PI3KAKT and AR Signalling axis

The role of the PI3K/AKT pathway in CaP cell proliferation, survival and progression from AD to AI disease has been linked to androgen receptor (AR) transcriptional activity, stability and expression. This section of the review will discuss the various modes of crosstalk between the PI3K-Akt and AR axis.

4.1. PI3K/PTEN and AR

The activation of Akt has provided a mechanistic link between PI3K and AR transactivation. However, other modes of interaction have been shown. AR can directly interact with the p85 regulatory subunit of PI3K. Upon their binding, the AR enhances PI3K enzyme activity to ultimately upregulate Akt phosphorylation [141]. Conversely, EGFR stimulated PI3K activity was decreased in PC-3 cells transfected with wildtype AR relative to AR null PC-3 cell line [141-142]. PI3K activity was further reduced upon R1881 treatment, suggesting AR activation as a negative regulator of PI3K stimulation. The role of androgens within the AR-PI3K axis has also shown their significance in modulating cell proliferation and growth. Multiple reports have demonstrated that androgens enhance PI3K activity and increase downstream Akt phosphorylation. In NIH3T3 fibroblasts, there was rapid activation of the PI3K/AKT pathway upon androgen stimulation. While this required the presence of the AR, AR transactivation was not essential [141, 143]. Low concentration of androgen further stimulated the association of the AR with Src and PI3K, which triggered the cells into S-phase entry [141, 143].

The negative regulator of the PI3K pathway, the PTEN tumor suppressor gene, as discussed previous is frequently inactivated in CaP as well as in CaP cell lines which include PC-3 and LNCaP. Functional loss of PTEN is associated with increased AKT phosphorylation, higher Gleason score, and poor prognosis [120, 144]. The development of conditional mouse models of PTEN -/- has shown that PTEN alone can drive the progression of CaP through invasion, metastasis and AI proliferation [82]. Although there was heightened PI3K activity, there was continued evidence of AR gain-of-function despite reduced steroid ligand levels [45, 82]. For example, prostatic epithelium in PTEN-/- mice was still sensitive to androgen withdrawal. As such, Mullholland et al., (2006) suggest that while the AR remains functional and sensitive to androgens, the PI3K/AKT and AR oncogenic signaling may complement and compensate for one another during the time of androgen ablation therapy. This is supported by studies demonstrating cells lacking PTEN had elevated PI3K/AKT axis activity upon androgen withdrawal [45, 46, 82]. Hence, PTEN loss may allow for epithelium with sufficient PI3K/AKT signaling to maintain cell proliferation and promote AR gain of function [82].

Currently, there is no direct method that supports PTEN loss for promoting AR-specific gene activation. However, CaP xenograft studies have shown that amplification of AR does occur [82, 147] under a PTEN null background, which can be correlated to increased AR stabilization [82]. Li et al, (2001) demonstrated that PTEN itself can negatively regulate AR gene targets such as PSA, reduce the nuclear localization of the AR and promote receptor degradation through caspase 3 or the proteosome [82]. Alternative to AR amplification and stabilization, the loss of PTEN may also contribute to AR activation through the various coregulators of the AR, this includes ARA70 [149] and ARA54 [150]. Such regulation by PTEN would allow for heightened AR response to low androgen concentrations or responsiveness to non-androgen ligands. On the other hand, at low androgen levels, AR expression alone can also stimulate cell proliferation (Denmeade et al., 1996) while PTEN restoration induced apoptosis and growth arrest [151]. However, in the presence of androgens, PTEN expression was sufficient to reduce cell proliferation but not induce apoptotic response [82, 148, 152]. PTEN loss then, results in an indirect AR gain-of-function phenotype by establishing an environment that may increase AR oncogenicity and CaP metastatic potential.

4.2. AKT and AR cross-talk

The regulation of the AR by Akt through direct and indirect modes of interactions has been demonstrated in literature. These include, but not limited to 1) direct phosphorylation of the AR by AKT, 2) AKT/mTOR dependent regulation of the AR, 3) AR interaction with FOXO family of transcription factors downstream AKT, and 4) AR regulation by AKT via the Wnt/ GSK3ß/ß-catenin cross-talk.

4.2.1. AKT and direct AR phosphorylation

AKT binds to the AR to directly phosphorylate at AR consensus sites, S213 and S791. Upon phosphorylation by AKT, the AR becomes transcriptionally active under physiological androgen concentrations. However, a study Xin et al, (2006) demonstrated that AR phosphorylations at S213 and S791 were not critical for tumor progression, which indicated that AR phosphorylation may not be the sole regulatory event inducing AR transcriptional activity [153, 141]. Alternatively, at high androgen concentrations, AKT can protect CaP cells from apoptosis and suppress AR transcriptional activity by phosphorylation at S210 and S790 [49]. Lin et al, (2002) also demonstrated that AR phosphorylation by AKT resulted in MDM2 mediated ubiquitlyation of the AR, leading to its proteosomal degradation. Taken together, these data indicate AKT-mediated regulation of AR activity is dependent on the external environment.

