The PI3K-AKT-mTOR Pathway and Prostate Cancer: At the Crossroads of AR, MAPK, and WNT Signaling

Oncogenic activation of the phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB/AKT), and mammalian target of rapamycin (mTOR) pathway is a frequent event in prostate cancer that facilitates tumor formation, disease progression and therapeutic resistance. Recent discoveries indicate that the complex crosstalk between the PI3K-AKT-mTOR pathway and multiple interacting cell signaling cascades can further promote prostate cancer progression and influence the sensitivity of prostate cancer cells to PI3K-AKT-mTOR-targeted therapies being explored in the clinic, as well as standard treatment approaches such as androgen-deprivation therapy (ADT). However, the full extent of the PI3K-AKT-mTOR signaling network during prostate tumorigenesis, invasive progression and disease recurrence remains to be determined. In this review, we outline the emerging diversity of the genetic alterations that lead to activated PI3K-AKT-mTOR signaling in prostate cancer, and discuss new mechanistic insights into the interplay between the PI3K-AKT-mTOR pathway and several key interacting oncogenic signaling cascades that can cooperate to facilitate prostate cancer growth and drug-resistance, specifically the androgen receptor (AR), mitogen-activated protein kinase (MAPK), and WNT signaling cascades. Ultimately, deepening our understanding of the broader PI3K-AKT-mTOR signaling network is crucial to aid patient stratification for PI3K-AKT-mTOR pathway-directed therapies, and to discover new therapeutic approaches for prostate cancer that improve patient outcome.


Introduction
Prostate cancer is the second leading cause of cancer-related deaths in men worldwide, despite extensive efforts to raise awareness and significant advancements in detection, screening, and treatment approaches [1][2][3]. Although patients with localized prostate cancer generally have a good prognosis, the 5-year relative survival rate is significantly reduced for patients that present with metastatic prostate cancer at diagnosis [4]. ADT and/or radiotherapy remains the mainstay treatment for patients that relapse post-surgery. ADT involves blocking the production of androgen in the testes via the hypothalamus-pituitary-gonadal axis with luteinizing hormone releasing hormone (LHRH) agonists (e.g., Leuprolide) or antagonists (e.g., Degorelix). Although prostate tumors respond initially to ADT, the emergence of androgen-independent, castration-resistant prostate cancer (CRPC) invariably occurs and the outcome is poor [5][6][7][8]. Treatment options for CRPC and patients with metastatic disease at diagnosis include chemotherapy, radium-223, second generation anti-androgens (e.g., the Cytochrome P450 17A1 (CYP17A1) inhibitor abiraterone acetate that prevents androgen biosynthesis, Table 1. Frequency of common genetic alterations in PI3K-AKT-mTOR pathway genes in prostate cancer. respectively. Like Class IA, Class IB PI3Ks generate PIP3 from PIP2 to stimulate downstream effectors [19]. Class IB PI3Ks transmit Gβγ-GPCR and RAS signals to coordinate immune, inflammatory and allergic responses, predominantly within hematopoietic cells [18][19][20]22]. However, Brazzatti and colleagues have shown that knockdown of p110γ or p101 in 4t1.2 and MDA-MB-231 triple-negative breast cancer cell lines reduces migration in vitro and metastatic potential in xenograft mouse models, whereas p87/p84 knockdown had the opposite effect [75]. PIK3CG mutation and amplification are frequent in multiple malignancies, including 9-11% of melanomas and uterine, stomach and squamous cell lung cancers, while genetic alterations in PIK3R5 and PIK3R6 are prevalent in uterine cancer and melanoma, occurring in 4-8% of cases [50,[76][77][78][79]. In prostate cancer, Class IB PI3K genetic aberrations are less common, and include PIK3CG mutation and amplification (1.4-1.8% and 0.6-3.6% incidence respectively) as well as PIK3R5 and PIK3R6 deep deletions (0-3.3% incidence) that are indicative of a homozygous deletion (Tables S1-S3).

Class II PI3Ks
In comparison to Class I PI3Ks, the Class II family of PI3Ks (PI3KC2α, β and γ, encoded by PIK3C2A, PIK3C2B, and PIK3C2G respectively) is less well-characterized. Class II PI3Ks are generally considered to catalyze the production of lipid secondary messengers phosphatidylinositol 3-phosphate (PtdIns3P or PI(3)P) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) to mediate cell migration, channel regulation, endocytosis, and exocytosis [18,80]. The frequency of PIK3C2A, PIK3C2B and PIK3C2G mutation is generally low (0.2-1.4% incidence, Tables S1-S3), however PIK3C2B amplification has been observed in as many as 10% of cases (Table S3). Although the role of PIK3C2B amplification in prostate cancer is not clear, a recent study identified that PI3KC2β is highly expressed in PTEN-negative PC3 and LNCaP prostate cell lines compared to PTEN-positive DU145 prostate cancer cells (PTEN +/− ), and PNT2 immortalized "normal" prostate epithelial cells (PTEN +/+ ) [81]. This study also reported that PI3KC2β regulates MAPK signaling to mediate prostate cancer cell invasion, thus the PI3KC2β-MEK-ERK signaling axis may present a novel therapeutic target for invasive prostate cancer [81].

Class III PI3Ks
The Class III PI3K subfamily is comprised of the catalytic subunit vacuolar protein sorting 34 (VPS34) encoded by PIK3C3, and the regulatory subunit vacuolar protein sorting 15 (VPS15, or p150) encoded by PIK3R4. VPS34 catalyzes the phosphorylation of phosphatidylinositol (PI) to produce PI(3)P, which plays a central role in the regulation of intracellular trafficking [82]. To regulate the fusion and maturation of endosomes, VPS34 binds to VPS15 and Beclin-1 to form either VPS34 Complex I or VPS34 Complex II that differ by binding to Autophagy Related 14 (ATG14) or UV radiation resistance associated protein (UVRAG) respectively [83]. The Class III PI3K family has also been shown to mediate autophagy, endosome-lysosome maturation, membrane trafficking, and AMPK-dependent insulin sensitivity [82,[84][85][86][87][88][89].
PIK3C3 mutations are most frequently observed in uterine and gastric cancer patients (7% and 3.5% respectively), and PIK3R4 gene mutation or amplification occur in up to 10% of squamous cell lung cancer and uterine cancer patients [50,76,77]. Although PIK3C3/PIK3R4 mutation and PIK3C3 gene amplification are infrequent events in prostate cancer (<1% of cases), PIK3R4 high-level gene amplification is observed in up to 6.5% of cases and could potentially facilitate prostate cancer growth (Tables S1-S3).
Taken together, these data highlight the emerging diversity of genetic alterations within the PI3K family in prostate cancer, and emphasize the need for future work to gain further insight into the functional importance of these different genetic alterations during prostate cancer formation, progression, and recurrence. This is particularly important, as determining their non-redundant roles may present novel therapeutic targets and could aid patient stratification for future clinical trials.

Loss of Function of Phosphoinositide Phosphatases
Phosphoinositide phosphatases are a family of enzymes that dephosphorylate phosphoinositides to diminish phosphoinositide signals and regulate cellular functions [90]. The PI3K-AKT-mTOR pathway is regulated by multiple phosphoinositide phosphatases, including the tumor suppressor PTEN that dephosphorylates PIP3 into PIP2 to reduce PI3K-AKT-mTOR pathway activity (Figure 1). Genetic alterations in phosphoinositide phosphatases are strongly associated with human malignancies, and PTEN is one of the most frequently deleted genes in prostate cancer [91][92][93][94][95]. Here we review the frequency of genetic alterations in prostate cancer for genes encoding key phosphoinisitide phosphatases known to regulate the PI3K-AKT-mTOR cascade.
In primary prostate adenocarcinoma, PTEN mutation and deep deletion occur in 2% and 18% of cases respectively (Table S1), and the frequency appears to increase in metastatic disease (6% and 26% respectively, Table S3). Although the majority of PTEN mutations identified in prostate cancer are truncating mutations, missense mutations are also observed, which could differentially impact PTEN lipid and/or protein phosphatase function [108]. Thus, determining how each PTEN genetic alteration impacts PTEN function may inform clinical trial design.
PTEN heterozygous deletion and epigenetic silencing can also deplete PTEN expression/function [92,98,106,109]. Importantly, mono-allelic deletion of PTEN has been reported in up to 68% of prostate cancer surgical specimens and PTEN immunohistochemistry (IHC) and/or fluorescent in situ hybridization analysis has revealed PTEN loss may occur in as many as 60% of advanced/CRPC cases [92]. A subset of patients with prostate cancer have also been found to harbor intratumoral heterogeneous PTEN loss [92], which could have significant implications for therapeutic strategies.

