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0 DNA-PK, Nuclear mTOR, and The Androgen Pathway in Prostate Cancer
0 DNA-PK, Nuclear mTOR, and The Androgen Pathway in Prostate Cancer
0 DNA-PK, Nuclear mTOR, and The Androgen Pathway in Prostate Cancer
of Pages 11
Trends in Cancer
Review
Androgen and its receptor (AR) are major drivers of prostate cancer (PCa), a lead- Highlights
ing cause of mortality in aging men. Thus, understanding the numerous mecha- DNA-PK and mTOR, large proteins that
nisms by which AR can promote the growth and proliferation of PCa cells and share structural and functional similarities
as PIKKs, have also been shown to
enable their escape from hormone-dependent therapies, eventually leading to me-
share a noncanonical function as tran-
tastasis and death of the patient, is essential to discover alternative therapeutic ap- scription cofactors.
proaches. Recently, two structurally related members of the phosphatidylinositol
3-kinase-like protein kinase (PIKK) family, DNA-dependent protein kinase When localized at specific regulatory
sites on chromatin, DNA-PK and
(DNA-PK) and mammalian target of rapamycin (mTOR), were shown to have a di- mTOR act as cofactors for the AR in
rect role in modulating AR activity on chromatin of PCa cells. In this review, the PCa cells.
common features of DNA-PK and mTOR and the similarities in their noncanonical
roles as transcription coregulators of the AR are highlighted. An outlook on how DNA-PK expression and mTOR localiza-
tion in the nucleus are both elevated in
these findings could be translated into new approaches to manage and treat advanced PCa and are predictors of me-
PCa is provided. tastasis and reduced overall survival.
vation therapy (ADT) is the first line of defense in the treatment of PCa [4]. However, ADT is not a
cure for PCa because the disease ultimately evolves toward castration-resistant PCa (CRPC).
Even at this advanced stage, AR hyperactivation continues to have a major role in the disease
via several mechanisms, including, among others, amplification of AR and/or enhancers regulating
its expression, mutation, and expression of variant spliced isoforms of the receptor, as well as
increased intratumoral androgen synthesis [5–9]. Therefore, identification of upstream effector
pathways and transcriptional coregulators modulating AR activity in PCa cells and their potential
crosstalk during progression of the disease are essential steps toward the development of novel
therapeutic strategies to treat CRPC.
Recently, DNA-PK and mTOR were shown to mediate transcriptional regulation by the AR in PCa
cells on a global scale. More importantly, the two PIKKs were shown to act as direct modulators
of large transcriptional networks implicated in cell migration and invasion, as well as in metabolic
programs supporting AR-dependent PCa progression and metastasis [10–12]. In this review, the
common features of DNA-PK and mTOR and the similarities in their noncanonical roles as tran-
scriptional coregulators are highlighted. Their crucial function and clinical significance as
coregulators of AR activity in PCa and the potential for new pharmacological approaches to manage
1
CRPC is discussed. Goodman Cancer Research Centre,
McGill University, Montréal, QC, H3G
1Y6, Canada
AR and PIKK Crosstalk in Prostate Cancer
A critical mediator of PCa cellular proliferation and survival is the phosphatidylinositol 3-kinase
(PI3K) pathway that controls metabolism and cell growth downstream of receptor tyrosine ki- *Correspondence:
nases (RTK) in response to multiple growth factors and hormones [5,13–15]. PI3K enzymes vincent.giguere@mcgill.ca (V. Giguère).
Functional interactions between the core activity of DNA-PK in DNA repair and the AR signaling
axis have also been reported. First, activation of transcription by several nuclear receptors that in-
cluded the AR, as well as by other classes of transcription factor, led to an induction of double-
strand breaks (DSB) and recruitment of topoisomerase IIβ and DNA-PK, two major components
of the damage response machinery [22]. These findings demonstrated that the mechanisms of
hormone-dependent gene transcription and DSB DNA repair are closely linked. More recently,
activation of AR in response to genotoxic insult was shown to drive the expression of genes im-
plicated in DNA damage repair, including that encoding DNA-PK [23]. Furthermore, genetic and
pharmacological inhibition of DNA-PK activity resulted in a significant decrease in the expression
of several AR target genes, representing a set of genes incorporating essential components of
androgen metabolism pathways, such as UGT2B15 and UGT2B17 [10]. These observations
imply that DNA-PK can modulate AR transcriptional activity and, conversely, that androgen-
dependent DNA-PK expression is required for AR-mediated DSB DNA repair. Together, these re-
sults demonstrate the existence of yet another level of crosstalk between the AR and PIKKs, that
of an androgen/AR/DNA-PK-positive feedback circuit driving PCa cell survival after DNA damage
[24].
