Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor

Cellular Signalling 23 (2011) 507–515

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Review

Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor Dennis Merkle ⁎, Ralf Hoffmann ⁎ a r t i c l e

i n f o

Article history: Received 24 July 2010 Received in revised form 14 August 2010 Accepted 20 August 2010 Available online 8 September 2010 Keywords: Androgen receptor PKA cAMP Prostate cancer Androgen-dependence Androgen-independence Neuroendocrine differentiation Phosphodiesterases β-Adrenergic receptor Adenylyl cyclase

a b s t r a c t Prostate carcinomas are among the most frequently diagnosed and death causing cancers affecting males in the developed world. It has become clear that the molecular mechanisms that drive the differentiation of normal prostate cells towards neoplasia involve multiple signal transduction cascades that often overlap and interact. A critical mediator of cellular proliferation and differentiation in various cells (and cancers) is the cAMP-dependent protein kinase, also known as protein kinase A (PKA), and its activating secondary messenger, cAMP. PKA and cAMP have been shown to play critical roles in prostate carcinogenesis and are the subject of this review. In particular we will focus on the cross-talk between PKA/cAMP signaling and that of the androgen receptor. © 2010 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 2. cAMP and PKA signaling . . . . . . . . . . . . . . . . . . . . 3. PKA in prostate cancer . . . . . . . . . . . . . . . . . . . . . 4. The androgen receptor . . . . . . . . . . . . . . . . . . . . . 5. AR in androgen-dependent and -independent prostate carcinomas 6. cAMP/PKA can regulate AR activation in prostate carcinomas . . . 7. PKA activation in prostate cancer, the cause of effect . . . . . . . 8. Androgen and AR regulation of β2-AR and PKA . . . . . . . . . 9. Other factors affecting cAMP and PKA signaling in prostate cancer 10. PKA and neuroendocrine cell differentiation . . . . . . . . . . . 11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Prostate cancer is the most commonly occurring non-skin malignancy in men, with an estimated 190,000+ new cases expected

⁎ Corresponding authors. Philips Research, High Tech Campus, 5656AE, Eindhoven, The Netherlands. Tel.: + 31 40 2749484 (DM), +31 40 2749487 (RH); fax: + 31 40 2742944. E-mail addresses: [email protected] (D. Merkle), [email protected] (R. Hoffmann). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.08.017

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for 2010 in the USA alone [1]. These statistics are expected to increase in concordance with our aging society since prostate cancer incidence also increases with age [2]. Androgens are required for the development, growth and function of normal prostate tissue, and the development of prostatic intraepithelial neoplasias is also initially under androgenic control, and as a result, androgen ablation therapies, such as chemical or surgical castration, have become standard against locally advanced and metastatic prostate cancers [3]. Despite the fact that approximately 80% of advanced prostate tumors respond favorably to the effects of anti-androgen treatments, the

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majority of these prostate cancers progress into an aggressive and, most often, incurable hormone-refractory phenotype within 2 years [4]. A significant fraction of patients diagnosed in early stages of disease, and treated radically, also end up with relapsed aggressive cancer and this is further complicated by the fact that approximately 70% of men older than age 80 present localized prostate cancer upon post-mortem examination [3]. This highlights the fact that many prostate cancers are slow growing and non-life threatening, while other subsets of tumors are highly aggressive, and despite being detected at early stages, result in significant patient morbidity and mortality. It follows that significant numbers of men with local earlystage prostate cancers may be over treated, while other patients may benefit from more aggressive therapies. Cancer development involves a number of signal transduction cascades, and prostate cancer is no exception (reviewed in [5,6]). A complex series of events occurs to activate and deactivate various oncogenes and tumor-suppressor genes respectively, driving the dedifferentiation of normal prostate epithelial cells and upsetting the balance between proliferation and apoptosis. As mentioned above, most prostate cancers are under androgenic control, at least in their initial stages, so it follows that the androgen receptor (AR) is a critical mediator in this neoplastic process. AR regulated genes, such as prostate specific antigen (PSA) are commonly used markers for monitoring treatment responses, prognosis, and progression in patients with prostate cancer (reviewed in [7]). Another classical signal transduction pathway, cAMP-dependent protein kinase A (PKA) and its activator cyclic adenosine monophosphate (cAMP) have also been extensively studied in the context of carcinogenesis and while it is clear that PKA/cAMP can have gross effects on cellular growth and proliferation, independently of other signaling cascades, there is an interesting cross-talk between PKA and AR, that is highly relevant with respect to prostate cancer progression. This review will discuss the current understanding of PKA/cAMP in prostate carcinogenesis, with a particular focus on their cross-talk with AR signaling. 2. cAMP and PKA signaling It has been over 50 years since cAMP was first discovered [8]. Since then it has become one of the best understood secondary messengers, controlling a diverse array of processes including; metabolism, cell growth and differentiation, apoptosis and gene expression (reviewed in [9]). Cyclic AMP is synthesized from ATP by the plasma-membranebound adenylyl cylcase, and it is rapidly broken down via hydrolysis to adenosine 5′-monophosphate (5′-AMP) by cAMP phosphodiesterases (PDEs). There are numerous extracellular signals that can increase the cellular concentrations of cAMP by increasing the activity of adenylyl cyclase via stimulatory G proteins (Gs) and calcium (Ca2+). Cyclic AMP can then directly activate or interact with various proteins, such as, ion-channels, transcription factors, guanine nucleotide exchange factors and protein kinases [10]. Some of the major cellular effects of cAMP are actuated via PKA, a heterotetrameric holoenzyme consisting of two regulatory subunits (R) bound to two catalytic kinase subunits (C) (reviewed in [9,11–13]). In the cAMP unbound state the R subunits dimerize via their Nterminals, which then bind the C subunit's substrate binding site via a pseudosubstrate motif, keeping PKA inactive. Upon cAMP binding to the R subunits, the major intracellular cAMP receptor, a conformational change leads to the dissociation of R and C subunits, resulting in free and active catalytic subunit monomers that can phosphorylate numerous cellular targets. Full PKA activity in mammalian cells also requires phosphorylation at Thr197 by phosphoinositide-dependent protein kinase 1 (PDK1). There are four R subunit genes, RIα, RIβ, RIIα and RIIβ, along with three C subunit genes, Cα, Cβ and Cγ. The two major isoforms of PKA, termed PKA-I and PKA-II, are a result of the different R subunits (RI and RII) interacting with identical C subunits. R isoforms are differentially expressed in tissues, and also demon-

