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.