CWR22 xenografted tumor was established from human primary prostate tumor in the patient with bone metastases [21,22
]. CWR22Rv1 is a CRPC cell line derived from the CWR22R subline, which was isolated from the recurrent tumor of the androgen-dependent CWR22 cells in castrated mice [23,24
]. The origin of CWR22Rv1 cells was different from other CRPC cell lines, such as C4-2 or C81, that were from metastatic lymph nodes of mice xenografted with human PCa cell line, LNCaP [25–28
]. CWR22Rv1 expresses an exon 3-duplicated fAR in addition to the multiple spliced forms [7–9,29
]. Recent studies [30
] revealed a duplicated AR locus mediated by repetitive elements, which may account for the aberrantly spliced forms of AR. The truncated receptor has been suggested to be derived from two possible pathways, the aberrant splicing as is the case for AR3 and protease cleavage of exon 3-duplicated fAR [29,31
]. A different CWR22-derived relapsed cell line, CWR22R1 [32
], also displayed abundant truncated AR [9
]. Thus, in the evolution of CRPC such as with CWR22, receptor truncation typified by AR3 seems to be a dominant underlying mechanism. Although the detailed truncation points may be different, most the reported species lack the LBD, and are expected not to respond to androgen and antiandrogens. As such, drugs which target the LBD may not work as effectively. The present study is designed to investigate the efficacy of drugs that target degradation of both fAR and the truncated receptor.
In this study, we used various PCa cell lines, including C81, C4-2, and CWR22Rv1 cells, as well as C81/AR3, C4-2/AR3, and CWR22Rv1-fARKD cells to study AR3 in vitro
function. The expression level of fAR and AR3 in these PCa cells was different: in C81 and C4-2 cells, most AR expression was fAR (fAR
AR3). In C81/AR3 and C4-2/AR3 cells, fAR expression level was equivalent to AR3 (fAR = AR3). In contrast, AR3 expression level was higher than fAR (fAR < AR3) in CWR22Rv1 cells, but most AR expression was AR3 in CWR22Rv1-fARKD cells (fAR
AR3). By characterizing these various PCa cells with differential expression ratios of fAR to AR3, we concluded that AR3 plays a critical role to promote PCa cell growth at some selective stages, which is similar to early findings showing that overexpression of AR3 in LNCaP cells promoted the cell growth [9
]. Furthermore, our data showed DHT-induced AR-targeted genes were obviously enhanced with addition of AR3 in C81 and C4-2 cells, suggesting that AR3 might be able to cooperate with fAR to promote DHT-induced AR-targeted genes. This is in agreement with a previous report showing that constitutively active AR splice variants (AR-V7) required fAR to promote AR-targeted genes and cell growth in LNCaP cells in the presence of androgen [12
However, our data also showed that cell growth of CWR22Rv1-fARKD was substantially increased compared to that of CWR22Rv1 cells under androgen-free conditions. Specifically knocking down AR3 in CWR22Rv1 and CWR22Rv1-fARKD cells resulted in significant growth inhibition, suggesting that AR3 might have dual roles: AR3 by itself might be able to promote PCa cell growth particularly in the absence of androgen at some selective PCa stages and the other role is that AR3 could cooperate with fAR to modulate AR-targeted genes and cell growth in the presence of androgen at many other PCa stages. These conclusions strengthened our central hypothesis and led us to believe that it is necessary to target both fAR and AR3 to have better therapeutic efficacy, especially at castration-resistant stages when AR3 expression is increased.
We demonstrated that Casodex failed to suppress the cell growth of CWR22Rv1 cells, which is in agreement with an early report showing that flutamide, another antiandrogen, had little suppressive effect on the transactivation of ARv567es
(another AR splice variant with deletion of AR exon 5–7 regions) [10
]. In this present study, we provided a new therapeutic approach through using ASC-J9, which was able to degrade both fAR and AR3 leading to the suppression of their mediated targeted genes and cell growth in various CRPC cells in vitro
and in vivo
In conclusion, our data suggest that AR3 may have its essential roles to promote PCa growth at selective PCa stages and targeting both fAR- and AR3-mediated cell growth through ASC-J9 may become a new useful therapeutic strategy to target PCa cells, which express AR splice variants such as AR3.