Cancer Therapy Vol 3, 383-396, 2005

Neuroendocrine differentiation in prostate cancer

Review Article

Siamak Daneshmand¹, Marcus L. Quek², Jacek Pinski^3,*

¹Division of Urology, Oregon Health and Science University, Portland, Oregon

²Department of Urology

³Division of Medical Oncology, Keck School of Medicine at the University of Southern California, Los Angeles, California

__________________________________________________________________________________

*Correspondence: Jacek Pinski, M.D., Ph.D., Assistant Professor, Division of Medical Oncology, USC/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Suite 3449, Los Angeles, CA 90089, USA; Tel: (323) 865-3929; Fax: (323) 865-0061; E-mail: pinski_j@ccnt.hsc.usc.edu

Key words: Neuroendocrine differentiation, prostate cancer, hormone refractory prostate cancer, Small cell (neuroendocrine) carcinoma of the prostate

Abbreviations: androgen receptor, (AR); benign prostatic hyperplasia, (BPH); human chorionic gonadotropin, (hCG); immunohistochemical, (IHC); interleukin-6, (IL-6); lymphocyte conditioned medium, (LCM); Neuroendocrine, (NE); neuron-specific enolase, (NSE); parathyroid hormone-related protein, (PTHrP); prostate-specific antigen, (PSA); prostatic intraepithelial neoplasia, (PIN); serotonin, (5-HT), neuron-specific enolase, (NSE); short-interfering RNA, (siRNA); somatostatin, (SST); thyroid-stimulating-like peptide, (TSH)

Received: 19 May 2005; Accepted: 6 June 2005; electronically published: July 2005

Summary

Neuroendocrine (NE) cells likely play a role in both normal development and pathologic conditions of the prostate. Neuroendocrine expression has attracted increasing attention in prostate cancer research as a potential mechanism for regulation of growth and differentiation. This review discusses the role of NE cells in normal and malignant prostatic tissue and examines the current literature on the topic. NE cells are thought to originate from basal stem cells and are known to produce a number of secretory factors that may act through endocrine, paracrine, and autocrine mechanisms. Virtually all benign prostatic tissue and prostatic adenocarcinomas show some degree of NE differentiation, which appears to vary with age and ethnic background. Clinical studies suggest that the extent of NE differentiation increases with tumor progression and the development of androgen insensitivity; however, there is controversy regarding its prognostic significance. NE cells lack the androgen receptor and appear to increase in response to androgen ablation. In vitro studies have shown that they can derive through a process of transdifferentiation from tumor cells. Some studies suggest that serum chromogranin A measurements in prostate cancer patients correlate with tissue expression and may provide prognostic information. Recent work from our laboratory has shown that NE cells may in fact have an inhibitory effect on prostate cancer cells. Lastly, we discuss small cell neuroendocrine carcinomas of the prostate and their clinical features. The current review underscores the need to improve our understanding of neuroendocrine cells, their regulatory products and their influence in prostate carcinogenesis. Further studies are needed to elucidate the contribution and significance of NE cells to prostate cancer growth and progression.

I. Introduction

The epithelial cells of the human prostate are composed of three principal cell types: secretory cells, basal cells, and neuroendocrine (NE) cells (Hansson and Abrahamsson, 2001). The NE cells are thought to be involved in cell regulation through the release of numerous secretory products that may act in an endocrine, paracrine or autocrine manner (di Sant'Agnese, 1992a). NE cells likely play a role in both normal as well as pathologic conditions of the prostate. It is controversial whether NE cells originate from the neural crest during embryogenesis or from a common precursor for NE and prostatic epithelial cells. The first hypothesis is supported by the finding that chromogranin A-positive cells are observed in the urethral epithelium and the surrounding mesenchyme of the fetus at very early stages of gestation (Abrahamsson, 1999a). On the other hand, the stem cell theory is supported by the presence of intermediate cells expressing both endocrine and exocrine markers (basal cell-specific cytokeratins and prostate-specific antigen [PSA]), which occur frequently in prostate cancer with NE differentiation (Bonkhoff, 1998). Moreover, NE cells have been shown to manifest simultaneous expression of chromogranin A and basal cell-specific cytokeratins (Bonkhoff, 1998; Rumpold et al, 2002).

Based on morphology, di Sant’Agnese (1992b) described two types of NE cells the “open” type, which resembles an open flask-shaped form with long slender luminal extensions, and the “closed” type, which lacks these extensions. Both types characteristically have irregular dendritic processes extending between adjacent epithelial cells (Figure 1). Despite these morphologic classifications, the different functional roles for these cells remain unclear. NE cells produce a variety of neurosecretory products that regulate cellular growth and differentiation, while also providing useful markers for their identification. Most studies have focused on growth stimulatory factors such as serotonin, calcitonin, and parathyroid hormone-related peptides (di Sant'Agnese, 1992a, 1998). Some of these regulatory peptides, such as bombesin, calcitonin, and parathyroid hormone-related protein, have been shown to stimulate tumor cell proliferation in vitro (Bologna et al, 1989; Iwamura et al, 1994b; Shah et al, 1994). It has been suggested that NE cells may also produce and secrete inhibitory factors based on in vitro co-culture experiments with prostate cancer cell lines (Wang et al, 2004b). Whatever their functional role, several of these markers, including chromogranin A, neuron-specific enolase, synaptophysin, and serotonin, provide useful marker proteins for immunohistochemical localization in both human and animal models (Angelsen et al, 1997a; Rodriguez et al, 2003). Furthermore, heterogeneity and variation in the neurosecretory products expressed by these cells suggest that there are actually several populations of prostatic NE cells, each with its own set of secretory factors (Abrahamsson, 1999a).