A potent nutrient and growth/survival pathway kinase, mTOR has been implicated in the progression of several types of cancer. Interestingly, it has been identified as a regulator of AI-CaP growth, but not CaP growth, by Ghosh et al. (2005). Further, it has been implicated in regulating the progression to androgen-independent disease [155-156] alongside AR signaling. There are three key routes through which mTOR could potentially interact with AR signaling: through its kinase activity as mTORC1 or mTORC2, post-transcriptional regulation via regulation of translation factors, or indirectly through a signaling cascade. Several broad studies have implicated a connection between the two molecules. Sircar et al. (2009) showed that AKT activation, mTOR activity and AR nuclear expression concurrently occur in several PTEN-null patient samples. Additionally, Müller et al. (2012) demonstrated that loss of phosphorylation of Ser2448 of mTOR, an inhibitory alteration, resulted in decreased levels of cellular AR in ERG-fusion-positive prostate cancer cells. Kaarb0 et al. (2010) also show a reduction of AR by mTOR in LNCaP cells. Furthermore, they demonstrate that mTOR inhibtion increases ligand-dependent AR activity.

Work by Wang et al. (2008) further demonstrated evidence of a connection between mTORC1 signaling and AR by demonstrating an induction of AR activity by rapamycin. The application of rapamycin to LNCaP and C4-2 cells resulted in inhibition of the mTORC1 pathway, but an increase in AR transcriptional activity. They further determined that this relationship was through AKT. Inhibition of mTORC1 by rapamycin was shown to activate a parallel ARmediated survival pathway putatively downstream of mTORC2. This potentially matches results by Müller et al. (2012), who showed that loss of mTOR deactivation resulted in AR reduction, suggesting an inverse relationship between the two molecules. Upon dual inhibition using rapamycin and bicalutamide, apoptosis occurred [159], signifying that there is a parallel compensatory effect for mTORC1 signaling through AR. This relationship was expanded on by Wu et al., (2010) who determined that the relationship between AR and mTORC1 is dependent upon testosterone availability, and generates a self perpetuating cycle that promotes cell survival. In low testosterone conditions, application of bicalutamide repressed mTOR activity, as did siRNA against AR in both low and high testosterone conditions. This was shown to be through AR repression of TSC2, a negative regulator of mTORC1. Conversely, rapamycin treatment induced AR activity, as was previously shown by Wang et al. (2008). Furthermore, in low testosterone conditions, where AR activity is low and thus TSC2 is not repressed, bicalutamide leads to apoptosis [160].

These data support the compensatory AR-mediated growth pathway found by Wang et al. (2008), as the lack of TSC2 repression would inhibit mTOR signaling while bicalutimide inhibits AR, leading to a similar dual inhibition of mTORC1 and AR which Wang et al. (2008) reported to induce apoptosis. The interactions between mTOR and AR have been shown to play a key role in progression to AICaP. The mechanism elucidated by Wu et al. (2010) demonstrated that LNCaP cells can become attuned to low testoterone by mTOR/AR crosstalk. When testosterone is low, AR activity drops, but so then does AR-mediated inhibition of TSC2. This represses mTOR activity, leading to induction of AR activity once more. If this cycle is perpetuated, the cells will become able to self-induce AR signaling, and thus also reestablish mTORC1 activity, leading to the development of an androgen-resistant and highly prolifera-tive tumor. Additionally, LNCaP cells acclimatized to low-testosterone conditions showed resistance to glucose deprivation, another way of repressing mTOR. The mTORC1 repression leads to higher levels of AR transactivity, activating a postulated second survival pathway that can compensate for the canonical PI-3K/AKT/mTOR axis. Thus, inhibition of one pathway would sensitize to inhibition of the other. Only by inhibition of both mTOR and AR was apoptosis achieved in both studies [159, 160]. Squillace et al. (2012) show similar results using bicalutamide and ridaforolimus, an mTOR inhibitor. In their study, they [161] also demonstrate that the combination therapy does not induce apoptosis in healthy RWPE-1 PTEN-expressing prostate cells. This is important, as it indicates that the targeted AR/mTOR system is an aberrancy in the tumour.

