Introduction
Prostate cancer (PCa) is the second most common malignancy in men and
the fifth leading cause of death worldwide (Rawla, 2019). Patients with
early-stage prostate cancer have a good prognosis (Trewartha and Carter,
2013). However, advanced prostate cancer is associated with high
mortality with the development of
invasion and metastasis
(Zong and Goldstein, 2013). Since Huggins and Hodges discovered that PCa
responds to androgen therapy (R, 2002), androgen-deprivation therapy
(ADT) has become the first-line treatment use to suppress PCa
progression. Despite an initial therapeutic efficiency of 80-90%
(Trewartha and Carter, 2013), nearly
all patients treated with ADT will ultimately
develop castration-resistant prostate cancer (CRPC)
(Davies, Conteduca et al., 2019).
Patients with CRPC show a sustained increase in serum prostate-specific
antigen (PSA) levels after castration treatment (Saad, Chi et al.,
2015), which is generally attributed to the reaction of the androgen
receptor (AR), which may be the result of AR gene amplification or other
mechanisms (Cai and Balk, 2011 , Tran, Ouk et al., 2009). The androgen
biosynthesis inhibitor abiraterone and AR antagonist enzalutamide (Enz)
are two novel agents targeting androgen action to treat patients with
CRPC and were approved by the Food and Drug Administration (FDA) in 2012
(Nelson and Yegnasubramanian, 2013). Enz is a potent AR signalling
inhibitor without agonistic activity. It suppresses the nuclear
translocation of activated AR to prevent its localization to androgen
response elements and coactivator recruitment, which induces apoptosis
while inhibiting proliferation of the CRPC cells (Erdogan, 2018 , Jiang,
Chen et al., 2020). A phase III clinical trial reported that patients
treated with Enz survived months longer than those in the placebo group
(Niu, Guo et al., 2018). Despite the
initial therapeutic effect of Enz, CRPC patients will eventually become
resistant to it (Erdogan, 2018). Therefore\sout, it is urgent to
explore new strategies to resolve resistance to Enz.
In this review, we summarized the emerging information on Enz-resistance
mechanisms in CRPC, including AR-related signalling pathways, lineage
plasticity, cytokines dysregulation, and
gene polymorphisms.
Furthermore, new potential therapeutic strategies to treat resistant
CRPC which are promising for better
therapeutic prognoses are introduced. We hope that
these recently discovered mechanisms and emerging solutions provide some
inspiration for the treatment of Enz-resistant CRPC.
Mechanisms ofEnz
resistance in CRPC
Restoration ofa ndrogen receptor
signalling
Androgen
receptor reactivation
AR belongs to the steroid hormone receptor family and functions as a
transcription factor. It contains three different functional domains:
the N-terminal domain (NTD), the DNA-binding domain (DBD), and the
carboxy-terminal ligand-binding domain (LBD). The main role of AR is to
regulate genes encoding protein transcription needed for prostate
function and the promotion of cellular differentiation in the normal
prostate (Wong, Ferraldeschi et al., 2014).
Recently, several studies have demonstrated that AR is highly expressed
and transcriptionally active in CRPC despite Enz treatment (Yuan, Cai et
al., 2014). David Y. et al. (Takeda, Spisák et al., 2018) utilized an
integrative analysis of ChIP-seq in primary specimens and identified a
somatically acquired AR enhancer located 650 kb centromeric to the AR,
which is frequently amplified in CRPC. Under low androgen conditions,
additional copies inserted into this region are sufficient to increase
cell proliferation and reduce cell sensitivity to Enz. Targeting this AR
enhancer may become a potential method for treating resistant CRPC.
Galectin-3, a member of the animal lectin family, significantly
inhibited the therapeutic effect of Enz by increasing the expression of
several AR target genes, such as
kallikrein-related peptidase 3
(KLK3) and
transmembrane protease serine 2
(TMPRSS2). Such enhancement of AR
transcriptional activity and expression of AR-related genes leads to
cell resistance to Enz (Dondoo, Fukumori et al., 2017). In contrast to
its direct influence on AR expression,
BMI1,
a polycomb group protein, binds
the androgen receptor, preventing ubiquitin E3 ligase-mediated AR
protein degradation, which results in sustained AR signalling in CRPC
cells (Zhu, Zhao et al., 2018). By constructing an Enz-resistant
xenograft model, the authors found that the
BMI1 inhibitor
PTC209 significantly decreased
Enz-resistant CRPC tumour growth. Thus, BMI1 is a novel therapeutic
target for CRPC.
Recently, Hwang et al. (Hwang, Seo et al., 2019) indicated that
resistant prostate cancer overexpressed or amplified cAMP-response
element-binding protein 5 (CREB5), mediating the enhancement of AR
activity at a subset of promoters and enhancers, including Myc and genes
involved in the cell cycle. Notably, the pioneering factor forkhead box
protein A1 (FOXA1) was also bound to CREB5/AR-binding sites and was
necessary for CREB5-mediated resistance, which was not eliminated by
Enz. The expression of the proto-oncogene c-Myc is positively associated
with AR expression (Bai, Cao et al., 2019). Alex Bainbridge et al.