strate distinct subcellular localizations due to A-kinase anchoring proteins (AKAP) that can specifically bind the unique N-terminal regions of the respective R subunits. Numerous AKAPs have been identified, and they are suggested to facilitate the compartmentalization of PKA isoforms, and hence signaling, to discrete intracellular localizations. The localization of PKA plays a crucial role in determining which substrates are phosphorylated, and as a result, PKA can mediate a diverse array of physiological responses elicited by hormones and GPCRs (G-protein coupled receptors). While the cellular ratio of C subunits to R subunits is fairly constant around 1:1, the RI and RII subunits show considerable differences in expression between tissues. Furthermore, changes in the ratios of PKA-I and PKA-II play roles in important processes like cellular differentiation and cancer development. In general, PKA-I appears to be transiently over-expressed in normal cells, only in response to cell proliferation stimuli, while being constitutively over-expressed in cancer cells and associated with poor prognosis. PKA-II seems to be preferentially expressed in normal differentiated tissues. Numerous overviews of PKA and cAMP signaling can be obtained in the literature [9,11–13]. 3. PKA in prostate cancer In 2000, Cho et al. demonstrated that PKA-I was over-expressed in various cancer cells, including prostate adenocarcinoma cells [14]. Interestingly, over-expression of the RII subunit, which switched the dominant isoform of PKA from PKA-I to PKA-II, resulted in a reversion of the transformed phenotype of these various cancer cells. In addition, cancer cells over-expressing PKA-I also produce an extracellular active catalytic subunit that was also detected in the serum of cancer patients. These studies have been followed by investigations into PKA expression in prostate tumor tissues for diagnostic and prognostic purposes. In a phase III randomized clinical trial, the overexpression of PKA RIα was examined as a biomarker for predicting the outcome of men with locally advanced prostate cancer treated with radiation therapy (RT) alone, or in addition to short-term androgen deprivation therapy (STAD) [15]. PKA RIα over-expression was observed in 80 of 456 eligible and analyzable men (17.5%) and was significantly related to poor patient outcome (i.e. a higher rate of distant metastasis (DM)). These studies were extended to an independent cohort of men treated with radiation therapy and either STAD or long-term androgen deprivation therapy (LTAD) [16]. In this study PKA RIα over-expression was an independent predictor for both DM and biochemical failure (BF), and furthermore, the benefit of LTAD over STAD was significantly diminished when PKA RIα expression was high. These studies not only revealed PKA to be a potentially suitable biomarker for identifying high-risk prostate cancer patients who may benefit from anti-PKA therapies, but also highlighted an interesting relationship between the androgenic state of prostate cancer and PKA signaling. 4. The androgen receptor The androgen receptor belongs to the superfamily of nuclear receptors that mediates the actions of lipophilic ligands, such as steroids, retinoids and thyroid hormones. Upon ligand binding in the cytosol, the AR dissociates from heat shock proteins and translocates to the nucleus where it can effectively bind to specific androgen response elements (AREs), regulating a host of genes, including PSA (reviewed in [17–19]). 5. AR in androgen-dependent and -independent prostate carcinomas As mentioned above, both healthy prostate and most early-stage prostate cancers are dependent upon androgens for growth.

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Unfortunately, while androgen ablation therapies do slow tumor progression, the majority of patients undergoing LTAD eventually relapse into an aggressive androgen-independent form of disease [3]. The exact molecular mechanisms that control this switching of prostate cancer cells from androgen-dependent to androgen-independent are still poorly understood, however, it has been demonstrated that a functional AR is critical for progression into hormonerefractory disease. Most androgen-independent tumors continue to express the AR and the AR dependent gene product, PSA. In fact, when prostate tumors become androgen-independent, PSA mRNA is constitutively up-regulated. 25–30% of androgen-independent tumors progressing past initial androgen ablation therapies demonstrate AR gene amplifications, and xenograft studies have suggested that AR over-expression also occurs in progression from androgen dependence to androgen independence. Mutations of the AR gene and cleavage of the AR protein by proteases have also been suggested to activate AR in advanced cancers within a low androgen environment (reviewed in [5]). Perhaps even more significant, numerous growth factors, neuropeptides, and interleukins acting on various signaling pathways can significantly activate AR or alter expression of AR coactivators despite low androgen conditions (reviewed in [5,6]). This aberrant activation of AR through signaling pathways independent of androgen likely plays significant roles in the progression of prostate tumors to the androgen-independent state. 6. cAMP/PKA can regulate AR activation in prostate carcinomas Several studies in the mid-late 1990s demonstrated that AR could be stimulated by elevated cAMP levels. In 1994, Ikonen et al. demonstrated that rat AR transactivation by testosterone could be enhanced by the PKA stimulators; forskolin (an activator of adenylate cyclase) and 8-bromo-cAMP (8-Br-cAMP, a cAMP analogue) in monkey kidney CV-1 cells cotransfected with an AR expression plasmid [20]. Synergistic stimulation of AR by androgen and PKA activators was dependent upon intact AR DNA- and ligand-binding domains. Two years later, Nazareth and Weigel demonstrated androgen-independent stimulation of human AR by forskolin in both CV-1 cells, and PC-3 prostate carcinoma cells transfected with AR [21]. Nazareth and Weigel observed that neither R1881 (methyltrienolone, a synthetic androgen analogue) nor forskolin increased AR protein levels, but rather enhanced the AR activity by increasing AR– DNA binding. These results suggested that other aspects of AR interactions with additional co-activators and/or transcription factors may also be affected by forskolin/cAMP, however the exact mechanisms underlying this androgen-independent stimulation remained unclear. To further delve into the mechanisms of androgen-independent AR cross-talk with PKA, Sadar investigated PSA gene expression via PKA stimulation in prostate cancer cells [22]. It was shown that PKA activation (by forskolin) led to increased PSA expression and this increased expression was dependent upon the presence of functional AR. The observed PKA induction of androgen-responsive reporter genes was promoter specific and appeared to occur via a mechanism involving the amino terminal region of AR. Sadar demonstrated that induction of PSA and other ARE reporter gene constructs by forskolin was inhibited by bicalutamide (an anti-androgen that prevents AR dissociation from heat shock proteins, thus preventing DNA-binding activity) in LNCaP prostate carcinoma cells (which express functional AR), and was dependent upon AR transfection in PC-3 prostate carcinoma cells (which do not normally express AR). Furthermore, nuclear extracts from forskolin-treated LNCaP cells showed an increase in AR–ARE complex formation. An interesting observation was that the levels of AR–ARE complexes were increased in forskolintreated cells versus R1881-treated cells, despite the fact that R1881 increased the amount of nuclear AR by 40-fold compared to a 5-fold increase in nuclear AR levels from forskolin-treated cells [22]. This

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suggested that the forskolin transformed AR may have a greater affinity for the PSA–ARE as compared to the AR activated by R1881 via differential interactions with other proteins, or modulation of AR (such as changes in phosphorylation state). Previous studies have demonstrated that there are multiple phosphorylated AR isoforms and that increased androgen concentrations can increase these phosphorylation levels [23]. A detailed analysis of AR phosphorylation sites in LNCaP cells revealed that PKA stimulation by forskolin resulted in phosphorylation of AR at serine 650, located in the hinge region between the DNA-binding domain and the ligand-binding domain. Mutation of serine 650, or any of the other identified phosphorylation sites, had no noticeable effect on AR transactivation in a PSA– luciferase reporter system [23]. It has been suggested that the in vivo phosphorylation sites on AR may regulate nuclear transport rather than transcription. Taken together, these results suggested that changes in the phosphorylation state of other proteins, which could then interact with the N-terminal of AR, were likely involved in the PKA regulation of AR, and potentially also responsible for the increased androgen-independent AR promoter specificity upon PKA stimulation. In a 2005 study by Kim and others, a novel mechanism of PKA–AR cross-talk was uncovered and confirmed that PKA activation can enhance androgen-dependent transcription [24]. It was shown that cAMP responsive element-binding protein (CREB) can cooperate with AR via CREB-binding protein (CBP) and p300. Previous studies demonstrated that cAMP stimulation leads to phosphorylation of CREB by PKA at serine 133, which subsequently results in the binding of CREB to cAMP responsive elements (CRE) and the recruitment of the transcriptional co-activators CBP and p300 (both of which are histone acetylases) [25,26]. In this study, Kim et al. did not observe androgen-independent stimulation of PSA reporter transcription by forskolin treatment alone in LNCaP cells, however, the transcription induced by dihydrotestosterone (DHT), an AR ligand, was significantly enhanced by forskolin, and was inhibited by bicalutamide treatment. Upon over-expression of CBP or p300 in LNCaP cells, forskolininduced transactivation of the PSA reporter was observed, and this was no longer inhibited by bicalutamide, indicating that AR does not mediate the androgen-independent stimulation [24]. These results demonstrated that CBP and/or p300 over-expression resulted in an AR-independent mechanism of PSA reporter transcription, that is stimulated via the PKA pathway while in the presence of androgens and active AR, this PKA stimulation is additive. Furthermore, when the PKA inhibitor, H89, was applied to LNCaP cells, both DHT- and DHT/ forskolin-induced activities were abolished, indicating that PKA signaling was required for AR signaling. Since the treatment of forskolin and DHT did not affect the expression or stabilization of AR, it was suggested that an additional transcription factor mediated both the androgen-independent and -dependent induction of PSA transcription. CREB was a logical candidate due to the fact that it is activated by PKA and can interact with both p300 and CBP. Next, Kim et al. demonstrated that combined DHT and forskolin treatment significantly elevated the level of phosphorylated CREB. Phosphorylated CREB was able to bind to a CRE at the 5′ regulatory region of the PSA gene only after DHT and forskolin exposure, and was concomitant with histone acetylation (although forskolin did not have an effect on the levels of histone acetylation, indicating this process was mainly androgen dependent). Finally, Kim et al. observed that CBP/p300 was capable of mediating an interaction between CREB and AR. While it was previously known that both AR and CREB can bind to CBP/p300, this was the first demonstration that there is an interaction between AR and CREB that is mediated, or “bridged” by CBP/p300. A point of inconsistency remained between the observations of androgen-independent AR stimulation by forskolin observed by Nazareth and Weigel and Sadar, versus the co-stimulation of AR by androgens and forskolin observed by Ikonen et al. and Kim et al. [20– 22,24]. These contradicting results may be due to the use of low versus

