Discussion
The present study realized a specific blockade of the AR and its target
gene IGF-1, which induces both apoptosis and cell cycle arrest by a
novel antiandrogen SBF-1. This unique compound was used for the
treatment of prostate cancer in a xenograft model. As we know, AR is a
hormone inducible transcription factor, which drives expression of tumor
promoting genes and represents an important therapeutic target in
prostate cancer (Dalal et al., 2018). Higher expression of AR in
prostate cancer with shorter overall survival was found in patients
(Fig. S3). Currently, small molecule drugs used in the treatment of
prostate cancer mainly interfere with steroid recruitment to prevent
AR-driven tumor growth (Caboni & Lloyd, 2013; Lallous, Dalal, Cherkasov
& Rennie, 2013). However, those kinds of small molecules are rendered
ineffective in the advanced prostate cancer by emergence of LBD
mutations or expression of constitutively active variants such as AR-V7
that lack the LBD. As the cell lines of prostate cancer, LNCaP cells
express AR with a novel mutation T878A in the AR-LBD, which is similar
to human prostatic adenocarcinoma, and their growth can be inhibited by
androgen withdrawal. Another cell line PC3/AR+ cells
have a stable expression of ARWT, which is originally a
sub-line from the parental cells AR negative PC3 cells. By using these
two cell lines, we aimed at finding a novel inhibitor against prostate
cancer in both ARWT and ARmutant levels
through targeting AR.
As the result, SBF-1 strongly inhibited proliferation of both LNCaP and
PC3/AR+ cells, suggesting a possibility that this
compound can be used for the treatment of prostate cancer with both
ARWT and ARmutant. For investigating the
mechanisms underlying its effect, we next found that SBF-1 showed a
significant inhibition on the adhesive ability of both LNCaP and
PC3/AR+ cells to fibronectin and laminin, increased
the percentage of apoptotic cells, and caused the cell cycle arrest in
G1 and G2/M phase.
AR can be activated by the binding of endogenous androgens, including
testosterone and DHT (Gao, Bohl & Dalton, 2005). The AR mediates the
growth of benign and malignant prostate in response to DHT. In patients
undergoing androgen deprivation therapy for prostate cancer, AR drives
prostate cancer growth despite low circulating levels of testicular
androgen and normal levels of adrenal androgen (Mohler et al., 2011).
Usually, prostate cancer cells gradually lose their dependence on
androgen signaling through the AR and become resistant to hormonal
therapy. It is now established that throughout various hormonal
manipulations, castrate-resistant prostate cancers continue to express
the AR (Hobisch, Culig, Radmayr, Bartsch, Klocker & Hittmair, 1995;
Hobisch, Culig, Radmayr, Bartsch, Klocker & Hittmair, 1996; Sadi &
Barrack, 1991; Tilley, Lim-Tio, Horsfall, Aspinall, Marshall & Skinner,
1994; van der Kwast et al., 1991) and IGF-1 as the transcription product
of AR has been shown to independently activate the AR in the absence of
DHT, with a mechanism that involves downstream phosphorylation of either
the AR or its associated proteins (Culig et al., 1994; Gregory et al.,
2004). It has also been reported that inhibition of IGF-1 could suppress
prostate cancer cell growth (Zheng et al., 2012). On the other hand,
IGF-1 will also activate its downstream proteins such as AKT kinase,
which regulates the cell proliferation and survival. AKT is usually
activated by hormones, growth factors, and chemical drugs (Zheng et al.,
2015; Zheng et al., 2012; Zhu et al., 2015). The downstream forkhead
transcription factor family FOXO plays a vital function in cell
apoptosis and survival in variety of cell types, which could be
phosphorylated by AKT kinase (Tzivion, Dobson & Ramakrishnan, 2011).
Those findings inspired us to test the effect of SBF-1 on the AR
signaling while stimulated with DHT. As shown in Fig. 2A, the
expressions of IGF-1, PCNA, Bcl-2, pARS515,
pAKTS473 and pFOXO1S256 were greatly
decreased in both LNCaP and PC3/AR+ cells by SBF-1
without effects on the AR, AKT1, and FOXO1 expressions. This result
suggests that SBF-1 may downregulate both AR/IGF-1 and
IGF1/AKT/FOXO1/PCNA pathways. When we used DHT to stimulate AR, DHT
greatly increased the AR expression as well as the AR phosphorylation
and the expressions of downstream proteins IGF-1 and PCNA. Against this,
SBF-1 down-regulated DHT-increased expressions of AR,
pARS515, IGF-1, and PCNA in both cell lines (Fig. 2B).