Key Figure
Cooperative Crosstalk between Androgen/Androgen Receptor (AR)
Signaling, the Phosphatidylinositol 3-Kinase/Phosphatase and Tensin
Homolog/Protein Kinase B (PI3K/PTEN/AKT) Pathway and the PI3K-
like protein kinase (PIKK) DNA-Dependent Protein Kinase (DNA-PK)
and Mammalian Target of Rapamycin (mTOR) in Prostate Cancer
(PCa) Cells
Trends in Cancer
Figure 1. Testosterone is first metabolized to dihydrotestosterone (DHT) by 5α-reductase. DHT can be excreted from the cell
following glucuronidation, the transfer of glucuronic acid (GA) to DHT by UDP-glucuronosyltransferase (UGT) enzymes
(e.g., UGTB15 and UGBT17). Androgens activate the PI3K/AKT/mTOR pathway via an uncharacterized mechanism,
which likely involves nongenomic AR activity. Upon binding to DHT, AR dissociates from heat shock proteins (HSPs) and
translocates to the nucleus. The presence of androgens also promotes the accumulation of mTOR in the nucleus, a process
that requires an activated AR. The AR acts as a regulatory partner of mTOR to allow its localization at precise sites in chro-
matin. The accessory transcription factors Forkhead box protein A1 (FOXA1) and Homeobox B13 (HOXB13) make the chro-
matin competent for AR binding and participate in AR-dependent gene regulation. The ligand-bound AR then recognizes
androgen-response elements (AREs) in the regulatory regions of androgen-dependent genes. Coactivators (CoA) and core-
pressors (not shown) are components of the AR complex, stimulating or inhibiting, respectively its interaction with the tran-
scription machinery (not shown). Both DNA-PK and mTOR are components of AR complexes on chromatin and
necessary for the regulation of gene programs (e.g., metabolism, cell cycle, and DNA repair) that are sensitive to the action
of kinase inhibitors, represented by Torin 1 and NU7441 for mTOR and DNA-PK, respectively. Some of these programs
are regulated by both PIKKs. The PI3K/PTEN/AKT pathway downstream of activated receptor tyrosine kinases (RTK) is
also subject to feedback regulation by androgen signaling via regulation of AKT activity. Androgens stimulate the expression
of FK506 binding protein 51 (FKPB5), a chaperone of PH domain and leucine rich repeat protein phosphatase (PHLPP), lead-
ing to downregulation of AKT (broken line). Therefore, loss of androgen signaling during androgen deprivation therapy (ADT)
may lead to upregulation of phospho-AKT. Abbreviation: HSP, heat shock protein.
PIKK family, can also localize to the nucleus and interact with nucleic acids and/or chromatin
[11,28,31,33,34].
DNA-PK and mTOR also share a domain organization and overall architecture that is common to
all PIKKs [35]. The conserved kinase (KIN) domain is flanked at its N-terminal end by the
Huntingtin, EF3A, ATM, TOR (HEAT) and Frap, ATM, TRRAP (FAT) domains, a long series of var-
iable α-helical domains forming an α-solenoid, and at its C-terminal end by the PIKK regulatory
domain (PRD) and FAT C-terminal (FATC) domains (Figure 2). Recent structural studies revealed
that the FAT domain is closely associated with the KIN and PRD domains and that, together,
these domains form a single FATKIN unit that constitutes the core of the PIKKs [35–37]. While
the FATKIN domain has a similar architecture among PIKKs, the long N-terminal solenoid repeats
diverge in both length and structure between members of the family, a feature allowing for inter-
action with specific partners to direct them to particular pathways and the regulation of distinct
cellular function. Partners of PIKKs are either proteins or nucleic acids that regulate PIKK function
upon a specific biological stimulus via a combination of conformational changes in the PIKK alter-
ing KIN activity, cellular localization of the enzyme, and/or interaction with a particular substrate
[38]. DNA-PK associates with Ku70/80 heterodimers to form an active DNA repair complex.
