MOLECULAR AND CELLULAR PATHOGENESIS OF BENIGN PROSTATIC HYPERPLASIA

0022-5347/04/1725-1784/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 172, 1784 –1791, November 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000133655.71782.14

MOLECULAR AND CELLULAR PATHOGENESIS OF BENIGN PROSTATIC HYPERPLASIA KEITH L. LEE

AND

DONNA M. PEEHL*,†

From the Department of Urology, Stanford University School of Medicine, Stanford, California

ABSTRACT

Purpose: Symptomatic benign prostatic hyperplasia (BPH) is one of the most common ailments seen by the urologist. Significant advances have occurred in medical and surgical therapy, and in the understanding of the biology of this disease. However, the basic science literature is often conflicting and confusing, without a unified voice. We report the current state of knowledge of the molecular and cellular basis of BPH. Materials and Methods: We compiled and interpreted basic science studies relevant to BPH pathogenesis. Results: Cellular alterations that include changes in proliferation, differentiation, apoptosis and senescence in the epithelium and stroma are implicated in BPH pathogenesis. Molecular analyses have yielded numerous candidate genes important in disease progression. Differential expression of cytokines and growth factors in BPH tissue suggests roles for inflammation and hypoxia. Through the use of cell culture models the complex regulatory mechanisms of growth control in BPH are becoming defined. Conclusions: The scientific endeavor has resulted in great strides in our understanding of BPH on a molecular and cellular level. It is hopeful that basic science and translational research will improve treatment and prevention strategies for this common disease of elderly men. KEY WORDS: prostate, prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is a common age related proliferative abnormality of the human prostate.1 The prostate in man is a composite organ whose glandular tissue comprises 3 histologically and anatomically distinct zones— the peripheral zone, central zone and transition zone (TZ). The TZ is the smallest and normally makes up 5% of the glandular prostate, and it is the only zone where BPH develops.2 The disease process is multifocal with expansile nodules filling the right and left TZs. Detailed autopsy and radical prostatectomy studies have shown that these nodules are caused mainly by budding and branching of epithelial glandular tissue (ie ducts and acini), and to a lesser degree by glandular unit enlargement or proliferation of prostatic stromal elements (ie smooth muscle and fibroblasts).2, 3 The prostates of other mammals occasionally become hyperplastic or can be stimulated to undergo excessive growth. However, in no species besides man and perhaps the chimpanzee have the cardinal features of BPH, multinodularity and architectural proliferation, been demonstrated.4 Consequently, this review is focused primarily on the findings of studies, mostly recent, of molecular and cellular features of human prostatic hyperplasia. Other species are excluded on the grounds that they possibly have little relevance to human biology. The colloquial term “BPH” should probably not be used except in humans until biological differences underlying multinodular versus diffuse hyperplasia are better understood. CELLULAR ALTERATIONS IN STROMA OF BPH

Morphologically, BPH is characterized by the formation of new architecture by budding of the epithelium from preexisting ducts.2 Although the formation of new architecture is a

central feature of embryonic development, this process is normally repressed in adult life. The appearance of the mesenchyme in periurethral nodules, the earliest manifestation of BPH, is reminiscent of embryonic mesenchyme. Morphological evidence of this type led to the hypothesis that BPH is intrinsically a mesenchymal disease, and results from a reawakening of embryonic inductive interactions between the prostatic stroma and epithelium.2 Bierhoff et al undertook a detailed comparison of fetal prostatic stroma and the stroma of BPH nodules.5 The fetal stroma displayed a developmental program of immature mesenchyme followed by the appearance of fibroblastic and fibromuscular components, culminating in mainly smooth muscle at the end of gestation. Similarly, individual BPH nodules displayed each of these phenotypes. These investigators concluded that ontogenetic processes are recapitulated in the development of BPH nodules, supporting the theory of embryonic “reawakening” in the pathogenesis of BPH. Given that stromal cells serve important paracrine regulatory functions in epithelial cell homeostasis, alterations in the stroma can lead to changes in stromal-epithelial interactions and BPH progression.6 Lin et al searched for evidence of phenotypic modulations in BPH stroma and found that smooth muscle elements are altered. Smooth muscle myosin heavy chain was significantly decreased in BPH without a concomitant increase in nonmuscle myosin heavy chain (NMMHC).7 This result was surprising, since, morphometrically, BPH had an increased content of smooth muscle compared to normal tissues. Another difference noted was the presence of cell clusters expressing NMMHC in BPH stroma that were not seen in normal stroma. These investigators concluded that active proliferation of smooth muscle probably occurs early in BPH development. In later stages smooth muscle is not actively proliferating (as indicated by no increase in NMMHC), but decreased expression of smooth muscle myosin heavy chain is evidence of previous proliferation. The heterogeneous nature of BPH nodules in different

