In this study, we demonstrated that consumption of low genistein doses (250 mg/kg diet) accelerates CaP progression in TRAMP-FVB mice when consumed after PIN initiation. This phenotype was characterized by Akt activation, OPN upregulation and occurrence of pelvic LN metastases. Furthermore, the previously-established chemopreventive dose (1000 mg/kg diet) (12
) lost its efficacy when consumed at 12 weeks of age. To delineate the mechanisms underlying these observations, in vitro
studies using PC3 cells treated with physiological versus pharmacological genistein doses recapitulated the proliferation and invasion increases observed in vivo
. These increases were dependent on OPN upregulation and required active estrogen and PI3K signaling, involving MMP-9 activation. To the best of our knowledge, this is the first report documenting an increase in metastasis by genistein in the TRAMP-FVB model while pinpointing an estrogen and PI3K-dependent induction of OPN as a necessary mechanism.
Considered potentially chemopreventive, increased usage of soy products prompted the examination of effects of physiologically achievable concentrations of genistein on breast and uterine cancer cell lines and animal models (20
). At low doses (<10 μM), genistein stimulates the growth of estrogen-sensitive cell lines (21
); while decreasing proliferation at higher doses (>10-20 μM) (22
). This suggests that genistein exerts a biphasic effect on growth and proliferation of cancer cells, similarly to what we have observed in prostate tumors of TRAMP-FVB mice and human PC3 cells; with low nanomolar concentrations inducing growth (14
) and metastasis of prostate tumor cells when treatment started after PIN in vivo
as well as proliferation and invasion in vitro
, and a higher dose (50,000 nM) reducing proliferation and invasion.
Increased tumor growth, metastasis and OPN upregulation were not seen when genistein is consumed (same dose) by 4-weeks old, tumor-free TRAMP-FVB mice or 12-weeks old non-transgenic C57BL/6xFVB mice (12, unpublished data); suggesting a dependence on exposure time or tumor presence and is not an inherent issue in the model used.
OPN expression concomitant with pelvic LN metastases in TRAMP-FVB mice and proliferation/invasion induction in PC3 cells by low genistein doses is significant in the context of CaP metastasis. CaP cells preferentially metastasize to bone, a process facilitated by OPN in various ways (24
). Here, we present data with reference to LN metastasis as opposed to bone, since the former occurs more frequently in the TRAMP model with 100% incidence in mice over 28 weeks as opposed to 25% incidence of bone metastases in 32-weeks old TRAMP mice (26
), and LN metastases were the most striking observation upon mice dissection. Interestingly, OPN expression has been correlated with LN metastases in a variety of cancers (28
). However, the possibility that bone metastasis occurs upon OPN induction by genistein in other models of CaP or humans cannot be eliminated.
We have observed OPN increases as early as 9-weeks of age, coinciding with PIN initiation and a highly proliferative stage (12
) in TRAMP-FVB mice, suggesting a role in proliferation. In fact, OPN induction promoted tumor growth, and its knockdown reduced Ras-transformed 3T3 cell growth in soft agar and animal implants (32
). This is also confirmed by the decreased proliferation of PC3(OPN-)
cells. Importantly, PC3 proliferation and invasion were no longer enhanced by genistein (500 nM) to the same extent in PC3(OPN-)
cells, indicating that OPN and/or its downstream effectors mediate at least the stimulatory part of the proposed biphasic genistein effect.
MMP-9 activity was also increased by low genistein doses (500 and 1,000 nM). Interestingly, elevated OPN expression correlate with MMP-9 levels (33
) and MMP-9 mediates OPN-induced cell migration and chemoinvasion in B16F10 cells (34
). In PC3 cells, OPN overexpression increases MMP-9 activity (17
), presenting strong evidence that MMP-9 is a downstream effector of OPN in invasion. Our results are in agreement with these reports in that PC3(OPN-)
exhibit reduced MMP-9 activity, and inhibition of genistein-induced OPN induction by ICI 182, 780 or Wortmannin, abrogated MMP-9 activation by genistein, suggesting that genistein (500 nM) may stimulate invasion via upregulation of OPN and subsequent activation of MMP-9.