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Fig. 1. Summary of reviewed prostate cancer pathways and their interactions: AR — androgen receptor, GPCR — G-protein coupled receptor, Gsα — G protein s alpha; FSK — forskolin; AC — adenylate cyclase; PDE4 — cAMP phosphodiesterase 4; PKA — cAMP-dependent proteins kinase; EPAC — exchange protein activated by cAMP; PKI β — protein kinase a inhibitor, isoform beta; CREB — cyclic AMP response element-binding protein; Rap — GTP binding protein/small GTPase; B-Raf — V raf murine sarcoma viral oncogene homolog B1; MEK — mitogen activated protein kinase kinase; ERK — extracellular regulated kinase; mTOR — mammalian target of rapamycin.

high passage number LNCaP cells. Since CBP/p300 over-expression is capable of inducing androgen-independent and PKA-mediated transcription, it could be that higher passage number cells may express more CBP/p300 overtime to compensate for a low androgen environment. Indeed, a previous study by Comuzzi et al. showed that androgen treatment of LNCaP cells resulted in decreased expression of both CBP protein and mRNA, and this effect was antagonized by the anti-androgen, bicalutamide [27]. Furthermore, CBP expression was detected in numerous therapy-resistant prostate tumor specimens analyzed with IHC and Heemers et al. also observed that androgen stimulation resulted in decreased expression of various co-regulators; including CBP and p300 [28,29]. Hence, “enhanceosomes” of CREB (and potentially other transcription factors) and AR, mediated by CBP and p300 (and potentially other co-regulators), likely play critical roles in the regulation of AR-dependent genes during the progression of prostate cancer through androgen-deprivation therapies [24,30,31] (For a summary of the different pathways discussed being relevant for prostate cancer development progression see Fig. 1). 7. PKA activation in prostate cancer, the cause of effect Thus far we have focused on how cAMP and PKA stimulation can affect AR activity. This section discusses how PKA is stimulated in prostate cancer cells. In a classic example of signal transduction, signal relay from G-protein coupled receptors (GPCRs) (such as β2adrenergic receptors) activate stimulatory G proteins (Gs) which can then activate AC to produce cAMP which can then stimulate PKA [10]. This cascade also holds true in the case of prostate cancer cells. Kasbohm et al. demonstrated that the activated α subunit of heterotrimeric guanine nucleotide-binding Gs protein activates AR in prostate cancer cells and synergizes with low concentrations of androgen to fully activate the AR via PKA [32]. Transfection of constitutively activated Gα subunits along with an ARE-regulated luciferase reporter system showed that Gs increased basal AR activity in LNCaP cells approximately 10-fold, whereas androgen stimulation with DHT resulted in an approximate 20-fold increase in reporter activity. Gene expression analysis demonstrated that LNCaP cells

expressed about 100-fold more β2-adrenergic receptor (β2-AR) than β1-AR, and stimulation of β2-AR with isoproterenol (ISO) increased ARE-regulated luciferase reporter activity in a dose-dependent manner and this effect was abolished by addition of the β2-AR antagonist, propranolol. In addition, ISO treatment of LNCaP cells resulted in time-dependent nuclear accumulation of AR, indicating AR activation, and also resulted in time-dependent increases in PSA expression (which is also ARE regulated). Next, Kasbohm investigate if there was a cooperative cross-talk between Gαs and AR stimulation under low androgen conditions. Under low DHT concentrations, which only resulted in minimal AR activation, ISO treatment further activated the AR to similar levels as achieved by saturating concentrations of androgen. Control experiments showed that a peptide inhibitor of Gαs abolished the ISO stimulation of AR as did expression of phosphodiesterase 4D (PDE4D). These experiments demonstrated that stimulation of endogenous β2AR-Gs signaling can cooperate with castrate levels of androgen to maximally activate AR via cAMP and both the peptide inhibitor of Gαs and PDE4D expression significantly impacted the direct activation of AR by androgen in LNCaP cells, suggesting that cAMP also plays a role in androgen mediated AR stimulation [32]. Finally, Kasbohm et al. demonstrated that PKA was directly involved in the ISO mediated stimulation of AR. ISO treatment of LNCaP resulted in VASP phosphorylation [33], a well known PKA substrate, and was inhibited by the PKA inhibitor H89. Interestingly, androgen stimulation of LNCaP cells also resulted in VASP phosphorylation that was H89 inhibitable. Furthermore, H89 attenuated both ISO and androgen induced expression of PSA, as well as inhibiting nuclear localization of AR in LNCaP cells, indicating that PKA regulates both the transactivation of AR via ISO and activation of AR by androgen. It was also shown that PKA-induced phosphorylation of AR on S650 is not required for the observed ISO- or androgen-regulated activation of AR [32]. The study by Kasbohm was pivotal since it clearly linked β2-AR-Gs signaling to AR via cAMP and PKA and these ideas were reinforced in another study demonstrating that regulator of G-protein signaling 2, an inhibitor of GPCR function, attenuated androgen-independent AR signaling and cell growth in prostate cancer cells [34]. In fact, GPCRs are the most commonly expressed

