Function and molecular mechanisms of neuroendocrine cells in prostate cancer

Benign prostate contains luminal epithelial cells, basal cells and a minor component of neuroendocrine cells whose function may be to regulate the growth, differentiation and secretory function of the prostate gland. Neuroendocrine (NE) cells are also present in prostate cancer (PC), and many studies have shown that their number increases in high-grade and high-stage tumors, particularly in hormonally treated and hormone-refractory (androgen independent) PC. Unlike the non-neuroendocrine secretory-type PC cells, NE cells lack androgen receptor and are likely androgen independent. Therefore it is conceivable that hormonal therapy for advanced or metastatic prostate cancer, which consists of inhibiting androgen production or blocking androgen function, will not eliminate NE cancer cells. Instead, these cells may be enriched after the therapy and they may establish paracrine networks to stimulate androgen-independent proliferation of PC, leading to tumor recurrence. This article reviews the major functions of NE cells in PC, including stimulation of cancer proliferation and invasion, apoptosis resistance and angiogenesis. It also discusses molecular pathways involved in NE differentiation and the effectors of the NE cells.     

A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase

Prostate cancer progresses from a hormone-sensitive, androgen-dependent stage to a hormone-refractory, androgen-independent tumor. The androgen receptor pathway functions in these androgen-independent tumors despite anti-androgen therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines expressed higher levels of the HER-2/neu receptor tyrosine kinase than their androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent prostate cancer cells allowed ligand-independent growth. HER-2/neu activated the androgen receptor pathway in the absence of ligand and synergized with low levels of androgen to 'superactivate' the pathway. By modulating the response to low doses of androgen, a tyrosine kinase receptor can restore androgen receptor function to prostate cancer cells, a finding directly related to the clinical progression of prostate cancer.     

5alpha-androstane-3alpha,17beta-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is reduced from the potent androgen, 5alpha-dihydrotestosterone (5alpha-DHT), by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) in the prostate. 3alpha-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5alpha-DHT. However, 3alpha-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3alpha-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3alpha-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3alpha-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3alpha-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3alpha-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3alpha-HSD expression are significantly elevated in localized and advanced prostate cancer, 3alpha-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

5α-androstane-3α,17β-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5α-Androstane-3α,17β-diol (3α-diol) is reduced from the potent androgen, 5α-dihydrotestosterone (5α-DHT), by reductive 3α-hydroxysteroid dehydrogenases (3α-HSDs) in the prostate. 3α-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5α-DHT. However, 3α-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3α-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3α-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3α-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3α-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3α-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3α-HSD expression are significantly elevated in localized and advanced prostate cancer, 3α-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

雄激素受体共调节因子与雄激素非依赖性前列腺癌

雄激素介导的雄激素受体（AR）信号途径对雄性胚胎的发育及雄激素依赖性靶组织的分化发育是必需的。异常的AR活性与前列腺癌由雄激素依赖转变为雄激素非依赖性密切相关。已证实AR共调节因子参与前列腺癌的发生和发展,并在雄激素非依赖性前列腺癌细胞的增殖中扮演着重要角色。它们的表达失衡,可导致AR转录活性的改变,促进晚期前列腺癌的进展。简要综述了AR共调节因子的类型和功能,及其与雄激素非依赖性前列腺癌的关系。     

Protein kinase A-mediated phosphorylation of RhoA on serine 188 triggers the rapid induction of a neuroendocrine-like phenotype in prostate cancer epithelial cells

Whilst androgen ablation therapy is used to treat locally advanced or metastatic forms of prostate cancer, side-effects can include the emergence of an androgen-independent neuroendocrine cell population which is associated with poor prognosis. Here we have examined how cyclic AMP elevation regulates early events in the neuroendocrine differentiation process. We demonstrate that selective activation of protein kinase A is necessary and sufficient for cyclic AMP (cAMP) elevation to rapidly promote a neuroendocrine phenotype in LNCaP cells independent of de novo protein synthesis. Furthermore, the effects of cAMP could be recapitulated by inhibition of RhoA signalling or pharmacological inhibition of Rho kinase. Conversely, expression of constitutively active Gln63Leu-mutated RhoA acted as a dominant-negative inhibitor of cAMP-mediated NE phenotype formation. Consistent with these observations, cAMP elevation triggered the PKA-dependent phosphorylation of RhoA on serine 188, and a non-phosphorylatable Ser188Ala RhoA mutant functioned as a dominant-negative inhibitor of cAMP-mediated neuroendocrine phenotype formation. These results suggest that PKA-mediated inhibition of RhoA via its phosphorylation on serine 188 and the subsequent inhibition of ROCK activity plays a key role in determining initial changes in cellular morphology during LNCaP cell differentiation to a neuroendocrine phenotype. It also raises the possibility that targeted suppression of this pathway to inhibit neuroendocrine cell expansion might be a useful adjuvant to conventional prostate cancer therapy.     

