New Developments in the Pathobiology of Prostate Disease

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New Developments in the Pathobiology of Prostate Disease Jack Schalken * Department of Experimental Urology, Radboud University Nijmegen Medical Center, Geert Grooteplein 30, 6525 GA Nijmegen, The Netherlands

Article info

Abstract

Keywords: Androgen receptor Benign prostatic hyperplasia Biomarkers 5a-reductase Prostate cancer Prostate-specific antigen

Our understanding of the cellular and molecular basis of prostate disease has increased substantially in the last decade. This review discusses the most recent advances in five key areas: ontogeny of prostate cancer, differential gene expression in prostate cancer, biomarkers for prostate cancer and benign prostatic hyperplasia (BPH), role of the androgen receptor in prostate cancer, and 5a-reductase isoenzyme expression in prostate disease. Knowledge of the ontogeny of prostate cancer cells has led to a greater understanding of the molecular and cellular basis for the transition from normal epithelium to prostatic intraepithelial neoplasia and carcinoma. Our understanding of oxidative stress and inflammation suggests a role for antioxidative and anti-inflammatory agents in preventing or treating prostate cancer. The underlying androgen drive for prostate cell growth and differentiation also supports the role of inhibition of 5a-reductase to reduce the risk of progression to cancer. Significant evidence indicates that synthesis of dihydrotestosterone in prostate cancer is driven by types 1 and 2 5a-reductase. This suggests that a 5a-reductase inhibitor that inhibits both isoenzymes may have a role in preventing and/or treating prostate cancer; the former is being tested in the large-scale Reduction by Dutasteride of Prostate Cancer Events study. Work on biomarkers of prostate disease has advanced and several viable candidates exist for diagnosing prostate cancer and discerning those likely to recur or progress. These markers should have a significant impact on the diagnosis and staging of prostate cancer, delineate men with BPH or indolent prostate cancer, and reduce the need for invasive testing. # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Tel. +31 24 3614146; Fax: +31 24 3541222. E-mail address: [email protected].

1.

Introduction

Our understanding of the cellular and molecular basis of prostate disease has expanded considerably during the last decade. This article reviews the most recent advances in five key areas: the ontogeny of

prostate cancer cells, differential gene expression in prostate cancer, biomarkers for prostate cancer and benign prostatic hyperplasia (BPH), the role of the androgen receptor in prostate cancer, and 5areductase isoenzyme expression in prostate disease. In doing so, it also examines the implications for

1569-9056/$ – see front matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.eursup.2006.06.012

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future diagnostic and therapeutic interventions that may make the transition from bench to bedside.

2.

Ontogeny of prostate cancer cells

One of the key areas of research in prostate cell biology is the characterisation of prostate cancer precursor cells. Prostate secretory epithelium consists of a bilayer of basal cells overlaid by secretory luminal cells and interspersed neuroendocrine cells. The basal cells are undifferentiated, self-renewing stem and transiently amplifying cells with a high proliferation index that differentiate into exocrine and neuroendocrine cells with a low proliferation index. During the process of epithelial cell differentiation, different cellular markers are expressed, and the initially androgen receptor(AR)-negative basal cells become androgen-sensitive luminal cells. The prostatic epithelial compartment is surrounded by a fibromuscular stroma, which contains AR-positive fibroblasts and smooth muscle cells that are pivotal to the survival of the epithelial compartment. Binding of dihydrotestosterone (DHT) to the AR in these cells stimulates the production of paracrine growth and survival factors (basic fibroblast growth factor [bFGF], keratinocyte growth factor [KGF], human growth factor [HGF], transforming growth factor b1 [TGF-b1], insulin-like growth factor 1 [IGF-1], epidermal growth factor [EGF], and vascular endothelial growth factor [VEGF]) that inhibit apoptosis and recruit epithelial cells into the cell cycle. There is cross-talk between the epithelial cells and the stromal cells via, for instance, VEGF and TGF-b1 [1– 4]. The pivotal role of androgens in promoting paracrine growth and survival factor secretion is evident when production of these factors is reduced; prostatic transiently amplifying cells become proliferatively quiescent, and luminal cells terminally differentiate. The effects of these factors on prevention of apoptosis is mediated via mitogen-activated protein kinases (MAPKs) and the protein kinase Akt [5]. However, it is known that TGF-b1 induces epithelial apoptosis, although to a lesser extent than androgen deprivation [6], and is inhibited by androgens, demonstrating that a balance exists between paracrine factors that modulate prostate growth and cell death. During the shift to an androgen-refractory state in prostate cancer, a change occurs in the dependency of malignant cells on these factors, with MAPKs and Akt acting in concert to inhibit apoptosis. Preliminary evidence suggests that prostate cancer cells are derived from both early and late progenitor cells, which are part of the transit amplifying cell pool [7]. These intermediate or