AR/mTOR dual inhibition was shown to be key to regulation of CaP progression by Schayo-witz et al. (2010), who demonstrated that usage thereof can prolong androgen sensitivity of a tumour. Using mouse models, Schayowitz et al. (2010) showed that combination therapy had significant effect on both xenografted androgen-dependent (LNCaP) and androgen-independ-ent (HP-LNCaP) tumours. In both LNCaP and HP-LNCaP xenografts, treatment with a single inhibitor caused no significant decrease in tumour volume, while combination therapy reduced tumour volume significantly. Bicalutamide or everolimus (an mTOR inhibitor) did not decrease tumour volume in LNCaP xenografts, while combination treatment offered significant reduction after 15 days [162]. Likewise, in HP-LNCaP xenografts bicalutamide, everolimus or VN124-1 (a novel androgen/AR inhibitor) did not significantly decrease tumour volume, while dual inhibition did. Interestingly, combination everolimus/VN124-1 treatment reduced tumour volume far more than did everolimus/bicalutamide treatment. Everolimus/ VN124-1 treatment also resulted in significant decreases in AR, p-mTOR, p-S6K and p-S6 levels as visualized by western blotting [162], signifying an inhibition of the AR/mTOR pathways. Single inhibition often led to increased pathway activation, further evidencing compensatory crosstalk. This study is notable because treatment of xenografts with the combination treatment was not overcome, nor was its efficacy as compared to tumour volume decreased over a 45-day period. This indicates that sensitivity to the dual inhibition was maintained, and compensatory crosstalk did not rescue the xenograft. Suppression of CaP progression to AI disease in this manner was also shown by Friedrichs et al. (2011), albeit through a different treatment. Omega-3 polyunsaturated fatty acids (PUFAs) were shown to inhibit CaP progression through suppression of mTOR and AR signalling. Application of DHA, an omega-3 PUFA, inhibited AKT signaling and decreased cell growth in AI clones of LNCaP. Friedrichs et al. (2011) also observed the effect of omega-3 PUFAs on suppression of CaP progression using an assay that mimics progression to androgen-independent disease with androgen ablation. In the control group, ~35% of the cells initally underwent growth arrest and then recovered, while ~42.5% stayed arrested. In the omega-3 PUFA groups (+DHA, +EPA), a majority of cells stayed arrested, as did cells treated with an AKT inhibitor. Treatment with DHA was also accompanied by suppression of AR and p-mTOR expression, along with downregulation of p-S6 and of p-TSC2, an AKT target. This data taken together with data by Wang et al. (2008), Wu et al. (2010), Schayowitz et al. (2011) and Squillace et al. (2012) suggest that suppression of both mTOR and AR signaling is key to inhibition of CaP progression, and that single inhibition leads to activation of the other in a compensatory mechanism.

Such a mechanism was postulated by Wang et al. (2008) to be downstream of mTORC2. Facompre et al., (2012) report that that mTORC2 is involved in an AR-mediated growth pathway. Addition of 5|M p-XSC, or 1,4-phenylenebis(methylene)selenocyanate, a known AKT and AR inhibitor, was shown by Facompre et al. (2012) to inhibit mTORC2 kinase activity in vitro, supporting Wang et al. (2008) in proposing that mTORC2 plays a role in AR-mediated crosstalk. This dual inhibition of AR and mTORC2 could indicate that either p-XSC is an mTORC2 inhibitor and inhibits AR downstream of TORC2, or that AR is upstream of mTORC2 and thus suppression of AR inhibits mTORC2 signaling. Treatment with p-XSC resulted in decreased growth of both androgen-dependent LNCaP and androgen-independent C4-2 CaP cell lines by ~25%, with rapamycin showing similar results [163]. As indicated by several other sources, dual inhibition had a far more marked effect. Addition of 1nM rapamycin in combination with the mTORC2/AR inhibitor p-XSC heavily decreased cell viability by ~50% in LNCaP calls, and by ~60% in C4-2 cells. The dual inhibitor treatment resulted in extremely efficient repression of phosphorylation downstream of both mTOR complexes, further supporting the postulation that an AR survival pathway is related to mTORC2. The role of mTORC2 in AR signaling has been investigated by Fang et al. (2011), who report that an mTORC2-mediated growth pathway is downstream of AR. Treatment of CWR22R3 cells with

DHT led to proteosome-mediated degradation of p27, a protein that induces cell cycle arrest through CDK inhibition. Such an action would contribute to the inhibited growth noted in CaP progression. This degradation was shown to be through mTORC2-mediated phosphorylation of AKT at Ser473, but not Thr308. AKT has previously been shown to be phosphorylated at Thr308 by PDK1 [165], so the modification of AKT in this context seems to be purely mediated by mTORC2 without influence from PDK1 molecules, leading to selective activation of only certain downstream substrates, such as SGK and PKCa. Fang et al. (2011) went on to demonstrate that DHT stimulation of AR is inducing nuclear accumulation of SIN1, a factor required for complexing of mTORC2, which signals for the assembly of mTORC2 and subsequent partial phophorylation of AKT. The actual phosphorylation of p27, required for its degradation, could be mediated by AKT or one of its selectively activated substrates. Growth pathways downstream of AKT, such as the SGK pathway, would also lead to increased viability and proliferation of CaP. Xu et al. (2006) implicated mTOR in a similar manner, showing that AR induces cyclin proteins, especially cyclin D1, D2 and D3. RT-PCR results did not indicate a similar increase at an mRNA level. This was because AR regulation of cyclin D was at a post-tran-scriptional level, through mTOR. Co-activation of cyclin proteins together with degradation of p27, a CDK inhibitor, could lead to potent activation of a CDK-Cyclin growth pathway.

