Abstract 1390: In utero estrogenic exposures increase tamoxifen resistance in a new pre-clinical model of estrogen receptor positive breast cancer.
Authors: Nguyen Nguyen , Warri Anni , Clarke Robert , Cook Katherine , Cruz Idalia , de Assis Sonia , Kim Dominic , Lu Jin , Zwart Alan , Xuan Jason , Hilakivi-Clarke Leena 
Published: August 2014

Abstract
Maternal estrogenic exposures during pregnancy increase breast cancer risk among daughters in humans and in animal studies, possibly through epigenetic mechanisms. In addition, our findings obtained using systems biology tools to analyze mRNA microarray data suggest that in utero estrogenic exposures reduce the efficacy of antiestrogen treatment. Here we tested whether development of tamoxifen (TAM) resistance is pre-programmed in the offspring by maternal exposure to estradiol during pregnancy. Pregnant rats were exposed either to 0 or 0.1 ppm ethinyl estradiol (EE2) during pregnancy, and their offspring were treated with DMBA to induce ER+ mammary tumors.
Untreated mammary tumors in the EE2 offspring and in women who subsequently recurred (in Loi dataset), exhibited reduced expression of genes in the ATF6 pathway of unfolded protein response (UPR), including an apoptosis-inducing gene CHOP compared with tumors in the control rats or in women who did not recur. In addition, gene microarray analysis identified SEMA3b (p&lt;.012) as down-regulated and KAL1 (p&lt;.02) as up-regulated in the mammary tumors in the EE2 offspring and in TAM resistant LCC9 human breast cancer cells. SEMA3b is a tumor suppressor, perhaps because it induces apoptosis through Akt-mediated pathway, and is found to be methylated in clinical breast cancer and LCC9 cells. In turn, TGF-β regulated KAL1 gene is overexpressed in breast cancer and suggested to induce cell survival and cancer metastasis.
TAM treatment (15 mg/kg TAM via feed) was started when first tumor reached a size of 1.3±0.2 cm in diameter. In rats that exhibited a TAM-resistant tumor, valproic acid (HDAC inhibitor) and hydralazine (DNMT inhibitor) were added as a 2nd line treatment. Control rats were more likely to exhibit complete response to TAM (54%) than in utero EE2 exposed rats (38%), and de novo resistance was also lower in the controls (38%) than in the EE2 group (53%). Acquired resistance (re-growth after a complete response to TAM), characterized by over-expression of UPR gene GRP78, was seen in 23% of the tumors in the control and 42% in the EE2 group (p&lt;0.007). Adding HDACi-DNMTi as a 2nd line of treatment significantly reduced de novo (from 53% to 19%, p&lt;0.001) and acquired TAM resistance (from 42% to 6%, p&lt;0.001) in the EE2 offspring, but did not affect TAM resistance in the controls. Treatment with HDACi-DNMTi also reversed the changes in SEMA3b and KAL1 in the rat tumors and reversed TAM resistance in LCC9 cells.
Our findings suggest that in utero estrogenic exposures increase TAM resistance by inducing epigenetic changes leading to down-regulation of specific tumor suppressor genes and up-regulation of pro-survival genes. These changes could be reversed by adding HDACi-DNMTi to TAM treatment. Our results may have important clinical implications for treatment of antiestrogen refractory ER+ breast cancer.
Citation Format: Nguyen Nguyen, Anni Warri, Robert Clarke, Katherine Cook, Idalia Cruz, Sonia de Assis, Dominic Kim, Jin Lu, Alan Zwart, Jason Xuan, Leena Hilakivi-Clarke. In utero estrogenic exposures increase tamoxifen resistance in a new pre-clinical model of estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2013-1390