Induction of autophagy
Autophagy is a highly conserved catabolic process that regulates
cytoplasmic biomass, organellar abundance, and organellar distribution,
removing harmful protein aggregates and intracellular toxins. Moreover,
autophagy provides regenerated metabolites to promote tumour cell
survival. Autophagy can be regulated in multiple ways. In nutrient-rich
conditions, the PI3K/AKT pathway inhibits autophagy through the
activation of the mammalian target of rapamycin (mTOR). In the absence
of nutrients or growth factors or under hypoxic conditions,
autophagy is activated by
the
AMPK pathway, resulting in the upregulation of the transcription of
autophagy genes and the inhibition of mTOR/S6K/4EBP activity by the
phosphorylation of TSC2. Autophagy contributes to Enz resistance through
the activation of AMPK and inhibition of the mTOR pathway (Farrow, Yang
et al., 2014 , Smith and Macleod, 2019 , Nguyen, Yang et al., 2014).
Inhibition ofapoptosis
Apoptosis is a form of programmed cell death that plays a critical role
in organism development and tissue homeostasis, resulting in the removal
of damaged cells. However, cancer cells have lost their ability to
undergo apoptosis, which lead to their uncontrolled proliferation. These
cancer cells usually overexpress proteins related to the resistance of
apoptosis cascade activation (Pistritto, Trisciuoglio et al., 2016).
BCL2 proteins are frequently overexpressed in cancers and are associated
with disease progression and treatment resistance. The BCL2 family
contains anti-apoptotic proteins, including BCL-2, BCL-XL, MCL-1, and
BCL-w, and pro-apoptotic proteins, including BAX, BAK, and BIM
(Siddiqui, Ahad et al., 2015).
It has been reported that the anti-apoptotic
BCL2 protein family members
BCL-XL and MCL-1 are critical factors
mediating Enz resistance. BCL-XL and MCL-1 bind and separate
pro-apoptotic proteins BIM and BAX, resulting in the inhibition of
apoptosis. Moreover, PI3K/AKT signalling is activated by Enz and can
suppress BAD, a BH3-only protein that activates pro-apoptotic signalling
through the inhibition of BCL-XL,
mediating apoptosis evasion. However, different prostate cell lines
showed differences in the expression of pro-apoptotic and anti-apoptotic
BCL2 family proteins. Another study found that BCL-2 was exclusively
upregulated in CRPC cells, but not BCL-XL and MCL-1, and was directly
induced by Enz, which enhanced cancer stem cell (CSC)-enriched
holoclones (Pilling and Hwang, 2019). The BCL-2-selective inhibitor
ABT-199 combined with Enz in LNCaP cells
drastically inhibited Enz-resistant CRPC. Thus,
targeting BCL2 protein signalling plus Enz is a promising therapeutic
treatment for ENZ resistance (Li, Deng et al., 2018). The novel small
molecule PAWI-2 reduced the protein levels of BCL-2, BCL-XL, and MCL-1
to normal levels in the presence of Enz. PAWI-2 can inhibit tumour
growth either as a single agent or in combination with Enz to overcome
Enz resistance in CRPC (Cheng, Moore et al., 2019).
The inhibitor of apoptosis protein (IAP) family also inhibits apoptosis
by regulating the activity of caspases, and some IAPs are overexpressed
in cancers, mediating tumour pathogenesis, progression, and resistance.
The IAP family contains eight members, including NAIP (BIRC1), cIAP1
(BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), survivin (BIRC5), Apollon/BRUCE
(BIRC6), ML-IAP/LIVIN (BIRC7), and
ILP-2 (BIRC8) (Krajewska, Krajewski et al., 2003). BIRC6 played a vital
role in Enz resistance in a high-fidelity, Enz-resistant,
patient-derived CRPC tissue xenograft model named LTL-313BR. BIRC6 is an
exclusive IAP member among the eight proteins that were upregulated in
both Enz-resistant systems. Furthermore, an BIRC6-targeting antisense
oligonucleotide (ASO-6w2) inhibited the growth of LTL-313BR xenografts
and increased the apoptosis rate (Luk, Shresth et al., 2016). Another
IAP inhibitor, AEG40995, expanded the caspase-mediated apoptotic
response to Enz by inducing TNF-α signalling. Combining Enz with IAP
antagonists may overcome Enz resistance in CRPC (Pilling, Hwang et al.,
2017).
Lineage
plasticity and phenotype switching
Cancer cell plasticity can be defined as the ability of a cell to
substantially alter its characteristics and exhibit a new phenotype that
is closer to a unique developmental lineage. Cell plasticity allows
tumour cells to reversibly convert to cellular properties independent of
the drug-targeted pathways. The increased diversity of tumour cells is
associated with therapeutic resistance and metastasis, suggesting that
the retention of pluripotent progenitors and the persistence of
progenitor cells can re-proliferate resistant and metastatic tumour
cells with different phenotypes. PCa is driven by androgens and developsvia ligand-mediated AR signalling. Although early treatment is
effective, PCa cells can adapt to androgen deprivation and restore AR
signalling, eventually progressing to CRPC, which can be treated by the
AR antagonist Enz. However, after long-term AR inhibition, the tumour
archetype changes, which results in histological de-differentiation and
cell lineage alterations in the form of the EMT and/or neuroendocrine
differentiation (Davie, Beltran et al., 2018).