transit amplifying cells have common features with secretory and neuroendocrine cells, but are androgen independent. One of the key pieces of evidence for this lies in the expression of c-met, which has been identified in a population of basal cells in nonmalignant epithelium, and is also expressed in a subpopulation of prostate cancer cells. The phenotypically intermediate cells that are believed to be progenitor cells to prostate cancer are concentrated in areas of focal glandular atrophy, which occur at sites of inflammation, termed proliferative inflammatory atrophy (PIA) [8]. The current working hypothesis for the origin of prostate cancer is that prostatic inflammation, with associated increases in oxidative stress, results in damage to both proteins and DNA. The resultant genetic instability in a proliferative environment over time may lead to the development of high-grade prostatic intraepithelial neoplasia (HGPIN) and eventually to the development of prostate cancer, where androgens initially drive the expansion of the cancer cells [9].

3. Differential gene expression in prostate cancer Another fruitful area of research in prostate cancer biology has been the comparative analysis of gene expression in prostate cancer cells and nonmalignant prostate epithelial cells. During the development and progression of prostate cancer, a number of genes are differentially expressed (reviewed by Nelson et al. [10]). These include GSTP1, which encodes a carcinogen detoxification enzyme (decreased expression); NKX3.1, which encodes a prostate-specific homeobox gene that is essential for prostate development (decreased expression); PTEN, which encodes a phosphatase active against both proteins and lipids (decreased expression and/ or function); CDKN1B, which encodes p27, a cyclindependent kinase inhibitor (decreased expression); and the AR gene, in which many separate mutations have been identified (see section 5). A recent study utilised array comparative genomic hybridisation (aCGH) to analyse prostate tumours from 64 men at intermediate-to-high risk of recurrence following a radical prostatectomy [11]. Half of these men had biochemical (prostatespecific antigen [PSA]) progression following prostatectomy and half did not. Tumours from men with progressive disease had significantly more genetic aberrations than those from men without progressive disease (median 20.5 vs. 10.5; p = 0.006). Two prominent alterations that had predictive value for recurrence were deletion of 8p23 and a gain

