Abstract 4754: An ancestral link between tumor immune microenvironment and tamoxifen resistance in breast cancer
Authors: Hilakivi-Clarke Leena A. , Bouker Kerrie , Andrade Fabia Oliveira De , Jin Lu 
Published: August 2018

Maternal exposures to endocrine disrupting chemicals (EDCs) are known to increase daughter's breast cancer risk in humans and animal models. We have found that these exposures also pre-program mammary tumors to exhibit increased resistance to antiestrogen therapy (Hilakivi-Clarke et al. JNCI 109, 2016). Consistent with the idea that in utero EDC exposures alter later disease susceptibility by epigenetic means, we discovered that adult treatment of in utero EDC exposed rats with valproic acid (VA, non-specific HDAC inhibitor) and hydralazine (H, non-specific DNMT inhibitor) prevented tamoxifen (TAM) resistance. In contrast, in the control offspring VA+H promoted TAM resistance. Here we investigated if maternal exposure to synthetic ethinyl estradiol (EE2) alters tumor immune environment in the offspring, and if treatment with VA+H reverses the changes. Pregnant Sprague Dawley rats received 0 or 0.1 ppm EE2 during gestation days 10-20. Estrogen receptor positive (ER+) mammary tumors in the offspring were induced with 7,12-dimethylbenz[a]anthracene, and when a tumor reached 13 mm in diameter, core needle biopsies were obtained. Thereafter, offspring were treated with 15 mg/kg TAM in diet, with or without 1.2 g/kg VA and 5 mg/kg H via drinking water. Antigen processing and activating proteins CD74 and HLA-DRB5 were significantly elevated in pre-treatment tumor biopsies from in utero EE2 exposed rats. Ingenuity pathway analysis of iTraq protein data from the biopsies indicated that T cell receptor and IL15 were upstream regulators of most of the signaling changes between EE2 and control offspring. Further, non-treated mammary tumors of EE2 offspring exhibited higher mRNA levels of PD-L1, PD-1, Foxp3 and Tgfβ1 than tumors in the control offspring. TAM treatment further upregulated Foxp3 and Tgfβ1 as well as CD8a and PD-1 in the EE2 offspring. However, VA+H reversed all these changes in TAM treated in utero EE2 exposed animals. In the control offspring, a combination of TAM and VA+H increased tumor PD-1, PD-L1, Foxp3 and TGFβ1 levels, compared with non-treated tumors. Since elevated levels of Foxp3 and TGFβ1 are indicative of immunosuppression, and PD-1 and its ligand PD-L1 induce effector T cell exhaustion, our data suggest that in utero EE2 exposure promotes an immunosuppressive tumor microenvironment that allows cancer cells to evade elimination by cytotoxic T lymphocytes, especially during TAM therapy. Our findings also indicate that adding HDAC + DNMT inhibitors to the treatment regimen prevents these changes, and thus possibly explains the ability of the combination treatment to prevent antiestrogen resistance in the in utero EE2 exposed rats. Future studies will determine if the combination therapy benefits ER+ breast cancer patients exhibiting high PD-1/PD-L1 expression in pre-treatment tumors, and/or if these patients should receive checkpoint inhibitors with endocrine therapy.

Citation Format: Leena A. Hilakivi-Clarke, Kerrie Bouker, Fabia Oliveira De Andrade, Lu Jin. An ancestral link between tumor immune microenvironment and tamoxifen resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4754.