AR has also been shown to be post-transcriptionally modified by mTOR [167] in an EGF/PI-3K/ AKT-dependent manner. This study elucidates the manner in which mTORC1 regulates and rapamycin induces AR. Through modulation of the interactions between translation initiation factor eIF4E and scaffolding protein eIF4G, mTOR putatively regulates the rate of translation of AR. Thus, rapamycin inhibition of mTORC1 would lead to an increased rate of translation of AR, leading to increased expression and revitalization of mTORC1 signaling. This combined pathway contains a failsafe in the form of the mTOR-mediated repression of AR and the AR regulation of TSC2, leading to cyclic and self-perpetuating support of two growth pathways. This pathway has consistently been implicated as important to CaP survival, growth and progression to AICaP. The data suggests that this crosstalk leads to maintainance of parallel mTORC1 and mTORC2 survival pathways. The pathway is protected from itself: induction of mTOR signaling decreases AR activity, which would enhance TSC2 and thus return mTORC1 to normal levels, rescuing AR. Repression of mTORC1 induces AR through attenuation of its post-transcriptional inhibition, leading to downstream mTORC2/CDK-Cyclin signaling. Additionally, AR represses TSC2, revitalizing the mTORC1 pathway. Repression of AR increases TSC2 activity, leading to inhibited mTORC1 and thus increased AR, hypersensitizing the cell and possibly leading to progression towards AI disease.

FOXO, a family of apoptosis-promtoing transcription factor, has shown relevance to AR and prostate cancer progression. AR has previously been shown to be a positive regulator of the PI-3K pathway, which represses FOXO family transactivity. Additionally, tissue microarray data from TM3-AR CaP cells treated with testosterone display marked downregulation of 65 FOXO-family proteins [168]. Sixteen of these have been shown to be important in development, including FOXO1. These data suggest that AR and FOXO are antagonistic towards one another. Li et al. (2001) first indicated an antagonistic nature of AR and PTEN/FOXO.

Ma et al. (2008) characterized one side of this antagonism, showing that FOXO1 mediates PTEN inhibition of AR. By expressing fragments of FOXO1 and determining their ability to repress AR activity, they discerned that the FOXO1 inhibition of AR required its AD and forkhead box. Further, the inhibition of AR was found to be through disruption of its NTD/CTD interaction. This disruption was found to be mediated by FOXO1 binding the AR NTD and repressing interaction with SRC1, a promoter of AR activity [169]. Completing the dichotomy, Li et al. (2003) showed that AR can also disrupt the activity of FOXO-family transcription factors, including FKHR. Androgen treatment and subsequent AR activation in PTEN-null cells was shown to repress FKHR and related FOXO-family protein activity in a manner independent of transcriptional coactivators. This repression was found to be by complexing of activated AR and FKHR, leading to an inability of FKHR to bind DNA. Two points of interaction were found for each molecule: AR binds to the FKHR C-terminus and binds weakly to the forkhead domain, while FKHR binds to the AR NTD and weakly to the LBD. Through flow cytometry, Li et al. (2003) demonstrated that FKHR when bound by AR can no longer induce cell cycle arrest, thereby leading to an attenuation of its role in growth control. This suggests that androgen ablation therapy might reintroduce FKHR and related FOXO activity, leading to arrest of cell growth. Thus, the progression to androgen independence would require compensation for androgen deprivation, such as those discussed earlier. In particular, mTOR and FOXO seem to have related roles. AR inhibition of FOXO combined with mTOR-mediated growth signaling could lead to potent CaP proliferation. Additionally, mTOR-related crosstalk could rescue FOXO-mediated AR inhibition.