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at 11q13.1. Deletion of 8p23 was more common in those with progression than those without (50% vs. 31%), with a significant association between deletion and advanced tumour stage (pT 3). The deletion overlaps exons of the CUB and sushi multiple domains 1 (CSMD1), which has been cloned as a candidate tumour suppressor or progression gene for tumours of the upper aerodigestive tract, ovary, and bladder, as well as for the prostate [12]. A gain at 11q13.1 was significantly associated with biochemical recurrence ( p < 0.002), a relationship that was even stronger in men with negative surgical resection margins. 11q12 is a gene-rich region, and from the six possible genes, multiple endocrine neoplasia 1 (MEN1) was identified as being the gene in question. In a separate analysis, five candidate gene locations were identified that were associated with metastasis: 3q26.2, 7q11.23, 22q13.1, 10q23.31, and 13q14.2. The authors concluded that these biomarkers have potential application in determining men likely to have progressive/metastatic disease, but additional prospective work will be needed to confirm their value. Recurrent chromosomal rearrangements are an important feature of the development of cancer, especially in haematologic malignancies. These rearrangements occur when promotor and/or enhancer components of one gene are juxtaposed to an oncogene, resulting in altered expression of an oncogenic protein, or where two genes are fused to create an abnormal protein with different activity. These rearrangements have been observed in a number of tumour types including lymphomas, leukemias, and myeloma, but although nonspecific gene rearrangements are common in carcinomas, recurrent rearrangements occur less frequently [13]. Recent studies have demonstrated several genes are commonly involved in fusion-type recurrent chromosomal translocations in prostate cancer. ERG and ETV1 and ETV4, all members of the ETS transcription factor family, are overexpressed in a mutually exclusive fashion in prostate cancer, but not in PIN or benign epithelium [14,15]. These genes fuse with TMPRSS2 [15], a type 2 transmembranebound serine protease that is highly expressed in the prostate and prostate cancer [16] and whose expression is androgen sensitive [17]. A recent study has also shown that the fusion allows androgens to stimulate the overexpression of the ERG/ETV1 and TMPRSS2 fusion gene. The implication is that androgen stimulation is directly responsible for promotion of a key aberrant fusion gene involved in prostate cancer tumourigenesis. This provides crucial new insights into the ‘‘oncogenic’’ role of the AR and has implications for endocrine therapy. Likewise, reduc-

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tions in the development of prostate cancer with inhibition of 5a-reductase (5-AR), reducing DHT synthesis, may, at least in part, be explained by this observation.

4.

Biomarkers for prostate cancer and BPH

The use of serum PSA has evolved from its original indication as a marker of disease progression in men with diagnosed prostate cancer to its more widespread use as a biomarker for malignancy. Although it has been understood for some time that the sensitivity and specificity of PSA at either commonly used threshold value of 2.5 or 4.0 ng/ml for prostate cancer is less than optimal, recent evidence also suggests that this pattern has changed with the more widespread use of PSA for cancer screening. It is now believed that PSA may be a better marker of BPH than of prostate cancer [18] although this has been disputed [19]. Much attention has focussed on the relative merits of different PSA molecular forms for prostate cancer: total PSA (tPSA), free PSA (fPSA), ‘‘nicked’’ PSA, PSA velocity, PSA density, PSA doubling time, age-adjusted PSA, and free/total PSA (f/ tPSA). The multicentre European Prostate Cancer Detection study evaluated the value and performance of the molecular forms of PSA and their derivatives in the early detection of prostate cancer [20]. The study concluded that complexed PSA (cPSA) performs better than tPSA in the differentiation between benign disease and prostate cancer, while providing similar information to the f/tPSA ratio. In addition, cPSA and cPSA volume-related parameters further improved the specificity of PSA in the detection of prostate cancer. However, area under the curve (AUC) values for all forms of PSA varied between 56.9 and 62.8, reinforcing the need for novel approaches. For BPH, the aim of PSA evaluation is clearly different. Considerable evidence indicates that total PSA correlates with prostate volume and future prostatic growth, but perhaps more importantly, that it has value in predicting clinical progression [21,22]. Recent evidence from the Medical Therapy of Prostatic Symptoms (MTOPS) study suggests that ‘‘benign PSA,’’ a form of free PSA that is associated with BPH, but not total PSA, is a significant predictor of BPH progression to acute urinary retention (AUR) or surgery [23]. From the above discussion, it is clear that there is a need for better biomarkers in the evaluation of both benign and malignant prostate disease. For the detection of prostate cancer, the lack of sensitivity of PSA can result in missed diagnosis; the likelihood of a positive second biopsy following an initially

