Abstract 1853: Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling
Authors: Valdez Kelli E. , Hong Yan , Fan Fang , May Lisa , Cusick Therese , Inciardi Marc , Redick Mark , Gatewood Jason , Behbod Fariba 
Published: August 2015

Abstract
Introduction: The ability of the ovarian steroid hormones estradiol (E) and progesterone (P) to influence cancer stem cell (CSC) self-renewal and progression of human ductal carcinoma in situ (DCIS) is a critical but understudied area or research. The objective of this study is to characterize steroid hormone signaling involved in CSC self-renewal and invasion in ER+PR+ DCIS. We have found that these hormones can increase CSC self-renewal in a subset of DCIS patients, and that this may be facilitated by activation of the IL-6 pathway.
Methods: Mammosphere (MS) assays (an in vitro assessment of CSC self-renewal) and the mouse intraductal (MIND) xenograft model were used to characterize hormonal regulation of the CSC population and invasion potential of primary human ER+PR+ DCIS cells. Additionally, ovarectomized mice were used to create MIND xenografts of ER+PR+ DCIS, treated with E+P or vehicle for 8 weeks, and used for RNA sequencing and immunofluorescent staining.
Results: Only a subset of cases responded to E+P in vitro by increasing MS efficiency, indicating increased CSC self-renewal. RNA sequencing of MSs identified genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing MS efficiency. Many of these changes were associated with epithelial mesenchymal transition (EMT), including Twist1, Snai1, MMP9. In E+P-treated MIND xenografts, DCIS cases that respond to E+P by increasing MS efficiency also had higher IL-8 and vimentin (markers of EMT) compared to non-responders (P&lt;0.05). This suggests that the cases which responded to E+P by increasing CSC self-renewal may have undergone EMT. Additionally, IL-6 and IL-8 gene expression and protein increased (P&lt;0.05) in a DCIS case which also responded to E+P treatment by increasing MS efficiency. In an ER+PR+ DCIS case which did not increase MS efficiency in response to E+P, IL-6 and IL-8 secretion remained unchanged. Finally, RNA sequencing analysis of E+P vs. control-treated mammary glands from ovarectomized MIND-transplanted mice indicated the IL-6 signaling pathway was one of the top signaling pathways to be activated. Expression of IL-8 and vimentin in E+P compared to control-treated mice also increased (P&lt;0.05).
Conclusions: Our results indicate that steroid hormone treatment can enrich the CSC population by increasing MS efficiency in some but not all DCIS cases, and that IL-6 may be involved in this selective ability. Therefore, DCIS cases with higher MS efficiency in response to E+P treatment may also show higher invasion potential in vivo. Our current model is that steroid hormones induce increased secretion of IL-6, leading to increased self-renewal and invasion potential. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma and allow development of novel therapeutic targets.
Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Lisa May, Therese Cusick, Marc Inciardi, Mark Redick, Jason Gatewood, Fariba Behbod. Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1853. doi:10.1158/1538-7445.AM2015-1853