AKT Gain of Function
AKT isoforms 1, 2, and 3 (encoded by AKT1, AKT2, and AKT3 respectively) form a subfamily of serine/threonine protein kinases that possess both overlapping and distinct cellular functions to regulate a variety of cellular processes during normal tissue homeostasis and cell transformation [121,122]. PI3K activity elevates PIP3 levels to recruit AKT to the plasma membrane where is it activated (Figure 1). AKT is activated by multiple kinases, including PDK1 and mTORC2 that phosphorylate AKT at residues Thr308 and Ser473 respectively, triggering a wave of phosphorylation through multiple downstream targets that stimulate cell survival, proliferation, metabolism and differentiation to promote tumor growth [19,20,32,123,124]. AKT downstream targets include PRAS40 (a component of mTORC1), BAD, FOXOs, and MDM2 (reviewed in [31]). AKT signaling is negatively regulated by several protein phosphatases that dephosphorylate and inactivate AKT, including protein phosphatase 2 (PP2A), and PH domain and leucine-rich repeat protein phosphatase-1 and -2 (PHLPP1 and PHLPP2) [125,126]. Below, we outline the various genetic alterations within the AKT isoforms and their regulators that have been detected in prostate cancer, and discuss their potential to activate AKT signaling and promote prostate tumor growth.

AKT Mutation and Amplification
AKT genetic aberrations that increase AKT activity have been detected in multiple malignancies and are especially common in breast cancer, where AKT3 amplification and AKT1 E17K oncogenic mutation have been reported in up to 24% and 1-8% of cases respectively [127][128][129]. AKT1, AKT2, and AKT3 activating mutations are rare in prostate cancer (≤0.9%, predominantly in AKT1 at E17K), whereas AKT1, AKT2, and AKT3 high-level gene amplification that can increase AKT activity is more common, particularly in advanced disease (up to 4.5%, 2%, and 4.7% incidence respectively, Tables S1-S3). Moreover, AKT activation in prostate cancer has been shown to positively correlate with Gleason score and invasive progression [37,130], and over-expression of myristoylated AKT (which causes constitutive AKT activation) causes prostate neoplasia in mice [131]. In support of an oncogenic role in prostate cancer and therapeutic resistance, conditional activation of AKT in either the LNCaP human prostate cancer cells or a transgenic mouse results in increased cell proliferation and inhibits cell death to promote tumor growth and castration-resistance in vivo [132]. Chen and colleagues have also demonstrated a requirement for AKT in PTEN-deficient prostate cancer, as Akt1 haplodeficiency was found to suppress high-grade prostate intraepithelial neoplasia development within Pten heterozygous mice [133]. AKT inhibitors are being widely explored in the clinic to treat prostate cancer and have shown promise in PTEN-deficient patients [16,134].

Genetic Alteration of AKT Regulators
A number of genetic alterations in genes that encode AKT regulators have been linked to prostate cancer, including kinases (e.g., PDK1), binding proteins (e.g., FKBP5), and phosphatases (e.g., PHLPP1, PHLPP2, and PP2A) [42][43][44][45][46]. PDK1 (encoded by PDPK1) is recruited to the membrane by PIP3 to phosphorylate and activate multiple targets, including AKT at residue T308 ( Figure 1). PDPK1 amplification and PDK1 over-expression are observed in several human cancers, including breast cancer [135]. In prostate cancer, PDPK1 mutations are rare (≤0.2%), yet PDPK1 amplification occurs in up to 8.1% of patients (Tables S1-S3). Interestingly, PDK1 RNAi-mediated knockdown does not impair Pten-deleted prostate cancer growth in mice, possibly reflecting mTORC2-mediated activation of AKT, and/or compensatory augmentation of the MAPK cascade [136]. These findings suggest that PDK1 inhibitors are not likely to be efficacious against PTEN-deficient prostate cancer in the clinic as a single agent.
FKBP5 (also known as FKBP51) is an AR target gene that plays a key role in mediating the cellular distribution of steroid hormone receptors and has been shown to negatively regulate AKT signaling by stabilizing PHLPP1/2 ( Figure 1) [11,24,137]. During androgen/AR-directed therapy, FKBP5-PHLPP1/2-AKT signaling forms a negative feedback loop between the AR and PI3K-AKT-mTOR pathways to facilitate ADT resistance [11,24,137], discussed in Section 3.2. Mutation and deep deletion of FKBP5 are fairly infrequent in prostate cancer (≤1.22%, Tables S1-S3), however FKBP5 down-regulation has been linked to CRPC and increased AKT signaling [11].
PHLPP1 and PHLLP2 (encoded by PHLPP1 and PHLPP2) are protein phosphatases that dephosphorylate and inactivate AKT. PHLPP1 and PHLPP2 deep deletion occurs in up to 3.9% and 6.5% of patients with prostate cancer respectively (Tables S1-S3), which could potentially sustain AKT-signaling. Interestingly, Chen and colleagues reported a strong tendency for PTEN, PHLPP1, PHLPP2, and TP53 co-deletion in metastatic prostate cancer and that low PHLPP1 expression correlates with reduced patient survival and relapse after surgery [138]. Additionally, the tumor suppressive function of PHLPP1 has been demonstrated in vivo, as Phlpp1 loss causes prostate neoplasia in mice and promotes invasive carcinoma progression in Pten +/− transgenic mice [138]. In contrast, Phlpp2 loss impairs Pten/p53-deleted prostate tumor growth in mice [139], indicating PHLPP1 and PHLPP2 mediate differential AKT-independent functions. Indeed, PHLPP2 can dephosphorylate MYC at residue Thr58 to prevent MYC degradation and promote tumor progression [139]. Consequently, in PHLPP2-positive MYC-driven advanced prostate cancer, it has been suggested that PHLPP2 may present a valuable therapeutic target [139].
In addition, genetic alterations in PPP2CA (protein phosphatase 2 catalytic subunit alpha) that encodes the negative AKT regulator PP2A have also been observed in prostate cancer [42][43][44][45][46], and PP2A loss has been linked to prostate cancer progression and metastatic potential in the clinic [140]. PPP2CA mutation and deep deletion events occur in 0.4-1.4% of patients with prostate cancer (Tables S1-S3), further highlighting the diversity of genetic aberrations in AKT regulators that could promote oncogenic PI3K signaling.

SGK Deregulation
The serum/glucocorticoid-regulated kinase isoforms SGK1, SGK2, and SGK3 belong to a subgroup of the AGC (cAMP-dependent, cGMP-dependent, and protein kinase C) family of protein kinases that play a role in multiple cellular processes including cell growth, proliferation, metabolism, intracellular trafficking and survival [141][142][143]. SGK1 and 3 are considered to be ubiquitously expressed, while SGK2 expression is prominent in the liver, kidney, pancreas, and brain [144]. SGKs share structural similarities, upstream regulators, substrates and functions with the AKT isoforms (reviewed in [142]). For instance, all SGKs are phosphorylated and activated by PDK1, and SGK1 is a downstream target of mTORC2 [26,143,[145][146][147][148] (Figure 1). SGKs are also activated by PI3K/PDK1-independent mechanisms, for example SGK1 is regulated by big mitogen-activated protein kinase-1 (BMK-1) and p38 mitogen-activated protein kinase in response to epidermal growth factor (EGF) and interleukin-6 (IL6) respectively [149,150]. Although the role of SGKs during prostate cancer is currently unclear, SGK1 over-expression has been shown to facilitate CRPC transition in a prostate cancer xenograft model, indicating that SGK1 can promote ADT-resistance [151]. Furthermore, the SGK1 inhibitor GSK650394 has been shown to induce autophagy and apoptosis in PC3, LNCaP, DU145, and CWR22RV1 prostate cancer cells in vitro [152]. Interestingly, SGK1 and SGK3 have also been linked to PI3K/AKT-targeted therapy resistance in breast cancer [145,148]. Gasser and colleagues have also shown that INPP4B over-expression leads to enhanced SGK3 activation in ZR-75-1 breast cancer cells, triggering a switch from AKT-to SGK-dependent signaling downstream of PDK1 [153].
Mutation of the SGK isoforms is a rare event in human cancers, however gene amplification is commonly detected [154]. In keeping with this, SGK1, SGK2, and SGK3 are rarely mutated in prostate cancer (≤0.41%), whereas amplification occurs in up to 2.5%, 2.0%, and 20.3% of cases respectively (Tables S1-S3). Of note, the frequency of SGK3 gene amplification is particularly high in the SUC2/PCF IDT metastatic prostate adenocarcinoma dataset (Table S3), underlining the need for future studies to establish how SGKs contribute to prostate cancer and metastatic progression.