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Table 1 – Potential prostate cancer biomarkers [26,27] 1. Genomic alterations a. Prostate cancer-associated gene mutations  HPC1 (1q24)  CAPB (1p36)  PCAP (1q42)  ELAC2 (17p11)  HPC20 (20q13)  8p22–23  HPCX (Xq27–28) b. Mitochondrial DNA alterations c. Microsatellite alterations 2. Prostate cancer-specific biological processes a. Kallikreins: 15 members of the gene family on chromosome 19q13.3–13.4, which may be over-expressed in prostate cancer progression 3. Epigenetic modifications  RASSF1A  GSTP1 4. Genes uniquely expressed in prostate cancer  TMPRSS2  a-methylacyl-CoA racemase  Hepsin  Telomerase  Prostate-specific membrane antigen  Delta-catenin  DD3PCA3  p53  HSP-27 and protein kinase C isoenzymes

negative one in a man with a persistently elevated PSA level is 10–20% [24]. Furthermore, if PSA, digital rectal examination (DRE), and transurethral ultrasound biopsy do indicate clinically confined disease, 40% of men have extracapsular penetration evident by radical prostatectomy [25]. Potential biomarkers can be divided into several categories: hereditary genomic alterations, somatic (prostate cancer cell) DNA changes, epigenetic modifications (alterations in DNA without changes in base sequence) and

differentially expressed genes (Table 1) [26,27]. These markers have a number of potential uses: reduction in the number of unnecessary biopsies by distinguishing benign from malignant disease, discrimination of organ-confined disease from locally advanced disease, prediction of tumour grade and aggressiveness, prediction of tumours likely to progress; and highlighting tumour cells for imaging purposes (Table 2) [26]. The most important opportunities for new biomarkers are distinguishing clinically significant from insignificant disease and better evaluating treatment response to predict clinical outcome (prognosis). Although many markers are currently being evaluated and more are likely to emerge in the next few years, it is unclear how many will offer the sensitivity and specificity needed for an adequate diagnosis of prostate cancer. For prostate cancer diagnosis, a number of markers appear to have better sensitivity/specificity than PSA; further studies should elucidate their value in larger populations (Table 3). One of the markers that has received considerable recent attention is DD3PCA3. This gene is the most prostate cancer-specific gene described to date; it is overexpressed in >90% of tumours [28]. Specimens with <10% of cancer cells can be accurately discriminated from noncancer tissues [29], providing a rationale for the use of a test on urinary sediments after an extended DRE. In a study of 108 men with a serum PSA value >3 ng/ml, 24 of whom had prostate cancer on biopsy, 16 were shown to be positive for DD3PCA3, demonstrating a sensitivity of 67% using a cut-off value of 130  10 3 RNA copies. The use of DD3PCA3 has been further examined using a newly developed nucleic acid sequence based amplification assay (uPM3) for detecting RNA in

Table 2 – Potential applications of biomarkers for prostate cancer [26] Goal Reduction in the number of unnecessary biopsies, by distinguishing benign from malignant disease

Discrimination of organ-confined disease from locally advanced disease

Biomarker

Application

50.3-kDa protein, PSMA, hK11/tPSA, hK2/fPSA. Combination of tPSA, %fPSA and %sum-pro-PSA GSTP1 DD3PCA3

Serum

Urine Urine

hK2, fPSA/(tPSAxhK2)

Serum

Prediction of Grade 3 (poorly differentiated) tumours

hK2/fPSA, fPSA/(tPSAxhK2)

Serum

Distinguish more aggressive tumours from indolent ones

Hepsin RASSF1A

Serum Urine

Prediction of disease progression (circulating tumour cells)

PSMA DD3PCA3

Tissue, blood Blood

Candidate for molecular probe in imaging modalities

AMACR

Tumour site

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Table 3 – Application, area under the curve (AUC), sensitivity, and specificity of biomarkers under evaluation for prostate cancer diagnosis [26] Biomarker hK2 fPSA/(tPSAxhK2) hK2/fPSA hK11/tPSA GSTP1 Telomerase DD3PCA3 AMACR