The origin of NE cells in the prostate remains a subject of controversy. A general stem cell theory postulates that NE cells share a common origin with other epithelial cell types from pluripotent stem cells within the basal layer (Bonkhoff, 1998). This concept is based on the finding of epithelial cells of intermediate differentiation based on morphologic and immunohistochemical marker analysis. Furthermore, these cells do not show proliferative activity (lack the proliferation-associated antigens, Ki-67 and MIB-1), as would be expected of postmitotic terminally-differentiated cells. Aumuller et al (1999) proposed that NE cells originate from the neural crest during embryogenesis. This hypothesis is supported by the finding of NE cells in the urogenital epithelium early in gestation, and by studies demonstrating a preponderance of NE cells in the periurethral and ductal regions. Cohen et al, (1990) raise the possibility of 2 functionally distinct populations of NE cells with potentially different embryonic origins, with peripheral NE cells deriving from a pluripotent stem cell and periurethral NE cells originating from the neural crest. Still another theory suggests that the increased NE cell

Figure 1. Typical neuroendocrine cell with its dendritic-like process found in the epithelial cell layer of the prostate. (Immunohistochemical staining for chromogranin A, 1:1000 dilution, mouse monoclonal antibody from Dako, Denmark, in a benign prostate sample taken from a cystoprostatectomy specimen, developed with diaminobenzidine tetrahydrochloride solution and counterstained with hematoxylin; original magnification X 400).

expression seen in prostatic malignancies, may be a result of a process of transdifferentiation from prostatic adenocarcinoma cells in response to various cytokines (Wang et al, 2004b).

Several studies report that NE cells lack expression of the androgen receptor (Bonkhoff et al, 1993; Krijnen et al, 1993; Abrahamsson, 1999a), while others suggest that a separate subpopulation of NE cells exist that may be responsive to androgens (Cohen et al, 1990; Nakada et al, 1993). In either case, the effects of hormonal manipulation on NE cell expression are not clearly understood. Angelsen et al. (1999) demonstrated that testosterone administration during the pubertal period results in an increased number of NE cells in proliferative lesions in the dorsal lobe of the rat prostate. On the other hand, other studies looking at both dog and human prostate tissue have shown promotion of NE differentiation under androgen ablative conditions (Ismail et al, 2002). In the dog model, the administration of androgens after castration restores NE cell density to normal levels, implying that NE differentiation is hormonally repressed and potentially reversible. Other studies looking at radical prostatectomy specimens following neoadjuvant hormonal ablation have also noted increased NE cell expression (Jiborn et al, 1998; Ahlgren et al, 2000). Collectively, these studies suggest that the hormonal milieu may affect NE differentiation in the human and animal prostate, and that further systematic studies are needed to establish the exact nature of the relationship.

Though their functional role remains largely unknown, these cells likely affect cellular growth and differentiation and exocrine secretions of the prostate through the local release of various neurosecretory products. Commonly found secretory products include bombesin, serotonin (5-HT), neuron-specific enolase (NSE), a thyroid-stimulating-like peptide (TSH), somatostatin (SST), parathyroid hormone-related protein (PTHrP), calcitonin as well as other members of the calcitonin family (di Sant'Agnese et al, 1985; Hansson and Abrahamsson, 2001). More products likely exist which have yet to be characterized. These cell products have the potential to regulate the growth, differentiation and homeostasis of normal as well as pathologic prostatic conditions. They are seen in both benign as well as malignant prostatic tissue (Figure 2). Increasing attention has focused on the potential clinical and prognostic implications of NE differentiation in prostate cancer. However, in order to appreciate the impact of NE expression in prostatic malignancies, it is imperative that we first understand its role in the development and function in normal benign prostate tissue.

II. Neuroendocrine cells in normal prostatic tissue

NE cells have been detected immunohistochemically as early as 13 weeks gestation, and in nearly all prostates by 21 weeks (Cohen et al, 1993). Xue et al, (2000) evaluated the number and distribution of NE cells in autopsy-collected prostates from fetuses, prepubertal males and young adults. NE cells were found primarily in the acinus/ductal regions, while the budding tips, the areas of highest proliferation, lacked NE staining. Although there was great variability in the number of NE cells in prenatal prostates, the ratio of NE cells to epithelial cell area appeared to be relatively constant through adult life. Another autopsy series by Cohen et al, (1993) looked at the distribution of NE cells in different prostatic structures from infancy to elder adulthood. The periurethral and ductal areas had the highest number of NE cells, while the peripheral acini had the least. Interestingly, this group noted age-dependent NE expression in the peripheral acini, such that NE cells were noted in this area during the first few months of life, conspicuously absent between 4 and 13 years, then reappearing at approximately 14 years and through adult life. This was in contrast to the relatively constant expression in the periurethral and ductal areas in all age groups. The authors suggest that 2 functionally distinct subpopulations of NE cells exist during normal development, with the ones in the peripheral zone being hormonally-responsive.

