The present study shows that 5α-reductase inhibition can enhance the expression of some androgen-responsive genes, particularly those with growth suppressive properties such as U19/Eaf2, during the regrowth of LNCaP xenograft tumors. This suggests that testosterone is more potent than DHT in the induction of U19/EAF2 in regressed prostate tumors in the castrated host. This phenomenon is similar to the observation that 5α-reductase inhibition enhanced the expression of a subset of androgen-responsive genes in the regressed rat ventral prostate upon testosterone replacement [24
]. Thus, the mechanism responsible for the differential regulation of androgen-responsive genes by testosterone and DHT exists in regressed androgen-sensitive LNCaP tumors, and is a phenomenon conserved from rodent to human.
Interestingly, the expression level of most of the androgen responsive genes we studied was not significantly affected by castration when compared to testes-intact controls. This could be attributed to the low concentration of serum testosterone that is present in nude mice. According to our previous publication [12
], serum testosterone in testis-intact nude mice is approximately 0.2 ng/ml, while in the Sprague–Dawley rat and in men are 3 and 3–10 ng/ml, respectively [24
]. The serum testosterone level in nude mice is slightly higher than the castrated testosterone levels in men [12
], which could be responsible for the weak down-regulation in androgen-responsive gene expression observed following castration. The testosterone delivery system used in the present experiments has been previously validated [11
] and is expected to achieve serum testosterone concentration of ~3 ng/ml, which corresponds to physiological levels in men and ~10 times higher than in testis-intact nude mice.
PSA gene expression was the most sensitive transcript to androgen manipulation, being significantly down-regulated by castration and by dutasteride treatment in testes-intact animals. However, during androgen stimulated tumor regrowth dutasteride exerted no effect on PSA expression levels. Contrary to PSA, U19/EAF2 and calreticulin both demonstrated significant up-regulation in response to dutasteride during tumor regrowth. The mechanism behind the transcript specific differential response to 5α-reductase inhibition is still unclear. It is likely due to differences in promoters and/or enhancers present in each gene.
Both dutasteride and finasteride enhanced the expression of U19/Eaf2 during the regrowth of LNCaP xenograft tumors ( and ), suggesting that the effect of these 5α-reductase inhibitors on U19/Eaf2 gene expression is mediated through the inhibition of the 5α-reductase enzyme rather than other unknown off-target effects. Our studies also showed that dutasteride did not influence the expression of androgen-responsive genes in LNCaP tumors in castrated animals without testosterone replacement (), suggesting that the influence of dutasteride on U19/Eaf2 and other androgen-responsive genes requires testosterone and is mediated by blocking the conversion of testosterone to DHT. While inhibition of 5α-reductase enzyme by either dutasteride or finasteride can enhance U19/Eaf2 expression, we are not able to state which inhibitor works more effectively. Dutasteride administration caused a slightly higher elevation in U19/Eaf2 expression in LNCaP model than finasteride. However, this difference in level of expression could be due to the fact that these experiments were carried out using different batch of animals.
Regressed LNCaP xenograft tumors in castrated mice responded to androgens differently from LNCaP tumors naїve to castration in testes-intact animals. We observed elevated expression of U19/Eaf2 and calreticulin genes by dutasteride treatment during the regrowth of regressed LNCaP tumors, but not in LNCaP tumors naїve to castration. The mechanism responsible for this difference is not clear. Variability in the expression of cofactors after castration and their recruitment by the androgen receptor in response to testosterone or DHT might explain the differential response to dutasteride of genes like U19/EAF2 and calreticulin in a regressed versus naїve tumor. However, presently this is only speculative and further studies are needed to elucidate the exact mechanisms involved. Nonetheless, this differential expression provides a potential explanation for the observation that finasteride significantly retarded the regrowth of regressed LNCaP xenograft tumors upon testosterone replacement, but not the growth of LNCaP tumors in testes-intact mice [11
]. U19/Eaf2 is a potent growth inhibitor and a potential tumor suppressor [22
]. The elevated expression of U19/Eaf2 as well as other growth suppressive androgen-responsive genes by 5α-reductase inhibition during testosterone-induced regrowth likely contributes to the retardation of LNCaP tumor regrowth observed with finasteride. In contrast, 5α-reductase inhibition did not increase the expression of U19/Eaf2 or other androgen-responsive genes in testes-intact animals, which correlates with the lack of LNCaP tumor growth inhibition observed in this group of animals upon treatment with 5α-reductase inhibitor [12
]. Since testosterone-stimulated tumor regrowth occurs during the off-cycles in intermittent androgen deprivation therapy (IADT), the off-cycles provide an opportunity for using 5α-reductase inhibitor to enhance the expression of tumor suppressive androgen-responsive genes such as U19/Eaf2, and may improve the therapeutic efficacy of IADT.
Our studies show that 5α-reductase inhibition only elevated the expression of a subset of androgen-responsive genes during LNCaP xenograft tumor regrowth. Among five assayed androgen-responsive genes, U19/Eaf2 and calreticulin displayed elevated expression during LNCaP tumor regrowth in the presence of dutasteride. The expression of the other three androgen-responsive genes was not increased by dutasteride. While dutasteride may increase the expression of additional androgen-responsive genes, it is not clear whether it will increase the expression of a majority of the growth-suppressive androgen-responsive genes during LNCaP xenograft tumor regrowth. Since 5α-reductase inhibition retarded LNCaP tumor regrowth, the elevated expression of a subset of growth suppressive androgen-responsive genes is likely to be in part responsible for the growth inhibition. Given the limited number of genes studied, it is also possible that other genes could behave similarly to U19/EAF2 and further contribute to the growth inhibitory effects associated with dutasteride treatment during tumor regrowth. In addition, it will be important to determine the time frame of this selective gene up-regulation tomaximize the benefits of 5α-reductase inhibition in IADT.
In summary, our studies indicate that 5α-reductase inhibition can upregulate tumor suppressor U19/Eaf2 during testosterone-induced regrowth of LNCaP xenograft tumor in castrated mice, but not in LNCaP tumors naїve to androgen-deprivation in testes-intact animals. This finding has potential clinical implications, particularly pertaining to use of 5α-reductase inhibitors in IADT, as the off-cycles in IADT involve testosterone-stimulated prostate tumor regrowth. The possibility of enhancing the expression of growth suppressive androgen-responsive genes by inhibiting testosterone conversion to DHT during prostate tumor regrowth is likely beneficial to patients with prostate cancer. Therefore, further studies are warranted to elucidate the mechanism by which growth suppressive androgen-responsive genes are induced to a higher level by testosterone than DHT during regrowth of normal and cancerous prostate.