Most breast cancer deaths occur in women with recurrent, estrogen receptor (ER)-α(+), metastatic tumors. There is a critical need for therapeutic approaches that include novel, targetable mechanism-based strategies by which ERα (+) tumors can be resensitized to endocrine therapies. The objective of this study was to validate a group of nuclear transport genes as potential biomarkers to predict the risk of endocrine therapy failure and to evaluate the inhibition of XPO1, one of these genes as a novel means to enhance the effectiveness of endocrine therapies. Using advanced statistical methods, we found that expression levels of several of nuclear transport genes including XPO1 were associated with poor survival and predicted recurrence of tamoxifen-treated breast tumors in human breast cancer gene expression data sets. In mechanistic studies we showed that the expression of XPO1 determined the cellular localization of the key signaling proteins and the response to tamoxifen. We demonstrated that combined targeting of XPO1 and ERα in several tamoxifen-resistant cell lines and tumor xenografts with the XPO1 inhibitor, Selinexor, and tamoxifen restored tamoxifen sensitivity and prevented recurrence in vivo. The nuclear transport pathways have not previously been implicated in the development of endocrine resistance, and given the need for better strategies for selecting patients to receive endocrine modulatory reagents and improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role these pathways play in reducing cancer recurrences.

Peptidylarginine deiminase (PAD) enzymes convert histone tail arginine residues to citrulline resulting in chromatin decondensation. Our previous work found that PAD isoforms are expressed in female reproductive tissues in an estrous cycle-dependent fashion, but their role in the anterior pituitary gland is unknown. Thus, we investigated PAD expression and function in gonadotrope cells. The gonadotrope-derived LβT2 cell line strongly expresses PAD2 at the protein level compared with other PAD isoforms. Consistent with this, PAD2 protein expression is highest during the estrous phase of the estrous cycle and colocalizes with the LH β-subunit in the mouse pituitary. Using the GnRH agonist buserelin (GnRHa), studies in LβT2 and mouse primary gonadotrope cells revealed that 30 minutes of stimulation caused distinct puncta of PAD2 to localize in the nucleus. Once in the nucleus, GnRHa stimulated PAD2 citrullinates histone H3 tail arginine residues at sites 2, 8, and 17 within 30 minutes; however, this effect and PAD2 nuclear localization was blunted by incubation of the cells with the pan-PAD inhibitor, biphenyl-benzimidazole-Cl-amidine. Given that PAD2 citrullinates histones in gonadotropes, we next analyzed the functional consequence of PAD2 inhibition on gene expression. Our results show that GnRHa stimulates an increase in LHβ and FSHβ mRNA and that this response is significantly reduced in the presence of the PAD inhibitor biphenyl-benzimidazole-Cl-amidine. Overall, our data suggest that GnRHa stimulates PAD2-catalyzed histone citrullination in gonadotropes to epigenetically regulate gonadotropin gene expression.