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type of receptor in mammalian cells and recognize a diverse array of ligands including various peptides and growth factors [35]. These studies open the door to potential GPCR targeting therapies for prostate cancer, especially in the context of hormone-refractory disease, and stimulated further investigations on the nature of PKA activation in prostate cancer. Since most androgen independent prostate cancers express AR and ARE-controlled genes even under castrate androgen levels, the targeting of GPCRs may provide novel avenues to treat progressive disease in these patients. In line with the previous results, Xie et al. showed that low nanomolar concentrations of vasoactive intestinal peptide (VIP), a neuropeptide, can induce growth of LNCaP cells, in low androgen conditions via AR transactivation [36]. This finding was interesting since advanced prostate tumors often contain increased levels of neuroendocrine cells (discussed in more detail below [37]) that secrete various neuropeptides, including VIP. VIP is expressed in human prostate tissues, usually in association with hypogastric and pelvic nerves that modulate prostate function, and can bind two Gs-coupled protein receptors, VPAC1-R and VPAC2-R. VIP was previously demonstrated to stimulate PSA secretion as well as potentiating the invasive capacity of LNCaP cells. Xie et al. showed that VIP can stimulate androgen-independent growth of LNCaP cells that is dependent upon the presence of functional AR, and also results in a 2.5-fold increase in androgen-independent PSA production. In line with these observations, Xie also observed nuclear localization of AR and subsequent ARE binding upon VIP treatment of LNCaP cells [36]. Xie et al. then went on to demonstrate that VIP transactivated AR via PKA. H89 and PKI (a peptide inhibitor of PKA) treatment of LNCaP cells resulted in dose-dependent reduction of VIP-induced AR transcriptional activation. Furthermore, it was shown that mitogen activated protein kinase kinase (MEK) inhibitor, U0126, also caused a dose-dependent reduction in VIP-stimulated AR activation, but not in the case of androgen-stimulated AR activation. H89, PKI and U0126 also attenuated the androgen-independent, VIP stimulated PSA production in LNCaP cells. Xie then showed that VIP treatment of LNCaP cells resulted in ERK1/2 activation, which was blocked by H89, PKI and U0126, and that blocking ERK1/2 activation (by transfecting dominant-negative MEK1 and ERK2 mutants into LNCaP) also attenuated the VIP-induced AR activation. Expression of a constitutively activated ERK2 in LNCaP cells resulted in AR activation in the absence of androgens. Finally, Xie showed that VIP-stimulation of LNCaP cells resulted in PKA-dependent activation of the Rap1 G-protein. Rap1 has been shown to play roles in hormonal activation of ERKs via Gs-coupled receptors in a number of cell lines, but this was the first demonstration of such activity, via PKA, in prostate cancer cells. AR activation by PKA action on Rap1 was cAMP dependent and occurred via a Src tyrosine kinase dependent mechanism, as the Src kinase inhibitor, PP2, attenuated the Rap1 activation. Transfection of the dominant-negative Rap1 mutant, Rap1-AGE, and the GTPase activating protein Rap1GAP (which inactivates Rap1) into LNCaP cells attenuated the observed VIP-stimulated, androgen-independent ERK and AR activities [36]. This study was significant since it was previously shown that MAPK/ERKs can directly phosphorylate and activate AR, promoting AR-dependent but androgen-independent growth [34,38]. Activated ERKs are commonly found in advanced androgen-independent prostate cancers, and Xie et al. demonstrated an important link between ERK, PKA and AR signaling in prostate carcinogenesis and proliferation. 8. Androgen and AR regulation of β2-AR and PKA Thus far we have focused our discussion on how extracellular signals are transduced via Gs-protein coupled receptors to AC, producing cAMP, which can activate PKA and subsequently AR in an androgen-independent context. But AR–PKA signaling cross-talks are

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truly bidirectional with hormones and androgens also affecting the regulation of β2-AR and PKA. Ramberg et al. examined the effect of androgens and other hormones on expression of β2-AR in prostate cancer cells [39]. It was observed that LNCaP cells incubated in 10% fetal calf serum (FCS) expressed about 5-fold more β2-AR (both mRNA and protein) as compared to LNCaP cells treated with 10% charcoal-striped FCS (CSS, which contains no androgens or growth factors). The decrease in β2AR expression in CSS treated LNCaP was also accompanied by abolished cAMP production in response to ISO stimulation and a 60% decrease in proliferation rates as compared to normal FCS grown LNCaP. Over-expression of β2-AR restored the ISO response in these CSS treated LNCaP cells. It was also demonstrated that androgendependent cells (such as LNCaP) expressed significantly less β2-AR than androgen-independent cells (such as Du145). While CSS treatment could decrease β2-AR mRNA levels in androgen-dependent cells, it had no effect on androgen-independent cells. Next Ramberg examined the effects of hormone treatment on β2AR regulation in LNCaP cells. Cells were incubated in CSS or FCS medium in the presence of triiodothyronine (T3), androgen (R1881), insulin, IGF-1, leptin, TNF-α, growth hormone, dexamethasone, retinoic acid, follicle-stimulating hormone, or ISO and β2-AR expression was examined. Interestingly, T3 was able to prevent the β2-AR down-regulation caused by CSS treatment in LNCaP cells, while R1881, dexamethasone and retinoic acid only produced minor effects. Furthermore, the T3 treatment was able to completely restore β2-AR protein levels in LNCaP cells cultured in CSS back to the levels of those observed under FCS conditions. Another study previously demonstrated that the β2-AR gene was a target for both thyroid hormone receptor alpha, the co-repressor SMRT, and the androgen receptor, explaining the observed effects in β2-AR expression. While R1881 only had slight effects on β2-AR expression, incubation of LNCaP cells (in FCS) with bicalutamide reduced the levels of β2-AR (both protein and mRNA) to similar levels as observed with CSS treatment. Furthermore, the bicalutamide treatment also prevented the T3induced up-regulation of β2-AR in these cells. Finally, Ramberg et al. also examined the levels of β2-AR expression in human prostate tissues. β2-AR was significantly increased in malignant tissues compared to normal and benign prostate epithelial cells. Epithelial cells expressed higher amounts of β2-AR mRNA as compared to stromal tissues. Importantly, Ramberg observed that primary prostate cancers expressed higher levels of β2-AR than primary tumor patients first treated with androgen ablation therapy. Furthermore, metastatic and androgen-refractory prostate tumor tissues showed increased expression of β2-AR compared to the androgen ablation treated samples, with the androgen-refractory samples showing levels of β2AR comparable to, and possibly higher, than those of the primary tumor samples [39]. This may suggest that while anti-androgen therapies initially down-regulate β2-AR signaling, in the case of androgen-independent cancers, the levels of β2-AR are somehow restored, and potentially over-expressed, which may in turn have effects on PKA activation and can then affect ARE-regulated genes. In line with this idea, Ramberg et al. did not observe significant differences in PSA mRNA expression between the primary tumor, primary tumor with androgen ablation therapies, metastatic tumors, and androgen-refractory tumor samples. In another study by Bagchi and associates, the effects of androgens on PKA activation were examined [40]. Treatment of LNCaP cells with testosterone induced dose-dependent increases in PKA activity that was H89 inhibited. It was also demonstrated that LNCaP stimulation with testosterone resulted in significant increases in cellular cAMP, while transient expression of PDE4D abrogated the testosterone stimulated PKA activity. In a next step, Bagchi et al. demonstrated that Gs protein (α subunit) signaling is responsible for the PKA activation by androgen. Gsα was either knocked down using small inhibitory RNAs (siRNAs) or inhibited using the RGS domain of Axin (a known

