Introduction
Prostate cancer is considered as one of the most common cancers in men.
There are 174,650 estimated new cases in 2019 while 31,62 estimated
deaths in USA (Siegel, Miller & Jemal, 2019). Although the etiology of
prostate cancer remains unclear, aging and a familial history are the
two most significant risk factors that increase a man’s chances of
developing the disease. Prostate cancers are reliant on male sex
hormones called androgens for growth and survival. Androgens exert their
effects throughout the body by binding to the androgen receptor (AR), a
ligand inducible transcription factor that is critical not only for the
growth and maintenance of the normal prostate, but also the development
and progression of prostate cancer. Thus, manipulations of androgens in
the body through lowering its levels or prevent androgens from binding
to AR is considered as reliable strategy in the treatment of prostate
cancer, which could effectively reduce symptoms and tumor burden in
almost all the patients. It is now accepted that despite various
hormonal manipulations, the AR continues signaling throughout prostate
cancer progression and remains the key therapeutic target. Androgens are
crucial for the growth, development and maintenance of the prostate. The
main androgen circulating in the male bloodstream is testosterone, which
is converted via an enzyme called 5-α reductase to androgen
dihydrotestosterone (DHT) in the prostate. Although similar in
structure, DHT has a higher dissociation constant with AR (Deslypere,
Young, Wilson & McPhaul, 1992; Dupaul-Chicoine et al., 2010; Kaufman &
Pinsky, 1983). Like other hormone receptors, the exons of the AR code
for functionally distinct regions of the protein. Exon 1 codes for the
amino terminal transactivation domain (NTD), which plays a key role in
transactivation, dimerization, and recruitment of co-regulator(s)
involved in transcriptional function. Following NTD is the DNA binding
domain (DBD) and ligand binding domain (LBD) of AR. The DBD of AR
contains PBox recognition helix and DBox site that control DNA
specificity and dimerization (Shaffer, Jivan, Dollins, Claessens &
Gewirth, 2004). AR inside the nucleus binds to specific recognition
sequences, androgen response elements (AREs), in the promoters and
enhancers of target genes (Claessens, Denayer, Van Tilborgh, Kerkhofs,
Helsen & Haelens, 2008). One of its main transcription products is
insulin-like growth factor 1 (IGF-1), which acts on its receptor IGF-1R
and activates MAPK and PI3K signaling to mediate cell proliferation and
growth (Guntur & Rosen, 2013). The LBD of AR takes part in the
posttranslational modifications of AR. The strong ligand-independent
activation function 1 (AF1) in N terminus of AR interacts with the LBD.
In response to ligand binding i.e. DHT, LBD becomes phosphorylated and
triggers translocation of AR into the nucleus (Denayer, Helsen, Thorrez,
Haelens & Claessens, 2010; Kuiper & Brinkmann, 1995; Schaufele et al.,
2005; Wong et al., 2004).
From above findings, the hormonal therapy is usually conducted against
prostate cancer to block AR signaling, either reducing androgen
production or antagonizing AR. Current clinically used antiandrogens
such as flutamide, bicalutamide, and enzalutamide mainly target the
hormone binding pocket (HBP) that is located in the LBD of AR. However,
the drug resistance rises from the fact that tumors often develop
various mechanisms to reactivate androgen receptor signaling. This
frequently involves sensitizing the tumor to low level of androgens
through overexpression of AR, which in roughly 30% of
castrate-resistant prostate cancer (CRPC) occur through amplification of
the receptor (Linja, Savinainen, Saramäki, Tammela,
Vessella & Visakorpi, 2001; Visakorpi et al., 1995). Antiandrogens can
lose their antagonism for AR and behave as partial agonists in the
setting of AR overexpression (Chen et al., 2004).
Although mutation of AR does not seem to be common in early stages of
prostate cancer, mutations have been well documented upon relapse in the
setting of antiandrogen therapy (Gottlieb, Beitel,
Nadarajah, Paliouras & Trifiro, 2012). There has been a wide degree of
difference in reports of incidence of AR mutations, but recent next gene
sequencing studies confirmed that they occur in 20% of patients with
metastatic CRPC (Beltran et al., 2013). These mutations often increase
ligand promiscuity, allowing other endogenous androgens (or hormones) to
activate AR signaling, and some of them can convert antiandrogens into
agonists of the mutant receptor (Hara et al., 2003). The clinical
observation of antiandrogen withdrawal syndrome, where prostate-specific
antigen (PSA) levels decrease or tumors regress upon withdrawal from
bicalutamide or flutamide treatment, correlates with the presence of AR
mutations (Paul & Breul, 2000).
Recent work has also shown that expression of AR splice variants, that
have distinct C-terminal extensions encoded by cryptic exons from the
intron regions between canonical coding exons of AR, occurs in
castration resistance (Hu et al., 2009). These variants often happen in
the LBD and may exhibit constitutive AR activation. Although reports
shown that these variants still require full-length AR to function,
recent studies have suggested that expression of these variants is
sufficient to elicit transcription of AR target genes in the absence of
androgen and confers resistance to the novel antiandrogen enzalutamide
(Li et al., 2013; Watson et al., 2010). As mentioned above, prostate
cancer is continually undergoing AR mutations that switch the
antiandrogen from antagonist to agonist and eventually relapses to
lethal CRPC. To combat the mutation driven drug resistance, several
rational antiandrogen design strategies have been developed, such as
targeting HBP of AR (Tian, He & Zhou, 2015). However, it seems like
ongoing running race and remains a big challenge.
In this study, we report a novel antiandrogen, a synthetic steroidal
glycoside SBF-1, directly targeting AR-DBD but not AR-LBD to exhibit a
strong cytotoxicity towards two prostate cancer cell lines, LNCaP and
PC3/AR+ cells, with considerably low
IC50. This compound strongly attenuates
IGF1/AKT/FOXO1/PCNA signaling, and eventually leads to apoptosis and
cell cycle arrest. Interestingly, SBF-1 shows a significant binding to
AR-DBD of whether the wild type AR or different mutant AR isoforms and
blocks the interaction between the AR (AR mutants) with their target
genes. SBF-1 is among such a handful developed antagonists against the
binding of AR to the genes, in our knowledge, which is quite different
from those blocking androgen-AR interaction, and can be considered as a
potential compound for treating advanced prostate cancer cases with AR
mutation.