Loss of FOXO Transcription Factors
The mammalian forkhead box O (FOXO) family consists of four transcription factors (FOXO1, 3, 4, and 6) that are highly similar in structure and function [155]. In response to insulin and growth factors, FOXOs modulate the transcription of several target genes to mediate key cellular processes including proliferation, apoptosis, autophagy, inflammation, metabolism and stress resistance, and they form an important regulatory circuit within the AKT and mTOR signaling cascades [156][157][158] (Figure 1). FOXOs are regulated by several kinases, including AKT and SGK isoforms, which phosphorylate and inactivate FOXO-mediated gene transcription by inhibiting FOXO DNA binding and triggering FOXO nuclear-to-cytoplasm translocation [157][158][159]. FOXOs are generally regarded as tumor suppressors, and are reported to inhibit mTORC1 via sestrins, however a number of oncogenic functions are emerging in the literature [156][157][158]. For instance, FOXO-mediated transcription of the mTORC2 component RICTOR in response to physiological stress is reported to promote mTORC2 signaling [156]. FOXOs also provide a reciprocal negative feedback loop between PI3K-AKT-mTOR pathway and AR signaling [12] (discussed in Section 3.2).
In prostate cancer, FOXO mutations are rare (<0.5% incidence), however FOXO1 and FOXO3 deep deletion is a frequent event, occurring in up to 15.2% and 13.4% of patients respectively (Tables S1-S3). FOXO3 lies within the 6q21 locus that is frequently lost in prostate cancer [160], and reduced FOXO3 (also FOXO3a) activity via peptide driven inhibition is reported to accelerate prostate cancer progression in the transgenic adenocarcinoma mouse prostate (TRAMP) neuroendocrine prostate cancer model [161]. FOXO1 has also been shown to bind and inhibit the transcriptional activity of E26 transformation-specific (ETS) transcription factor ERG, which is over-expressed in 50% of prostate cancers owing to TMPRSS2-ERG (transmembrane protease, serine 2: ERG fusion) gene rearrangements [162]. Furthermore, Foxo1 bi-allelic deletion and ERG overexpression can cooperate to cause prostate neoplasia in mice [162]. Together, these findings suggest FOXO1/3 act as tumor suppressors during prostate cancer.
FOXO4 gene amplification occurs in up to 8.8% of patients with metastatic prostate cancer (Table  S3), however the functional importance of this genetic alteration remains to be clarified. Although FOXO4 down-regulation is reported to correlate with reduced prostate cancer metastasis-free survival, conversely FOXO4 knockdown in LNCaP cells can increase metastatic potential [163]. Thus, future work addressing the role of FOXO4 during prostate cancer progression is warranted.
TSC1 and TSC2 are frequently mutated/deleted in a variety of solid tumors, including lung (22%) and liver (16%) cancers, leading to deregulated PI3K-AKT-mTOR signaling [169,170]. In prostate cancer, the frequency of TSC1 and TBC1D7 mutation or deep deletion is low (≤0.8% incidence, Tables S1-S3), whereas TSC2 mutation and deep deletion are more frequent (1-1.8% and up to 4.2% of cases respectively, Tables S1-S3). Interestingly an inactivating splice variant of TSC2 unique to African American patients with prostate cancer has also recently been linked to aggressive prostate cancer and therapeutic resistance [60]. In mice, Tsc1 conditional deletion in murine prostate epithelium is reported to cause prostate neoplasia associated with elevated mTORC1 signaling [171], and combined Tsc2 and Pten heterozygosity has been shown to promote invasive prostate carcinoma relative to single mutants [172]. In lung cancer, TSC1 and TBC1D7 have been shown to function as oncoproteins [173], possibly reflecting mTORC1-independent functions such as TSC1-mediated activation of TGFβ-SMAD2/3 signaling [174]. Remarkably, up to 3%, 4%, and 7% of patients with prostate cancer also display TBC1D7, TSC1, and TSC2 high-level amplification respectively (Tables S1-S3), yet the functional consequence is currently unclear. RHEB GTPase has also been shown to act as a proto-oncogene in prostate cancer and up to 4% of patients with prostate cancer carry RHEB gene amplification, however RHEB oncogenic mutations are rare (≤0.1% incidence, Tables S1-S3). RHEB GTPase is over expressed in several prostate cancer cell lines and transgenic mice over-expressing Rheb specifically within the prostate epithelium develop low-grade prostatic intraepithelial neoplasia lesions by 10 months of age, accompanied with increased mTORC1 activity [175]. Rheb over-expression can also cooperate with Pten haploinsufficiency to promote prostate tumorigenesis [175], indicating RHEB amplification is likely to be a genetic driver of prostate tumorigenesis in the clinic.

Amplification of mTORC1 and mTORC2 Complex Components
The mTORC1 and mTORC2 protein complexes are functionally and structurally distinct, originally distinguished by their sensitivity to the mTOR inhibitor rapamycin [176][177][178]. Both mTORC1 and mTORC2 complexes contain mTOR, MLST8 (also known as G-protein beta-subunit like, GβL), TEL2, TTI1, and the negative regulator DEPTOR [179,180]. RAPTOR and PRAS40 (encoded by AKT1S1) are additional members of mTORC1 complex, whereas RICTOR, mSIN1, and PROTOR1/2 form the mTORC2 complex [180] (Figure 1). mTORC1 and mTORC2 are downstream effectors and regulators of PI3K/AKT signaling that mediate key cellular processes in response to growth factors and hormones [179,[181][182][183]. mTORC1 is sensitive to rapamycin treatment and functions to regulate cell growth, autophagy, protein translation machinery, and cell-cycle progression by phosphorylating substrates such as ULK1, S6K and 4EBP1 [179,[183][184][185]. The mTORC2 complex plays a critical role in PI3K/AKT signaling by increasing the activity of AKT, SGK1 and PKCα to regulate cell survival, metabolism and cytoskeletal dynamics [184] (Figure 1). mTORC2 is generally insensitive to rapamycin [179], however chronic exposure to the drug has been shown to impair mTORC2 assembly [185]. Crucially, mTORC1 and mTORC2 can also regulate each other via multiple mechanisms, including AKT regulation of PRAS40 to block suppression of mTORC1 activity and S6K regulation of mSIN1 to modulate mTORC2 activity [186].
In general, the frequency of genetic alterations in mTORC1 and mTORC2 components is low in prostate cancer. Genomic profiling data have shown that mTOR mutation occurs in 0.6-1.6% of cases, and the frequency of mutation or deep deletion in the other components of mTORC1/2 is ≤1% (Tables S1-S3). However, DEPTOR gene amplification is comparatively frequent, occurring in 5.1-21.4% of cases, with the highest incidence observed in the SUC2/PCF-IDT metastatic prostate adenocarcinoma dataset (Tables S1-S3). In addition, DEPTOR amplification directly correlates with worse disease/progression-free survival in the TCGA Firehose Legacy prostate adenocarcinoma dataset ( Figure S4), indicating DEPTOR amplification may provide a valuable predictive biomarker in the clinic. DEPTOR is an endogenous suppressor of mTOR kinase activity, yet DEPTOR upregulation can reduce S6K1 activation, thus relieving feedback inhibition from mTORC1 to PI3K and mTORC2 signaling that results in increased AKT activation [187]. Nevertheless, DEPTOR knockdown in colorectal cancer cells reduced cell proliferation and induced differentiation [188], raising the possibility that DEPTOR can promote tumorigenesis in other epithelial cancers. DEPTOR has also been shown to exert mTORC1/2-independent functions in the nucleus as a transcriptional regulator in multiple myeloma cells [189] and is a transcriptional target of WNT/β-catenin/MYC signaling in colorectal cancer cells [188], adding further complexity to PI3K-AKT-mTOR and WNT pathway crosstalk.
Intracellular amino acids can also activate mTORC1 signaling by stimulating vacuolar H + -ATPase (v-ATPase) to activate Ragulator, a guanine exchange factor that converts RAGA/B·GDP to RAGA/B·GTP, enabling formation of the active RAG complex where RAGA·GTP or RAGB·GTP form heterodimers with either RAGC·GDP or RAGD·GDP [36,[191][192][193][194][195]. Similarly to RAGA/RAGB, RAGC/RAGD are functionally redundant and are 80-90% homologous [195]. When amino acids are sufficient, mTORC1 is recruited to the lysosome where it binds to the active RAG complex via RAPTOR, followed by its localization to RHEB that leads to mTORC1 activation [36,191,195] (Figure 1). Recent evidence also suggests that amino acids such as glutamine can activate mTORC1 in a RAG-complex independent manner, for example via the GTPase adenosine ribosylation factor 1 (ARF1) [196], highlighting the complex nature of mTORC1 regulation.
In prostate cancer, genetic alterations in RRAGA and RRAGC genes that encode RAGA and RAGC respectively are uncommon, however RRAGB (encoding RAGB) is amplified in up to 7.7% of cases and RRAGD (encoding RAGD) deep deletion occurs in 6.5-14.4% of cases (Tables S1-S3). Interestingly, RRAGD deep deletion in prostate adenocarcinoma strongly correlates with FOXO3 deletion (one-sided Fisher's Exact test, p-value < 0.001; data sourced from the cBioPortal platform, TCGA Firehose Legacy prostate adenocarcinoma dataset, n = 492), however the functional consequence of RRAGD/FOXO3 co-deletion and RAGB amplification during prostate cancer growth and therapeutic resistance is currently unknown and merits further investigation.