Regional differences in the distribution of NE cells noted in adult prostates may be associated with the predilection for particular areas to develop pathologic processes. Santamaria and colleagues, (2002) evaluated the distribution of NE cells in various prostatic zones. A predominance of NE cells was noted in the transition zone, scarce involvement in the central zone, and intermediate in the peripheral zone. This group hypothesized that the NE cells noted in the transition zone could play a stimulatory role in the development of benign prostatic hyperplasia (BPH) often noted in this area, while peripheral zone NE cells could potentially induce androgen-independent growth of prostate cancer. Similarly, Islam et al, (2002) noted NE cell density to be greater in the verumontanum and main prostatic ducts than in the acini of the peripheral zone, regardless of age. Taken together, these studies agree that more NE cells are found in glandular structures closer to the urethra and less prominent in the periphery of the gland. Others studies have suggested a causal link between NE cell expression and BPH. Although most adenomatous nodules lack significant amounts of NE cells, Cockett and others, (1993) distinguished between proliferating foci in smaller BPH nodules with numerous NE cells and larger “mature” nodules that lacked NE cells, suggesting that NE cells provided mitogenic stimuli for areas of active hyperplasia.

There appears to be a difference in the distribution of NE cells among different ethnic backgrounds. We determined the relative distribution of NE cells in the benign prostate tissue of men from four different ethnic backgrounds to determine whether NE expression levels mirror the degree to which incidence and mortality vary across racial groups. We observed a 6-8 fold decrease in the mean number of NE cells in the prostates from African American men when compared to other races (Figure 3). There was a trend toward higher NE expression in Asians as compared to Caucasians and Hispanics, however, this difference did not reach statistical significance (Daneshmand et al, 2005). Given their potential role in regulation of growth and carcinogenesis, it is conceivable that decreased NE cell expression in the prostates of African American men may

Figure 2. Neuroendocrine differentiation seen in benign prostatic hyperplasia from a cystoprostatectomy specimen (A) and primary Gleason pattern 4 prostatic adenocarcinoma from a radical prostatectomy specimen (B). (Immunostaining for chromogranin A was performed as described in Figure 1; original magnification X 400).

Figure 3. Distribution of neuroendocrine cells in benign prostates taken from cystoprostatectomy specimens from four different ethnic backgrounds: Asian (n=15), Caucasian (n=16), Hispanic (n=13), and African American (n=15).

have a significant influence on the higher incidence of prostate cancer observed in this population.

III. Neuroendocrine differentiation in prostate cancer

In 1984 di Sant'Agnese and De Mesy Jensen, in 1984 described the endocrine-paracrine cells of the prostate and suggested that they play a role in various pathologic conditions. Since then, more than 300 articles have been published on the topic. Recent studies suggest that prostatic NE cells may be involved in carcinogenesis, however the influence of NE cells in the regulation and progression of prostate cancer is not well understood. Virtually all prostate carcinomas contain at least focal areas of NE cells believed to be quiescent, non-proliferative cells that do not stain with the human mitotic indicator antibodies Ki-67 or MIB1 (Cox et al, 1999). NE cells in prostate cancer are easily identified using immunohistochemical markers. Serotonin and chromogranin A appear to be the best markers for identifying NE cells in formalin-fixed sections of the prostate (Bostwick et al, 2002). NE differentiation can also be identified in 25% of prostate cancer needle biopsies though it does not appear to provide any useful prognostic information (Casella et al, 1998).

Clinical studies to date have suggested that the extent of NE differentiation increases with tumor progression and the development of androgen insensitivity. Hirano et al, (2004) examined 72 prostate cancer specimens obtained at radical prostatectomy (38 from patients with no neoadjuvant therapy and 34 patients with neoadjuvant therapy for 3-6 months) and 21 prostate cancer autopsy specimens from patients who died from hormone refractory prostate cancer after androgen deprivation therapy for more than one year. They found that NE differentiation increased with longer duration of hormone therapy, but the study could not attribute the increase in NE differentiation to the condition of hormone refractoriness. Whether NE cells have any prognostic significance in primary prostate adenocarcinomas or lymph node metastases is controversial. Several studies have reported a significant correlation between NE differentiation and pathologic stage or survival (Bostwick et al, 2002). Conversely, other studies have found no significant association between neuroendocrine differentiation and patient survival or prostate cancer progression (Noordzij et al, 1995).