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inhibitor of Gsα signaling) resulting in decreased phosphorylation of VASP in response to both ISO and androgen. Furthermore, treating LNCaP cells with the nuclear AR antagonist bicalutamide did not abrogate the VASP phosphorylation induced by androgen, but did inhibit AR-induced expression of PSA. These results were re-enforced by examination of additional prostate epithelial cell lines; RWPE1, which represents normal prostate epithelial cells, LAPC4, which is an androgen-dependent prostate cancer cell line, and PC3M, which is an androgen-independent prostate cancer cell line that lacks expression of endogenous nuclear AR. In all three of the cell lines stimulation with androgen resulted in stimulation of PKA and VASP phosphorylation, and in the case of PC3M, this stimulation is not dependent upon nuclear AR. As a control, Bagchi et al. also showed that stimulation of PKA in LNCaP cells, via cAMP induction by the PDE inhibitor rolipram, resulted in nuclear AR activation and PSA expression, and this AR activation was inhibited by H89. Furthermore, androgen stimulation of LNCaP proliferation was also inhibited by H89, demonstrating that the androgen induced PKA activity plays a role in cell proliferation via nuclear AR, as previously demonstrated [40]. These studies demonstrate interesting interplay between PKA, androgen and AR. Not only does PKA activate nuclear AR leading to gene expression and prostate cancer cell proliferation, but androgen can also activate PKA via a Gsα dependent pathway that is independent of nuclear AR. Furthermore, androgens can also induce the expression of β2-AR which can in turn increase cellular cAMP levels, leading to PKA activation and downstream AR-related growth and proliferation. These signaling interplays are very important and may make interesting therapy targets in prostate cancer patients, especially in the context of when androgen-deprivation therapies are administered and for how long they are employed. 9. Other factors affecting cAMP and PKA signaling in prostate cancer Thus far we have focused on the cross-talk between cAMP/PKA signaling and the androgen receptor. But the control and regulation of PKA is far more complex than simple β2-AR activation, G-protein signaling, AC activation, and subsequent cAMP production. In the following section we will discuss other factors that can influence the activity and levels of PKA/cAMP, hence influencing their signaling with AR, that have been shown to play roles in prostate cancer cell function. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMPdependent protein kinase from the nucleus (reviewed in [41]). Three distinct PKI isoforms exist (PKI-α, PKI-β, and PKI-γ) and PKI-β has been shown to be over-expressed in prostate cancer, promoting its growth and aggressiveness. In a recent study by Chung et al. expression levels of PKI-β were examined in various prostate cancer cells microdissected from patient tissues compared to that of normal prostatic epithelial cells [42]. Over-expression of PKI-β was observed in hormone-refractory prostate cancer cells as compared to the normal prostate cells and androgen-sensitive prostate cancer cells. Interestingly, samples from androgen-sensitive tumor patients with Gleason scores of 8 or higher (indicative of aggressive disease) also showed strong expression of PKI-β, as compared to androgensensitive tumor samples with Gleason scores of 6 or lower. This may indicate that PKI-β is involved in aggressive tumor phenotypes and poor prognosis of prostate cancer [42]. To further examine the effects of PKI-β in prostate carcinogenesis, Chung and colleagues performed PKI-β knockdown experiments using short-hairpin RNAs (shRNA) on various prostate cancer cell

lines. Expression of the shRNAs correlated with decreased expression of PKI-β and drastically attenuated the growth and viability of prostate cancer cells. PKI-β knockdown also induced G0/G1 cell cycle arrest in LNCaP cells and decreased the cellular invasion potential in PC-3 cells, as gauged by Matrigel invasion assays. In a next step, Chung et al. expressed PKI-β in Du145 cells (that normally express very little or no PKI-β) and observed that these cells had higher proliferation rates than non-transfected cells. NIH3T3 cells transfected with PKI-β also demonstrated increased Matrigel invasion as compared to nontransfected cells. These results indicated that PKI-β over-expression promoted aggressive and invasive phenotypes in prostate cancer cells [42]. PKI-α was previously shown to bind to the catalytic subunit of PKA (PKA-C) via a pseudosubstrate motif, inhibiting PKA kinase activity and promoting nuclear export [43,44]. The same pseudosubstrate motif exists in PKI-β however it has been shown that the inhibitory activity of PKI-β is much smaller than that of PKI-α. Also, it was not known whether or not PKI-β is involved in the cellular translocation of activated PKA. Performing co-immunoprecipitation on COS-7 cells transfected with PKI-β and PKA-C, Chung et al. demonstrated that PKA-C and PKI-β interact. Furthermore, the cellular localization of PKA-C, examined in PC-3 cells expressing PKI-β, showed that PKA-C accumulated in the nucleus and upon siRNA induced knockdown of PKI-β, no or very little PKA-C was observed in the nucleus, indicating that PKI-β could potentially facilitate the nuclear import of PKA-C, or prevent PKA-C nuclear export [42]. This is in contrast to the other PKI family member PKI-α which functions to export PKA-C. Finally, Chung examined the role of PKI-β on Akt phosphorylation in prostate cancer cells. PTEN-PI3K-Akt signaling has been linked to prostate cancer progression. Knockdown of PKI-β with siRNAs in PC-3 and LNCaP cells resulted in diminished Akt phosphorylation at serine 473, a site commonly phosphorylated by PKA-C and linked to metastatic progression of cancers (reviewed in [45]). Over-expression of PKI-β enhanced the phosphorylation of Akt at Ser473 and in in vitro kinase assays using PKA-C and Akt, the addition of PKI-β also enhanced the phosphorylation at Ser473 of Akt by PKA-C revealing that PKI-β can enhance the activity of PKA for Akt-Ser473. Looking into PKI-β expression and Akt-Ser473 phosphorylation in human prostate cancer tissues also revealed a positive correlation between the two events [42]. These results again highlight the complexity of signaling pathways in prostate cancer, where multiple pathways interact, beyond AR and PKA, to influence how prostate cancer progresses. In some of the above discussed studies we also mentioned control experiments, where phosphodiesterases (PDEs) were expressed in prostate cancer cells to diminish cellular cAMP levels. PDEs cleave cAMP and are expressed in a wide variety of tissues. In a recent study by Rahrmann et al., employing a transposon-based somatic mutagenesis screen, identified candidate cancer genes that may play a role in the initiation of prostate cancer [46]. By inducing somatic mutations in prostate epithelial cells, the authors produced foci of altered histology (hyperplasia) and increased proliferation (based on the Ki-67 proliferation marker), that resembled precursor lesions of prostate cancer. Analysis of these transposon insertion sites identified PDE4D over-expression as a potential prostate cancer-related gene. The authors then investigated the expression of PDE4D in prostate cancer samples. Examining immunohistochemical stains on human prostatic carcinoma tissue microarrays demonstrated that stronger PDE4D staining correlated with adenocarcinoma tissues versus benign prostatic hyperplasia tissues, although the PDE4D signals did not differ significantly between differing tumor grades of adenocarcinoma. The study was extended to examining PDE4D RNA expression in pooled samples of pathologically normal prostate versus prostatic adenocarcinoma and confirmed increased PDE4D expression in the cancer tissues. PDE4D is a gene encoding 9 different phosphodiesterase isoforms and by sequencing the predominant PCR product from

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the pooled prostatic adenocarcinoma RNA samples, Rahrmann et al. identified the PDE4D5 isoform as the major PDE4D isoform overexpressed in the cancer tissues. They then examined the effects of PDE4D knockdown on the growth rate of prostate cancers. Using shRNAs targeting PDE4D in Du145 and PC-3 cells lines, the authors observed decreased proliferation rates both in in vitro cell experiments, as well as in xenograft tumor models [46]. These studies demonstrate how other protein factors that affect the cellular levels of cAMP and activity of PKA can also contribute to the pathogenesis of prostate cancer. Thus far we have mainly discussed how cAMP regulation occurs via PKA activation, however, in 1998 a cAMP-responsive guanine nucleotide exchange factor (cAMP-GEF) was identified that can activate the Ras superfamily small GTPases Rap1 and Rap2 [47,48]. This cAMP-GEF is also called exchange protein directly activated by cAMP (Epac). Epac contains a cAMP binding domain that is identical to that of the PKA R subunits, and thus far there are two known isoforms of Epac, Epac1 and Epac2,, that play roles in numerous cellular processes such as, cell adhesion, cell–cell junction formation, exocytosis and secretion, cellular differentiation and proliferation, gene expression, and apoptosis (reviewed in Cheng et al. [49]). In a recent study by Misra and Pizzo it was shown that selective stimulation of Epac1 via the cAMP analogue 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cAMP (8-CPT) increased DNA synthesis and proliferation of human 1-LN prostate cancer cells [50]. Treatment with 8-CPT elevated the phosphorylation of B-Raf1, MEK1/2 and ERK 1/2, as well as, increased the phosphorylation of mTOR, TSC2 and Rheb proteins. This implicating raised cAMP levels in the activation of Epac1 and increased proliferation in prostate cancer cells via both B-Raf1/MAPK and mTOR signaling pathways. When Misra and Pizzo knocked down Epac1 with siRNAs, activation of Rap1, B-Raf and ERK 1/2 and mTOR by 8-CPT was attenuated. Misra and Pizzo went on to show that 8-CPT also induces the activation of B-Raf and mTOR signaling in the Du145 and PC-3 cells lines. In a conflicting study by Grandoch et al. it was shown that Epac inhibits the migration and proliferation of Du145 and PC-3 cells [51]. Here the authors demonstrated time-dependent decreases in ERK 1/2 phosphorylation when PC-3 cells were treated with 8-CPT, and DNA synthesis and migration potentials of both PC-3 and Du145 were also decreased by the 8-CPT treatment. It will require further studies to elucidate the details of how Epac1 and Epac2 exact their cellular effects in the context of prostate cancer, however the likely explanation for the contrasting results comes from the concentrations of 8-CPT used in the respective studies. The study by Misra and Pizzo used a range of 25–200 μM 8-CPT, while the study by Grandoch et al. employed a high concentration of 300 μM [50,51]. As we will discuss below, the levels of cAMP stimulation in cells can have drastic effects on the behavior and proliferation of cells. Specifically, chronic induction of cAMP levels can result in neuroendocrine-like differentiation of prostate cancer cells, and this is accompanied by loss of mitogenic activity. It will remain to be seen whether high level and long-term treatments of prostate cancer cells with 8-CPT result in neuroendocrine differentiation. 10. PKA and neuroendocrine cell differentiation Neuroendocrine (NE) cells have been observed in prostate tumors with their occurrence considered a potential indicator for disease progression towards an androgen-independent state. There is evidence to suggest that prostate cancer cells, or their precursors, can differentiate into NE-like cells, both in vitro and in vivo, and some clinical studies have associated prostatic NE cells as prognostic markers for tumor progression (reviewed in [37]). While NE cells in normal prostate tissues are believed to be derived from neural crest cells, which form the urogenital cavity during embryonic development, some studies have demonstrated that NE-like cells can