CAMKKβ Amplification
CAMKKβ is encoded by CAMKK2 and phosphorylates AMPK in response to Ca 2+ signaling. In prostate cancer, CAMKK2 is amplified in up to 6.3% of patients (Tables S1-S3), however it is currently unknown if CAMKK2 amplification promotes AMPK activity in the clinic. In a Pten-deleted prostate cancer mouse model, Camkk2 deletion or CAMKKβ pharmacological inhibition has been shown to suppress prostate tumorigenesis and reduce de novo lipogenesis, whereas Prkab1 (AMPK-β1) and Pten co-deletion accelerates tumor progression [210]. These findings indicate that CAMKKβ plays an oncogenic role in this setting and that CAMKKβ and AMPK-β1 play opposing roles in Pten-deficient prostate cancer, possibly reflecting their differential regulation of lipogenesis [210]. CAMKKβ has also been shown to activate AMPK in response to androgen signaling, and AMPK can subsequently inhibit AR function to form a negative feedback loop [210]. However, the impact on the PI3K-AKT-mTOR signaling cascade remains unclear. Interestingly, a recent report has shown that CAMKKβ can directly phosphorylate AKT at residue Thr308 in ovarian cancer cells [211], indicating CAMKKβ may regulate AKT/mTOR signaling both directly and indirectly via AMPK.

LKB1 Loss
LKB1 (encoded by serine/threonine kinase 11, STK11) is a multifaceted enzyme that plays a tumor suppressive role by phosphorylating multiple substrates (e.g., AMPK and PTEN) to regulate crucial cellular processes including cell metabolism, polarity, differentiation, and proliferation [212,213] ( Figure 1). While STK11 deletion or inactivating mutations are frequent in lung cancer (occurring in up to 50% of patients) [208], STK11 mutations are rare in prostate cancer (0.2% incidence, Tables S1-S3) and the frequency of STK11 deep deletion is also comparatively low (0-3.4% incidence, Tables S1-S3). We have previously shown that LKB1 exerts a tumor suppressive function in the prostate, as Lkb1 homozygous deletion in murine prostate epithelial cells causes prostate intra-epithelial neoplasia (PIN), associated with elevated PI3K/AKT signaling [214]. The relatively mild effects of LKB1 loss are greatly enhanced when combined with Pten heterozygosity in the mouse prostate, which causes lethal metastatic prostate cancer [215]. Interestingly, the expression of either wild-type LKB1, or a kinase-dead form of LKB1 (LKB1 K78I ) is sufficient to reduce tumor burden and impair metastatic potential of DU145 prostate cancer cells that lack LKB1, indicating LKB1 may also elicit a kinase-independent tumor suppressive function [215]. These in vivo findings indicate that deregulation of the LKB1-AMPK signaling axis is a potential mechanism whereby AKT/mTOR signaling is potentiated to facilitate prostate tumor formation and/or progression. Furthermore, a recent study has shown that LKB1 protein levels are reduced in immortalized prostate cancer cell lines relative to normal prostate epithelial cells, and siRNA-mediated STK11 knockdown correlated with elevated hedgehog signaling and increased proliferation and invasion of prostate cancer cells in vitro, however PI3K-AKT-mTOR signaling was not assessed [216].

Sestrin Deletion
Sestrins are a family of stress inducible antioxidant proteins comprising of SESN1, SESN2, and SESN3, which play a key role in regulating autophagy, mitophagy, metabolic homeostasis, inflammation, hypoxia and oxidative stress [217][218][219]. SESN1 and SESN2 are p53 target genes that are induced upon DNA damage and oxidative stress [217]. SESN1 and SESN2 can directly bind to both the TSC1:TSC2 complex and AMPK, which leads to AMPK activation/autophosphorylation in a p53-dependent manner and stimulates AMPK-mediated phosphorylation of TSC2 to negatively regulate mTORC1 signaling [217]. In addition, sestrins are reported to negatively regulate mTORC1 signaling via GATOR2/RAG, indicating that sestrins can also mediate PI3K-AKT-mTOR signaling in response to energy stress (e.g., nutrient starvation) [220,221].
Genetic alterations in the genes encoding sestrins have been linked to non-small cell lung carcinoma (NSCLC) and colorectal cancer, and recent evidence in the literature has indicated sestrins play a tumor suppressive role [221,222]. Although sestrin mutations are rare (≤0.8%), SESN1 deep deletion is a frequent event in prostate cancer occurring in 4.7-13.4% of cases (Tables S1-S3), potentially leading to increased mTORC1 signaling through alleviation of SESN1-mediated negative regulation of mTORC1. Interestingly, similarly to FOXO3, SESN1 is located within the 6q21 locus that is commonly lost in prostate cancer [160]. SESN1 is also reported to be transcriptionally repressed by AR [223], whereas p53 and FOXOs are known to mediate SESN1 transcription [156,217]. Thus, future work exploring the functional significance and predictive value of SESN1 depletion in prostate cancer could identify new therapeutic avenues or biomarkers to aid patient care.

MAP3K7 Deletion
MAP3K7 (also known as transforming growth factor (TGF) β-activated kinase 1, TAK1) is a serine/threonine protein kinase that mediates cell survival via NF-κB-dependent and NF-κB-independent signaling in response to TGFβ and cytokines [224]. Recent evidence in the literature has indicated that MAP3K7 may also mediate AMPK-AKT-mTOR signaling, as MAP3K7/TAK1 inactivation is associated with AMPK activation and reduced p-mTOR levels in skeletal muscle [209]. However, MAP3K7 is reported to mediate mTOR signaling independently of AMPK in hepatocellular carcinoma, possibly via p38 activation [225].
In prostate cancer, MAP3K7 is a putative tumor suppressor gene and MAP3K7 deletion has been shown to directly correlate with prostate cancer progression, lymph node metastasis, and biochemical recurrence [226,227]. MAP3K7 deep deletion is a frequent event in prostate cancer, occurring in up to 14.8% of patients (Tables S1-S3). Furthermore, loss of Map3k7 in mice has been shown to promote prostate tumorigenesis [227], suggesting MAP3K7 plays a tumor-suppressive function in the prostate. However, in an AML xenograft model MAP3K7 inhibition was found to attenuate leukemia development [228], indicating that MAP3K7 plays a dual role as a tumor suppressor and an oncogene depending on the malignancy.

The PI3K-AKT-mTOR Pathway Intersects with Multiple Oncogenic Signaling Cascades to Facilitate Prostate Cancer Growth
The PI3K-AKT-mTOR signaling cascade is one of the most frequently upregulated pathways in prostate cancer, which potentiates multiple downstream signaling events to mediate a plethora of cellular processes that promote tumor growth and therapeutic resistance to current treatment regimens. Targeting the PI3K-AKT-mTOR pathway using small molecules, such as pan-PI3K, PI3K-isoform specific, AKT, mTOR and dual PI3K/mTOR inhibitors has been challenging owing to their limited efficacy and poor tolerability (reviewed in [14][15][16][17]134,229,230]). Many clinical trials involving PI3K-AKT-mTOR-directed therapies have failed owing to incomplete inhibition of the pathway, reflecting the multiple modes of pathway redundancy and numerous positive/negative feedback loops that exist both within the PI3K-AKT-mTOR cascade and via crosstalk with other signaling pathways [15,[231][232][233][234][235][236] (Figure 1). Here, we review PI3K-AKT-mTOR interactions with the RAS/MAPK, AR, and WNT signaling pathways, illustrating the need to improve our molecular understanding of the broader PI3K-AKT-mTOR signaling network. Delineating the complexity of the PI3K-AKT-mTOR pathway interactions with other signaling cascades during normal tissue homeostasis, tumorigenesis and therapeutic resistance is crucial for the discovery of new, efficacious personalized treatment approaches that overcome PI3K-AKT-mTOR inhibitor resistance.