These studies however do not elucidate or reflect the role of NE cells in carcinogenesis. The increase in NE cells seen during androgen deprivation is not necessarily attributable to the hormone refractory state of the tumor. Ismail et al, (2002) studied NE differentiation in the dog and human prostate following androgen ablation. They found that castration induced NE differentiation in the normal prostates of dogs and that this was reversible with the addition of androgen or estrogen supplements. To verify whether similar changes are seen in humans, the investigators examined NE differentiation in radical prostatectomy specimens obtained from patients who received neo-adjuvant androgen ablative therapy prior to surgery and compared them to a similar group of untreated patients. The short course of androgen ablation significantly increased the extent of NE differentiation in the benign glands of the human prostate. Similarly in a prostate cancer xenograft model, short-term androgen withdrawal resulted in an increased number of NE cells. A time course experiment with these PC-295 tumor bearing mice provided evidence that this increase occurred by induction of NE differentiation rather than by rapid proliferation and subsequent differentiation (Noordzij et al, 1996). Other investigators have shown that the androgen receptor (AR) is responsible for the repression of NE transdifferentiation. In vitro experiments have shown that AR silencing through short-interfering RNA (siRNA) directed against AR induces NE transdifferentiation. Furthermore, AR silencing inhibits the growth of LNCaP cells in vitro (Wright et al, 2003). This confirms that the AR actively represses the NE transdifferentiation process in prostate cancer cells.

NE cells produce a number of hormonal growth factors such as serotonin, bombesin-related peptides, PTHrP, neurotensin, and calcitonin, that may act through endocrine, paracrine, and autocrine mechanisms (di Sant'Agnese, 1998). Several of these growth factors including bombesin and neurotensin, have been shown to stimulate the proliferation of some prostate cancer cell lines in vitro (Burchardt et al, 1999; Cox et al, 1999; Cox et al, 2000). Although NE tumor cells themselves do not proliferate, they have been implicated in the progression of prostate cancer through the production of mitogenic factors that maintain cell proliferation in adjacent tumor cells through a paracrine mechanism. Extensive and multifocal NE differentiation in prostatic tumors has been reported to be more aggressive and resistant to hormonal therapy (Abrahamsson, 1996; Bonkhoff, 1998).

The origin of NE cells in prostate cancer is unknown. The androgen sensitive human prostate cancer cell line, LNCaP, has been documented to transdifferentiate into a NE phenotype in response to the cytokine interleukin-6 (IL-6), increasing levels of cyclic AMP, or following culture in steroid-deprived medium (Abrahamsson, 1999b; Burchardt et al, 1999; Cox et al, 1999; Spiotto and Chung, 2000; Zelivianski et al, 2001). The observation that conditions associated with transformation of prostate cancer cells in vitro, such as androgen deprivation or exposure to IL-6 also increases NE differentiation in prostatic tumors in vivo, suggest that NE cells may be derived from adenocarcinoma cells by a process of transdifferentiation in response to changes in the hormonal and growth factor milieu of the microenvironment (Jiborn et al, 1998; Wang et al, 2004a,b).

The implication of NE cells in the progression of prostate cancer is largely based on the fact they produce growth factors which stimulate the proliferation of prostate cancer cell lines in vitro (Jongsma et al, 2000; Amorino and Parsons, 2004). Most studies describing the products of prostatic NE cells have been derived from prostatic carcinoma. NE cells have also been implicated in the upregulation of Bcl-2, which is known to have anti-apoptotic activity (Segal et al, 1994). However, recent work from our laboratory has shown that NE cells may actually have an inhibitory effect on prostate cancer cells (Wang et al, 2004b). In cell culture studies, the proliferation of 3 prostate cancer cell lines (LNCaP, PC-3 and DU-145) was significantly inhibited when exposed to IL-6-induced NE cell conditioned medium or NE cell co-culture. These results imply that NE cells may be releasing inhibitory factors, which could dominate the mitogenic effects of growth factors characteristically associated with NE cells.

A number of publications have presented evidence, which not only questions the presumed mitogenic influence of NE cells, but suggests that they may potentially protect the prostate from carcinogenesis. For example, Algaba et al, (1995) noted that with advancing age there is a gradual decrease in the number of NE cells in the peripheral zone of the prostate, the area that is most susceptible to carcinogenesis. Others have noted that as BPH development progresses, the NE cells are greatly diminished in number or completely lost from most adenoma nodules (Islam et al, 2002). Bostwick et al, (1994) reported that the number of NE cells is decreased around areas containing high-grade prostatic intraepithelial neoplasia (PIN), a premalignant lesion. The same investigators also found that benign prostatic epithelium and primary prostate cancer express a significantly greater number of NE cells than lymph node metastases, suggesting that decreased expression of NE cells may be involved in progression of prostate cancer (Bostwick et al, 2002). Autopsy studies have also revealed that in the developing fetus, NE cells are found only in the acinous/ductal compartment of the prostate and conspicuously absent in the budding tips, an area of active growth (Xue et al, 2000).

The association between neuroendocrine elements in relapsed prostate cancer and sensitivity to chemotherapy has also been studied. In a study by Steineck et al, (2002), about one-half of progressive metastatic androgen-independent prostate cancers showed measurable response to cytotoxic therapy regardless of degree of neuroendocrine differentiation. The exact role of NE cells and contribution to the progression of prostate cancer is still unclear. Although more NE cells are seen in the androgen-independent prostate carcinomas, whether these cells have the potential to induce androgen-independent cell growth is not known.