* Correspondence: Department of Urology, Stanford University Medical Center, Stanford, California 94305-5118 (telephone: 650725-5531; FAX: 650-723-4200; e-mail: [email protected]). † Financial interest and/or other relationship with Jenapharm GmbH. 1784

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prostatic regions makes generalization difficult. Early nodules in the periurethral area are mostly stromal. This finding is in contrast to nodules in the TZ, where epithelial glands predominate with minimal stroma.2 In addition, tissue composition of BPH can change with time.3 Some investigators report that smooth muscle is decreased rather than increased in BPH compared to normal tissue, with an overall increase in fibrous elements.8 Whether the overall stromal-toepithelial ratio is altered in BPH compared to normal tissue is still debated. Some have claimed that the normal ratio is 2:1, and that the ratio increases to about 5:1 in BPH.7, 8 In contrast, Doehring et al found that the stromal-to-epithelial ratio was equivalent in patients with hereditary BPH (characterized by autosomal dominant inheritance and early onset of BPH severe enough to require prostatectomy) and age matched subjects.9 CELLULAR ALTERATIONS IN EPITHELIUM OF BPH

Human prostatic epithelium consists of 3 major cell types—luminal secretory epithelium, basal epithelium and neuroendocrine. The luminal epithelia are fully differentiated, while the basal layer consists of putative epithelial stem cells and transit proliferating/amplifying cells.10 Neuroendocrine cells are scattered throughout the epithelium. All cell types are affected during the development of BPH. In the BPH secretory epithelium overall protein expression seems depressed. RNA transcript analysis of whole tissues revealed that expression of several genes is decreased in BPH.11 One of these genes is the prostate specific membrane antigen (PSMA) gene, which encodes a membrane glycoprotein that is abundant in luminal epithelial cells of normal prostatic tissues. Furthermore, Wright et al confirmed that PSMA protein expression was also decreased in BPH luminal cells.12 Production of a splicing variant of the PSMA transcript by BPH cells is a possible explanation for lower detection of protein by immunohistochemical analysis. In addition, differential expression of ␣1-antichymotrypsin (ACT) between luminal epithelia of normal and BPH tissues is striking. ACT is a serine protease inhibitor to which the major proportion of prostate specific antigen is complexed in serum. Using immunohistochemical analysis and in situ hybridization, Bjork et al observed that BPH epithelia expressed little or no ACT, whereas normal prostatic epithelia expressed abundant amounts.13 This finding may be relevant to increased serum prostate specific antigen levels in individuals with BPH. Given the prominent role of ␣1-adrenergic antagonists in medical treatment of BPH, it is interesting to consider the localization and expression of ␣1-adrenergic receptor subtypes.14 While adrenoceptor blockade in the stroma is believed to mediate the clinical effects of ␣1-adrenergic antagonists, studies by Walden et al suggest that adrenoceptors in the epithelia might also be relevant to the pathophysiology of BPH.15 Using adrenoceptor subtype specific antibodies, these investigators observed decreased expression of ␣1Badrenergic receptors in BPH epithelium. The medical and biological significance of this finding is not yet known. In contrast, vimentin is an example of a protein whose expression is enhanced in the BPH epithelium.16 Expression of this mesenchymal marker may be evidence of an epithelial to mesenchymal transformation. Wright et al also identified an uncharacterized member of the super immunoglobulin (IgG) gene family that was over expressed in BPH.17 Since this protein appears to be secreted and is also lower in cancer compared to BPH tissues, levels of this IgG related factor in body fluids may be useful for diagnosing BPH versus adenocarcinoma. The basal epithelium is attenuated in BPH, as is readily apparent with the use of antibodies against basal cell markers, such as keratin 5.18 Similarly, immunohistochemical staining