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differentiate from exocrine and basal epithelial cells within prostatic tumors. Hence, in addition to the de-differentiation of normal prostate epithelial cells into tumor cells, there are also differentiation events towards NE-like cells that can affect the tumor phenotype. NE cells produce a variety of neuro-secretory products that can promote cell growth, including parathyroid hormone-related peptides, neurotensin, serotonin, calcitonin and bombesin-related peptides, suggesting that these cells can function in paracrine and/or endocrine signaling within the prostate, possibly promoting progression of tumors. It has been shown that a significant amount of prostate tumor tissues contain scattered NE-like foci and, although the exact mechanisms that drive NE differentiation in vivo are poorly understood, there are numerous in vitro studies with prostate cancer cell lines that demonstrate the acquisition of NE characteristics when the cells are stimulated by agents naturally occurring in the tumor microenvironment. NE characteristics include: the appearance of dense core granules in the cytoplasm, the extension of neuron-like processes, loss of mitogenic activity and the expression and secretion of NE markers, such as neuron-specific enolase (NSE) and the neurosecretory products mentioned above [37]. While the observed increasing amounts of NE foci observed in prostate tumors have reduced proliferation, it has also been shown that proliferating carcinoma cells are found in close proximity, strengthening the idea that NE cells can provide paracrine stimuli to promote progression and proliferation of the surrounding tumor cells. In the following section we will discuss the roles of cAMP and PKA in the acquisition of NE-like phenotypes in prostate cancer cells. Early studies into NE cells in prostate cancer utilized chronic elevation of cAMP levels by various agents including; AC stimulators (forskolin), cAMP analogues (dibutyryl cAMP), β-AR agonists (ISO and epinephrine (epi)) and phosphodiesterase inhibitors (isobutylmethylxanthine (IBMX)) to induce NE-like differentiation of various prostate carcinoma cell lines showing that cAMP increases alone were able to induce NE differentiation in both androgen-dependent and androgen-independent cells [52,53]. In addition, removal of the differentiating agents reversed the NE-phenotype back to an epithelial carcinoma cell-phenotype, indicating that NE-like differentiation of prostate cancer cells can be reversible, and therefore dynamic, depending upon the tumor microenvironment [53]. Cox et al. then demonstrated that the cAMP mediated NE differentiation of LNCaP cells was induced by activated PKA [54]. In LNCaP cells stimulated with forskolin, ISO and epi, PKA was shown to have increased kinase activity and transfection of these cells with constitutively active PKA catalytic subunit was also sufficient to induce NE-like morphologies, mitotic arrest, and the expression of NSE. Furthermore, the expression of a dominant-negative mutant of the PKA regulatory subunit, RIα, abolished the β-AR and AC mediated NE differentiation induced by forskolin and ISO. In another study, NE differentiation of LNCaP cells led to increased expression of PKA catalytic subunit Cβ supporting the idea that activated PKA is the critical mediator in NE-like differentiation in LNCaP cells caused by increased cAMP levels [55]. In addition to elevation of cAMP, there are additional stimuli that can induce NE differentiation of prostate cancer cells, including, longterm androgen ablation and treatment with certain proteins or peptides, such as interleukin-1β and -6, and VIP [56–59]. In the case of VIP, NE differentiation of LNCaP cells occurred partially via a PKAmediated mechanism, which was partially inhibited by H89 [59]. At first glance these results may seem contradictory to the discussions above of studies that demonstrated PKA activation leading to AR stimulation and increased proliferation of prostate cancer cells. The important difference between these respective studies is the differences in degrees of cAMP and PKA stimulation. In the studies by Nazareth, Sadar and Kasbohm employed ranges of 0.1 to 50 μM forskolin for 16 to 24 h, or 10 μM ISO for a period of 10 min to stimulate cAMP production and PKA activity [21,22,32]. In the NE-

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differentiation studies by Bang and Cox, chronic cAMP elevation was achieved with combined treatments of db-cAMP (0.1 to 1 mM) or forskolin (10 μM) with the phosphodiesterase inhibitor IBMX (100 to 500 μM) for periods of 3 to 7 days [52–54]. In the study by Xie et al. VIP-induced PKA stimulation was performed for 2 to 4 days at a concentration of 20 nM VIP, whereas in a study by Gutierrez-Canas et al., NE differentiation of LNCaP cells was achieved using 100–200 nM VIP over 4 days [36,59]. These are important differences that illustrate that short-term, low level PKA stimulation can activate AR and increase cellular proliferation, while chronic PKA stimulation over prolonged periods of time may lead to cellular differentiation to NE-like phenotypes. Kvissel et al. examined the effects of androgen-deprivation and cAMP induced NE differentiation of LNCaP cells with respect to the expression of PKA Cβ isoforms [55]. There are 4 different splice variants of the Cβ PKA subunit (Cβ1, Cβ2, Cβ3, and Cβ4) and they seem to play unique roles with respect to proliferation versus NE differentiation [60–62]. When LNCaP cells were induced to differentiation via 4 day androgen-deprivation treatments, Cβ1, Cβ3 and Cβ4 mRNA expression levels increased 3, 7 and 4 fold, respectively, while Cβ2 levels slightly decreased about 0.8 fold. Similar results were seen at the protein level, although Cβ2 protein levels decreased much more significantly compared to mRNA levels (approximately 75%). These trends in protein and mRNA expression were reversed when androgen-depleted LNCaP cells were cultured in the presence of androgens suggesting that the PKA Cβ2 isoform plays roles in cellular proliferation, while Cβ1, Cβ3 and Cβ4 play roles in the NE-phenotype of LNCaP cells. These ideas were reinforced when Kvissel investigated the expression of the different Cβ isoforms in prostate cancer tissues. Indeed Cβ2 was up regulated in tumor compared to normal tissues further implicating this PKA catalytic isoform in proliferative cellular processes [55]. Taken together, these results show that not only is the degree of PKA activation important with respect to growth and

