Abstract B66: Life-time and adult genistein consumption have opposing effects on tamoxifen's ability to prevent recurrence of mammary tumors and on unfolded protein response pathways in rats

We recently found that starting intake of isoflavone genistein (GEN) present in soyfoods at the time when mammary tumors are treated with tamoxifen (TAM) increases de novo resistance to TAM and the risk of recurrence in a new rat model of estrogen receptor positive mammary tumor dormancy. However, rats which started consuming GEN before puberty and continued through their life time were highly responsive to TAM and exhibited reduced rate of mammary tumor recurrence. In this study, we investigated potential mechanisms that may explain the findings. 95 female Sprague-Dawley rats consuming AIN93G diet were supplemented with 0 or 500 ppm GEN between postnatal days (PND) 15 to 30 (prepubertal exposure) and then were fed AIN93G diet with no GEN between PND 30 and 55. On PND 48, rats received 10 mg DMBA to induce mammary tumors, and were then divided to four dietary groups on PND 55: prepubertal GEN-adult GEN group (GG, lifelong GEN, n=20); prepubertal GEN-adult control group (GC, prepubertal GEN, n=20); prepubertal control-adult GEN (CG, adult GEN, n=20); and control-control (CC, n=35). When mammary tumor reached a size of 1.4 cm in diameter, 337 ppm TAM citrate was added to rats' diet. GG and CG rats continued also consuming GEN, and all rats in GC and 20 rats in CC groups (post-diagnosis GEN) started consuming GEN one week after the TAM treatment started. 15 CC rats were never fed GEN (C, no GEN control, n=15). Mammary tumor latency was longer in GG (p<0.003) and GC rats (p<0.095), compared to CC rats. The response of tumors to TAM treatment was classified as complete or partial response, or de novo resistant. Prepubertal and life-time GEN intake increased, and intake starting during TAM treatment reduced response to TAM (p<0.001). Mammary cancer recurrence rate was highest in the rats which started consuming GEN during TAM treatment, and significantly lower in life-time, prepubertal and adulthood only GEN intake groups (p<0.001). In the mammary gland, no changes in estrogen receptor alpha (ERα) or HER2/ErB2 protein expression in the mammary gland were seen among the groups, but estrogen receptor beta (ERβ) protein level was significantly up-regulated (p<0.05) in the lifelong GEN exposed animals, when compared to no GEN control or prepubertal GEN exposed animals. We also investigated changes in the unfolded protein response (UPR) pathway. Surprisingly, the active form of ATF6, ATF6 p50, was highest in the mammary gland of lifelong GEN fed rats and lowest in that of CG and C animals (p<0.05). Target gene expression of ATF6 p50 was investigated in the mammary gland by quantitative real-time PCR and up-regulation of Xbp1 and GRP78 mRNA level was confirmed (p<0.05). In the tumor, ERα and progesterone receptor (PgR) protein expression did not differ among the five groups, but HER2/ErB2 and ERβ expression were higher in GC rats when compared with rats exposed to GEN lifelong (p<0.05). Further, protein levels of several important components in the UPR pathway - GRP78, IRE1α, ATF4 and Beclin -were significantly lower in the mammary tumors of lifelong GEN exposed rats than in the tumors of rats that started consuming GEN as adult (p<0.05). Our findings suggest that the effect of prepubertal GEN exposure in increasing TAM responsiveness may be related to the differentially regulated UPR pathway: members of this pathway were activated in the normal mammary gland and downregulated in the tumors. Elevated expression of GRP78 and other UPR genes in the mammary tumors of rats that started consuming GEN for the first time during TAM treatment may be associated with high de novo resistance and recurrence rate. Citation Format: Xiyuan Zhang, Leena A. Hilakivi-Clarke. Life-time and adult genistein consumption have opposing effects on tamoxifen's ability to prevent recurrence of mammary tumors and on unfolded protein response pathways in rats. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B66.