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with antibody against a basal epithelial cellular adhesion molecule (C-CAM) has been informative. Kleinerman et al observed C-CAM staining of a continuous basal epithelial cell layer in normal tissues, whereas only scattered or no staining occurred in BPH.19 Cellular adhesion molecules are believed to have a critical role in maintaining the differentiated status of epithelia. With the loss of C-CAM in BPH one wonders if epithelial cells may undergo functional change or if the loss of C-CAM only reflects attenuation of basal cells. Neuroendocrine cells secrete numerous peptide hormones and biogenic amines, and may have a regulatory role in prostatic growth and differentiation in addition to exocrine activity.20 Xue et al evaluated neuroendocrine cell density in the prostatic epithelium from fetal through adult life and observed that neuroendocrine cells were mostly absent from active growth foci of budding tips of glands.21 Perhaps this observation is relevant to findings regarding the number of neuroendocrine cells in BPH nodules. Cockett et al reported that neuroendocrine cells were significantly less frequent in BPH nodules compared to normal tissues, and this finding was especially true for large nodules.22 These investigators suggested that neuroendocrine cells in the smaller nodules contribute to more rapid growth that is presumably occurring as small nodules develop. In contrast, the absence of neuroendocrine cells in large nodules was postulated to be related to the mature and less active nature of large nodules. However, the observations of Xue et al regarding the absence of neuroendocrine cells from active foci of growth in budding tips raises an alternative possibility that large BPH nodules are still undergoing active growth.21 Finally, the histological appearance of epithelium is different in BPH. In a morphometric analysis Babinski et al found that the luminal secretory epithelium in BPH is flattened, with a 28% decrease in mean epithelial height compared to normal TZ.23 In addition, the intraluminal space is increased in BPH by 125%. As a result, epithelial glands appear more cystic in BPH. The authors suggest that locally expansile BPH nodules could obstruct the epithelial ducts, leading to secretory stasis as a mechanism for this morphological change. Cellular changes in the epithelium and stroma of BPH are summarized in Appendix 1. GENE EXPRESSION AND GENETIC CHANGES IN BPH

With progress in medical informatics and microarray analysis investigators have compared differential global gene expression between BPH specimens, histologically normal tissues and high grade prostate cancer.24 Not surprisingly, gene expression patterns in BPH are different from normal and cancer. For example in a cluster analysis of more then 6,500 genes by Stamey et al 22 genes were up-regulated and 64 were downregulated in high grade cancer compared to BPH controls.25 Others have categorized BPH tissue based on clinical symptoms in an effort to identify genes that may be associated with a higher risk of symptomatic BPH.26 Using the comprehensive gene array approach, numerous (ie greater than 500) candidate genes were identified. However, the relative significance of each of these genes remains to be determined. Walden et al used mRNA differential display to identify differences in genetic expression between BPH and nonhyperplastic tissues of the TZ.27 They performed separate analyses with BPH containing a high stromal-to-epithelial ratio versus that with a high epithelial-to-stromal ratio. Two BPH associated genes were identified. One, the gene for chondroitin/dermatan sulfate proteoglycan, was over expressed in BPH with the high stromal-to-epithelial ratio. The other, B cell translocation gene 2, was over expressed in the sample with a greater epithelial than stromal content. However, immunohistochemical labeling of prostatic tissues with antibodies against proteins of these 2 genes did not confirm increased expression in BPH nodules. This finding concurs