The function of the Ku70/80 partners is to recruit DNA-PK at free double-stranded ends,
where the enzyme phosphorylates multiple targets with a role in the nonhomologous end-joining
DNA repair pathway. mTOR participates in the formation of two distinct complexes, referred to as
mTORC1 and mTORC2, which respond to nutrient availability and growth factor signaling. Both
complexes contain LST8, and mTOR bound to LST8 forms the common core of the mTOR dimer
complexes [39]. In mTORC1, RAPTOR directs the mTOR/LST8 dimer to the lysosome, while, in
Trends in Cancer
Figure 2. Common Functional Domains of DNA-dependent protein kinase (DNA-PK) and Mammalian Target
of Rapamycin (mTOR) and Interactions with Regulatory Partners and the Androgen Receptor (AR). The
Huntingtin, EF3A, ATM, TOR (HEAT) repeats connect to the FATKIN fold, comprising the Frap, ATM, TRRAP (FAT),
conserved kinase (KIN), fragment rapamycin binding (FRB), and PIKK regulatory domain (PRD) domains present in both
DNA-PK and mTOR. The DNA-PK regulatory partners Ku70/80 interact with the HEAT repeats, bringing the N-terminal end
of the HEAT region toward the FATKIN domain. The mTOR regulatory partners LST8 and RAPTOR (mTORC1) and LST8,
RICTOR, and SIN1 (mTORC2) also make contact with both the HEAT and FATKIN domains. Bitopic drugs simultaneously
target both the KIN and FRB domains, altering mTOR function at two distinct sites. The structure of the AR is shown here as
a putative direct regulatory partner of both DNA-PK and mTOR. As a phosphatidylinositol 3-kinase-like protein kinase (PIKK)
partner, the AR is responsible for their localization at specific androgen-regulated sites in chromatin. The exact mechanism
by which the AR interacts with the two PIKKs is currently unknown, as indicated by ‘?’. Abbreviations: DBD, DNA-binding
domain; FATC, FAT C-terminal; Hinge, region connecting the DBD to the LBD; LBD, ligand-binding domain; LST8,
mammalian lethal with SEC13 protein 8; NTD, N-terminal domain; SIN, stress-activated map kinase-interacting protein 1.
mTORC2, RICTOR/SIN1 guides the complex to the plasma membrane. Both RAPTOR and
RICTOR also participate in the selection of specific substrates for mTOR at these locations.
DNA-PK and nuclear mTOR also share the AR as a common accessory partner (Figure 2). As
such, the AR escorts the two PIKKs to regulatory sites of androgen-dependent genes in the chro-
matin of PCa cells [10,11,40]. In addition, both genetic and pharmacological inhibition of AR ac-
tivity can prevent the observed increase in nuclear mTOR levels following androgen stimulation,
suggesting a direct involvement of the AR in mTOR translocation from the cytoplasm to the nu-
cleus in PCa cell lines [11]. Androgen treatment also leads to an increase in the nuclear levels
of the mTORC1- and mTORC2-specific partners RAPTOR and RICTOR in PCa cells [11], consis-
tent with previous reports describing their nuclear localization in other cell types [33,41–43].
However, it is not known whether RAPTOR and/or RICTOR act as active partners in the yet to
be defined AR/mTOR complex(es) on chromatin.