This result indicates a dual effect of SBF-1 on AR/IGF1 axis and their
down-stream signaling. SBF-1 also strongly suppressed the mRNA
expression of IGF-1 and PCNA even in the case of DHT-doubled expression
(Fig. 2C). These findings suggest a possibility of SBF-1 to directly
block the gene transcription mediated by AR that is known to bind the
promoter of its target genes.
To examine how SBF-1 affects AR and its subsequent signaling pathways,
we hypothesized that SBF-1 might directly bind to AR since it is a
steroidal glycoside. Both MST and ITC assays were used to examine the
binding affinity between SBF-1 and purified ARWT. After
a strong binding was concluded (Fig. 2D, and E), we performed the
polarity shift assay to check whether the AR-LBD is the binding site of
SBF-1 because AR-LBD is recognized as the vital domain to be targeted in
prostate cancer therapy (Wang et al., 2006). However, despite the
significant binding to AR-LBD by DHT as a positive control, what we
hypothesized for the binding of SBF-1 to AR-LBD is absolutely negative
(Fig. 2F). Above findings suggest that SBF-1 does bind to the AR protein
but AR-LBD is not the binding site.
AR signaling in CRPC tumor epithelial
cells could be caused by activating AR point mutations. Such mutations
are rare in untreated PC, but detected in 15–20% of CRPC patients
(Grasso et al., 2012; Robinson et al., 2015; Taylor et al., 2010), and
in up to 40% of CRPC patients treated with AR antagonists (Balk, 2002).
Activating AR point mutations generally affects the c-terminal LBD,
while about one-third occur in the transactivating NTD (Gottlieb,
Beitel, Nadarajah, Paliouras & Trifiro, 2012; Steinkamp et al., 2009),
resulting in broaden ligand specificity. The first and most frequently
identified AR point mutation is the flutamide-driven T878A mutation
(Fenton et al., 1997; Taplin et al., 1999; Taplin et al., 1995;
Veldscholte et al., 1990), while W742C also has been reported after
treatment with first-generation AR antagonists (Lallous et al., 2016;
Steketee, Timmerman, Ziel-van der Made, Doesburg, Brinkmann & Trapman,
2002; Tan et al., 1997; Taplin et al., 2003; Watson, Arora & Sawyers,
2015; Yoshida et al., 2005). The T878A and L702H mutations have been
observed in CRPC patients during abiraterone treatment (Lallous et al.,
2016; Steketee, Timmerman, Ziel-van der Made, Doesburg, Brinkmann &
Trapman, 2002; Tan et al., 1997; Taplin et al., 2003). Also, F876L
mutation confers an antagonist-to-agonist switch that drives phenotypic
resistance (Korpal et al., 2013). Taken together, we decided to cover
these frequently occurring mutations in CRPC through the construction of
AR mutants as shown in (Fig. 3) and determined the effect of SBF-1 on
those mutants. As the result, SBF-1 showed a strong binding to all the
mutant constructs, L702H, W742C, F876L and T878A (Fig. 3A). It should be
emphasized that SBF-1 showed an almost complete inhibition against the
DHT-increased risen activity of ARWT and the mutants
(Fig. 3B). Above results suggest that SBF-1 may have a novel binding
site of SBF-1 on AR.
Growing evidence suggests that castration resistance of prostate cancer
may be partially mediated by AR splice variants lacking the LBD coding
sequence, which leaves only the NTD and DBD as viable domains that are
targetable by small molecules (Dehm, Schmidt, Heemers, Vessella &
Tindall, 2008; Guo et al., 2009; Hu et al., 2009; Sun et al., 2010a;
Watson et al., 2010). Inhibition of splice variant transcriptional
activity would be a significant breakthrough in the development of a new
class of anti-AR drugs (Dalal et al., 2014). To find the binding site of
SBF-1 in AR, we constructed an ARE-1 sequence, which known to be
consensus recognition site for the AR (Denayer, Helsen, Thorrez, Haelens
& Claessens, 2010). This constructed sequence was incubated with each
purified ARWT or AR∆DBD (Androgen
receptor lacking DBD). As the result, SBF-1 did show a binding to
ARWT but not AR∆DBD (Fig. 4A, and B),
suggesting that AR-DBD might be the target domain for SBF-1. The ITC
technique confirmed that SBF-1 totally failed to show any binding signal
with the purified AR∆DBD (Fig. 4C). To understand the
result of SBF-1 binding to AR-DBD, furthermore, the AR-induced gene
expression enrichment assay demonstrated that DHT induced a significant
increase in the AR target gene IGF-1 enrichment while SBF-1 diminished
this enrichment (Fig. 4E). These results suggest that SBF-1 might block
AR from binding to its target genes by binding to its DBD.