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with that of Goulas et al, who reported that the total content of chondroitin and dermatan sulfates did not differ between BPH and normal tissues.28 However, these investigators found that the ratio of chondroitin to dermatan sulfate was substantially increased in BPH, implicating altered enzymatic activity involved in post-translational modification of the core protein. The ability of chondroitin sulfate to promote proliferation could be relevant to BPH growth. In addition, the protein product of the B cell translocation gene 2 was abundantly expressed in atrophic glands, which concurs with its activity in quiescent cells. Since tissues were assessed before applying the mRNA differential display technique to avoid an excess of atrophic glands in BPH tissue, the authors interpret their findings to suggest that increased RNA transcription of B cell translocation gene 2 may occur in BPH epithelium before accumulation of protein and atrophic gland development. One of the major limitations of gene array analyses as indicated by Stamey et al is the heterogeneous nature of the prostate.29 Different zones are architecturally distinct, and gene expression patterns may differ due to tissue location rather than BPH alterations. Genetic analyses of BPH that require large volumes of tissues also suffer from the possibility of undetected cancer present in the specimens. Furthermore, gene expression even in noncancerous lesions such as proliferative inflammatory atrophy can be quite different from that in normal tissues and BPH.30 Thus, reports of somatic mutations in BPH must be interpreted with caution.31–33 However, the results of one striking study cannot be attributed to inaccurate evaluation of tissue composition. Bedford and van Helden found that global levels of genomic DNA methylation were considerably lower in BPH than in normal tissues or in primary adenocarcinomas of the prostate.34 Methylation of genomic DNA is a dynamic process involved in the regulation of gene expression. Although the exact role for global genomic DNA hypomethylation in BPH is the subject of continued research, the differences seen in normal, BPH and cancer tissue highlight the importance of tissue homogeneity in genetic analysis. An intriguing publication in 1997 described methodology that achieved almost perfect discrimination between normal, BPH and malignant prostatic tissues.35 Models of DNA structure based on logistic regression of infrared spectral data were used to calculate the probability of the histological nature of the tissue. The models had a sensitivity and specificity of 100% for distinguishing normal versus cancer and normal versus BPH, and almost 100% for distinguishing BPH versus cancer. The authors believed that the hydroxyl radical was likely the main contributor to the structural alterations in DNA that were recognized by this technology. The presence of hydroxyl radicals is evidence of radical induced mutagenic base modifications. Therefore, these findings suggest that genetic damage is involved in the etiology of BPH. In an effort to be more tissue selective investigators are using laser capture microdissection (LCM) in conjunction with microarray analysis and primer directed methylation studies.36 Using LCM, Nakayama et al showed that normal and BPH tissues differ significantly from proliferative inflammatory atrophy and cancer.30 Thus, future genetic studies must build on the cell selective principle and avoid confounders generated by tissue heterogeneity. As is apparent from our analysis, a variety of studies have compared levels of genetic transcripts by measurement of RNA from whole tissues, and in many of these studies the RNA or corresponding protein has not been localized to specific cells in the tissue by in situ hybridization or immunohistochemical analysis. In the absence of such additional information it is difficult to rule out the possibility that observed differential expression is not solely due to a difference in cellular composition of the tissues being compared.

Nevertheless, several studies suggest that components of extracellular matrix are differentially expressed between normal and BPH tissues. Using competitive reverse transcription polymerase chain reaction analysis, Djavan et al found that elastin expression was significantly decreased in BPH tissues.37 Alterations in elasticity of the TZ may relate to bladder outlet obstruction associated with BPH, and changes in the extracellular matrix such as decreased elastin suggest structural remodeling (that may be a cause or effect of BPH). DISCOVERIES FROM CELL CULTURE MODELS

Cell culture systems provide an experimental environment where genetic expression, protein interactions and other intercellular events can be studied in a controlled setting. Establishing primary cultures of stromal and epithelial cells from normal, malignant and BPH tissues is now a widespread practice38, 39 Different groups have reported several features in common among stromal cells cultured from different zones of the prostate or from pathological tissue specimens, such as the expression of smooth muscle actin, smooth muscle myosin, fibronectin and vimentin.39 Furthermore, smooth muscle differentiation can be enhanced by manipulating culture conditions.39 Similarly, cultured epithelial cells generally maintain a basal and/or transit amplifying cell phenotype unless special complex culture methods are used.38 Cultured prostatic stromal and epithelial cells express numerous autocrine and paracrine growth factors such as fibroblast growth factors (FGFs), epidermal growth factors, insulin-like growth factors (IGFs) and others. Steroid hormone receptors such as androgen receptors may be expressed, although functional responses may be limited. Comparative studies have demonstrated a number of differences between normal and BPH cells that may be biologically significant. Hormones. Androgens have an important role in normal prostate and BPH development. Men with genetic diseases that block or impair androgen activity or who undergo castration before puberty do not have development of BPH.1 Moreover, medical therapies such as androgen withdrawal and inhibition of testosterone metabolism can yield clinical responses in patients with symptomatic BPH.1 Within the prostate testosterone is converted to the biologically more potent androgen dihydrotestosterone via 5␣reductase. Enzymatic activity of 5␣-reductase activity is about 7-fold higher in cultured BPH compared to normal stromal cells.40 Interestingly, 17␤-hydroxysteroid dehydrogenase activity (which metabolizes testosterone to the inactive ⌬4-androstenedione) is 250-fold higher in BPH stromal cells. The potential biological significance of these findings is not yet clear. Certainly increased production of dihydrotestosterone could contribute to increased growth in BPH. However, the production of the inactive ⌬4-androstenedione would decrease testosterone levels. Nonetheless, it is important to note that the conversion of testosterone to ⌬4-androstenedione is reversible. Thus, it is possible that increased 17␤-hydroxysteroid dehydrogenase activity might result in overall higher testosterone levels in BPH. Interpretation of these results is clouded because normal tissues used in this study were acquired from organ donors and BPH tissues were obtained from transurethral resection of the prostate. There was likely a significant difference in age between these groups, and androgen metabolism can change with age. Furthermore, tissues from transurethral resections are usually heterogeneous and are not a good source of BPH nodular tissue. Despite efforts to correlate circulating levels of androgen or estrogen with BPH, investigators have not conclusively detected any associations. Intraprostatic levels of hormones or hormone receptors may be more relevant but are challenging to measure.41 Bonnet et al measured prostatic tissue levels of