Another similarity between FOXO and mTOR crosstalk with AR is the role of p27. Unlike FOXO1 and FKHR, FOXO3a has been shown to transcriptionally upregulate AR by binding its promoter [171], while AR deactivates FOXO3a [172]. FOXO3a also promotes transcription of p27 via its promoter [173]. These interactions lead to a system where AR deactivates FOXO3a, leading to inhibition of p27. If AR were reduced, FOXO3a would become active, promote AR expression and thus reinstate the same state. Disruption of this cycle was shown by Li et al. (2007), using a DIM compound called B-DIM. Treatment with B-DIM repressed FOXO3a binding to the AR promoter while maintaining its binding to the p27 promoter, leading to cell cycle arrest. In CaP, redundant repression by mTOR might prevent this method of rescue. Additionally, AR disruption of FOXO proteins in conjunction with AR/mTOR crosstalk would lead to a deadly regulatory loop whereby cell cycle regulation is suppressed and growth is promoted. Zhang et al. (2010) also showed potential for FOXO-based therapy in response to AR signaling. Methylseleninic acid (MSA) can be metabolized to methylselenol, which has been shown to have anticancer effects and to induce apoptosis [175]. Zhang et al (2010) found that treatment of LNCaP cells with MSA leads to induction of FOXO1 expression and transactivity. Additionally, knock down of FOXO1 after MSA treatment was found to nullify its apoptotic effects. The critical role of FOXO1 in this context was found to be repression of AR activity, though the mechanism for this inhibition remains unknown. Based on results from Ma et al. (2008), FOXO1 could be attenuating NTD/CTD interactions of AR. Results from treatments with FOXO-associated drugs such as B-DIM and MSA implicate it as an important molecule in AR-mediated CaP growth and its role as an antagonist of AR could potentially implicate it in CaP progression. Since AR/FOXO crosstalk is seemingly similar but opposite to AR/mTOR crosstalk, investigation of both AKT-dependent and AKT-independent crosstalk between the two pathways could elucidate important mechanisms of CaP progression.

4.2.4. PI-3KJAKT/GSK3P and AR

The evidence thus far clearly shows that increased activation of the PI-3K/AKT signaling pathway and transcriptional activity of AR are closely intertwined. The role that AKT plays in modulating AR activity, however, remains obscure. One of the many downstream substrates of AKT, GSK3p has also been shown to play a role in AR regulation [176-179], and is ubiquitously expressed in CaP cell lines, including COS-1, PC-3, LNCaP and DU145 (Wang et al., 2004). It has increased expression in AICaP cell lines [176, 179], and appears to be a key player in the progression of CaP to androgen-independent disease. However, the nature of this role is at present ambiguous. In varied contexts, GSK3p has been shown to both promote and antagonize AR transactivaton independently of its interactors and other substrates in both a ligand-dependent and ligand-independent manner.

A repressor of several EMT pathways, GSK3p has been shown by Salas et al. (2004) to be capable of repressing ligand-dependent AR activity by phosphorylation. Transfection of wild type GSK3p or constitutively active GSK-3BA9, a mutant of GSK3p devoid of its first 9 amino acids, into AR-expressing LNCaP, A103 and V28 cells significantly increased phosphorylated AR compared to transfection of empty pCMV4 or inactivated tyrosine 216 mutated GSK3p (GSK3pY216F). Furthermore, treatment of cells with LiCl, a GSK3p inhibitor, significantly decreased phosphorylation of AR (Salas et al., 2004), indicating that altered modification of AR is indeed due to the activity of GSK3p. In AR- COS-1 prostate cancer cells, co-transfection of wtGSK3p or GSK3pA9 with AR lead to increased phosphorylation of AR, as opposed to cotransfection of AR with empty pCMV4 or GSK3pY216F which did not elevate phosphorylation of AR. This suggests that the activity of GSK3p is essential to the phosphorylation of AR, as the active forms of GSK3p were the only ones to display an effect. Upon treatment of AR-expressing COS-1 cells with LY294002, phosphorylation of AR increased [176]. This is due to reduced deactivation of endogenous GSK3p through indirect inhibition of AKT via the PI3K pathway. When these cells were treated with LiCl, AR phosphorylation decreased in a dose-dependent manner, indicating that these results are related to increased GSK3p activity. In following with the increased phosphorylation of AR, Salas et al. (2004) also reported that ARmediated transactivation in the presence of R1881 (metribolone), a synthetic nonmetabolizable androgen, was decreased with increased GSK3p activity. This was shown through luciferase reporter assays both with an ARE-driven ARE2LUC construct as well as with a PB-LUC (a promoter from an endogenous AR target). By using C-terminal and N-terminal domain mutants of AR, Salas et al. (2004) were able to determine that GSK3p preferentially phosphor-ylates AR on its CTD. Furthermore, usage of a GST-tagged ARLBD revealed that GSK3p could phosphorylate AR on its LBD. This may provide a mechanism by which AR-driven transcription is decreased by GSK-3p. The effect of GSK3p being through the AR LBD is further evidenced by the fact that Salas et al. (2004) did not note any suppression of the ligand-independent, constitutively active AR5 and AR104 constructs.