The EMT is a process in which the epithelial phenotype is lost and
mesenchymal characteristics are acquired. The expression of EMT drivers
(ZEB1, ZEB2, Snail, Twist, and FOXC2) and mesenchymal markers
(N-cadherin, fibronectin, and vimentin) in PCa cells was significantly
enhanced by Enz treatment, promoting the migration of PCa cells and
inducing the transformation of prostate cancer into spindle and
fibroblast-like forms (Miao, Yang et al., 2017). Transforming growth
factor β
(TGF-β)
is one of several molecular drivers contributing to the EMT process
(Quintanal-Villalonga, Chan et al., 2020). RNA-seq data from CTCs have
shown that TGF-β and cyclin D1
(CCND1)
signalling pathways are significantly upregulated in drug-resistant
CTCs. Moreover, the key regulators of the TGF-β pathway, namely,
SMAD family member 3 (SMAD3) and
CCND1, participate in the resistance to Enz (Pal, Patel et al., 2018). A
new study found that the loss of the transcription factor FOXA1 led to
the distinct upregulation of TGF-β3, inducing TGF-β signalling and the
EMT. Compared with a single Enz treatment, adding the
TGF-β receptor I kinase inhibitor
galunisertib (LY2157299) enhanced
the efficacy of Enz in inhibiting CRPC xenograft tumour growth and
metastasis (Song, Park et al., 2019 , Paller, Pu et al., 2019). The
transcription factor STAT3 is also associated with the EMT and ENZ
resistance. Metformin, a
TGF-β1/STAT3 axis inhibitor,
alleviated resistance to Enz by suppressing the EMT (Liu, Tong et al.,
2017). Recently, the quinazoline-derived agent DZ-50 was found to
reverse EMT to MET by targeting insulin-growth factor binding protein-3
(IGFBP-3), resulting in the re-differentiation of prostate cancer.
Phenotype reversal in prostate cancer cells contributes to
re-sensitizing PCa cells to Enz to overcome resistance to anti-androgen
therapy (Hensley, Cao et al., 2019).
Previous studies have demonstrated that the loss of the retinoblastoma
tumour suppressor gene RB1 promotes lineage plasticity and the
metastasis of PCa with PTEN mutations.
Simultaneously losing RB1 and the
tumour suppressor
gene TP53 facilitated resistance to anti-androgen
therapy. RB1 and TP53
suppress
epigenetic reprogramming factors
such as EZH2 and SOX2, which play
critical roles in generating induced pluripotent stem cells (Ku, Rosario
et al., 2017 , Ge, Wang et al., 2020). The
enhancer of EZH2 is usually
overexpressed in PCa and associated with poor prognosis. EZH2 directly
binds to the promoter of prostate-specific antigen (PSA) and suppresses
its expression in Enz-resistant PCa cells. EZH2 inhibition/depletion
boosts the efficacy of Enz in resistant CRPC (Xiao, Tien et al., 2018).
Studies have proven that AR inhibition drives the transformation of
prostate cancer into a neuroendocrine phenotype (Carceles-Cordon, Kelly
et al., 2020). The understanding of neuroendocrine prostate cancer
(NEPC) biology remains poorly understood. Zhang et al. (Zhang, Zheng et
al., 2018) demonstrated that neuroendocrine differentiation (NED) and
angiogenesis were regulated by ADT-activated CREB, which in turn
enhanced EZH2 activity. Notably, anti-angiogenic factor
thrombospondin-1(TSP1) was a
direct target of EZH2 epigenetic repression. Castration activated the
CREB/EZH2 axis, thereby upregulating NE markers and downregulating TSP1.
The CREB/EZH2/TSP1 axis was found to be a new pathway for CRPC/NEPC
treatment. The long non-coding RNA-p21
(lncRNA-p21), as a TP53
co-repressor, was upregulated upon Enz treatment to induce NED. Enz
promoted the transcription of lncRNA-p21 by changing the AR binding
affinity for different AREs, converting the function of EZH2 from being
a histone-methyltransferase to a non-histone methyltransferase, which
led to STAT3 methylation resulting in NED. The
Enz/AR/lncRNA-p21/EZH2/STAT3 signalling pathway provides novel targets
for developing new therapeutic methods for
Enz-resistant CRPC (Luo, Wang et al., 2019). SOX2 is a reprogramming
transcription factors and is upregulated after the function of TP53 and
RB1 is lost, leading to drug resistance. However, previous studies did
not indicate whether elevated SOX2 was sufficient to induce NE marker
expression or Enz resistance (Mu, Zhang et al., 2017). By utilizing
LNCaP cells engineered for the inducible elevation of SOX2, Metz et al.