PATHOGENESIS OF BENIGN PROSTATIC HYPERPLASIA

androgen receptor, estrogen receptor, 5␣-reductase types I and II, testosterone and ␤-estradiol, and found that the steroid hormone receptors and 5␣-reductase were lower in BPH compared to normal.42 On the other hand, nuclear androgen receptor was up-regulated in BPH basal epithelium compared to normal.43 Hiramatsu et al observed that aromatase was increased in BPH, especially in proliferative stroma, suggesting local increase of estrogen levels.44 Additional modification of hormonal response may occur via steroid receptor co-factors. Mestayer et al found that in cultured cells several androgen receptor co-activators that could enhance androgen activity (ie ARA54, ARA55, SRC1) were up-regulated in BPH stromal cells compared to normal.45 Similarly, the expression of corepressors affects receptor activity. Agoulnik et al found that DAX-1, a repressor of androgen receptor activity, is markedly decreased in BPH.46 Low DAX-1 expression in BPH could also contribute to increased androgen mediated growth. Growth factors and cytokines implicated in BPH. A number of peptide growth factors are reportedly over expressed in BPH. Members of the FGF, IGF and transforming growth factor (TGF) families in particular have been implicated. Certainly the known mitogenic activities of FGF-1, 2 and 7 on stromal and/or epithelial cells are consistent with a growth promoting role in BPH. FGFs are produced for the most part by stromal cells, with some small amount of FGF-2 production by epithelial cells, suggesting autocrine and paracrine modes of action. Local hypoxia in BPH may be the initial event that induces FGF production. As BPH tissue grows, oxygen consumption increases and exceeds supply.47 This condition can lead to hypoxia and subsequent up-regulation of hypoxia inducible factor 1 (HIF-1). Binding of HIF-1 to hypoxia response elements then activates hypoxia response genes and release of FGFs.47 Berger et al reported that when cultured stromal cells were exposed to hypoxia HIF-1 became up-regulated in a time dependent manner, and the secretion of FGF-2 and FGF-7 also increased.47 Quantitative analysis of FGF-2 and FGF-7 revealed over expression of both in BPH.48 Furthermore, analysis of cellular proliferation showed a strong correlation between the epithelial proliferation index and FGF-7 content, consistent with the known mitogenic activity of FGF-7 in epithelial cells.49 There was a weak correlation of FGF-2 content with the proliferative index of stromal cells, the major target of FGF-2. Further investigations by these researchers demonstrated that cells expressing FGF-7 were preferentially localized to the stroma immediately adjacent to the epithelium.49 This finding suggests that epithelial cells may induce the expression of FGF-7 in nearby stromal cells. The paracrine factor responsible for this induction of FGF-7 is believed to be the cytokine interleukin (IL)-1␣. IL-1␣ is known to induce FGF-7 expression in cultured prostatic stromal cells.49 Furthermore, IL-1␣ levels were increased in BPH and correlated with FGF-7 levels. Therefore, a “double paracrine loop” of epithelial IL-1␣ induction of stromal FGF-7, which stimulates growth of epithelial cells and increases IL-1␣ expression, may lead to increased tissue mass in BPH. A similar paracrine loop may exist for FGF-2 and IL-8.50 Epithelial cells secrete IL-8 as they do IL-1␣, and IL-8 induces the production of FGF-2 in prostatic stromal cells. Also, similar to IL-1␣, IL-8 is secreted by epithelial cells or inflammatory cells in vivo and is increased in BPH tissues. Another member of the FGF family, FGF-9, was recently discovered to be abundant in the prostate and present at higher levels in BPH than in normal. Similar to FGF-7, FGF-9 is secreted by stromal cells. However, unlike FGF-7, FGF-9 is mitogenic to prostatic stromal and epithelial cells.51 Regulatory derangement in BPH also occurs at another level via the pro-inflammatory cytokine IL-17. This cytokine, which is increased in activated T lymphocytes and perhaps in epithelial and smooth muscle cells in BPH, stimulates epi-

