Several studies dating back to the 1980s have suggested that mechanisms other than androgen suppression may be involved in the estrogen-mediated inhibition of CaP growth. Estrogens appear to be slightly more effective in treating CaP than other means of androgen suppression [4
]. Compounds with estrogenic activity are capable of exerting direct cytotoxic effects on androgen-independent CaP cells in vitro
]. Our data, obtained from the androgen-deficient environment of female mice [24
] and from the present work, show that estrogens have powerful growth-inhibitory effects on CaP in vivo
In the present study, we have shown that E2 and DES both inhibit the growth of androgen-independent CaP tumors in the androgen-depleted environment of castrated male mice. These data clearly demonstrate that E2 exhibits effects on CaP cells that are unrelated to the suppression of the hypothalamic-hypophyseal axis and the subsequent decrease in testosterone. This novel observation prompted us to characterize the effects of E2 on androgen-independent CaP at the molecular level by profiling transcript alterations. Although many of the genes differentially regulated by estrogen in this system are of unclear significance, others have quite plausible roles in the observed growth inhibition on the basis of their established functions. Among these are genes involved in signal transduction, cellular metabolism, and the control of transcription and translation. We also observed substantial changes in genes that function to regulate immune responses—a mechanism that may contribute to tumor growth-inhibitory effects resulting from estrogen treatment.
Among immune response-related genes altered by E2 treatment in CaP are those modulating cellular responses to IFNs. This group was found to be significantly enriched in the set of genes upregulated by E2 when tested by GSEA using an independently generated list of IFN-regulated genes. The increased expression of IFN-regulated genes is of particular interest due to the direct antitumor activities reported for these cytokines [43–50
]. Our results are in keeping with the results on the upregulation of IFN-regulated genes in LNCaP CaP cells following exposure to the estrogenic herbal preparation PC-SPES [51
] and the induction of IFNγ-regulated genes after E2 treatment in other tissues [52
]. In addition, tamoxifen has been shown to enhance IFN-regulated gene expression in breast cancer cells [53
]. Specifically, IRF1, whose expression was increased three-fold by E2 (qRT-PCR data), has been described as a negative regulator of proliferation [54
] and has exhibited tumor-suppressor activities in breast cancer cells [55
]. These published observations and our results are consistent with a model in which IFN and genes regulated by IFN modulate a component of the growth-inhibitory activity of E2 toward androgen-independent CaP cells.
E2 treatment significantly increased the expression of several MHC class I/II transcripts in the androgen-independent LuCaP 35V xenograft. Similarly, the upregulation of MHC class I transcripts has been observed in LNCaP cells on PC-SPES exposure [51
]. MHC class I molecules are expressed in most human cells and play a pivotal role in the immune response to viruses and tumor cells. Tumor cells often evolve mechanisms to modulate or escape immune surveillance through the downregulation of MHC class I molecules [56–60
]. IFNγ treatment, like E2 treatment in our studies, has been reported to upregulate the expression of MHC class I/II molecules in CaP cell lines [44,58,59
]. According to this evidence, the treatment of advanced CaP patients with E2 might result not only in direct inhibitory effects but also in the stimulation of T-cell attack on tumors by the upregulation of MHC proteins. Such a mechanism could not be directly tested in our study, which employed immune-compromised SCID mice, but it represents an independent potential benefit of E2 treatment that could be exploited in the context of clinical therapies employing vaccine or other immunomodulatory treatment strategies.
DES has been reported to be ineffective in inhibiting LuCaP 35 growth in intact male mice [61
]. We also observed that E2 did not inhibit LuCaP 35 growth in intact male mice (data not shown). These results suggest that phenotypic changes caused by E2 treatment are specific to an androgen-depleted environment. In contrast to our E2 data, raloxifene, an estrogen receptor antagonist, has been reported to inhibit the growth of both androgen-sensitive and androgen-independent CaP in vitro
]. Raloxifene has also been reported to delay CaP development in probasin/SV40 Tantigen transgenic rats [62
] and to inhibit the growth of both androgen-sensitive and androgen-independent variants of the CWR22 CaP xenograft [63
]. Thus, the emerging picture of estrogenic effects on androgen-independent CaP is complex, possibly involving multiple mechanisms, some of which may involve signal transduction by estrogen receptors. Additional preclinical studies are clearly warranted to deconvolute these effects.
A potential mechanism whereby E2 may cause alterations of the gene expression profile we have observed in CaP cells is signal transduction through ERβ expressed by CaP cells. It has been reported that ERβ expression declines as CaP develops in the prostate gland, but we and others have shown that it reappears in lymph node and bone metastases [27
]. This apparent discrepancy is probably explained by the recent findings of the reversible epigenetic regulation of ERβ in CaP metastases [64
]. We have shown previously that the xenografts used in this study express ERβ [24
]. In the present study, we have shown that the androgen-independent LuCaP 35V xenograft expresses ERβ protein in a form that is capable of DNA binding, and that ERβ levels in nuclei and DNA-binding activities are increased on E2 treatment. Together, these results suggest the possibility that E2-mediated inhibition is, at least in part, transduced by ERβ signaling, but further studies are required to demonstrate direct involvement of ERβ with these phenomena. One important aspect of preclinical testing involves the use of models that mimic the disease in patients. If it is eventually found that E2 is beneficial in advanced CaP and that the effects are mediated by ERβ, then evaluation of the expression of ERβ in patient tumors could prove to be valuable in treatment decisions, as is the case with HER2/Neu and herceptin treatment today.
The E2-inhibitory effects observed cannot be caused by suppression of the hypothalamic-hypophyseal axis reduction in testosterone levels because the tumors were grown in castrated male mice. However, our data do suggest that AR signaling may be at least partially involved in the inhibitory effects observed. All of the xenografts, except LuCaP 49, express AR (data not shown), and the inhibition of LuCaP 49 by E2 was less pronounced than in other xenografts. Moreover, GSEA showed that genes in an independently generated list of genes downregulated by androgen deprivation were significantly enriched in the phenotype of E2-treated LuCaP 35V, with about half of the genes downregulated by E2 and half upregulated by E2. For example, the expression of heat shock protein 70, which is downregulated after castration [65
], was upregulated by E2 treatment (). These results illustrate the complexity of these signaling networks. Further studies are needed to delineate the action of E2 on AR signaling in CaP cells.
The results reported here support the multifaceted roles of estrogen in the inhibition of androgen-independent CaP growth. These observations extend the traditional view of estrogen activity beyond the suppression of circulating concentrations of androgens. Direct cellular effects and the modulation of immune responses represent additional potential mechanisms that could be further exploited through combination therapies. Given that estrogens also decrease bone lysis caused by androgen suppression [66
] and may ameliorate cognitive side effects associated with low testosterone [67
], the use of estrogens should be considered as a viable first-line treatment strategy for androgenindependent CaP.