Application

AUC

Sensitivity

Specificity

Serum Serum Serum Serum Urine Urine Urine Serum

0.68 0.75 0.86 0.77

90% 88% 100% 90% 73% 58% 67% 97%

20% 57% 48.2% 51.5% 98% 100% 83% 100%

urine samples. Sensitivity, specificity, positive predictive value, and negative predictive value were 82%, 76%, 67%, and 87% respectively, compared with 98%, 5%, 40%, and 83% for PSA at a cut-off of 2.5 ng/ ml. The AUC was 0.87 (CI, 0.81–0.92). For a PSA value of <4, 4–10, and >10 ng/ml, sensitivity was 73%, 84%, and 84% and specificity was 61%, 80%, and 70%, respectively [30]. Furthermore, a correlation between Gleason score and the PCA3/PSA mRNA ratio has been observed from biopsy samples, suggesting that DD3PCA3 may also have value as a prognostic, as well as a diagnostic, indicator (Fig. 1) [31]. DD3PCA3 is now under further evaluation in larger-scale studies with longer follow-up. Another marker of great interest is a-methylacylcoenzyme A racemase (AMACR). This enzyme is critical in peroxisomal b-oxidation of branchedchain fatty acid molecules found in beef and dairy products [32]. AMACR mRNA expression is increased by almost 1 log in prostate cancer versus normal tissue, whereas expression in needle biopsies was

Fig. 1 – Mean and median ratios of PCA3:PSA mRNA by Gleason score from biopsies of men with prostate cancer [31].

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originally reported to have a sensitivity of 97% and specificity of 100% for prostate cancer [33]. However, it is now known that AMACR is also expressed in HGPIN, and its utility in predicting progression from HGPIN to carcinoma is as yet unknown. Current recommendations are for its use in needle biopsy specimens ‘‘. . .to convert an atypical diagnosis to cancer in cases that are highly suspicious for cancer on hematoxylin and eosin stained sections and negative for basal cell markers, yet in which a confident diagnosis of cancer still cannot be made’’ [34].

5.

The AR axis in prostate cancer

The role of androgens, principally DHT, and the AR is an important focus for research in prostate pathobiology. DHT, via AR, up-regulates a wide array of genes in prostate epithelial cells. Gene regulation by androgens has been investigated by blocking DHT synthesis through inhibition of the types 1 and 2 5-AR enzymes with the dual 5-AR inhibitor (5-ARI) dutasteride [35]. Addition of dutasteride to LNCaP cells results in a change in expression of a wide array of genes involved in metabolism, catalytic activity, cell growth/maintenance, protein metabolism, signal transduction, and many other processes. Overall, these studies offer an insight into the central role of DHT and AR signalling pathways in the function of benign and malignant prostate cells. The AR plays an important role in the maintenance of prostate tumour cell growth [36]. Although androgen deprivation therapy has shortterm effects on tumour viability, most tumours become insensitive to the currently available endocrine therapies. This can be explained by a number of mechanisms. First, it is known that the number of AR-positive cells and the density of ARs decrease immediately following castration, but that AR density, and to a lesser extent cellular positivity, rises thereafter [37]. Recent evidence suggests that not only do androgen-refractory prostate cancer

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cells express ARs to a greater extent than androgensensitive cells, but also that increased AR expression and binding of androgen is required for a transition to a hormone-refractory state [38]. It may seem paradoxical that such a transition can occur during androgen deprivation therapy, but it is becoming clear that androgen levels, although significantly reduced in the prostate during such therapy, may be sufficient to activate ARs [39]. Second, 85 mutations in the ARs have been identified in different prostate cancer specimens, the majority of which are somatic, single-base substitutions [40]. However, the pathogenetic role in enhancing prostate cancer risk or tumour severity of the majority of these mutations has not yet been established. Third, evidence indicates that the expression of cofactors (activators and repressors) that modulate AR nuclear function is altered in prostate cancer [41]. Finally, evidence indicates that prostate cancer is associated with greater downstream redundancy of MAPKs, which are key mediators in signalling cascades for cell survival and proliferation [42]. Such redundancy in androgen-independent tumours may serve to ensure cell survival during androgen deprivation.