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thelial, endothelial and fibroblastic cells to secrete cytokines such as IL-8. Steiner et al found that IL-17 is negligible in normal prostates but increased in 79% of BPH specimens.52 Others have observed that mRNA levels of the proinflammatory cytokines IL-2 and IL-4 were up-regulated 10fold and 13-fold, respectively, in BPH tissue.53 These investigators localized increased expression to T lymphocytes, and concluded that chronic inflammation and T cell lymphocytic infiltration are important in the pathogenesis of BPH. Interestingly, patterns of cytokine expression can change with BPH progression as different subpopulations of T lymphocytes are activated.54 Type I T lymphocytes, which are associated with cell mediated inflammatory responses, are activated early in BPH pathogenesis, whereas type II T lymphocytes, which are responsible for humoral response and antibody production, are found more in chronically infiltrated nodular BPH.54 Over expression of IL-15 and IL-15 receptor ␣-chain in BPH epithelia also suggests a role for this cytokine in prostatic inflammation.55 Another family of growth regulators implicated in BPH includes the IGFs and their receptors, and binding proteins that regulate IGF availability (IGFBPs). Using reverse transcription polymerase chain reaction, Dong et al found that mRNA levels of the IGF-I receptor and IGF-II peptide growth factor were 3-fold and 10-fold higher, respectively, in stromal cells cultured from BPH compared to normal.56 Furthermore, expression of the Wilms tumor gene, a tumor suppressor that normally down-regulates IGF, was 7-fold lower in cultured BPH stromal cells.56 A decrease in IGFBP-2 and an increase in IGFBP-5 were also observed in BPH stromal cells.57 These alterations in the IGF axis may have important roles in stromal and epithelial cell proliferation and apoptosis in BPH. Others have reported different patterns of IGF-II and IGFBP expression among stromal cells cultured from different zones of the prostate and BPH. Boudon et al found that BPH and normal tissues from the periurethral zone were alike in expression pattern and suggested that the IGF axis may be related to BPH development, specifically in the periurethral zone.58 Despite convincing findings of IGF alterations in BPH, a direct causal role in BPH pathogenesis remains to be proved. In fact, Bonnet et al suggested that increased IGF-I, IGF-II and IGF-I receptor RNA levels in prostatic tissues may be associated with aging rather than BPH per se.42 One of the challenges of elucidating cause and effect in BPH is that myriad changes occur at the same time. Thus, the interdependence of different regulatory pathways must be addressed. TGF-␤ is implicated in BPH and serves as one example of the cellular complexity of BPH. TGF-␤ differentially affects growth of normal and BPH stromal cells, possibly via differential regulation of IGFBPs. When cultured cells were exposed to TGF-␤ IGFBP-3 expression increased 15-fold in normal cells but only 2-fold in BPH cells. This difference was associated with an overall inhibition of 60% of growth in normal cells compared to 20% in BPH cells.59 However, studies do not concur on tissue selectivity or cellular localization of TGF-␤ in BPH. Although Cohen et al59 found that inhibition of BPH stromal cell growth by TGF-␤ was attenuated compared to normal stromal cells, Story et al60 reported no difference. In terms of tissue localization Kyprianou et al observed TGF-␤ primarily in secretory epithelial cells of BPH, and maintained that expression was higher in BPH than in normal tissues.61 Thus, epithelial secretion of TGF-␤ could regulate stromal cell response in BPH pathogenesis, and this cell-cell interaction reflects but one of the complex stromal-epithelial interactions on a cellular level. In addition, different subtypes of TGF-␤ may be important, as Mori et al found that TGF-␤2, but not TGF-␤1, was increased in BPH compared to normal.62 Alterations in the renin-angiotensin system have also been