(Bainbridge, Walker et al., 2020) observed the significant inhibition of
AR-regulated gene expression upon siRNA-mediated
I-kappa-B
kinase (IKBKE) depletion or
pharmacological inhibition of IKBKE in an LNCaP-derived cell line
resistant to Enz. Further analysis showed that IKBKE regulated AR levelsvia Hippo pathway inhibition to reduce c-Myc levels at
cis-regulatory elements within the AR gene, leading to the formation of
a large pool of AR. Another study showed that
erythropoietin-producing human
hepatocellular (Eph) receptors
positively regulated AR by inducing
proto-oncogene c-Myc expression,
which was critical for Enz resistance. To date, several EphB4 inhibitors
have been tested in clinical trials (Li, Lanman et al., 2020).
Considering the low AR conditions after AR inhibitor treatments, Liu et
al. discovered that deficient AR altered the balance between
eukaryotic initiation factor
4E-binding protein 1(4EBP1) and eukaryotic translation initiation factor
4F (eIF4F) complex formation (Liu,
Horn et al., 2019). The loss of AR increased
eIF4F
assembly to drive the translation of a network of pro-proliferation
mRNAs that were needed for tumour growth. Thus, the suppressing eIF4F
may be a possible strategy for treating Enz-resistant CRPC.
Moreover, inhibitors of signal transducer and activator of transcription
3 (STAT3) and transactivation activity, namely, galiellalactone,
(GPA500) (Thaper, Vahid et al.,
2018), and triptolide (Han, Huang
et al., 2017), have been proven to overcome drug resistance by targeting
AR signalling. Tetra-aryl cyclobutanes are novel AR antagonists that are
structurally different from Enz and inhibit the growth of Enz-resistant
xenograft tumours (Pollock, Wardell et al., 2016).
Proteolysis-targeting chimaeras
(PROTACs) are used in an emerging approach that has recently attracted
considerable attention. They are small molecules that control
intracellular protein levels by recruiting targeted proteins into the
ubiquitin-proteasome system (UPS) for selective degradation (Toure and
Crews, 2016). Jemilat Salami et al. (Salami, Alabi et al., 2018)
performed a head-to-head comparison of Enz and the PROTAC
derivative
ARCC-4
by utilizing different cellular models of PCa drug resistance. The
results indicated that ARCC-4 was better at overcoming resistance in
CRPC cellular models. Furthermore, at low nanomolar concentrations
ARCC-4 inhibited the proliferation of AR-amplified PCa cells. Hence,
PROTAC-mediated AR degradation is a very promising method of
Enz-resistant CRPC therapy. In addition to protein degradation by
PROTACs, the orally bioavailable
AR degrader UT-34
and interferon regulatory factor 8
(IRF8)-induced drugs are alternative strategies for Enz-resistant CRPC
treatment (Ponnusamy, He et al., 2019 , Wu, You et al., 2020).
Androgen receptor
splice variants
AR is composed of NTD, DBD, and LBD. When LBD binds to a ligand, the
conformational changes of LBD allow AR to be transported into the
nucleus where it binds to DNA. AR can undergo alternative splicing to
generate isoforms that contain exons 1 to 3 with a deleted LBD. A
truncated AR protein without the LBD fails to respond to AR targeting by
Enz (J, Bubley et al., 2017).
AR variant 7 (AR-V7) is the most common variant that detected in
circulating tumour cells from patients with CRPC, and it is
significantly increased upon Enz therapy (Antonarakis, Lu et al., 2014).
The splicing factor hnRNPA1 and the long non-coding RNA (lncRNA) Malat1
have been identified as contributors to Enz resistance by promoting the
generation and expression of AR-V7 (Stone, 2017). Furthermore, it has
been reported that tyrosine kinase ACK1 (TNK2) phosphorylates histone H4
functioned as an epigenetic modifier
mediatingc a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit.
The transcription of AR and AR-V7 was increased, leading to Enz
resistance (Mahajan, Malla et al., 2017). Recently, Zhao et al. (Zhao,
Peacock et al., 2019) found that arginine vasopressin receptor 1a
(AVPR1A) was the most downregulated
gene after the depletion of AR-V7 or the versatile coactivator
VAV3. Therefore, suppressing
AVPR1A in CRPC cells can decrease cell proliferation. Utilizing AVPR1A
as a therapeutic target is effective not only for CRPC but also for
bone-metastatic CRPC. High AR/AR-V7 expression and a positive
correlation of AR-V7 with an
E2F score indicated a
supraphysiological testosterone (SPT) response (Lam, Nguyen et al.,
2020). SPT produced an enduring response as shown by the sustained
inhibition of AR-V7, E2F transcriptional output, and the
DNA damage response
(DDR) transcriptome. Hence, SPT
treatment is a potent candidate for treating Enz-resistant CRPC.