PI3K-AKT-mTOR and RAS/MAPK Signaling Crosstalk
The RAS/MAPK cascade transduces extracellular growth signals via transmembrane receptors (e.g., RTKs and GPCRs) and a series of intracellular protein kinases to regulate gene expression in the nucleus, and to mediate a range of cellular functions including cell proliferation, migration, differentiation, senescence, and survival [25,237,238]. Growth factors bind to the extracellular surface of RTKs (e.g., epidermal growth factor receptor, EGFR, and fibroblast growth factor receptor, FGFR) leading to a conformational change that enables RTK dimerization and autophosphorylation of several tyrosine residues within the RTK cytoplasmic tail. This creates docking sites for adaptor proteins that stimulate downstream effector cascades, such as growth factor receptor-bound protein 2 (GRB2) that recruits Son of Sevenless (SOS) and the GTPase RAS to activate the MAPK cascade (RAF-MEK-ERK signaling) and drive transcription of RAS/MAPK target genes [237,238] (Figure 1).
The RAS/MAPK cascade is frequently deregulated in human cancers, including prostate cancer [238]. Activating genetic alterations (i.e., mutation/amplification) in RAS (HRAS, NRAS, or KRAS) and BRAF have been reported in primary and metastatic prostate cancer (1-8% incidence), and augmented MAPK signaling is reported to correlate with castration-resistance and metastatic progression [43,45,46,101,239]. The PI3K-AKT-mTOR and RAS/MAPK pathways are interconnected at multiple levels ( Figure 1), predominantly owing to (a) shared upstream regulation mechanisms through RTKs/GPCRs and their associated adaptors, (b) the ability of respective cytosolic signaling components to interact and cross-regulate, and (c) the regulation of joint downstream targets (e.g., BAD and RPS6), reviewed in [25,240]. At the level of the receptor for example, the GRB2-SOS complex that is recruited to activated RTKs can bind to the scaffolding protein GAB1 (GRB2-associated binder-1), which interacts with RasGAP, SHP2, PI3K, and PIP3 to augment both RAS/MAPK and PI3K-AKT-mTOR signaling [25]. In addition, mTORC1 signaling can negatively regulate RTK signaling to reduce both PI3K-AKT-mTOR and RAS/MAPK activity, including mTORC1-S6K-mediated suppression of the insulin receptor substrate protein IRS1; a major IGF-1 receptor substrate and adaptor protein that can promote both PI3K and RAS activation by binding to p85 and GRB2 respectively [25,241]. S6K can also phosphorylate RICTOR to reduce mTORC2 signaling [25,242].
At the membrane, RAS-GTP can also bind to the RAS-binding domain (RBD) of p110α, p110δ, and p110γ to directly activate several Class I PI3K catalytic subunit isoforms [20,243]. Intracellular components of both cascades also interact to form multiple feedforward and feedback loops that enable PI3K-AKT-mTOR and RAS/MAPK pathway cross-regulation ( Figure 1) [25,232,240]. For instance, RAS/MAPK activation has been shown to stimulate mTORC1 signaling through ERK, which can directly phosphorylate TSC2, RAPTOR and 90 kDa ribosomal S6 kinase (RSK) to inactivate/dissociate the TSC1:TSC2 complex and regulate the recruitment of mTORC1 substrates [244][245][246]. ERK-RSK signaling can also phosphorylate serum response factor (SRF), cAMP response element-binding protein (CREB) and RPS6, thus promoting cap-dependent translation independently of mTORC1-S6K signaling [247,248]. In addition, AKT is also reported to directly phosphorylate and negatively regulate RAF to suppress the MAPK cascade [249,250], and activated RAS has recently been shown to directly interact with mSIN1 to stimulate mTORC2 signaling in cancer cells (including prostate cancer cell lines) [251].
3.1.1. RAS/MAPK-PI3K-AKT-mTOR Interactions Promote Resistance to PI3K-AKT-mTOR Pathway-Directed Therapies Clinical trials exploring the efficacy of inhibitors targeting the PI3K-AKT-mTOR pathway in prostate cancer have been extensively reviewed previously [14][15][16][17]. Despite promising results in early preclinical studies [252,253], allosteric mTORC1 inhibitors (e.g., rapamycin and rapamycin analogs/rapalogs such as Everolimus and Temsirolimus) have been ineffective in patients with prostate cancer, owing to their inability to suppress AKT activity and a number of adverse side effects [17,254]. Evidence in the literature has revealed several mechanisms of resistance, including activation of the RAS/MAPK pathway [235,[255][256][257]. Both normal and transformed prostate epithelial cells have been shown to augment RAS/MAPK signaling in response to mTORC1 inhibition [235,255], and administration of Everolimus (RAD001) has been shown to induce MAPK signaling in a Pten-deleted mouse model of prostate cancer [235,255]. Although the mechanisms underpinning resistance to mTORC1 inhibitors are not completely understood, several signaling events that involve PI3K/AKT/PI3K and RAS/MAPK crosstalk have been identified. For instance, mTORC1 inhibition is reported to promote AKT and RAS/MAPK signaling by blocking mTORC1-S6K-mediated negative regulation of IRS1 and mTORC2 signaling [258,259] (Figure 2A). Inhibition of mTORC1 has also been shown to prevent mTORC1 stabilization of growth factor receptor bound protein 10 (GRB10), an RTK adaptor protein that negatively regulates RTK signaling [260].
Additionally, the mTORC2 substrate SGK1 can replace AKT in response to PI3K/AKT inhibition, leading to the activation of shared AKT substrates that mediate oncogenic cellular processes such as cell growth, survival metabolism, and migration [145,267] (Figure 2B). In PIK3CA mutant breast cancer cells, PDK1-SGK1 signaling has been shown to sustain AKT-independent mTORC1 activation to promote resistance to the p110α-isoform-specific PI3K inhibitor BLY719, and PDK1 or SGK1 blockade can restore BYL719 sensitivity [145]. Furthermore, elevated SGK1 can predict for AKT inhibitor resistance in breast cancer cells [267]. Interestingly, Class I PI3K and AKT inhibition has also been shown to increase PI3K Class III hVsp34-SGK3 signaling in breast cancer cells, which can substitute for AKT by phosphorylating TSC2 to activate mTORC1 [148]. Whether SGK1/3 shares AKT's ability to phosphorylate and activate RAF is currently unknown.

Co-targeting RAS/MAPK and PI3K-AKT-mTOR Signaling in Prostate Cancer
Co-activation of the RAS/MAPK and PI3K-AKT-mTOR signaling pathways occurs frequently in human malignancies including prostate cancer, thus considerable research has been devoted to establishing how these two oncogenic cascades interact [101,256,257,[268][269][270]. Nearly all metastatic prostate cancer patients are reported to show deregulation of both cascades [43]. To model this in vivo, genetically engineered mouse models of prostate cancer with prostate specific Pten homozygous deletion harboring either a KRas G12D activating mutation or oncogenic BRaf V600E with NK3 Homeobox 1 (Nkx3.1) depletion, promotes rapid tumor growth and metastatic progression relative to the single mutants [101,268,269]. To our knowledge, these tumor models were the first immunocompetent transgenic mouse models of prostate adenocarcinoma to display reproducible metastatic disease.
Taken together, these findings indicate that PI3K-AKT-mTOR and RAS/MAPK signaling synergize to promote prostate cancer growth and metastatic progression, and given the frequency of co-activation of these cascades in the clinic, this provides a clear justification for exploring the combination of PI3K-AKT-mTOR and RAS/MAPK pathway inhibitors in patients with advanced prostate cancer. This notion is further supported by the fact that MEK inhibition is associated with elevated PI3K-AKT-mTOR signaling in mammalian cancer cells, including prostate cancer cells [271,272]. Preclinical studies have also shown that co-inhibition of MEK and mTORC1 can significantly reduce tumor burden relative to monotherapy in a mouse model of prostate cancer driven by simultaneous heterozygous deletion of Nkx3.1 and Pten [256], and can inhibit cell growth and increase cytotoxicity in the castration-resistant CWR22Rv1 human prostate cancer cell line [272]. However, MEK inhibition alone is reported to be sufficient to suppress the metastatic spread of Pten-deleted and KRas activated stem/progenitor murine prostate cancer cells orthotopically transplanted in vivo, similarly to combined mTORC1 and MEK inhibition [101]. This highlights the need to improve our molecular understanding of how these cascades interact during disease progression and in the presence of different genetic drivers to aid the stratification of patients that will benefit from (a) PI3K-AKT-mTOR inhibition, (b) MEK inhibition or (c) combined PI3K-AKT-mTOR and RAS/MAPK blockade.
Several prostate cancer clinical trials have been designed to investigate the therapeutic efficacy of targeting MEK (e.g.,MEK1/2 inhibitor trametinib, ClinicalTrials.gov identifiers: NCT02881242 and NCT01990196) or the PI3K-AKT-mTOR cascade (e.g., pan-AKT inhibitors including ipatasertib and capivasertib, ClinicalTrials.gov identifiers: NCT01485861/NCT03673787 and NCT02525068/ NCT02121639 respectively) [134,273]. Metformin (an oral type 2 anti-diabetic drug) is also currently being investigated in prostate cancer within the STAMPEDE trial [274]. Metformin targets the mitochondrial respiratory chain complex I, leading to reduced mitochondrial ATP production that causes cellular energy crisis with subsequent AMPK activation and mTORC1 inhibition [275]. Metformin has also been shown to inhibit MEK/ERK in response to growth factors, contrasting mTORC1 inhibitor treatment with rapamycin that increases MAPK signaling [276].
Although not currently specific to patients with prostate cancer, clinical trials exploring co-inhibition of the PI3K-AKT-mTOR and MAPK cascades to treat various advanced solid cancers have also been developed (e.g., ClinicalTrials.gov identifiers: NCT01390818, NCT01347866, and NCT02583542), although response rates appear to be low and are linked to RAS and RAF mutations [277]. For example, a recent Phase Ib study of combination therapy with the MEK1/2 inhibitor binimetinib (Mektovi) and the pan-PI3K inhibitor Buparlisib (BKM120) in advanced solid tumors reported promising efficacy in patients with advanced ovarian cancer with RAS/RAF genetic alterations, however continuous dosing resulted in intolerable toxicities and an intermittent schedule is suggested for future trials [278]. Additionally, the MATCH screening trial (targeted therapy directed by genetic testing in treating patients with advanced refractory solid tumors, lymphomas, or multiple myeloma, ClinicalTrials.gov identifier: NCT02465060) will investigate the efficacy of MEK and PI3K inhibitors as monotherapies in patients with progressive disease that carries a genetic alteration in either the RAS/MAPK or the PI3K-AKT-mTOR pathways respectively.