A more direct connection between nuclear mTOR and AR signaling in dictating specific transcrip-
tional programs was made through the use of ChIP coupled with high-throughput sequencing
(ChIP-seq) to monitor the genome-wide association of mTOR with DNA in PCa cells subjected
to androgenic stimulation [11]. Androgen treatment led to a robust reprogramming of mTOR as-
sociation with chromatin in LNCaP cells with N17 000 and N58 000 binding events negatively and
positively regulated, respectively. Of interest, regulated mTOR-binding events were associated
with KLK3, TMPRSS2, and UGT2B17, genes also regulated via modulation of DNA-PK activity
[10]. In addition, MTOR itself is a target of androgen-dependent nuclear mTOR DNA binding,
indicating the existence of a feedforward regulatory circuit involving AR and mTOR signaling
[31]. Mechanistically, positive association of mTOR with chromatin upon androgen treatment
was found to occur at androgen-response elements (ARE) and to require the presence of a func-
tional AR. In agreement with this observation, N80% AR-binding events overlapped with mTOR
sites in PCa cells treated with androgen. In addition, sequential ChIP experiments (known as
ChIP-re-ChIP) using antibodies against AR and mTOR allowed for the simultaneous detection
of the two proteins at the same genomic sites, thus demonstrating cobinding of AR and mTOR
at several loci. Furthermore, analyses of the LNCaP cell transcriptome showed that abrogation
of mTOR activity using the specific kinase inhibitor Torin 1 diminished the number of androgen-
regulated genes by N35%. Functional pathway enrichment analysis of the genes sensitive to
both androgens and Torin 1 showed them to be highly represented in biological processes
associated with cell cycle and metabolism. More specifically, common AR/mTOR targets
were found to be highly enriched for the processes of glycolysis and OXPHOS. Interestingly,
the classic androgen response signature was found to be unaffected by treatment with Torin 1,
demonstrating that the AR/mTOR common transcriptional program predominantly relates
to metabolic functions, a finding unifying the nuclear and canonical functions of mTOR [31].
In agreement with this observation, loss of mTOR activity abrogated the androgen-induced
metabolic reprogramming of LNCaP cells, as assessed through glucose uptake, extracellular
acidification rate, mitochondrial respiration, and de novo lipogenesis [11].
uncovered, the finding that the DNA-PK inhibitor NU7441 abrogates the androgen response in
PCa cells supports a direct role for the kinase function of DNA-PK in modulating androgen-
dependent gene expression [10,12]. The identification of the components of the DNA-PK/AR
complex at AR-bound regulatory sites and the targets of DNA-PK within this complex, which
may likely include AR, would provide valuable information on the molecular mechanisms dictating
the functional interactions between these factors in response to androgens in PCa cells. In this
regard, the DNA-PK partners Ku70 and Ku80 were identified using tandem mass spectroscopy
analysis as AR-interacting proteins more than a decade ago [63], but their role in this process has
not been investigated further.
As observed with DNA-PK, treatment of PCa cells with the specific mTOR kinase inhibitor Torin 1
did not affect mTOR interaction with chromatin but reduced AR-mediated transcriptional activity
[11]. The availability of ChIP-seq data sets for nuclear mTOR also allowed for the identification of
possible transcriptional partners of mTOR at targeted regulatory sites. Indeed, DNA motif analysis
of mTOR bound to chromatin provided important clues to how mTOR interacts with chromatin
[11]. First, the ARE motif is specifically enriched in mTOR-bound regions in the presence of andro-
gens, indicating that AR recruits mTOR to these sites. Second, the most enriched motif at mTOR-
bound sites was for FOXA1, regardless of whether or not the cells were exposed to androgens.
FOXA1 is a transcription factor known for its pioneer activity establishing competence for gene
expression that is often mutated in PCa [9,64–66]. Of interest, the FOXA1 motif is the transcription
factor motif most frequently enriched at enhancers in PCa-specific enhancer-promoter loops
[67]. Consistent with this observation, FOXA1 has been shown to dictate AR and other nuclear
receptor-binding profiles [68–73], although it is also present at non-AR-targets in PCa cells
[67,74]. Since the FOXA1 motif is present at androgen-independent mTOR binding sites, this
finding suggests that FOXA1 activity is required for mTOR interaction with chromatin regardless
of the transcription factor with which it is potentially associated with at these regulatory sites.
Third, the motif for HOXB13 is also enriched at mTOR androgen-induced binding sites.