The binding between SBF-1 and AR or its mutants leads to further insight
into where could SBF-1 binds to AR, and especially all the found mutants
are located in AR-LBD. This hints us that the current efforts for the
treatment of prostate cancer to target AR may become resistant after the
mutation accompanying with the malignant progression, and a novel
inhibitor targeting the new domain of AR is needed. To our knowledge, a
small molecule EPI-001 was reported to block transactivation of the NTD
and was specific for inhibition of AR without attenuating
transcriptional activities of related steroid receptors (Andersen et
al., 2010). On the other hand, targeting AR-DBD may be a new strategy
for CRPC treatment (Dalal et al., 2014). However, there is still little
development of inhibitors that specifically target the NTD or DBD of the
AR (Caboni & Lloyd, 2013; Lallous, Dalal, Cherkasov & Rennie, 2013).
Here we propose a solid proof of strong anti-tumor small molecule SBF-1
that inhibit the growth of advanced prostate cancer through the binding
to AR-DBD. That is why SBF-1 can bind both AR and its multiple mutants
for the inhibition of both ARWT and
ARmutant cells. In fact, IGF-1/AKT/FOXO1 axis has been
known to be of importance in prostate cancer castration resistance.
Stimulation of AR through androgens, i.e. DHT has shown to activate the
antiapoptotic IGF-1/AKT/FOXO1/PCNA pathway in LNCaP and
PC3/AR+ cells (Zhao, Tindall & Huang, 2014). The
activation of IGF-1/AKT/FOXO1/PCNA signaling is critical for mediating
cell survival and involved in the castration resistance through the
modulation of AR expression and its down-stream signaling. In this
study, we used LNCaP cells that express AR, including the common AR
mutation, ETV1 overexpression, and PTEN deletion, which serves as a good
model to examine late-stage prostate cancer with metastatic potential,
and also, PC3/AR+ cells that express AR but WT and has
normal PTEN expression (Kim, Park & Dong, 2006). These two cell lines
will give a broad spectrum of how mutant prostate cancer cases could be
affected by the treatment of SBF-1.
Currently, targeting AR-DBD has become increasingly in-focus as the
AR-DBD exists in all forms of AR and its mutants, also helps in directly
treating castration resistance in prostate cancer (Dalal et al., 2018).
Blocking AR from regulating its target gene IGF-1 could help overcome
castration resistance, and stimulation of IGF-1 using glucose will lead
to the over-expression of IGF-1, which could explain how SBF-1 works.
SBF-1 almost completely overcame the activated IGF-1 signaling through
glucose intake or DHT stimulation (Fig. 5). This characteristic suggests
that SBF-1 may have different mode of actions from the current agents
against prostate cancer despite the endogenous levels of androgens and
AR mutation, which is quite beneficial to various situations of PCa
patients. Finally, such unique mechanism of SBF-1 was tested on prostate
cancer growth in nude mice, where SBF-1 significantly reduced the tumor
size in mice bearing either ARWT or
ARmutant cells, and prolonged the survival rate at very
low doses in a dose-dependent manner, with a strong decrease in the
IGF-1 protein and its downstream signaling but without loss of body
weights of tumor-bearing mice (Fig. 6). These findings provided great
advantages for the targeting of AR-DBD against AR wild type and mutant
prostate cancer by SBF-1.
In summary, we present a novel antiandrogen targeting AR-DBD, for the
first time, which attenuates AR in a wide spectrum of different variants
for the treatment of prostate cancer especially the castration resistant
prostate cancer (summarized in Fig. 7). Our findings suggest a better
strategy in dealing with the development of advanced prostate cancer by
targeting AR-DBD rather than the conventional methods.