PI3K-AKT-mTOR and AR Signaling Crosstalk
AR signaling regulates cell growth, differentiation, migration and survival, and plays a critical role as a transcriptional regulator during prostate development, normal prostate tissue homeostasis, and prostate cancer [279][280][281]. AR is a steroid nuclear receptor that transmits androgen signals such as testosterone (T), or its more potent metabolite dihydrotestosterone (DHT), to regulate gene expression and coordinate cellular responses. T is derived from cholesterol through a cascade of biochemical reactions involving four enzymes: cytochrome P450 side-chain cleavage enzyme (P450scc), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) [282]. The conversion of cholesterol to pregnenolone is catalyzed by P450scc, and its subsequent conversion to progesterone is catalyzed by 3β-HSD. Pregnenolone and progesterone can be converted by CYP17A1 to 17-OH-pregnenolone and 17-OH-progesterone and subsequently to dehydroepiandrosterone (DHEA) and androstenedione (AD or A4). DHEA and AD may then be converted to androstenediol and T by 17β-HSD [282]. T synthesis and secretion predominantly occurs in the Leydig cells of the testes, and is stimulated by pituitary-derived luteinizing hormone (LH), which is secreted in response to hypothalamus-derived LHRH, (also known as gonadotrophin-releasing hormone, GnRH) [281,283]. In the prostate, 5-alpha-reductase converts T to DHT [281,283]. In addition, androgens can also be produced by the adrenal glands and in some instances by prostate tumor cells [284], which may contribute to prostate cancer growth post-orchiectomy [285].
In the absence of androgens, AR forms a cytoplasmic complex with chaperones (e.g., HSP90 and HSP70). Androgen binding displaces the chaperones and triggers a conformational change in AR, which enables AR homodimerization and nuclear translocation (Figure 1) [279,281,286]. Nuclear AR homodimers regulate the transcription of androgen-regulated genes (e.g., SLC43A1, FKBP5, CAMKK2, NKX3.1 and KLK3) by directly binding to an androgen responsive element (ARE) in the promotor/enhancer region of target genes [279,286]. However, growth factors (e.g., EGF and IGF-1), cytokines (e.g., IL6) and intracellular signaling kinases (e.g., AKT and SRC) can also independently stimulate AR dependent transcriptional activity when androgen levels are low, which can facilitate therapeutic resistance to androgen/AR blockade [12,281,287]. In addition to regulating gene transcription, AR can also mediate a number of intracellular signaling pathways through direct protein-protein interactions within the cytoplasm (known as non-genomic AR signaling) [12,281]. For instance, AR is reported to activate SRC family kinases, PKC, RAS, ERK, PI3K, and AKT [12,288].
Aberrant AR signaling is a common feature of prostate cancer [289], with up to 56% of primary cases and 100% of metastatic cases reported to carry genetic alterations within key AR pathway components [43]. While the majority of men with prostate cancer initially respond to androgen/AR-directed therapy, they inevitably develop castration-resistant prostate cancer (CRPC), as malignant cells develop therapeutic resistance [5,290]. Several inherent and acquired resistance mechanisms have been identified, including AR genetic alterations (e.g., activating mutations, gene amplification, androgen-independent constitutively active splice variants, AR loss), augmented androgen biosynthesis, adrenal androgens, AR-bypass signaling (e.g., glucocorticoid receptor (GR) regulation of shared AR target genes), trans-differentiation to neuroendocrine prostate cancer and ligand-independent activation via crosstalk with another signaling cascade, such as the PI3K-AKT-mTOR pathway [12,283].
The PI3K/AKT/mTOR and AR pathways have been shown to cross-regulate through several reciprocal inhibitory loops [11,12,24] (Figure 3). Consequently, the PI3K-AKT-mTOR pathway can be inadvertently activated in response to androgen/AR-directed therapies, and vice versa PI3K-AKT-mTOR pathway inhibition can augment AR signaling, leading to therapeutic resistance. Human patient samples (both primary tumor and bone metastases), human prostate cancer cell lines and transgenic mouse models of prostate cancer have consistently demonstrated that AKT-mTOR signaling is increased in response to androgen/AR-directed blockade [11,13,24,[291][292][293]. Mechanistically, it is reported that inhibiting AR signaling reduces expression of the AR target gene FK506-binding protein-5 (FKBP5), which leads to PHLPP destabilization and reduced PHLPP-mediated dephosphorylation of AKT at Ser473 to promote AKT signaling [11,24] (Figure 3A). Thus, compensatory activation of the PI3K-AKT-mTOR pathway in response to androgen/AR pathway inhibition can facilitate CRPC growth.
Conversely, PI3K-AKT-mTOR pathway inhibition is associated with augmented AR signaling that can contribute to drug resistance and promote prostate cancer progression [11,[293][294][295]. Carver and colleagues showed that PI3K/mTOR inhibition activates AR signaling in human xenograft and transgenic mouse models of prostate cancer, and that co-treatment with the PI3K/mTOR inhibitor BEZ235 and the antiandrogen MDV3100 (enzalutamide) significantly reduced tumor burden relative to monotherapy [11]. In corroboration, resistance to the AKT inhibitor capivasertib (AZD5363) in LNCaP prostate cancer xenografts is also associated with elevated AR signaling, and combining AZD5363 treatment with the antiandrogen bicalutamide prolonged disease stabilization [295]. Furthermore, mTOR and EGFR co-inhibition with everolimus and gefitinib has shown limited sensitivity in patients owing to enhanced AR activity and PSA levels [293], providing further rationale for combining AR and PI3K-AKT-mTOR blockade to treat prostate cancer. Despite the antagonistic crosstalk between AR and AKT (Figure 3), AR signaling can boost mTORC1 activation through an AR-dependent increase in amino acid transport during tumorigenesis [304]. AR mediates expression of L-type amino acid transporters (e.g., LAT3 encoded by SLC43A1) to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth (Figures 1 and 3) [304]. Moreover, LAT1 and LAT3 transport inhibition is sufficient to decrease cell growth and mTORC1 signaling in prostate cancer cells in vitro [304]. Recent in vitro data have also revealed that mTOR can directly interact with AR in the nucleus of prostate cancer cells to promote metabolic rewiring, and high levels of nuclear mTOR correlate with poor prognosis in patients with prostate cancer [293]. Additionally, AKT has been shown to directly bind and phosphorylate AR when T levels are low, although the functional significance of this event remains to be determined [16,305,306].
The reciprocal feedback loop between AR and PI3K-AKT-mTOR signaling may also be perturbed by Speckle-type BTB/POZ protein (SPOP) loss of function mutations that lead to the stabilization of the SPOP substrate SRC3 (e.g., p.F133V), which consequentially increases PI3K activity [307]. SPOP is an adaptor protein of the Cullin 3 family E3 ligases that can target SRC3 for Several distinct molecular mechanisms have been identified that underpin AR reactivation upon AKT inhibition. Notably, AKT inhibition can prevent AKT-mediated nuclear exclusion of FOXOs, which can lead to augmented transcription of FOXO-target genes such as RTKs (e.g., ERBB2/3 encoding HER2/3) ( Figure 3B) [11,262,296]. HER2/3 activity has been shown to promote AR signaling by protecting AR from ubiquitination and proteasomal degradation, and by enhancing AR binding to ARE target sequences and stimulating AR transcriptional activity [297][298][299] (Figure 3B). Nonetheless, the role of FOXO-dependent signaling in PI3K-AKT-mTOR and AR pathway crosstalk is complex. Although FOXO transcription factors upregulate the expression of RTKs [262] causing a subsequent increase in AR signaling [298], the ectopic expression of FOXO1 conversely dampens AR activity, which is further exacerbated when FOXO1 is co-transfected with the AR coregulator HDAC3 [300]. PTEN loss has also been shown to downregulate AR signaling via the upregulation of several factors that inhibit AR signaling through histone modification mechanisms such as early growth response 1 (EGR1), transcription factor AP-1 (c-JUN), and the catalytic subunit of polycomb repressive complex 2 enhance of zeste homolog 2 (EZH2) (Figure 1) [24]. PTEN protein-phosphatase activity has also been shown to protect the tumor suppressor NK3 homeobox 1 (NKX3.1) from degradation, which can derail the AR transcriptional network [301]. Of note, NKX3.1 and AR can cross-regulate [302], and enforced NKX3.1 expression can suppress Pten-deleted prostate tumorigenesis in transgenic mice [303].
Despite the antagonistic crosstalk between AR and AKT (Figure 3), AR signaling can boost mTORC1 activation through an AR-dependent increase in amino acid transport during tumorigenesis [304]. AR mediates expression of L-type amino acid transporters (e.g., LAT3 encoded by SLC43A1) to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth (Figures 1 and 3) [304]. Moreover, LAT1 and LAT3 transport inhibition is sufficient to decrease cell growth and mTORC1 signaling in prostate cancer cells in vitro [304]. Recent in vitro data have also revealed that mTOR can directly interact with AR in the nucleus of prostate cancer cells to promote metabolic rewiring, and high levels of nuclear mTOR correlate with poor prognosis in patients with prostate cancer [293]. Additionally, AKT has been shown to directly bind and phosphorylate AR when T levels are low, although the functional significance of this event remains to be determined [16,305,306].
The reciprocal feedback loop between AR and PI3K-AKT-mTOR signaling may also be perturbed by Speckle-type BTB/POZ protein (SPOP) loss of function mutations that lead to the stabilization of the SPOP substrate SRC3 (e.g., p.F133V), which consequentially increases PI3K activity [307]. SPOP is an adaptor protein of the Cullin 3 family E3 ligases that can target SRC3 for ubiquitin-mediated degradation and a known tumor suppressor [12,[308][309][310]. In prostate cancer, SPOP is frequently mutated (9-11% incidence) [45,46]. Remarkably, SPOP mutation can also stabilize AR and potentiate AR signaling whilst the PI3K-AKT-mTOR signaling pathway is activated, allowing coordinated and cooperative signaling that drives tumorigenic growth [307]. Conversely, wildtype SPOP can trigger E3 ligase mediated degradation of AR via hinge domain binding when androgens levels are low [311]. Furthermore, AR has been shown to positively regulate the PI3K-AKT-mTOR pathway via a direct interaction with the SH2 domain of the Class IA PI3K regulatory subunit p85α, which has been shown to activate the PI3K-AKT-mTOR cascade [312], further highlighting the complexity of the interactions between these two cascades.