(MetzWilder et al., 2020) showed that increasing SOX2 did not diminish
the growth inhibition of Enz or its ability to reduce the expression of
PSA. Thus, the identification of the exact mechanisms of the synergistic
effects of SOX2 elevation in NE plasticity and the discovery of
therapeutic targets to restore cell sensitivity to Enz are urgently
needed. SOX9 is a recently discovered SOX family member that is
upregulated in stem-like reprogrammed prostate cancer cells. Transient
SOX9 expression facilitated the resistance to Enz via NF-κB dimer
activation (Nouri, Massah et al., 2020). Another epigenetic remodelling
regulator, the RE1-silencing
transcription factor (REST),
mainly binds to chromatin in the proximity of neuron-specific genes.
REST silencing is a key driver of neuron-specific gene expression in
prostate cancer (Flores-Morales, Bergmann et al., 2019).
Serine/arginine repetitive matrix
4 (SRRM4) is an upstream regulator
of REST gene functions in PCa cells, promoting the progression of NEPC.
Both AR inhibition and RB1 and TP53 loss enhanced the SRRM4-induced
acquisition of the neuroendocrine phenotype in prostate cancer (Li,
Donmez et al., 2017). A new study (Tiwari, Manzar et al., 2020) showed
that
serine peptidase inhibitor Kazal
type 1 (SPINK1) was transcriptionally repressed by AR and its
corepressor REST. AR inhibition triggered SPINK1 upregulation, and
increased SOX2 expression directly transactivated SPINK1, which
facilitated the acquisition of the NE-like phenotype.
In addition, neuroendocrine lineage reprogramming is constitutively
associated with MYCN. N-Myc is
encoded by MYCN and overexpressed in NEPC. Epigenomic and transcriptomic
reprogramming can be induced by N-Myc overexpression and its subsequent
DNA binding, contributing to the lineage-plastic phenotype.
Additionally, EZH2 inhibitors reversed the N-Myc-induced inhibition of
epithelial lineage genes. N-Myc depended on the BET family of epigenetic
readers, especially BRD4, which promote the expression of the target
genes. N-Myc also overlapped with HOXB13, a lineage-specific
homeodomain-containing transcription factor, located predominantly at
genomic loci implicated in neural lineage specification (Dardenne,
Beltran et al., 2016 , Berger, Brady et al., 2019). Nerlakanti et al.
(Nerlakanti, Yao et al., 2018) found that
BRD4 epigenetically promotes HOXB13
expression and binds the enhancer of HOXB13. This BRD4-HOXB13 axis
activated AR-independent cell cycle programmes, promoting the
proliferation of CRPC. The neural
transcription factor BRN2 was also a
major driver of NEPC and was required for the expression of terminal NE
makers and the aggressive growth of Enz-resistant CRPC tumours (Bishop,
Thaper et al., 2017). Mucin 1
(MUC1) (Yasumizu, Rajabi et al.,
2020) is a heterodimeric protein that suppresses AR signalling and
induces BRN2 in association with the induction of MYCN and EZH2, and
upregulation of the NE markers related to NEPC progression. BRN4 was
found to be a novel transcription factor that drove neuroendocrine
differentiation by interacting with BRN2. Enz treatment contributed to
the amplified release of BRN2 and BRN4 in PCa extracellular vesicles
(EVs), facilitating neuroendocrine differentiation (Bhagirath, Yang et
al., 2019). The tumour suppresser
protein
kinase C
(PKC)λ/ι
also played a critical role in NEPC progression. The downregulation of
PKCλ/ι
promoted cancer cell plasticity and NEPC differentiation via the
mTORC1/activating transcription
factor 4
(ATF4)/PHGDH
axis. This metabolic reprogramming augmented intracellular
S-adenosyl methionine
(SAM) levels to meet the
epigenetic changes that facilitated the development of NEPC properties.
Notably, the loss of PKCλ/ι led to the activation of mTORC1, which drove
an ATF-dependent gene transcription programme that increased flux
through the one-carbon pathway (Reina-Campos, Linares et al., 2019).
Tumour heterogeneity has been widely considered to negatively affect
targeted cancer therapy, especially when genomic changes occur at the
same time, which reduces the reliance on proto-oncogene drivers. In an
in vivo small hairpin RNA (shRNA) screening of 730 prostate cancer
genes, CHD1 loss was found to be
the cause of
anti-androgen
resistance. ATAC-seq and RNA-seq analyses demonstrated that CHD1 loss
led to overall changes in open and closed chromatin and associated
transcriptomic changes, conferring resistance to Enz. Notably, four
transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) were found by
screening to participate in Enz resistance, and they can be future
therapeutic targets. To better
understand the genetic and epigenetic mechanisms of neuroendocrine
transformation, the CTC-derived explant (CDX) model is a distinct tool
that can also be used as an effective therapeutic
screening approach (Zhang, Zhou et al., 2020 , Faugeroux, Pailler et
al., 2020).