels showed lower levels in patients with prostate cancer.16 These studies did not necessarily imply that prostate cancer patients have a deficient intake of dietary vitamin A but may suggest a decrease in tissue uptake or an increase in catabolism of vitamin A in prostate cancer. Although the mechanism of loss is unknown, decreased expression of RARß mRNA may promote carcinogenesis. This hypothesis is supported by a report that lung cancer cell lines, head and neck cancer, and oral tumors have decreased expression of RARß mRNA.35,44 The reduced expressions of RXRß and RARß mRNAs in the normal tissue adjacent to the tumor suggests that this change is an early event in prostate carcinogenesis. Similar findings of decreased RARß mRNA in the adjacent normal tissue were reported in head and neck cancers.45 Further support for the importance of RARß comes from the demonstration that isotretinoin can increase expression of RARß mRNA in premalignant oral tissues and that this upregulation positively correlates with clinical response.46 Transfection of human epidermoid lung carcinoma in vitro with an RARß expression vector also resulted in decreased tumorigenicity in nude mice, suggesting that RARß expression can suppress tumorigenesis in lung carcinoma cells.47 The use of RARß and RXRß expression as a clinical marker is premature and needs further investigation, but the finding of depressed expression of RARß and RXRß mRNAs in histologically normal tissues adjacent to established cancer supports the concept of field of cancerization, meaning that independent premalignant foci develop concurrently at various rates to form independent primary cancers in the same tissue. If the loss of RARß and RXRß expression in normal prostates is proven to be a premalignant feature, its use as a surrogate marker in prostate cancer chemoprevention studies may assume great importance. Because 50% of normal or BPH prostates also lack RXRß expression, the lack of RARß expression may be a more significant correlate.
Our study is the first to investigate in situ hybridization for localization of RAR and RXR transcripts in prostate cancer. We are unaware of any data indicating a differential expression of RAR and RXR transcripts in animal models. Most normal and malignant prostate tissues express RAR In addition, 4-HPR may not act at the level of the nuclear retinoid receptors. Several possible mechanisms of action of 4-HPR have been proposed. Moreover, as evidenced by the decreased expression of RARß in the adjacent normal prostate tissue, this suppression may represent an early change that is not affected by 4-HPR. Other retinoids may prove to be more effective in causing a change in receptor status. Pienta et al39 reported a phase II chemoprevention trial of oral 4-HPR in patients with prostate-specific antigen > 4.0 ng/mL and negative prostate biopsies that was discontinued early because prostate cancer was diagnosed in eight of the 22 participants in the study. Although the significance of the study is limited by its small sample size, 4-HPR proved to be nontoxic to the patients. More studies are necessary to examine the effect of 4-HPR and other retinoids in vivo. In conclusion, RARß and RXRß mRNAs are selectively and significantly reduced in prostate cancer as well as in adjacent normal prostate tissue. The reduced expression of RXRß and the more significant loss of expression of RARß mRNA may be associated with prostate carcinogenesis. At 200 mg/d orally for 28 days, 4-HPR had no statistically significant effect on retinoid receptor expression in the prostate.
Supported by The National Cancer Institute (SPORE P50-58204) and an award from CaP CURE.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