Taken together, these findings support the rationale for combining pharmacological inhibition of the AR and PI3K-AKT-mTOR cascades to treat prostate cancer in the clinic, and highlight the need for further work to delineate the molecular mechanisms underpinning crosstalk between these two oncogenic cascades. Importantly, clinical trials exploring co-targeting AR and PI3K-AKT-mTOR signaling are beginning to show promise. A randomized Phase Ib/II study combining the pan-AKT inhibitor ipatasertib with abiraterone in mCRPC patients has reported ipatasertib + abiraterone prolongs radiographic progression-free survival (rPFS), improves overall survival and extends time to PSA progression compared to abiraterone alone, particularly in patients with PTEN loss (ClinicalTrials.gov identifier: NCT01485861) [134]. This study also reports that the adverse effects common to PI3K-AKT-mTOR blockade (e.g., hyperglycemia) were generally clinically manageable [134]. A Phase I dose escalation study combining enzalutamide and capivasertib to treat mCRPC has also recently reported 3/16 patients responded (ClinicalTrials.gov identifier: NCT02525068) [313]. In this study, patients who met the response criteria had PTEN loss or AKT activating mutations, low/absent AR-V7 protein levels and elevated p-ERK [313]. Nevertheless, several additional clinical trials investigating the combination of AR and PI3K-AKT-mTOR blockade in men with mCRPC did not demonstrate a therapeutic benefit and were associated with poor tolerability (ClinicalTrials.gov identifiers: NCT01385293, NCT01634061 and NCT01717898) [314][315][316]. Interestingly, D'Abronzo and colleagues also recently showed that eIF4E phosphorylation at residue Ser209 in human CRPC cell lines pre-treated with the antiandrogen bicalutamide underpins resistance to subsequent combination therapy with bicalutamide + rapamycin treatment. Remarkably, suppression of eIF4E phosphorylation by MNK1/2 (MAP kinase interacting serine/threonine kinase1/2) or ERK1/2 inhibition was shown to sensitize bicalutamide pre-treated CRPC cells to combined anti-androgen and mTORC1 blockade [317], presenting a novel avenue for overcoming therapeutic resistance. Thus, despite some promising results, it is evident that further investigation into the molecular mechanisms underpinning AR and PI3K-AKT-mTOR pathway crosstalk in prostate cancer is required to improve patient stratification and to discover new therapeutic approaches and predictive biomarkers that can inform future clinical trial design.
Activation of both the canonical and non-canonical WNT cascades has been reported in localized and advanced prostate cancer, and oncogenic deregulation of core WNT pathway components frequently occurs in primary and metastatic prostate cancer (up to 6% and 19% incidence respectively [45]), primarily via APC deep deletion/truncating mutations and CTNNB1/β-catenin activating mutations [45,46,320,322,323]. Furthermore, the WNT/β-catenin pathway is strongly linked to androgen/AR-directed therapy and chemotherapy resistance [324][325][326][327], thus WNT signaling presents an attractive therapeutic target for advanced prostate cancer. In addition, AR is in fact a WNT/β-catenin target gene, and AR and β-catenin can directly interact and co-localize in the nucleus to mediate transcriptional activity of AR-regulated genes [328][329][330] (Figure 1). WNT and AR signaling cascades have also been shown to reciprocally inhibit each other in murine prostate cancer [331].
Mouse models have been instrumental in determining the role of WNT signaling in prostate cancer, and we and others have shown that constitutive activation of β-catenin or Apc bi-allelic deletion predisposes to prostate adenocarcinoma in mice [269,[331][332][333]. Moreover, β-catenin activation can cooperate with Pten heterozygous or homozygous deletion to promote prostate cancer progression, CRPC transition and metastatic potential [269,[331][332][333], indicating a synergistic relationship exists between the PI3K-AKT-mTOR and WNT cascades.
Several molecular mechanisms that permit cross-regulation of the PI3K-AKT-mTOR and WNT signaling cascades have emerged in the literature, which could influence prostate cancer growth and resistance to anti-androgens and/or PI3K-AKT-mTOR pathway inhibitors [320,322,323,334,335] ( Figure 4). While canonical WNT signaling mediates cellular β-catenin levels, the level of active β-catenin (unphosphorylated at residues Ser37 and Thr41) in melanoma, breast and prostate cancer cells is reported to be regulated by the PI3K-AKT-mTOR cascade, in a process that is dependent on PP2A activity [335]. PP2A is known to negatively regulate AKT, however it is currently speculated that this phosphatase may also directly dephosphorylate and activate β-catenin [335]. Additionally, the PI3K-AKT-mTOR pathway has also been shown to mediate β-catenin localization [335], and several transcription factors that directly interact with β-catenin are co-regulated by the PI3K-AKT-mTOR pathway, such as FOXO3a [336] and SOX4 [337]. In prostate cancer cells, FOXO3a has been shown to suppress β-catenin transcriptional activity and can be inhibited by AKT [336], whereas SOX4 is a positive regulator of canonical WNT and is stimulated by AKT [338] (Figure 4). In addition to transmitting canonical WNT signals, β-catenin also form adherens junctions with α-catenin and E-cadherin at cell-cell junctions to maintain tissue architecture and facilitate cell-cell signaling (Figure 4). PTEN has also been shown to modulate β-catenin nuclear localization and transcriptional activity through caveolin-1 (CAV1)-dependent dissociation of β-catenin from Ecadherin at the membrane independently of PI3K-AKT-GSK3β signaling, leading to increased tumor formation and metastatic progression in melanoma [339].
Crosstalk between the PI3K-AKT-mTOR and WNT/β-catenin signaling pathways is also mediated via GSK3β and the TSC1:TSC2 complex [167] (Figure 4). GSK3β play a critical role in both cascades, serving as a core member of the β-catenin destruction complex that helps to maintain low level of cytosolic/nuclear β-catenin in the absence of WNT signal [340], and as a direct substrate of AKT [167]. AKT inactivates GSK3β by phosphorylating residue Ser9 [167]. GSK3β can also phosphorylate and activate TSC2 resulting in inhibition of mTOR activity [167], and can restrict cellular growth by suppressing glucose uptake via TSC2 and mTOR [341]. Active WNT signaling inhibits GSK3β, abrogates the suppression of mTOR and stimulates phosphorylation of S6K, S6, and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) [167]. Interestingly, in the absence of TSC1 or TSC2, S6K has also been shown to inactivate GSK3β by directly phosphorylating residue Ser9 and active GSK3β has also been shown to phosphorylate/activate S6K, adding further complexity to GSK3β signaling between the WNT and PI3K-AKT-mTOR cascades [342]. However, previous work has also indicated that GSK3β does not mediate crosstalk between the PI3K-AKT-mTOR and WNT/β-catenin pathways [343,344], raising the possibility that GSK3β function is In addition to transmitting canonical WNT signals, β-catenin also form adherens junctions with α-catenin and E-cadherin at cell-cell junctions to maintain tissue architecture and facilitate cell-cell signaling (Figure 4). PTEN has also been shown to modulate β-catenin nuclear localization and transcriptional activity through caveolin-1 (CAV1)-dependent dissociation of β-catenin from E-cadherin at the membrane independently of PI3K-AKT-GSK3β signaling, leading to increased tumor formation and metastatic progression in melanoma [339].
Crosstalk between the PI3K-AKT-mTOR and WNT/β-catenin signaling pathways is also mediated via GSK3β and the TSC1:TSC2 complex [167] (Figure 4). GSK3β play a critical role in both cascades, serving as a core member of the β-catenin destruction complex that helps to maintain low level of cytosolic/nuclear β-catenin in the absence of WNT signal [340], and as a direct substrate of AKT [167]. AKT inactivates GSK3β by phosphorylating residue Ser9 [167]. GSK3β can also phosphorylate and activate TSC2 resulting in inhibition of mTOR activity [167], and can restrict cellular growth by suppressing glucose uptake via TSC2 and mTOR [341]. Active WNT signaling inhibits GSK3β, abrogates the suppression of mTOR and stimulates phosphorylation of S6K, S6, and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) [167]. Interestingly, in the absence of TSC1 or TSC2, S6K has also been shown to inactivate GSK3β by directly phosphorylating residue Ser9 and active GSK3β has also been shown to phosphorylate/activate S6K, adding further complexity to GSK3β signaling between the WNT and PI3K-AKT-mTOR cascades [342]. However, previous work has also indicated that GSK3β does not mediate crosstalk between the PI3K-AKT-mTOR and WNT/β-catenin pathways [343,344], raising the possibility that GSK3β function is context/tissue dependent. Of note, WNT ligands can also activate mTOR through MYC-dependent suppression of TSC2 [345].
PI3K-AKT-mTOR and WNT signaling may also interact through Hippo signaling. Hippo signaling is tightly intertwined with cell size regulation and nutrient sensing through LKB1-AMPK, TSC1:TSC2 and mTOR [346,347], and the Hippo pathway signaling proteins yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are integral parts of both canonical and non-canonical WNT signaling [348,349]. YAP and TAZ are members of the β-catenin destruction complex, and in the presence of WNT signals, they dissociate from the complex and translocate to the nucleus to activate downstream targets [348] (Figure 4). AMPK activation has also been shown to negatively regulate YAP/TAZ activity [347].
Non-canonical WNT signaling has also been shown to activate the PI3K-AKT-mTOR pathway. For example, WNT/FZD7-dependent dissociation of Gβγ from Gαi enhances PI3K-AKT signaling and increases tumor cell invasive potential [350]. ROR1 can also activate PI3K-AKT signaling in response to trans-phosphorylation by tyrosine kinases, such as MET and SRC [351]. In addition, WNT receptor Frizzled2 (FZD2) can drive epithelial-to-mesenchymal transition (EMT) and cell migration through activation of Fyn [352], and activated Fyn kinase activity has been shown to suppress the AMPK-LKB1 signaling axis by blocking LKB1 redistribution into the cytoplasm [353].
Interestingly, the PI3K-AKT-mTOR, WNT, MAPK and AR signaling cascades all converge to regulate the transcription factor MYC, which is frequently amplified in prostate cancer/mCRPC [44,45,354]. While MYC is a WNT/β-catenin target gene [355], PI3K-AKT-mTOR signaling can mediate MYC mRNA stability, translation and protein stability [356][357][358][359][360]. AR signaling has also been shown to stimulate MYC in AR-driven prostate cancer, while in normal prostate tissue AR silences MYC to maintain normal homeostasis [361,362]. However, MYC is also reported to antagonize AR transcriptional activity in prostate cancer [363]. MYC upregulation is frequently observed in prostate cancer, and although targeting MYC remains a clinical challenge, preclinical studies have emphasized the potential efficacy of MYC blockade for patients with late-stage prostate cancer [364].
WNT inhibitors are beginning to enter clinical trials, including small-molecule inhibitors to the enzyme porcupine that block WNT ligand secretion, such as WNT974 (LGK974) [320,322]. Preliminary data from a WNT974 phase 1 clinical trial (NCT01351103) for a small range of human malignancies (excluding prostate cancer) report a manageable safety profile and suppression of canonical WNT/β-catenin target gene AXIN2 [320], and recent preclinical studies have indicated that WNT974 treatment is efficacious against prostate cancer [331,365]. β-catenin has also been reported to facilitate resistance to PI3K and AKT inhibition in colon cancer [334]. Thus, further work exploring the therapeutic benefit of targeting the WNT pathway in prostate cancer is warranted.