6. 5a-reductase isoenzyme expression in prostate disease The biologic activity of the AR is predominantly driven by DHT, a reduced form of testosterone, which is mediated by 5-AR isoenzymes. Thus, these

enzymes are of particular interest for prostate growth. The cellular localisation of the two isoenzymes of 5-AR, types 1 and 2, has been examined in a number of studies, with conflicting results. Early studies suggested that whereas type 1 5-AR was expressed by both epithelial and stromal cells, type 2 was present only in stromal cells [43]. However, evidence now shows that type 2 5-AR is also expressed in epithelial cells [44]. As with studies of cellular localisation, those examining the distribution of 5-AR isoenzymes in normal versus hyperplastic and malignant prostate tissue have produced differing results depending on the assessment methods used. However, it has become evident that expression of not only type 2 5-AR, but also type 1, is elevated in hyperplastic prostate tissue, and that, furthermore, type 1 expression may be enhanced in prostate cancer [45]. Most recently, evidence has emerged that immunostaining for type 1 5-AR in BPH is low to moderate in intensity and primarily nuclear, whereas in localised prostate cancer, high-intensity staining is observed in 28% of specimens and is primarily cytoplasmic [46]. An extension of this work has demonstrated that whereas type 1 5-AR expression in BPH cells is low, it rises steadily in PIN and primary, recurrent, and metastatic prostate cancer cells. In contrast, type 2 5-AR expression is lower in prostate cancer than in normal or hyperplastic tissue (Fig. 2) [47]. Taken together, these data reinforce the role of both isoenzymes of 5-AR in the development of prostate disease, and, in particular, the role of type 1 5-AR in prostate cancer.

Fig. 2 – 5a-reductase type 1 and 2 isoenzyme expression in normal tissue and that from men with BPH and prostate cancer (PCa) [47].

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7.

Conclusions

Our knowledge of the pathobiology of prostate disease is evolving rapidly. Knowledge of the ontogeny of prostate cancer and its cellular origin (cancer stem cell model/hypothesis) has led to a greater understanding of the cell biologic origin of prostate cancer. Our understanding of the role of oxidative stress and inflammation, both from infection and other sources, suggests a role for antioxidative and anti-inflammatory agents in the prevention or even the treatment of prostate cancer. The underlying androgen drive for prostate cell growth and differentiation also supports the role of inhibition of 5-AR to reduce the risk of progression to cancer. This is reinforced by new data demonstrating the androgen dependency of the overexpression of the ERG/ETV1 and ETV4 and TMPRSS2 fusion gene; these may be key aberrant fusion genes involved in prostate cancer tumorigenesis. Work on biomarkers of prostate disease has advanced substantially in recent years. Several viable candidates exist not only for the diagnosis of prostate cancer but also for discerning those likely to recur or progress to metastasis. The PSA era may not be over yet, but it is likely that new biomarkers, with a more favourable sensitivity and specificity profile, will become available for clinical practice within a decade. These markers should have a significant impact on the diagnosis and staging of prostate cancer, delineate men with BPH, and reduce the need for invasive testing with biopsies. Although the androgen dependency of prostate cancer and the propensity for escape from endocrine therapy have been recognised for quite some time, a more detailed understanding of androgen synthesis and receptor pathways is coming to fruition. A significant body of evidence now indicates that synthesis of DHT in prostate cancer is driven by both type 1 and type 2 5-AR, in contrast with BPH. This suggests that a 5-ARI that inhibits both isoenzymes may have a role in the prevention and/or treatment of prostate cancer; the former is currently being tested in the large-scale REDUCE study. We now have a greater understanding of the nature of escape from currently registered therapies for prostate cancer. Increased AR expression is not only a feature of an androgen-refractory state, it is a necessary event for it, best illustrated by AR amplifications being the most common event in advanced prostate cancer. Furthermore, the concentration of DHT in the prostate, rather than circulating androgen levels, may shed light on the stimulation of the AR in advanced prostate cancer.

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This has clear implications for the future development of strategies for the management of androgen refractory disease.

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