A partial differential equation model and its reduction to an ordinary differential equation model for prostate tumor growth under intermittent hormone therapy

Hormonal therapy with androgen suppression is a common treatment for advanced prostate tumors. The emergence of androgen-independent cells, however, leads to a tumor relapse under a condition of long-term androgen deprivation. Clinical trials suggest that intermittent androgen suppression (IAS) with alternating on- and off-treatment periods can delay the relapse when compared with continuous androgen suppression (CAS). In this paper, we propose a mathematical model for prostate tumor growth under IAS therapy. The model elucidates initial hormone sensitivity, an eventual relapse of a tumor under CAS therapy, and a delay of a relapse under IAS therapy, which are due to the coexistence of androgen-dependent cells, androgen-independent cells resulting from reversible changes by adaptation, and androgen-independent cells resulting from irreversible changes by genetic mutations. The model is formulated as a free boundary problem of partial differential equations that describe the evolution of populations of the abovementioned three types of cells during on-treatment periods and off-treatment periods. Moreover, the model can be transformed into a piecewise linear ordinary differential equation model by introducing three new volume variables, and the study of the resulting model may help to devise optimal IAS schedules.     

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.     

CaV3.2 T-type calcium channels are involved in calcium-dependent secretion of neuroendocrine prostate cancer cells

Because prostate cancer is, in its early stages, an androgen-dependent pathology, treatments aiming at decreasing testosterone plasma concentration have been developed for many years now. However, a significant proportion of patients suffer a relapse after a few years of hormone therapy. The androgen-independent stage of prostate cancer has been shown to be associated with the development of neuroendocrine differentiation. We previously demonstrated that neuroendocrine prostate cancer cells derived from LNCaP cells overexpress CaV3.2 T-type voltage-dependent calcium channels. We demonstrate here using prostatic acid phosphatase as a marker of prostate secretion and FM1-43 fluorescence imaging of membrane trafficking that neuroendocrine differentiation is associated with an increase in calcium-dependent secretion which critically relies on CaV3.2 T-type calcium channel activity. In addition, we show that these channels are expressed by neuroendocrine cells in prostate cancer tissues obtained from patients after surgery. We propose that CaV3.2 T-type calcium channel up-regulation may account for the alteration of secretion during prostate cancer development and that these channels, by promoting the secretion of potential mitogenic factors, could participate in the progression of the disease toward an androgen-independent stage.     

KLF9 suppresses cell growth and induces apoptosis via the AR pathway in androgen-dependent prostate cancer cells

Kruppel-like factors (KLFs) play an important role in many biological processes including cell proliferation, differentiation and development. Our study showed that the level of KLF9 is lower in PCa cell lines compared to a benign prostate cell line; the androgen-independent cell line PC3 expresses significantly lower KLF9 than the androgen-dependent cell line, LNCaP. Forced overexpression of KLF9 suppressed cell growth, colony formation, and induced cell apoptosis in LNCaP cells. We also found that KLF9 expression was induced in response to apoptosis caused by flutamide, and further addition of dihydrotestosterone antagonized the action of flutamide and significantly decreased KLF9 expression. Furthermore, activation of the androgen receptor (AR) was inhibited by the overexpression of KLF9. Our research shows that KLF9 is lower in androgen-independent cell lines than in androgen-dependent cell lines; Overexpression of KLF9 dramatically suppresses the proliferation, anchorage-independent growth, and induces apoptosis in androgen-dependent cells; KLF9 inhibition on prostate cancer cell growth may be acting through the AR pathway. Our results therefore suggest that KLF9 may play a significant role in the transition from androgen-dependent to androgen-independent prostate cancer and is a potential target of prevention and therapy.     

Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells.

Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G1-S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be partially initiated by the Ras pathway in androgen-dependent cells.     

Requirement of androgen-dependent activation of protein kinase Czeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells

A cell line that we designed, AILNCaP, proliferated in androgen-depleted medium after emerging from long-term androgen-depleted cultures of an androgen-sensitive prostate cancer cell line, LNCaP. Using this cell line as a model of progression to androgen independence, we demonstrated that the activity of the mammalian target of rapamycin/p70 S6 kinase transduction pathway is down-regulated after androgen depletion in LNCaP, whereas its activation is related to transition of this cell line to androgen-independent proliferation. Kinase activity of protein kinase Czeta is regulated by androgen stimulation in LNCaP cells, whereas it is activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with a protein kinase Czeta pseudosubstrate inhibitor reduced p70 S6 kinase activity and cell proliferation in both cell lines. We identified that both protein kinase Czeta and p70 S6 kinase were associated in LNCaP cells and this association was enhanced by the androgen stimulation. We examined the expression of phospho-protein kinase Czeta and phospho-p70 S6 kinase in hormone-naive prostate cancer specimens and found that the expression of both kinases was correlated with each other in those specimens. Significant correlation was observed between the expression of both kinases and Ki67 expression. Most of the prostate cancer cells that survived after prior hormonal treatment also expressed both kinases. This is the first report that shows the significance of this pathway for both androgen-dependent and -independent cell proliferation in prostate cancer. Our data suggest that protein kinase Czeta/mammalian target of rapamycin/S6 kinase pathway plays an important role for the transition of androgen-dependent to androgen-independent prostate cancer cells.     