Conclusions
In summary, the PI3K-AKT-mTOR cascade is frequently activated in prostate cancer, and genomic profiling has revealed that oncogenic genetic alterations occur within a diverse array of PI3K-AKT-mTOR pathway components. Significant research efforts have been devoted to delineating the mode of action of several of these aberrations (e.g., PTEN deletion and PIK3CA activating mutation), however our molecular understanding of how these events differentially mediate cell signaling programs is limited, and several genetic alterations remain to be studied functionally. Future work to gain novel insight into the functional consequence of these genetic alterations in prostate cancer is necessary to (a) identify passenger vs. driver alterations, (b) establish the ability of individual aberrations to synergize with additional oncogenic events, and (c) discover their mode of action during tumor growth, metastasis and therapeutic resistance. In conjunction with genomic and transcriptomic data, establishing the frequency and impact of post-transcriptional modifications and epigenetic events within core PI3K-AKT-mTOR pathway components during prostate tumorigenesis and disease progression/recurrence is also crucial, as these components are regulated at multiple levels and genomic/transcriptomic data do not consistently equate with protein activity.
Targeting the PI3K-AKT-mTOR pathway in prostate cancer remains a key clinical challenge. Therapeutic resistance emerges owing to various feedback/feedforward loops and redundancy mechanisms that prevent complete suppression of the pathway and cause compensatory augmentation of interacting signaling pathways, thus rationalizing the exploration of combination therapies. Encouragingly, clinical trials are beginning to report therapeutic efficacy when combining PI3K-AKT-mTOR and androgen/AR-directed therapies, particularly in patients with mCRPC that display PTEN loss. However, the mechanism of resistance to PI3K-AKT-mTOR pathway-targeted therapies is likely to vary dramatically between patients and within individual tumors owing to several factors. These include the activity status of the pathway components, the extent of intratumoral heterogeneity, the mode and concentration of upstream stimuli, the genetic alterations present and the composition of the tumor microenvironment. Furthermore, our ability to successfully translate preclinical findings to the clinic is currently hampered by the limited number of prostate cancer preclinical models available, which do not fully cover the broad range of prostate cancer subtypes or disease heterogeneity seen in the clinic. Accordingly, to discover newtherapeutic approaches that increase patient response rates and overall survival, further delineation of the complex signaling network that exists within the PI3K-AKT-mTOR pathway and the interacting MAPK, AR, and WNT pathways is needed, together with the development of a wider range of preclinical models that better recapitulate the clinic and a deeper understanding of the molecular biology underpinning prostate cancer disease subtypes and tissue heterogeneity.

Conflicts of Interest:
The authors declare no conflict of interest.