IV. Neuroendocrine differentiation in PIN

Neuroendocrine differentiation is also present in PIN, a precursor of prostatic carcinoma. Bostwick et al, (1994) determined the extent of neuroendocrine differentiation in high-grade PIN by examining the immunohistochemical expression of 10 markers in 26 radical prostatectomy specimens with PIN and adenocarcinoma. At least one of the markers was present in 88% of cases of PIN and 92% of carcinoma. Serotonin, NSE, chromogranin, and human chorionic gonadotropin (hCG) were expressed in all non-neoplastic epithelial cells with levels significantly greater than in PIN and cancer. They concluded that neuroendocrine differentiation is down regulated in prostatic carcinogenesis, with intermediate levels of expression in PIN compared with normal cells and carcinoma. Algaba et al. (1995) also found lower numbers of neuroendocrine cells in patients with foci of PIN and carcinoma. They suggested that the decrease in neuroendocrine cells make the prostate more susceptible to carcinogenic factors. di Sant'Agnese (1996) also agreed that the degree of neuroendocrine differentiation in PIN is intermediate between normal prostate (containing the most number of cells with neuroendocrine differentiation) and carcinoma.

V. Neuroendocrine serum tumor markers in prostate cancer

The various secretory products from NE cells not only function at the local tissue level of the prostate, but may also be secreted in the serum. Serum detection of NE proteins may provide some prognostic information, and may actually constitute a more representative indicator of overall neuroendocrine differentiation since it accounts for the entire tumor cell population (Abrahamsson, 1999b). Studies have correlated immunohistochemical tissue staining intensity for chromogranin A with serum levels of chromogranin A, suggesting that serum levels may be useful markers for NE differentiation in prostatic tumors (Angelsen et al, 1997b). Early studies correlated elevated serum chromogranin A and neuron-specific enolase (NSE) levels to hormone therapy resistance and poor prognosis (Kadmon et al, 1991; Deftos et al, 1996; Kimura et al, 1997; Wu et al, 1998; Tarle, 1999). In a review by Berruti and colleagues, (2001), serum chromogranin A levels were found to be higher in prostate cancer patients than in patients with benign or pre-malignant diseases, and correlated with advanced stage or hormone refractoriness. There was no apparent relationship to serum PSA levels, however supranormal levels of chromogranin A or NSE were correlated with decreased survival in the hormone refractory cases. Isshiki et al, (2002) also suggested a potential diagnostic or prognostic role for chromogranin A in patients with advanced stage or hormone-refractory disease independent of PSA. They found that poorly differentiated adenocarcinoma was associated with higher chromogranin A levels. The stage D cases with higher chromogranin A had a poorer prognosis than those with lower chromogranin A, however this only held true for those with a median PSA of 172 ng/ml. or less. Cussenot et al (1996) observed that plasma chromogranin A and neuron-specific enolase levels were elevated in 55% and 30% of patients with hormone independent prostate cancer. In patients with stage D3 disease, patients with elevated chromogranin A levels had statistically worse survival. Pre-treatment serum NSE levels have also been shown on multivariate analysis to be an independent prognostic factor for survival in metastatic prostate cancer on androgen ablative therapy (Kamiya et al, 2003), hormone-resistant prostate cancer (Hvamstad et al, 2003), as well as localized prostate cancer treated with external beam radiation (Lilleby et al, 2001).

The role of serum NE markers may prove to be a more useful indicator of prostatic NE differentiation than its corresponding tissue expression. Further studies are needed to accurately determine the diagnostic and prognostic significance of these markers during various stages of the disease and its response to different therapeutic modalities.

VI. Prognostic significance of neuroendocrine differentiation

There have been conflicting reports regarding the predictive value of neuroendocrine differentiation in prostate cancer. Although it appears that neuroendocrine differentiation is present at least focally in all cases of prostatic adenocarcinoma, reports of the percent of tumors containing NE differentiation vary from 24% to 99% in radical prostatectomy specimens (Aprikian et al, 1993; Theodorescu et al, 1997; Abrahamsson, 1999b; Bostwick et al, 2002). Bostwick et al, (2002) comprehensively reviewed published reports of the predictive value of NE differentiation by immunohistochemistry in patients with adenocarcinoma of the prostate. According to most reports neuroendocrine cells have no apparent clinical or prognostic significance in benign epithelium, primary prostate cancer or lymph node metastases. Allen et al (1995) examined 120 prostate tumors from different stages and reported no association with cancer grade, stage or survival. Noordzij et al (1995) studied NE differentiation in 90 patients who underwent radical prostatectomy (stages T2-T4) for prostate cancer. They found NE differentiation was not associated with Gleason sum, pathological stage or cancer specific death. Abrahamson et al, (1998) evaluated 87 patients with clinically localized prostate cancer who underwent radical prostatectomy and found no correlation between NE differentiation and disease progression with a mean follow-up of 4.2 years. Bostwick et al, (2002) determined the expression of chromogranin A and serotonin in 196 patients with lymph node positive prostate cancer. They found the greatest number of neuroendocrine cell in benign prostate epithelium with less expression in primary cancer and lymph node metastases. There was no significant association of chromogranin A expression with cancer specific or all cause survival, while serotonin expression was associated with cancer specific but not all cause survival. Aprikian et al, (1993) found no correlation of neuroendocrine differentiation with pathological stage or metastases.