differentiation, but also different isoforms of PKA can play important roles in the proliferative or differentiating process. It is interesting to speculate whether over-stimulation of PKA in prostate tumor cells, inducing NE differentiation, could be useful as an anti-tumor therapy. 11. Conclusions In this review article we have given a comprehensive overview of the roles that PKA and cAMP play in the development and progression of prostate cancer, specifically with respect to its cross-talk with AR signaling. Some of the major ideas of this review are illustrated in Fig. 2. This is a true example of signaling cross-talk, since not only does cAMP and PKA activation result in the stimulation of AR, but androgens can also regulate the activity of PKA. It is interesting that under low androgen conditions, PKA can activate AR to levels similar to those found under androgen-stimulated conditions. Furthermore, the complexity of the signaling cross-talk is highlighted when examining the effects of PKA and androgen depletion in the context of various tumor stages. Androgen depletion can result in downregulation of β2-AR that should result in decreased cAMP production and lowered PKA activity. This would suggest promise for antiandrogen therapies blocking direct AR stimulation as well as preventing PKA over-activation that could compensate for the lowered AR activity in these androgen-depleted environments. Indeed anti-androgen treatments do decrease tumor proliferation for some time, however advanced cancers seem to be inevitable, and in these advanced cancers we often observe increased expression of β2-AR and PKA despite castrate levels of androgens, that can then further stimulate AR-regulated gene expression. In addition to this, other protein factors such as PKI-β and PDE4D isoforms also seem to play roles in the activation or deactivation of PKA and these signaling cascades are further complicated by the potential for NE differentiation and paracrine signaling within tumors by the prostate cancer

Fig. 2. Prostate cancer progression and differentiation with respect to some of the major players in the PKA/cAMP signaling cascade. Normal prostate epithelial cells become neoplastic through a complex series of carcinogenic events that involve various oncogenes being activated, and tumor-suppressor genes being deactivated. During this process a host of proteins (including PKA-I, β2-AR, PKI-β, and PDE4Ds) can be over-expressed. When a patient presents with local advanced or metastatic disease, androgen-deprivation therapies are introduced, which can lead to the down-regulation of some proteins (such as β2-AR). Despite these castrate levels of androgen and initial slowing of tumor growth, the AR continues to function, which can be explained in part, by PKA-I, β2-AR, PKI-β, PDE4D and Epac over-expression or -stimulation, and eventually the cancer progresses into an aggressive androgen-independent phenotype. Chronic cAMP signaling can also lead to NE-like differentiation and loss of mitogenic activity, however, these NE-like cells have the ability to paracrine signal to surrounding prostate carcinoma cells via various peptides and growth factors (such as VIP) that can, in turn, continue to activate PKA, and hence AR, related pathways, and drive cell growth and cancer progression.

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cells. It could be speculated that a depleted androgen environment, while being highly effective at suppressing the growth of many prostate cancer cells, may create an environment that favors the selection of cells that already over-express certain GPCRs and/or PKA (or perhaps cells that contain constitutively active versions of these proteins or other partners in their signaling cascades), and perhaps the delay in onset of advanced cancers is related to the emergence of such cells as the dominant tumor cell. Further research into these avenues will hopefully yield important and clinically relevant answers to these questions. While the signaling pathways that are central to driving the carcinogenesis of prostate cells are complex and sometimes seem daunting, these studies also shed light on potentially novel avenues for therapeutic intervention. As better biomarkers emerge that allow us to discriminate aggressive from non-aggressive cancers, the therapies should also adapt accordingly. Surgery and radiation will likely remain effective mainstays in the treatment of early-stage localized prostate tumors and androgen-deprivation therapies will surely remain useful in advanced cancers. The questions that will require answering in the future will be to predict who really benefits from STAD versus LTAD and at what stage should they be administered, and as we discussed here, PKA may play a central role in such decisions. Furthermore, for patients who are identified as having aggressive and/or advanced cancers, the targeting of other signaling factors, such as, PKA, GPCRs, AC, PKI and PDEs, via monoclonal antibodies, siRNAs or small molecule inhibitors, may provide additional and powerful benefits in the treatment of prostate carcinomas. It will be exciting to see how these developments progress in the future. 12. Outlook It becomes clear that the development and progression of prostate cancer is driven by a very complex interplay of different pathways. The exact action of different modulators and molecules involved may often depend on the details of activity status of the surrounding molecules. Consequently, the same modulator/molecule may have different effects in different cellular environments. It seems obvious that we require more integrated approaches to investigate cancer pathway signaling from different angles including targeting as well as large scale approaches. The gathered data need to be integrated in a systems biology manner as only this will lead to a system understanding of cancer compared to analyzing individual and separated events that are inherently difficult to assemble back into a whole system view. Acknowledgements We thank Dr Ian Mills, Cambridge University, UK and Dr Guido Jenster, EMC Rotterdam, The Netherlands for their valuable comments. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, CA Cancer J. Clin. 59 (4) (2009) 225. [2] M. Quinn, P. Babb, BJU Int. 90 (2) (2002) 162. [3] R. Govindan (Ed.), The Washington Manual of Oncology, Second Edition, Lippincott Williams and Wilkins, Philadelphia, 2008. [4] J.J. Coen, C.S. Chung, W.U. Shipley, A.L. Zietman, Int. J. Radiat. Oncol. Biol. Phys. 57 (3) (2003) 621. [5] H.L. Devlin, M. Mudryj, Cancer Lett. 274 (2) (2009) 177. [6] M. Kaarbo, T.I. Klokk, F. Saatcioglu, Bioessays 29 (12) (2007) 1227. [7] D.V. Makarov, S. Loeb, R.H. Getzenberg, A.W. Partin, Annu. Rev. Med. 60 (2009) 139. [8] T.W. Rall, E.W. Sutherland, J. Biol. Chem. 232 (2) (1958) 1065. [9] K.V. Chin, W.L. Yang, R. Ravatn, T. Kita, E. Reitman, D. Vettori, M.E. Cvijic, M. Shin, L. Iacono, Ann. NY Acad. Sci. 968 (2002) 49. [10] B. Alberts, A. Johnosn, J. Lewis, R. Martin, K. Roberts, P. Walter, Molecular Biology of the Cell, Garland Science, Taylor and Francis Group, New York, 2002. [11] I. Bossis, C.A. Stratakis, Endocrinology 145 (12) (2004) 5452. [12] L.R. Pearce, D. Komander, D.R. Alessi, Nat. Rev. Mol. Cell. Biol. 11 (1) (2010) 9.