Wang et al. (2004) also showed a reductive effect of GSK3ß on AR transcription. Co-transfection of AR, wtGSK3ß and two reporter constructs in a dual luciferase system with an ARE-driven promoter (AREJ revealed that increased GSK3ß decreased AR transactivation. Further, usage of a constitutively active GSK3ß mutant (GSK3ßS9A) further restricted AR-driven transcription. These data taken together suggest that GSK3ß kinase activity regulates the level of AR transactivation. GSK3ß was also shown to decrease AR transcription in LNCaP cells, which express endogenous AR. These effects were shown to be reversible by LiCl treatment. However, in contrast to the work by Salas et al. (2004), Wang et al. (2004) demonstrated that GSK3ß phosphorylates AR on its NTD more significantly than its LBD or a DBD-LBD fragment by using GST-tagged fragments. Moreover, they showed that GSK3ß repressed transcription by GAL4-AR-N-terminal in the presence of a pG5-Luc reporter, which contains the ligand-independent AF-1 domain, while failing to repress activity of the AF-2 domain-containing GAL4-AR-LBD. These data suggest that GSK3ß inhibits ligand-independent activity of AR. Wang et al. (2004) also demonstrated that GSK3ß binds to the CTD and NTD of AR in both transfected and endogenously expressing CaP cell lines, leading to the postulation that GSK3ß-mediated suppression of AR transcription may be due to attenuated AR CTD-NTD interactions, which are required for transactivation. Supporting the interaction of the two molecules, Salas et al. (2004) noted that there was a physical co-distribution of the two molecules in CaP cell lines and in CaP tissue. Salas et al, (2004) reported that inhibition of GSK3ß by Ser9 phosphorylation is elevated in the androgen-dependent LNCaP in comparison to the androgen-independent PC3 and DU145 CaP lines, which may signify an increased role for GSK3ß in androgen-dependent tumours. Liao et al (2004) also show that GSK3ß Tyr216 phosphorylation is elevated in AICaP cells, especially 22-RV1. Aberrant activity of the PI-3KAKT signaling system has been demonstrated in AI 22-RV1 CaP cells, and has been associated with an increased Gleason grade [82], which in turn has been shown to be an accurate predictor of progression to AICaP [182]. These data taken together suggest that the increased activity of PI-3K/AKT in AICaP, usually due to PTEN deficiency, may have the effect of disabling GSK3ß and thus increasing activity of AR in a ligand-independent manner.

Work by Liao et al. (2004), Mazor et al. (2004) and Schütz et al. (2011) contradicts Salas et al. (2004) and Wang et al. (2004), reporting that inhibition of GSK3ß actually represses ARmediated transcription. Mazor et al. (2004) reported that GSK3ß sequestration or knockdown inhibits AR signaling, while transfection of a constitutively active form (GSK3ßS9A) into LNCaP cells with majority Ser9-phosphorylated GSK3ß increased AR transcriptional activity. Moreover, this effect was independent of its downstream substrate, the oncogenic ß-catenin. Liao et al. (2004) demonstrate that GSK3ß is necessary for ligand-dependent transcriptional activity to occur. In the presence of LiCl, two other GSK3ß inhibitors R0318220 and GF109203X and siRNA against GSK3ß, R1881-stimulated AR transcriptional activity as measured by a PSA-SEAP reporter was significantly reduced. This was not due to reduced nuclear translocation, as no inhibitors blocked AR nuclear localization with R1881 treatment. Knockdown of AKT and ß-catenin, another substrate of GSK3ß, did not yield any similar results, implicating that

GSK3ß activity is directly inducing the observed effect. Interestingly, in the presence of R1881 GSK3ß Tyr216 phosphorylation was also increased, signifying a synergistic relationship between GSK3ß and the androgen-dependent AR signaling cascade.

Schütz et al. (2011) further complicate the story, reporting that GSK3ß is necessary for androgen-independent AR activity, though not by directly affecting the AF-1 or AF-2 activity domains. Instead, this inhibition is in a CRM1-dependent manner, as discerned in an earlier study [179]. CRM1 is an export receptor for substrates containing an L-rich NES, likely acting in a RanGTP-dependent manner [182]. CRM1 activity is inhibited by leptomycin B (LMB). Upon treatment of 22-RV1 cells with SB216763, a GSK3ß inhibitor, AR was increasingly localized in the cytoplasm and experienced a two-fold drop in the nucleus. When LMB was added, the effects were reversed. AR transcriptional activity was also shown to drop with the inhibitor, and was rescued by LMB. Furthermore, AR association with CRM1 was shown to increase with SB216763, and a putative binding site was reported to be located within the C-terminal LBD. This was found using a mutant deleted of its LBD (Schütz et al., 2010). In their future work, Schütz et al. (2011) showed that unliganded AR in AI LNCaP lines, which was localized to the nucleus, is exported upon application of SB216763 in a CRM1-dependent manner, rescued by LMB. Decreased AR signaling with GSK3ß was also shown in vivo using a tumor-engrafted chick choriallantoic membrane model. Of note, knockdown (shRNA) or long-term inhibition (SB216763) reduced the nuclear and cellular levels of AR respectively. Mazor et al (2004) showed similar data, suggesting that GSK3ß may also play a role in maintaining stability of the AR protein.