Conversely Weinstein et al, (1996) studied 104 patients with clinically localized prostate cancer with a mean follow-up of 8 years and found that neuroendocrine differentiation was associated with patient survival. Cohen et al, (1990) found that neuroendocrine cells were an independent prognostic variable in prostate cancer. However, they examined only a small number of cases, and did not control for other predictive factors. Theodorescu et al, (1997) showed that neuroendocrine differentiation predicted patient survival in 71 patients with stages T1-2 prostate cancer but only on a univariate or multivariate analysis of 1 variable. Krijnen et al, (1997) reported that neuroendocrine cell density along with Gleason score was an independent prognostic factor for cancer progression in 72 transurethral prostate resection detected cancers followed by androgen deprivation.

Recently a consensus panel of the College of American Pathologists determined that there is no demonstrated clinical usefulness of evaluating neuroendocrine differentiation in prostate adenocarcinoma (Bostwick et al, 2000).

VII. Neuroendocrine differentiation in hormone refractory prostate cancer

Androgen deprivation has been shown to increase the number of NE cells in prostate tissue (Krijnen et al, 1997; Ahlgren et al, 2000; Hvamstad et al, 2003). A number of independent studies have shown that the NE cells are androgen receptor negative(Krijnen et al, 1993), thus androgen ablation therapy may lead to preferential growth of these cells, which would account for their increased concentration in hormone-refractory tumors. NE cells themselves are non-proliferative and exert their effects through secretion of neuropeptides. It has been suggested that NE cells contribute to the development of androgen-independent prostate cancers through the paracrine effects of growth factors (Abrahamsson, 1999b). This has led some investigators to conclude that NE cells promote aggressive, hormone-independent growth of prostate cancer, however sound evidence to support this theory is lacking.

It is well documented that withdrawal of androgen causes androgen-dependent prostate cancer cells to transdifferentiate into the NE phenotype in vitro and in vivo (Ismail et al, 2002; Wright et al, 2003). Ismail et al, (2002) showed that NE cell densities were within the same range in normal and hyperplastic dog prostates but that it significantly increased following castration. This transdifferentiation was reversible with the addition of androgens and estrogens. A number of studies have shown an induction of NE differentiation following neoadjuvant hormonal treatment of patients undergoing radical prostatectomy or transurethral resection of the prostate (Iwamura et al, 1994a; Van de Voorde et al, 1994; Guate et al, 1997; Pruneri et al, 1998). Ahlgren et al, (2000) performed immunohistochemical (IHC) analysis on 103 specimens of patients on a clinical protocol who were randomized to neoadjuvant LH-RH analogue versus radical prostatectomy alone. While NE cells were statistically more abundant in hormone-manipulated prostates, this was not associated with a difference in the degree of cancer regression or tumor-cell proliferation in response to treatment.

Wright et al, (2003) suggested that activation of the NE transdifferentiation process represents an early response to androgen receptor inactivation induced by androgen withdrawal. They showed that AR silencing induces a NE phenotype in both androgen-dependent LNCaP and androgen-independent LNCaP-AI human prostate cancer cells. Neuroendocrine differentiation in prostate cancers appears to increase with time during androgen withdrawal therapy (Jiborn et al, 1998; Hirano et al, 2004) although this has not been proven in a prospective manner in vivo. In the androgen-dependent human prostate cancer xenograft PC-310 cell line, androgen deprivation causes time-dependent NE differentiation (Jongsma et al, 2002). In the CWR22 human prostate cancer model, androgen deprivation induces a significant increase in tumor-associated NE cells and this precedes the increase in tumor cell proliferation signaling a recurrence (Huss et al, 2004). This has implications on the selection of androgen-independent tumor cells and subsequent progression of prostate cancer. It is as yet unclear whether the increase in NE differentiation can be attributed to the hormone refractory nature of the tumor or just long-term androgen deprivation.

Some published evidence suggests a suppressive effect of NE cells on prostate cancer cells. Bostwick et al, (2002) found only 37.5% of lymph node metastases had any cells which expressed NE markers, with an average of 2.2% of total cells staining positive, compared with 98.5% of primary tumors, in which an average of 6% of cells stained positive. Similarly, Roudier et al, (2003) analyzed multiple bone metastases of 14 prostate cancer patients and found that in the majority of bone metastases, fewer than 1% of cells expressed chromogranin A. This suggests that loss of NE differentiation may facilitate metastasis of prostate cancer cells.