515

[13] Y.S. Cho-Chung, M.V. Nesterova, Ann. NY Acad. Sci. 1058 (2005) 76. [14] Y.S. Cho, Y.N. Lee, Y.S. Cho-Chung, Biochem. Biophys. Res. Commun. 278 (3) (2000) 679. [15] L.Y. Khor, K. Bae, T. Al-Saleem, E.H. Hammond, D.J. Grignon, W.T. Sause, M.V. Pilepich, P.P. Okunieff, H.M. Sandler, A. Pollack, Int. J. Radiat. Oncol. Biol. Phys. 71 (5) (2008) 1309. [16] A. Pollack, K. Bae, L.Y. Khor, T. Al-Saleem, M.E. Hammond, V. Venkatesan, R.W. Byhardt, S.O. Asbell, W.U. Shipley, H.M. Sandler, Clin. Cancer Res. 15 (17) (2009) 5478. [17] N. Bennett, J.D. Hooper, C.S. Lee, G.C. Gobe, IUBMB Life 61 (10) (2009) 961. [18] S.S. Dutt, A.C. Gao, Future Oncol. 5 (9) (2009) 1403. [19] K.R. Lamont, D.J. Tindall, Adv. Cancer Res. 107 (2010) 137. [20] T. Ikonen, J.J. Palvimo, P.J. Kallio, P. Reinikainen, O.A. Janne, Endocrinology 135 (4) (1994) 1359. [21] L.V. Nazareth, N.L. Weigel, J. Biol. Chem. 271 (33) (1996) 19900. [22] M.D. Sadar, J. Biol. Chem. 274 (12) (1999) 7777. [23] D. Gioeli, S.B. Ficarro, J.J. Kwiek, D. Aaronson, M. Hancock, A.D. Catling, F.M. White, R.E. Christian, R.E. Settlage, J. Shabanowitz, D.F. Hunt, M.J. Weber, J. Biol. Chem. 277 (32) (2002) 29304. [24] J. Kim, L. Jia, M.R. Stallcup, G.A. Coetzee, J. Mol. Endocrinol. 34 (1) (2005) 107. [25] A.J. Shaywitz, M.E. Greenberg, Annu. Rev. Biochem. 68 (1999) 821. [26] B. Mayr, M. Montminy, Nat. Rev. Mol. Cell Biol. 2 (8) (2001) 599. [27] B. Comuzzi, C. Nemes, S. Schmidt, Z. Jasarevic, M. Lodde, A. Pycha, G. Bartsch, F. Offner, Z. Culig, A. Hobisch, J. Pathol. 204 (2) (2004) 159. [28] B. Comuzzi, L. Lambrinidis, H. Rogatsch, S. Godoy-Tundidor, N. Knezevic, I. Krhen, Z. Marekovic, G. Bartsch, H. Klocker, A. Hobisch, Z. Culig, Am. J. Pathol. 162 (1) (2003) 233. [29] H.V. Heemers, L.J. Schmidt, E. Kidd, K.A. Raclaw, K.M. Regan, D.J. Tindall, Prostate 70 (9) (2010) 959. [30] Y. Shang, M. Myers, M. Brown, Mol. Cell 9 (3) (2002) 601. [31] M.C. Louie, H.Q. Yang, A.H. Ma, W. Xu, J.X. Zou, H.J. Kung, H.W. Chen, Proc. Natl Acad. Sci. USA 100 (5) (2003) 2226. [32] E.A. Kasbohm, R. Guo, C.W. Yowell, G. Bagchi, P. Kelly, P. Arora, P.J. Casey, Y. Daaka, J. Biol. Chem. 280 (12) (2005) 11583. [33] M. Eigenthaler, C. Nolte, M. Halbrugge, U. Walter, Eur. J. Biochem. 205 (2) (1992) 471. [34] X. Cao, J. Qin, Y. Xie, O. Khan, F. Dowd, M. Scofield, M.F. Lin, Y. Tu, Oncogene 25 (26) (2006) 3719. [35] K.L. Pierce, R.T. Premont, R.J. Lefkowitz, Nat. Rev. Mol. Cell Biol. 3 (9) (2002) 639. [36] Y. Xie, D.W. Wolff, M.F. Lin, Y. Tu, Mol. Pharmacol. 72 (1) (2007) 73. [37] G.P. Amorino, S.J. Parsons, Crit. Rev. Eukaryot. Gene Expr. 14 (4) (2004) 287. [38] D. Gioeli, J.W. Mandell, G.R. Petroni, H.F. Frierson Jr., M.J. Weber, Cancer Res. 59 (2) (1999) 279. [39] H. Ramberg, T. Eide, K.A. Krobert, F.O. Levy, N. Dizeyi, A.S. Bjartell, P.A. Abrahamsson, K.A. Tasken, Prostate 68 (10) (2008) 1133. [40] G. Bagchi, J. Wu, J. French, J. Kim, N.H. Moniri, Y. Daaka, Cancer Res. 68 (9) (2008) 3225. [41] G.D. Dalton, W.L. Dewey, Neuropeptides 40 (1) (2006) 23. [42] S. Chung, M. Furihata, K. Tamura, M. Uemura, Y. Daigo, Y. Nasu, T. Miki, T. Shuin, T. Fujioka, Y. Nakamura, H. Nakagawa, Oncogene 28 (32) (2009) 2849. [43] W. Wen, A.T. Harootunian, S.R. Adams, J. Feramisco, R.Y. Tsien, J.L. Meinkoth, S.S. Taylor, J. Biol. Chem. 269 (51) (1994) 32214. [44] J.C. Wiley, L.A. Wailes, R.L. Idzerda, G.S. McKnight, J. Biol. Chem. 274 (10) (1999) 6381. [45] M. Qiao, S. Sheng, A.B. Pardee, Cell Cycle 7 (19) (2008) 2991. [46] E.P. Rahrmann, L.S. Collier, T.P. Knutson, M.E. Doyal, S.L. Kuslak, L.E. Green, R.L. Malinowski, L. Roethe, K. Akagi, M. Waknitz, W. Huang, D.A. Largaespada, P.C. Marker, Cancer Res. 69 (10) (2009) 4388. [47] J. de Rooij, F.J. Zwartkruis, M.H. Verheijen, R.H. Cool, S.M. Nijman, A. Wittinghofer, J.L. Bos, Nature 396 (6710) (1998) 474. [48] H. Kawasaki, G.M. Springett, N. Mochizuki, S. Toki, M. Nakaya, M. Matsuda, D.E. Housman, A.M. Graybiel, Science 282 (5397) (1998) 2275. [49] X. Cheng, Z. Ji, T. Tsalkova, F. Mei, Acta Biochim Biophys Sin (Shanghai) 40 (7) (2008) 651. [50] U.K. Misra, S.V. Pizzo, J. Cell. Biochem. 108 (4) (2009) 998. [51] M. Grandoch, A. Rose, M. ter Braak, V. Jendrossek, H. Rubben, J.W. Fischer, M. Schmidt, A.A. Weber, Br. J. Cancer 101 (12) (2009) 2038. [52] Y.J. Bang, F. Pirnia, W.G. Fang, W.K. Kang, O. Sartor, L. Whitesell, M.J. Ha, M. Tsokos, M.D. Sheahan, P. Nguyen, W.T. Niklinski, C.E. Myers, J.B. Trepel, Proc. Natl Acad. Sci. USA 91 (12) (1994) 5330. [53] M.E. Cox, P.D. Deeble, S. Lakhani, S.J. Parsons, Cancer Res. 59 (15) (1999) 3821. [54] M.E. Cox, P.D. Deeble, E.A. Bissonette, S.J. Parsons, J. Biol. Chem. 275 (18) (2000) 13812. [55] A.K. Kvissel, H. Ramberg, T. Eide, A. Svindland, B.S. Skalhegg, K.A. Tasken, Cell. Signal. 19 (2) (2007) 401. [56] M. Diaz, M. Abdul, N. Hoosein, Prostate Suppl. 8 (1998) 32. [57] Y. Qiu, D. Robinson, T.G. Pretlow, H.J. Kung, Proc. Natl Acad. Sci. USA 95 (7) (1998) 3644. [58] P.D. Deeble, D.J. Murphy, S.J. Parsons, M.E. Cox, Mol. Cell. Biol. 21 (24) (2001) 8471. [59] I. Gutierrez-Canas, M.G. Juarranz, B. Collado, N. Rodriguez-Henche, A. Chiloeches, J.C. Prieto, M.J. Carmena, Prostate 63 (1) (2005) 44. [60] A.K. Kvissel, S. Orstavik, P. Oistad, T. Rootwelt, T. Jahnsen, B.S. Skalhegg, Cell. Signal. 16 (5) (2004) 577. [61] S. Orstavik, A. Funderud, T.T. Hafte, S. Eikvar, T. Jahnsen, B.S. Skalhegg, FEBS J. 272 (7) (2005) 1559. [62] S. Orstavik, N. Reinton, E. Frengen, B.T. Langeland, T. Jahnsen, B.S. Skalhegg, Eur. J. Biochem. 268 (19) (2001) 5066.