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implicated in BPH. Following immunohistochemical analysis that revealed markedly decreased levels of angiotensin II receptors in normal periurethral smooth muscle compared to that of BPH Dinh et al,63 and Lin and Freeman64 showed that angiotensin II transactivated ErbB1 and ErbB2 receptors in cultured human prostatic stromal cells and modestly promoted growth. Besides its mitogenic effects, angiotensin II can increase muscle tone. The decreased expression of angiotensin receptors in BPH would imply less mitogenic activity of angiotensin II and decreased muscle tone, features that are not consistent with BPH. However, angiotensin converting enzyme (which converts inactive angiotensin I to the active form II) is increased in BPH, so perhaps increased ligand despite decreased receptor levels results in increased activity of angiotensin II.65 The reported over expression of ErbB2 in BPH could also contribute to increased mitogenic activity of angiotensin II.66 Finally, Doll et al measured angiogenic activity in conditioned media from normal versus BPH epithelial cultures and found that normal cell secretions were anti-angiogenic, whereas those from BPH tended to be angiogenic.67 This result was mainly due to decreased expression of the antiangiogenic factor, thrombospondin-1, and increased expression of the angiogenic factor, vascular endothelial growth factor, by some of the BPH cell cultures. PROLIFERATION, APOPTOSIS AND SENESCENCE

Kyprianou et al hypothesize that escape from normal apoptotic processes is responsible for growth in BPH.61 In support of this hypothesis they found that the proliferative-to-apoptotic ratio of epithelial cells was higher in BPH than in normal tissue. The observed increased expression of the anti-apoptotic factor bcl-2 in BPH epithelial cells was proposed to account for the decreased number of apoptotic cells. These findings were mirrored by those of Colombel et al, who also observed that proliferation was higher and apoptosis lower in BPH epithelia compared to normal TZ.68 Decreased apoptosis in BPH was similarly attributed to increased expression of bcl-2. An increased level of ornithine decarboxylase (ODC) activity in BPH as measured by Liu et al is also consistent with cellular proliferation.69 ODC is a key enzyme in the polyamine biosynthesis pathway, and polyamines are known to stimulate cellular proliferation. However, this particular study was marred by the fact that normal tissues were obtained from men 20 to 40 years old, and, therefore, age related changes in expression of ODC cannot be ruled out. Another group found increased mitogen activated protein kinases in BPH by several techniques.70 In particular, extracellular signal regulated protein kinase was detected only in nuclei of stromal cells in normal tissues and not in epithelia, whereas the majority of epithelial nuclei were also labeled in BPH tissues. The p38 kinase was present in stromal and epithelial nuclei in normal and BPH tissues but the percentage of cells expressing p38 increased significantly in BPH. These mitogen activated protein kinases could be involved in the increased proliferation of BPH cells and may reflect increased growth factor activity. Another finding related to increased cellular proliferation is the pattern of expression of p27KIP1 in BPH.71 The p27KIP1 protein is a negative regulator of the cell cycle, and epithelial and stromal cells in normal prostatic tissues express abundant amounts of p27KIP1 RNA and protein. In BPH p27KIP1 RNA and protein are almost undetectable in either epithelial or stromal cells. The absence of this growth suppressive factor may be causally linked to increased proliferation in BPH. Despite the perceived enhanced proliferative rate in BPH, proliferation remains similarly compartmentalized to the basal epithelium of BPH and normal glands.72 This finding is in distinct contrast to the premalignant lesion prostatic in-