VIII. In vitro models of neuroendocrine differentiation

Although the origin of the neuroendocrine cells is uncertain, a number of recent publications have demonstrated that prostate cancer cells can transdifferentiate into a neuroendocrine phenotype in vitro (Bang et al, 1994; Qiu et al, 1998; Burchardt et al, 1999; Zelivianski et al, 2001; Horiatis et al, 2004). The androgen sensitive human prostate cancer cell line, LNCaP, has been documented to transdifferentiate into a NE phenotype in response to IL-6, increasing levels of intracellular cyclic AMP, or following culture in steroid-deprived medium (Abrahamsson, 1999b; Cox et al, 1999; Cox et al, 2000; Spiotto and Chung, 2000). This transformation is reversible within a few hours of treatment with cAMP-inducing agents, such as forskolin and epinephrine. However, treatment with 7-10 days of continuous IL-6 leads to permanent NE transdifferentiation (Wang et al, 2004b). The observation that conditions associated with transformation of PCA cells in vitro, such as androgen deprivation or exposure to IL-6 also increase NE differentiation in prostatic tumors in vivo (Jongsma et al, 1999, 2002; Wang et al, 2004b), suggest that NE cells might be derived from adenocarcinoma cells by a process of transdifferentiation.

NE cells may in fact represent terminal differentiation of hormone refractory prostate cancer cells to a less malignant form. Hsieh et al (1995) studied the growth of LNCaP cells when cultured in lymphocyte conditioned medium (LCM). Prostate cancer cells in LCM acquired NE differentiation and their growth slowed, with cells halted in G1 rather than progressing further in the cell cycle into S phase. The induced differentiation was associated with ultimate termination of cells, rather than proliferation, even though androgen receptor expression was down regulated and PSA secretion decreased. These provocative results suggest there is more to learn about the relationship between NE cells and androgen-independent prostate cancer.

Recent work from our laboratory has shown that NE cells may in fact have an inhibitory effect on PCA cells. In our cell culture studies, the proliferation of 3 PCA cell lines tested (LNCaP, PC-3 and DU-145) was significantly inhibited when exposed to IL-6-induced NE cell conditioned medium or NE cell co-culture. These results imply that NE cells may be releasing inhibitory factors, which could dominate the mitogenic effects of growth factors characteristically associated with NE cells, such as bombesin or neurotensin (Wang et al, 2004b). In advanced PCA, increased NE differentiation might reflect a response of some cancer cells to changes in the microenvironment, such as increased release of IL-6 from osteoblasts, resulting in tumor growth inhibition rather than tumor progression. IL-6 treatment causes G₀ arrest through the induction of the cyclin-dependent kinase inhibitor p27. The paradox that these patients will eventually die due to progression of this disease despite enhanced NE differentiation, suggests that the suppressive effect of NE cells on the proliferation of surrounding PCA cells might delay advancement of PCA but is not potent enough to completely prevent tumor expansion and cancer spread.

Additional support for this hypothesis would be derived by documenting an increased amount of apoptosis in areas of prostate cancer with high-density NE expression compared to areas with few NE cells. To our knowledge, such estimation has not as yet been reported. Fixemer et al, (2002) reported that in 18 radical prostatectomy specimens apoptosis in NE cells was an extremely rare event, and the overall number of NE cells in a specimen did not correlate with the overall number of apoptotic cells, however they did not evaluate tumor cells in the immediate vicinity of NE cells for apoptosis, which is imperative given the likelihood of paracrine rather than endocrine activity of pro-apoptotic signals (Aprikian et al, 1993).

Other experiments have suggested that select compounds exert their inhibition of prostate cancer proliferative activity through NE transdifferentiation. Melatonin, the main secretory product of the pineal gland has been shown to dramatically reduce the number of prostate cancer cells and stop cell cycle progression in both LNCaP and PC3 cells and promote neuroendocrine differentiation (Sainz et al, 2004). Jolkinolide B, a compound found in Euphorbia fischeriana, a Chinese herbal medicine reported to possess chemotherapeutic effects has potent anti-proliferative activity in LNCaP cells in part by inducing G1 arrest and neuroendocrine differentiation (Liu et al, 2002).

The majority of investigators argue that NE differentiation in prostate carcinoma correlates with poor prognosis, tumor progression, and androgen-independence (Abrahamsson, 1999a; di Sant'Agnese, 2001; Bonkhoff and Fixemer, 2004). NE differentiation exclusively occurs in the G0 phase of the cell cycle and thus NE cells are resistant to radiation therapy and cytotoxic drugs (Bonkhoff and Fixemer, 2004). In addition, NE tumor cells are also resistant to apoptosis (Vanoverberghe et al, 2004). NE cells themselves do not proliferate and represent and immortal cell population within the prostate and are thought to exert their mitogenic effects through paracrine interactions. In vitro systems may not accurately reflect the behavior of prostate cancer cells in vivo and animal models are necessary to elucidate the role of neuroendocrine differentiation in prostatic tumor progression.