The mechanism underlying OPN induction by genistein is unknown. However, OPN increase was paralleled by an increase in Akt phosphorylation in vivo
and in vitro
and was consistently abrogated in vitro
by Wortmannin or ICI 182, 780. OPN is a transcriptional target of the ER-related receptor alpha (ERR-α) (35
) and is induced by estradiol in vivo
). Therefore, OPN induction in prostates of TRAMP-FVB mice consuming 250 mg/kg genistein might be of estrogenic nature. OPN is also under the control of the PI3K pathway and is induced by PTEN deletion in colon cancer, whereas Ras-induced expression of OPN is PI3K-dependent (37
), which is in agreement with our Wortmannin studies.
Estrogen sensitivity of cell lines/models used seems to determine the growth and metastasis-promoting effects of genistein. Studies reporting proliferation increase by genistein in estrogen-responsive cell lines have failed to observe similar effects in ER-negative cells (38
). Consumption of 250 mg/kg diet before orthotopic implantation of PC3-M, a metastatic subline of PC3 lacking ERs (39
) decreased lung metastases (40
), whereas LN weights increased in genistein-treated mice harboring PC3 xenografts (41
). Our study agrees with these observations in that TRAMP-FVB prostates (unpublished data) and PC3 cells express ER α and β (39
). The need for estrogen signaling was also highlighted by the administration of ICI 182,780, which reduced the induction of proliferation and invasion by genistein. However, the importance of estrogen signaling on the in vivo effects of genistein remains to be determined.
We and others have pinpointed Akt inhibition by genistein as one mechanism by which genistein exerts its chemopreventive actions (42
). Recently, an increase in Akt phosphorylation by 10 μM genistein was reported in porcine aortic endothelial cells in vivo
). Although not made in a tumor cell setting, this observation is in agreement with our findings regarding Akt activation by genistein in vitro
and in vivo
We have also observed that PI3K inhibition by Wortmannin abrogated the proliferation and invasion increase by genistein, suggesting that genistein acts in a PI3K-dependent manner in PC3 cells. Furthermore, the inhibitory effects on invasion by nanomolar doses of genistein in the absence of PI3K activity, suggest that there might be a balance between inhibitory and activating effects of genistein, with the balance shifted towards inhibition upon PI3K inactivation. Experiments are underway to determine whether PI3K/Akt and estrogen signaling activation by genistein are independent events or an interaction between both pathways.
The inhibition of metastasis by genistein in a chemopreventive setting has been reported extensively (44
). However, genistein increased the size of LN metastases but not tumor size when administered to PC-3/nude mouse xenograft model (41
); postulating that LN metastases increase is due to genistein’s antiangiogenic effects and subsequent hypoxia. However, in our study, genistein resulted in increased tumor size and metastasis, suggesting a direct effect on tumor cell proliferation when administered to TRAMP-FVB mice with PIN lesions. Furthermore, proliferation and invasion were potentiated in the same cells used for the xenografts in vitro, eliminating the hypoxia theory in our model at least.
Recent findings showed that PD carcinomas in the TRAMP-FVB strain are derived from neuroendocrine (NE) cells (45
). In this study, 250 mg/kg diet genistein increased the number of synaptophysin-expressing PD carcinomas, which was also expressed in pelvic LNs (data not shown). One hypothesis is that a low genistein environment (provided by 250 mg/kg diet) targets the synaptophysin-expressing NE population in the prostate resulting in the upregulation of OPN, the positive selection of this population (considered highly-proliferative and a candidate for the transit-amplifying population in the prostate (46
)) and emergence of a more aggressive phenotype in this group. More experiments characterizing the NE population in our model, its possible differences at 4 and 12-weeks of age, and its ER and Akt status would prove/disprove this hypothesis and further highlight the potential detrimental effects of low genistein doses.
In this work, we have shown that timing of genistein exposure as well as the dose used had a major impact on CaP outcome and progression in TRAMP-FVB mice. This highlights the importance of examining the effects of physiologically achievable levels of genistein and its deleterious effects on undiagnosed prostate cancer.