HOXB13 is required for prostate development, a rare G84E germ line mutation in HOXB13 is as-
sociated with increased risk of PCa, and its motif can also be found at relatively high frequency
within PCa-specific enhancers [67,75,76]. HOXB13 has also been shown to directly interact
with the AR, to confer androgen responsiveness to promoters containing a HOXB13-binding
site, and to synergize with the AR on regulatory sites with dual HOXB13/AR motifs [77]. In
human tissues, HOXB13 and FOXA1 colocalize to a set of AR-binding sites that are
reprogrammed in PCa tumors, and introduction of HOXB13 and FOXA1 into an immortalized
prostate cell line reprograms the AR cistrome to a state approximating that of a prostate tumor
[72]. Recent analysis of prostate tissue ChIP-sequencing data suggested that AR-FOXA1, and
AR-HOXB13 complex formation in particular, are crucial to the reprogramming of AR genome-
wide binding in PCa tumors [78]. Taken together, these findings strongly suggest that HOXB13
and FOXA1 have a similar role in dictating the composition of the mTOR cistrome during the pro-
gression of PCa and, conversely, that mTOR through its kinase activity influences the activity of
these factors and, thus, of the AR interaction with chromatin at shared locations.
factors the activities of which are modulated by DNA-PK [10]. DNA-PK inhibitors also delayed the
formation of metastases in xenograft assays, and tumors harvested at sacrifice showed signifi-
cant decrease in the expression of prometastatic factors (e.g., ITGB4, ROCK2, and VAV3) asso-
ciated with DNA-PK activity in PCa cell lines. Second, DNA-PK expression in a cohort of patients
with high-risk localized PCa showed elevated levels of the kinase, an observation correlating with
the enrichment of genes in the AR, MAZ, and SP1 pathways. Third, DNA damage-independent
hyperphosphorylation of DNA-PK was observed in fresh clinical specimens, indicating the likeli-
hood that DNA-PK activation in metastatic tissues correlates with its transcriptional activity rather
than with its canonical function in DNA repair. As for mTOR, the clinical relevance of its activity in
the nucleus is supported by two observations [11]. First, immunohistochemistry analysis of PCa
specimens showed higher mTOR nuclear expression in both aggressive localized PCa under
ADT and in metastasis samples, compared with primary untreated tumors, an observation that
nuclear mTOR localization is indicative of poor prognosis in patients. Second, an mTOR target
gene signature strongly discriminates between normal prostate, primary tumors, and ADT-
resistant metastatic tissues, and is also predictive of PCa recurrence. Collectively, these findings
support a significant correlation between the role of DNA-PK and nuclear mTOR as direct regu-
lators of gene expression and PCa progression.
While kinase inhibitors reduced the transcriptional activity of DNA-PK and nuclear mTOR, it can
be envisaged that, if crucial to their oncogenic activity, the function of these two PIKKs as tran-
scription factors could be modulated via other mechanisms. Those mechanisms could involve
targeting the sites of physical interaction between the PIKKs and their partners, such as the
AR, FOXA1, and HOXB13, or any other coregulators present in the complexes associated with
DNA-PK and mTOR on DNA and necessary for their transcriptional activity on chromatin. Indeed,
bitopic drugs that can simultaneously target two distinct domains of the same protein have been
developed to modulate mTOR activity [81]. The bitopic inhibitor RapaLink-1, which targets both
the FKBP12-rapamycin-binding domain and ATP-binding sites of mTOR, potently inhibited
tumor growth in cells that had acquired resistance to first-generation mTOR inhibitors (rapalogs)
and ATP-competitive inhibitors [81]. The development of bitopic drugs could be envisioned to tar- Outstanding Questions
get other allosteric sites in DNA-PK and mTOR required for their interactions with the AR and/or What are the physiological signals
the transcriptional machinery (Figure 2). Small-molecule inhibitors that reduce the transcriptional and mechanisms that dictate mTOR
translocation to the nucleus, and what
activity and concentration of the steroid receptor coactivator 3 (NCOA3) have already been devel-
is the role of nuclear mTOR in normal
oped [82], and a similar strategy could be used to target PIKKs at novel functional interfaces. development and physiology?
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