Activated 3',5'-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line

Neuroendocrine (NE) differentiation within prostate tumors is proposed to be a contributing factor in disease progression. However, the cellular origin and molecular mechanism controlling differentiation of prostatic NE cells are unresolved. The prostate tumor cell line, LNCaP, can reversibly acquire many NE characteristics in response to treatment with beta-adrenergic receptor agonists and activators of adenylate cyclase. In this study, we demonstrate that these treatments induce protein kinase A (PKA) activation in LNCaP cells and that ectopic expression of a constitutively activated form of the PKA catalytic subunit, CIalpha, results in acquisition of NE characteristics, including the extension of neuritic processes, cessation of mitotic activity, and production of neuron-specific enolase. Forskolin-, epinephrine-, and isoproterenol-dependent NE differentiation of LNCaP cells was significantly inhibited by expressing a dominant negative mutant of the PKA regulatory subunit, RIalpha. These results demonstrate that prostatic NE differentiation in response to these agents depends on PKA activation, and this signaling pathway may provide a therapeutic target for treating advanced forms of prostate cancer.     

Androgen Receptor Splice Variant AR3 Promotes Prostate Cancer via Modulating Expression of Autocrine/Paracrine Factors

Deregulation of androgen receptor (AR) splice variants has been implicated to play a role in prostate cancer development and progression. To understand their functions in prostate, we established a transgenic mouse model (AR3Tg) with targeted expression of the constitutively active and androgen-independent AR splice variant AR3 (a.k.a. AR-V7) in prostate epithelium. We found that overexpression of AR3 modulates expression of a number of tumor-promoting autocrine/paracrine growth factors (including Tgfβ2 and Igf1) and expands prostatic progenitor cell population, leading to development of prostatic intraepithelial neoplasia. In addition, we showed that some epithelial-mesenchymal transition-associated genes are up-regulated in AR3Tg prostates, suggesting that AR3 may antagonize AR activity and halt the differentiation process driven by AR and androgen. This notion is supported by our observations that the number of Ck5⁺/Ck8⁺ intermediate cells is increased in AR3Tg prostates after castration, and expression of AR3 transgene in these intermediate cells compromises prostate epithelium regeneration upon androgen replacement. Our results demonstrate that AR3 is a driver of prostate cancer, at least in part, through modulating multiple tumor-promoting autocrine/paracrine factors.     

MnSOD drives neuroendocrine differentiation, androgen independence, and cell survival in prostate cancer cells

An increase in neuroendocrine (NE) cell number has been associated with progression of prostate tumor, one of the most frequent cancers among Western males. We previously reported that mitochondrial manganese superoxide dismutase (MnSOD) increases during the NE differentiation process. The goal of this study was to find whether MnSOD up-regulation is enough to induce NE differentiation. Several human prostate cancer LNCaP cell clones stably overexpressing MnSOD were characterized and two were selected (MnSOD-S4 and MnSOD-S12). MnSOD overexpression induces NE morphological features as well as coexpression of the NE marker synaptophysin. Both MnSOD clones exhibit lower superoxide levels and higher H(2)O(2) levels. MnSOD-overexpressing cells show higher proliferation rates in complete medium, but in steroid-free medium MnSOD-S12 cells are still capable of proliferation. MnSOD up-regulation decreases androgen receptor and prevents its nuclear translocation. MnSOD also induces up-regulation of Bcl-2 and prevents docetaxel-, etoposide-, or TNF-induced cell death. Finally, MnSOD-overexpressing cells enhance growth of androgen-independent PC-3 cells but reduce growth of androgen-dependent cells. These results indicate that redox modulation caused by MnSOD overexpression explains most NE-like features, including morphological changes, NE marker expression, androgen independence, inhibition of apoptosis, and enhancement of cell growth. Many of these events can be associated with the androgen dependent-independent transition during prostate cancer progression.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

Induction of Cbeta splice variants and formation of novel forms of protein kinase A type II holoenzymes during retinoic acid-induced differentiation of human NT2 cells

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.     

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.     

Phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is essential for neuroendocrine differentiation of prostate cancer

Hormonal therapy of prostate cancer, by inhibiting androgen production and/or androgen function, is the treatment of choice for advanced prostate cancer. Although most patients respond initially, the effect is only temporary, and the tumor cells will resume proliferation in an androgen-deprived environment. The mechanism for androgen-independent proliferation of cancer cells is unclear. Hormonal therapy induces neuroendocrine differentiation of prostate cancer cells, which is hypothesized to contribute to tumor recurrence by a paracrine mechanism. We studied signal transduction pathways of neuroendocrine differentiation in LNCaP cells after androgen withdrawal, and we showed that both the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway and ERK are activated, but only the former is required for neuroendocrine differentiation. A constitutively active AKT promotes neuroendocrine differentiation and a dominant negative AKT inhibits it. Activation of AKT by IGF-1 leads to neuroendocrine differentiation, and neuroendocrine differentiation induced by epinephrine requires AKT activation. We also show that the AKT pathway is likely responsible for neuroendocrine differentiation in DU145, an androgen-independent prostate cancer cell line. Therefore, our study demonstrated a novel function of the AKT pathway in prostate cancer progression and identified potential targets that may be explored for the treatment of androgen-independent cancer.     

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.     

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.     

The Role of Estrogen Receptor β in Prostate Cancer

Although androgen receptor (AR) signaling is the main molecular tool regulating growth and function of the prostate gland, estrogen receptor β (ERβ) is involved in the differentiation of prostatic epithelial cells and numerous antiproliferative actions on prostate cancer cells. However, ERβ splice variants have been associated with prostate cancer initiation and progression mechanisms. ERβ is promising as an anticancer therapy and in the prevention of prostate cancer. Herein, we review the recent experimental findings of ERβ signaling in the prostate.     

Androgen receptor represses the neuroendocrine transdifferentiation process in prostate cancer cells

Androgen-ablation therapy is an effective method for treating prostate cancer. However, prostate tumors that survive long-term androgen-ablation therapy are classified as androgen-independent as they proliferate in the absence of androgens, and they tend to be enriched for neuroendocrine (NE) cells. Androgen withdrawal causes androgen-dependent prostate cancer cells to adopt a pronounced NE phenotype, suggesting that androgen receptor (AR) represses an intrinsic NE transdifferentiation process in prostate cancer cells. In this report we show that short interfering RNA-induced AR silencing induced a NE phenotype that manifested itself in the growth of dendritic-like processes in both the androgen-dependent LNCaP and androgen-independent LNCaP-AI human prostate cancer cells. Western blot analysis revealed that neuronal-specific enolase, a marker of the neuronal lineage, was increased by AR knockdown in LNCaP cells. The expression levels of the neuronal-specific cytoskeletal proteins beta-tubulin III, nestin, and glial acidic fibrillary protein were also characterized in AR knockdown cells. Most interestingly, AR silencing induced beta-tubulin III expression in LNCaP cells, while AR knockdown increased glial acidic fibrillary protein levels in both LNCaP and LNCaP-AI cells. Lastly, AR silencing reduced the proliferative capacity of LNCaP and LNCaP-AI cells. Our data demonstrate that AR actively represses an intrinsic NE transdifferentiation process in androgen-responsive prostate cancer cells and suggest a potential link between AR inactivation and the increased frequency of NE cells in androgen-independent tumors.     

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.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.