Moreover, CRPC cells can recruit more natural killer (NK) cells than
normal prostate epithelial cells in the PCa microenvironment. NK cells
altered microRNA-34 and microRNA-449 to suppress AR-V7 expression and
AR-V7-induced enhancer of zeste homologue 2 (EZH2) expression. Utilizing
these immune cells provides a novel angle for approaching Enz resistance
(Lin, Chou et al., 2017).
Protein homeostasis is another potential mechanism leading to drug
resistance. Here, Liu et al. (Liu, Lou et al., 2018) demonstrated that
UPS was inhibited in Enz-resistant prostate cancer. The interaction
between heat shock protein family member HSP70 and functional E3
ubiquitin ligase STUB1 was required for AR/AR-V7 proteostasis.
Suppressing HSP70 significantly inhibited tumour growth and resistance
to Enz by decreasing AR-V7 expression. Similarly, aldo-keto reductase
family 1 member C3 (AKR1C3) enhanced AR-V7 protein stability viathe alteration of the UPS. AKR1C3-targeted indomethacin greatly reduced
AR/AR-V7 protein expression in vitro and in vivo through the activation
of the ubiquitin-mediated proteasome pathway (Liu, Yang et al., 2019). A
preclinical study indicated that the
anti-apoptotic B cell lymphoma-2
(BCL2)
protein inhibitor ABT263 increased
ROS levels, leading to the inhibition of
ubiquitin specific protease 26
(USP26) activity. As a result, the
ubiquitination and ubiquitin-proteasome-dependent degradation of AR and
AR-V7 were increased (Xu, Sun et al., 2020). Other agents,
including fatty acid synthase
inhibitor (FASN) IPI-9119,
luteolin, triterpenoid antioxidant
drug bardoxolone-methyl (CDDO-Me), and GnRH antagonist degarelix
(Firmagon), have also been identified as inhibitors of the AR-V7 protein
in Enz-resistant CRPC (Zadra, Ribeiro et al., 2019 , Naiki-Ito, Naiki et
al., 2019 , Khurana, Chandra et al., 2020 , Cucchiara, Yang et al.,
2019).
In prostate cancer cells, c-Myc is one of the most overexpressed genes
and is frequently upregulated in CRPC. c-Myc promotes AR gene
transcription and enhances the stability of
full-length AR
(AR-FL) and AR-V proteins without
altering AR RNA splicing. The inhibition of c-Myc, such as with 5-Lox
inhibitor MK591/quiflapon, can interrupt oncogenic c-Myc signalling to
kill Enz-resistant cells (Bai, Cao et al., 2019 , Monga, Subramani et
al., 2020). GATA2 is also a
critical regulator of AR-Vs, and its cistromes considerably overlap with
bromodomain
and extra-terminal (BET) proteins
(Chaytor, Simcock et al., 2019). Small-molecule inhibitors of GATA2 are
potent agents that suppress the expression and transcriptional function
of both AR-FL and AR-Vs (He, Lanz et al., 2014). Moreover, the selective
inhibitors of the small-molecule
BET, namely, JQI and
I-BET762, have also been proven to
be effective in treating Enz-resistant CRPC by targeting the
amino-terminal bromodomains of
bromodomain-containing protein 4
(BRD4) (Asangani, Dommeti et al.,
2014 , Chan, Selth et al., 2015). In addition, later research
demonstrated that the conserved
zinc-finger transcription factor
(ZFX) interacted with AR-V7 and
co-occupied the unique AR-V7-binding sites in target gene promoters in
CRPC cells. Targeting this ZFX can provide a more effective
Enz-resistant CRPC treatment (Cai, Tsai et al., 2018).
Considering the difficulty in distinguishing the
activity of AR-Vs from that of AR-FL, there is urgent demand for AR-V
cell models that enable the identification of AR-V-specific functional
and phenotypic read-outs that are not influenced by
AR-FL.
Kounatidou et al. (Kounatidou, Nakjang et al., 2019) established
CWR22Rv1-AR-EK cells in which the expression of
AR-FL was abrogated and all
endogenous AR-Vs were retained through CRISPR/Cas9 editing. By utilizing
this model, they validated that AR-Vs regulated a DDR gene network,
including an AR-FL-like ‘BRCAness’ signature, which was essential for
cell survival after ionizing radiation treatment. Moreover, AR-Vs
interacted with poly (ADP-ribose) polymerase
(PARP) and depended on PARP
activity for transcriptional function. Notably, suppressing PARP
influenced AR-V gene expression and reduced the growth of CRPC cells.
The successful establishment of this model provided a platform for
accurate studies of AR-Vs in CRPC.