Neuropeptides bombesin and calcitonin induce resistance to etoposide induced apoptosis in prostate cancer cell lines

BACKGROUND: Neuroendocrine differentiation in prostatic carcinoma has been related to regulation of proliferation and metastatic potential and correlated with prognosis. More than 80% of prostate carcinomas initially respond to androgen ablation, but most relapse, due to the heterogeneous presence of androgen-dependent and independent clones. The pathways of cellular proliferation and apoptosis are inexorably linked to minimize the occurrence of neoplasia, and disfunction of apoptosis is proposed as a pathogenic process in malignant tumors. Androgen-dependent prostatic cancer cells undergo apoptosis after androgen deprivation, but not androgen-independent ones due to a defect in the initiation step. Anyway, they retain the basic cellular machinery to undergo apoptosis. We suggest a possible role of neuroendocrine differentiation in the onset and regulation of apoptosis in prostatic neoplasia. METHODS: LNCaP, PC-3 and DU 145 prostatic cancer cell lines were induced to undergo apoptosis after treatment with etoposide alone or plus androgen ablation. We tested the role of neuropeptides bombesin and calcitonin at modulating etoposide induced apoptosis. RESULTS: Etoposide-induced apoptosis in all cancer cell lines was achieved. In LNCaP androgen ablation was also required. Apoptosis is prevented in all three lines when bombesin was added. Calcitonin addition prevents apoptosis in PC-3, LNCaP and in an etoposide dose-dependent way in DU 145. CONCLUSION: Neuropeptides bombesin and calcitonin can modulate the apoptotic response of prostate cancer cells by inducing resistance to etoposide-induced apoptosis, suggesting that neuropeptides can be used as a target of therapeutical approach in prostatic carcinoma.     

Sphingosine Kinase-1 Is Central to Androgen-Regulated Prostate Cancer Growth and Survival

Background

Sphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival.

Methodology/Principal Findings

Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway—by negatively impacting SphK1 activity—could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity.

Conclusions/Significance

We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer.     

The significance of Her2 on androgen receptor protein stability in the transition of androgen requirement in prostate cancer cells

Androgen ablation therapy is the most common strategy for suppressing prostate cancer progression; however, tumor cells eventually escape androgen dependence and progress to an androgen-independent phase. The androgen receptor (AR) plays a pivotal role in this transition. To address this transition mystery in prostate cancer, we established an androgen-independent prostate cancer cell line (LNCaPdcc), by long-term screening of LNCaP cells in androgen-deprived conditions, to investigate changes of molecular mechanisms before and after androgen withdrawal. We found that LNCaPdcc cells displayed a neuroendocrine morphology, less aggressive growth, and lower expression levels of cell cycle-related factors, although the cell cycle distribution was similar to parental LNCaP cells. Notably, higher protein expression of AR, phospho-Ser(81)-AR, and PSA in LNCaPdcc cells were observed. The nuclear distribution and protein stability of AR increased in LNCaPdcc cells. In addition, cell proliferation results exhibited the biphasic nature of the androgen (R1881) effect in two cell lines. On the other hand, LNCaPdcc cells expressed higher levels of Her2, phospho-Tyr(1221/1222)-Her2, ErbB3, and ErbB4 proteins than parental LNCaP cells. These two cell lines exhibited distinct responses to Her2 activation (by heregulin treatment) on Her2 phosphorylation and Her2 inhibition (by AG825 or Herceptin treatments) on proliferation. In addition, the Her2 inhibitor more effectively caused AR degradation and diminished AR Ser(81) phosphorylation in LNCaPdcc cells. Taken together, our data demonstrate that Her2 plays an important role in the support of AR protein stability in the transition of androgen requirement in prostate cancer cells. We hope these findings will provide novel insight into the treatment of hormone-refractory prostate cancer.     

Steroid hormones, polypeptide growth factors, hormone refractory prostate cancer, and the neuroendocrine phenotype

The growth, development, and differentiation of the prostate gland is largely dependent on the action of androgens and peptide growth factors that act differentially at the level of the mesenchymal and epithelial compartments. It is our premise that to understand the emergence of metastatic and hormone refractory prostate cancer we need to investigate: (1) how androgen action at the level of the mesenchyme induces the production of peptide growth factors that in turn can facilitate the growth and development of the epithelial compartment; (2) how androgen action at the level of the epithelium induces and maintains cellular differentiation, function, and replicative senescence; and (3) how transformation of the prostate gland can corrupt androgen and growth factor signaling homeostasis. To this end, we focus our discussion on how deregulation of the growth factor signaling axis can cooperate with deregulation of the androgen signaling axis to facilitate transformation, metastasis, and the emergence of the hormone refractory and neuroendocrine phenotypes associated with progressive androgen-independent prostate cancer. Finally, we suggest a working hypothesis to explain why hormone ablation therapy works to control early disease but fails to control, and may even facilitate, advanced prostate cancer.