traepithelial neoplasia, in which luminal as well as basal epithelial cells proliferate.73 Claus et al detected proliferating cells in BPH stroma but could not detect any apoptotic cells.74 They hypothesized that increase in BPH volume is due to stromal growth that is not balanced by cell death. However, in this study normal tissues were not evaluated, so it is not possible to conclude whether lack of stromal apoptosis was peculiar to BPH. The same drawback applies to a study by Vacherot et al, in which rates of apoptosis did not differ between normal and BPH tissues in either the stroma or the epithelium, but the proliferative index was higher in BPH in stroma and epithelium.75 The normal tissues in this case were also obtained from young individuals. A third mechanism that should be considered in the development of BPH is cellular senescence. Senescence describes the process whereby somatic cells lose their ability to proliferate or to undergo apoptosis. The senescent cell remains metabolically active in a growth arrested state where the lack of response to apoptotic signals could lead to cellular accumulation.76 Using the biomarker senescence associated ␤-galactosidase (SA-␤-gal), Choi et al demonstrated that more than 80% of BPH tissues from prostate specimens larger than 55 gm stained positively, while only 12% of glands less than 55 gm were positive.77 These investigators concluded that senescence could be a significant mechanism in BPH pathogenesis. Recently Castro et al carried out additional studies regarding cellular senescence in BPH.78 As previously reported by Choi et al,77 SA-␤-gal was observed in a significant number of cells in BPH tissues. It is noteworthy that in both studies this biomarker of senescence was expressed only in epithelial cells and not in the stroma. These investigators attempted to link expression of IL-1␣ to the senescent phenotype by illustrating that SA-␤-gal activity strongly correlated with tissue levels of IL-1␣. They propose that one stromal-epithelial mechanism driving BPH progression is the accumulation of senescent epithelial cells expressing IL-1␣, which acts as a paracrine factor that increases expression of FGF-7 in stromal cells. As stated previously, FGF-7 then would stimulate the proliferation of nonsenescent epithelial cells and contribute to the enlargement of BPH tissue. Alterations in molecular signaling in BPH are summarized in Appendix 2.

CONCLUSIONS

Beginning with a histological appreciation of the zonal anatomy of the prostate and the cellular composition of BPH, investigators have sought to understand the molecular and cellular signals in BPH pathogenesis. From studies of early prostate development, the concept developed that cellular interactions between epithelial and stromal elements are important in BPH. Autocrine and paracrine pathways through stromal cells serve as focal areas for reawakening of epithelial budding and subsequent BPH nodule formation. Through genetic arrays and primary cell culture models, numerous gene products and growth factors associated with BPH have been identified. The relative importance of each will be the focus of continued research. Although the heterogeneous nature of BPH tissue makes comparison difficult between studies, research using cell selective LCM will likely improve our understanding of BPH. Finally, our appreciation of the paradigm of balanced cellular growth with apoptosis and senescence will stimulate research in novel areas such as senescence and BPH specific biomarkers. Drs. John McNeal, Larissa Nonn and Thomas Stamey provided input and critical review of this report.

PATHOGENESIS OF BENIGN PROSTATIC HYPERPLASIA APPENDIX 1: CELLULAR CHANGES IN BPH

Stromal Elements “reawakening” of embryonic mesenchyme a fibroblasts/fibromuscular components a nonmuscle myosin heavy chain (NMMHC) s smooth muscle myosin heavy chain (SMMHC) s elastin Epithelial Elements A. Luminal cells Flatten a intraluminal space a Vimentin s Prostate specific membrane antigen (PMSA) s Alpha1-antichymotrypsin (ACT) s Alpha1b-adrenergic receptor B. Basal cells Attenuated s Cellular adhesion molecule expression (eg C-CAM) C. Neuroendocrine cells s Numbers s Overall innervation APPENDIX 2: BPH ALTERATIONS IN MOLECULAR SIGNALING

Hormones a 5-alpha-reductase activity (stromal cells) a 17-beta-hydroxysteroid dehydrogenase a nuclear androgen receptors (basal epithelium) a androgen receptor co-activators s androgen co-repressor Growth Factors a Fibroblast growth factors (e.g., FGF-1, -2, -7, -9) a Hypoxia induced factor 1 (HIF-1) a Insulin growth factors (e.g., IGF-2) a Insulin growth factor receptors (eg IGF-I-R) a Vascular endothelial growth factor (VEGF) s IGF down-regulators (e.g., WT-1, IGFBP-2) Cytokines a Interleukins (e.g., IL-1-alpha, -2, -4, -8, -15, -17) Others a Mitogenic activated protein kinases (eg, ERK, p-38) a Angiotensin converting enzyme activity s Thrombospondin-1 s Vitamin D 1-alpha-hydroxylase activity REFERENCES

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