The evidence clearly indicates that GSK3ß plays a crucial role in regulation of AR; however the nature of that role is highly controversial. Upregulation of GSK3ß has been shown to be associated with an elevated Gleason grade [180], which would suggest that support of AR signaling would be likely. Moreover, as Gleason grade often indicates increased risk for AICaP progression [182], however, GSK3ß is also a target of AKT for Ser9 phsophorylation, which deactivates its kinase activity. Thus it would seem detrimental that GSK3ß induction and reduced inhibition of the PI-3K pathway occur concurrently, as is the situation in several AICaP lines such as 22-RV1. It may be important to note that while Salas et al. (2004) and Wang et al. (2004) made liberal usage of overexpression models, while Liao et al. (2004), Schütz et al (2010) and Schütz et al. (2011) used mainly endogenous protein. This is largely due to the nature of the work, using GSK3ß as a suppressant as opposed to studying the effects of repressed GSK3ß activity, however ectopic expression can alter a system from the in situ function. From the presented results, it becomes apparent that endogenous GSK3ß-AR interactions seem to be AR-promoting. Mullholland et al. (2006) suggests that a baseline level of GSK3ß may be necessary for AR activity, and ectopic expression may alter the nature of the system, causing an inhibition and suggesting that GSK3ß Tyr216 phosphorylation may ultimately be AR-inhibitory. However, the recent results by Schütz et al. (2011) contradict this, while results by Mazor et al. (2004) indicate that overexpression of GSK3ß in a system with active endogenous GSK3ß, such as 22-RV1 cells, has little effect. The mechanism outlined by Schütz et al. (2010) may be key to note: GSK3ß could have a higher affinity for CRM1, thereby preferentially preventing AR export and thus promoting AR transactivation. Further, it would seem that these interaction play a different role in ligand-dependent and AI disease, which may suggest a role in promoting the progression of CaP to an AI state. Alteration of GSK3p or alteration of its interactions in situ may play an important role in regulating GSK3p function with respect to CaP, as it has been shown to play a wide variety of tumour-suppressing and oncogenic roles when in different environments. Thus, a change in cellular context may be key to its role in CaP progression.

The Wnt pathway and the PI-3K have both been implicated in CaP progression. Additionally, crosstalk has been evidenced between the two systems, usually downstream of AKT. In particular, GSK3p is a common intermediary between the Wnt and PI-3K pathways through which crosstalk is often implicated. In the Wnt pathway, GSK3p phosphorylates p-catenin, the central effector of the pathway, to mark it for ubiquitination and subsequent proteosomal degradation [184]. p -catenin is a multifunctional protein that both aids in the stabilization of the adherens junction with E-cadherin and activates transcription of Wnt target genes. Wnt ligands activate the Wnt pathway by binding to their seven-pass transmembrane receptor frizzled (Fzd) and its co-receptors LDL receptor related proteins 5 and 6 (LRP5/6). The Wnt pathway is divided into the canonical Wnt pathway, which signals through p-catenin, and the non-canonical Wnt pathway. The non-canonical Wnt pathway includes the calcium dependent pathway and the planar cell polarity pathway, both of which play vital roles in development. [185,186]. The canonical Wnt pathway is stimulated when a member of a subset of Wnt ligands binds Fzd. This transduces a signal through dishevelled (Dvl) to disrupt the p-catenin destruction complex, made up of adenomatous polyposis coli (APC), casein kinase 1 (CK1) and GSK3 p unified by the scaffolding protein Axin. By sequential phosphorylation, ubiqui-tination and degradation in the presence of an active destruction complex, p-catenin is maintained at reasonable levels. Upon stimulation by a Wnt ligand, GSK3pis deactivated by Dvl and Axin is seuestered to the membrane by the now-phosphorylated LRP5/6. This allows p-catenin to accumulate unchecked, and translocate to the nucleus. The mechanism for p-catenin translocation remains unclear. In order to play its role as a transcativator, p-catenin must bind its nuclear interactor T-cell factor (TCF). In a cell unstimulated by Wnt activation, TCF is bound to its repressor, Groucho. With Wnt activation, p-catenin displaces Groucho, and the p-catenin/TCF complex transcribes a plethora of Wnt target genes, many of which play oncogenic roles. In this way, p-catenin itself is a potent oncogene.