IX. In vivo models of neuroendocrine differentiation

Much of the uncertainty about the pathogenic significance of NE differentiation derives from a paucity of reliable animal models. These cells are not common in the normal human prostatic epithelium and even rarer in the mouse prostate. In 1996, Noordzij et al established 2 prostate cancer xenograft models that contained NE cells. They showed that short-term androgen withdrawal resulted in a rapidly increased number of NE cells and suggested that this increase occurred by induction of NE differentiation rather than by rapid proliferation and subsequent differentiation or selective persistence. Since then other cell line xenograft and transgenic mouse models for neuroendocrine prostatic carcinoma have been described (di Sant'Agnese, 1998). Uchida et al, (2004) have established an immortalized cell line designated NE-CS, developed from an NE mouse prostate allograft (NE-10) that has characteristics of NE cells in vitro. When inoculated subcutaneously into athymic mice the NE-CS cells formed tumors with the NE phenotype and exhibited accelerated growth compared to the original NE-10 allograft. Other investigators have found that in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continue to grow, and have increased levels of nuclear AR, as opposed to the decrease in growth seen after castration in mice bearing LNCaP tumors alone (Jin et al, 2004). Hu et al, (2004) have established prostate NE cancer cell lines from CR2-TAg prostate tumors and metastases and have used GeneChip analyses to reveal factors that enhance survival by inhibiting apoptosis. Although these are useful models to understand the molecular mechanisms of NE tumors, they may not accurately reflect the role of benign NE cells seen in prostate cancers.

Conversely we have recently shown that IL-6 can significantly inhibit the growth of LNCaP xenografts in nude mice by the process of NE differentiation. IL-6 treatment results in G₀ arrest in over 90% of cells and NE differentiation with augmentation of neuron-specific enolase and ßIII tubulin. In prostate cancer, clones of IL-6 producing cells could induce paracrine NE differentiation of some surrounding cancer cells. Thus, enhanced tumor NE differentiation may not only indicate the presence of aggressive cancer cells but also reflect a suppressive and protective response of IL-6 transformable cancer cells to an aggressive tumor behavior.

Transdifferentiation into an NE phenotype does not necessarily require the presence of carcinoma. In an experiment to verify the presence of NE cells in the dog prostate and test their hormonal regulation, Ismail et al, (2002) showed that NE cell densities were within the same range in normal and hyperplastic dog prostates but significantly higher after castration. Androgens as well as estrogens given after castration restored NE cell density back to normal values. In human, the density of serotonin-positive NE cells was also significantly higher in benign glands after androgen ablation in prostate cancer patients subjected to androgen ablation prior to prostatectomy.

More animal models are needed to investigate the role of NE differentiation in prostate cancer. Experiments aimed at inducing NE differentiation in an animal model can be used to test the influence of these cells in a prostate carcinogenesis model.

X. Small cell (neuroendocrine) carcinoma of the prostate

Primary small cell carcinoma of the prostate is uncommon and is usually discovered incidentally coexisting with adenocarcinomas. Pure small cell carcinoma of the prostate is an extremely rare occurrence and is a highly aggressive tumor. There is typically no associated elevation of PSA, making early diagnosis difficult (Ro et al, 1987; Nadig et al, 2001). Several theories have been proposed to describe their origin. One theory suggests that small cell carcinomas of the prostate arise from amine precursor uptake decarboxylation cells of local endodermal origin. Another theory proposes that they arise from dedifferentiation of prostatic adenocarcinomas, suggesting that small cell carcinomas are part of a spectrum of prostatic adenocarcinomas rather than a separate disease entity (Sandhu et al, 1997; Yashi et al, 2002). Because of the histologic similarities between prostate and lung small cell carcinomas and the occurrence of similar neuroendocrine paraneoplastic syndromes, the most widely accepted view is that prostatic small cell carcinomas arise from totipotential stem cells of the prostate, which have the ability to differentiate into either epithelial or neuroendocrine type carcinomas (Rubenstein et al, 1997).

Whereas mixed small cell carcinomas and adenocarcinomas usually are aggressive recurrences of a primary adenocarcinoma, pure small cell carcinoma of the prostate often is associated with early metastatic disease because of its aggressive nature. Like adenocarcinomas, small cell prostate cancers arise in the periphery of the prostate gland and hence can occur without urinary symptoms. The disease has a propensity to metastasize to visceral organs, including the liver, bone, lungs, central nervous system, and pericardium, and regionally to the pelvic lymph nodes, rectum, and bladder. In addition, small cell prostate cancers have been reported to produce paraneoplastic syndromes associated with the production of adrenocorticotrophic and antidiuretic hormones. (Tetu et al, 1989; Kawai et al, 2003)

Despite treatment with chemotherapy, the prognosis of small cell prostate cancer is extremely poor, and the median survival is 7 months (Rubenstein et al, 1997). Because of the rarity of the condition, no standard therapeutic regime has been developed. Small cell carcinomas of the prostate are generally unresponsive to hormone therapy. Reported cases have generally been managed by chemotherapeutic regimens similar to those recommended for small cell lung cancer. Small case reports have shown poor responses to etoposide and cisplatin or cyclophosphamide (Debras et al, 1994; Steineck et al, 2002).

XII. Conclusion

Although much has been learned about the distribution and expression of NE cells in the prostate, we are still far from understanding the precise role of these cells in carcinogenesis. Studies have delineated the molecular pathways leading to neuroendocrine differentiation and many of the factors produced by the NE cell have been well characterized. More animal models are needed to clarify the role of these cells within the prostate and the effect of the released factors on neighboring cells. There is some controversy regarding the role of these cells in carcinogenesis and studies should focus on the mechanisms by which these cells interact with normal and/or malignant prostate cells.

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Jacek Pinski