Wang et al. (2008) used castration resistant mouse models to demonstrate that AR expression seems to be concurrently expressed with increased levels of cytoplasmic p-catenin that is unattached to the adherens junction. This is significant because free p-catenin has the potential to shuttle to the nucleus and activate transcription of Wnt target genes. Trucia et al. (2000) establish a direct significance for this co-expression: p-catenin and AR can directly interact, leading to enhanced AR signaling and hypersensitivity to androgens. In LNCaP cells p-catenin and AR were shown to complex both in the presence and absence of androgen, but binding was markedly enhanced in the presence of DHT. However, using a stabilized mutant of p-catenin (p-catenin S33F) it was shown that AR activity is only enhanced in the presence of androgen, signifying a ligand-dependent activation. This was measured using a luciferase reporter assay. Trucia et al. (2000) went on to show that |-catenin binds AR on its LBD, and reduces the effects of bicalutamide on AR. This was shown to be through alteration of the AR LBD, broadening the scope of AR-ligand interactions to include other ligands. In this way |-catenin was show by Trucia et al. (2000) to be a co-activator of AR, providing it with increased significance beyond its role as a coactivator of Wnt target genes with TCF. |-catenin's interaction with AR were shown to be increasingly important by Mullholland et al. (2002), who showed that the AR/|-catenin complex can serve as a vehicle for |-catenin translocation in a ligand-dependent manner. Treatment with androgen in LNCaP cells led to colocalization of AR and |-catenin to the nucleus. Mullholland et al. (2002) went on to show that there are several points of overlap between |-catenin-driven and AR-driven transcription by noting several common targets, including cell cycle proteins such as cyclin D1. Others since have showed interaction between the two molecules, both in support [67] and in contention [47, 189]. In fact, data by Chesire et al. (2002) indicates that ligand-dependent AR/ |-catenin interactions inhibit |-catenn/TCF activity.

The most obvious point of crosstalk between PI-3K and Wnt is their common intermediary, GSK3. Sharma et al. (2002) investigated the crosstalk between these two molecules and AR. Treatment of LNCaP cells with LY294002 resulted in inhibition of AR-driven PSA expression, demonstrating a regulation of AR activity similar to that seen by Li et al. (2001). Upon application of LY294002, phosphorylation of AKT decreased, as did inhibitory phosphoryla-tion of GSK3 |. In conjunction with the lack of deactivation of GSK3 |, nuclear accumulation of |-catenin was significantly reduced. Usage of a mutant |-catenin mutated at its GSK3 ^phosphorylation site attenuated the results, showing that the modulation of AR transactiva-tion by the PI-3K pathway occurs through |-catenin. This finding is contradicted by Liao et al. (2004) and Mazor et al. (2003), who demonstrate that GSK3 |is required for AR transactivation. Liao et al (2004) showed that knockdown of |-catenin by pooled siRNA does not affect the levels of R1881-stimulated AR transactivation as measured using a PSA-SEAP reporter construct. Mazor et al. (2003) also show that depletion of |-catenin levels by siRNA treatment does not inhibit transactivation by endogenous AR in 22-RV1, LNCaP and CWR-R1 cells. In fact, they demonstrate that knockdown of |-catenin leads to increased levels of AR activity. It is worthy to note that both Liao et al. (2004) and Mazor et al. (2003) worked with primarily endogenous proteins, using knockdown models, and demonstrating that endogenous |-catenin is not a co-activator of AR. Mazor et al. (2003) notes the importance of confirming results obtained using ectopic expression with studies of endogenous protein.

For the most part, this section focuses on crosstalk through GSK3|. However, it is important to note that AKT modulates a variety of substrates downstream of PI-3K, and a number of these could be means for crosstalk. Hoogeboom et al. (2008) noted that FOXO interrupts |-catenin/TCF transcription by binding and sequestering |-catenin. This type of interference could play a role in inhibiting |-catenin/AR transcription as well, should that interaction take place. If |-catenin Is truly a coactivator of AR, or if AR does act as a shuttle for |-catenin, a great depth of understanding could be arrived at. In order to understand CaP progression and the roles of the Wnt, PI-3K and AR pathways therein, these interactions must be studied and understood. Should p-catenin coactivate with AR, the question arises as to whether it might do the same with FOXO. Many other proteins adapt functions based on their interactors and p-catenin is no different, being responsible for maintaining anchorage dependence when interacting with E-cadherin at the adherens junction. Mazor et al. (2003) had justification in commenting that the endogenous interactions of a protein should be understood. Until the relationships underlying p-catenin and its interactors are characterized, its role in CaP progression will remain elusive.

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