Abstract C41: Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal

Introduction: Ductal carcinoma in situ (DCIS), the premalignant form of human breast cancer, only progresses to invasive breast cancer (IBC) in an estimated 50% of diagnosed cases. Elucidation of the mechanisms underlying this selective event could prevent unnecessary treatment and increase the accuracy of diagnosis. Progression to an invasive phenotype has been associated with an increase in mammary cancer stem cells (CSCs). The ability of the ovarian steroid hormones estradiol and progesterone to influence mammary stem cell self-renewal has been demonstrated in models of both normal and pathological mammary gland growth. The common theme appears to involve estradiol and progesterone acting on the ER, PR-expressing luminal cells, which in turn through paracrine signaling can induce proliferation and increased self-renewal of both normal and CSC. However, none of these studies have examined the ability of these steroid hormones to influence CSC self-renewal and progression of DCIS. Purpose: The objective of this study is to characterize steroid hormone paracrine signaling involved in aberrant stem cell self-renewal and invasion in human primary DCIS. Our hypothesis is that those DCIS cases which respond to hormonal treatment by increasing CSC self-renewal also have the greatest invasive potential and risk of malignant progression. Experimental design: Mammosphere assays, an in vitro assessment of self-renewal, and the in vivo mouse intraductal (MIND) xenograft model are being used to characterize hormonal regulation of the stem cell population and invasion potential of primary human DCIS cells obtained from patient biopsies. Results: Cells from primary human DCIS, atypical hyperplasia, and DCIS with microinvasion were cultured under non-adherent conditions to allow primary mammosphere (MS) formation in the presence of vehicle, 10 nM estradiol, 100 nM progesterone, or both. After 14 days, cells were passaged to allow secondary MS formation. We found that only a subset of cases responded to the steroid hormone treatment by increasing MS forming efficiency, indicating increased self-renewal ability. Using human growth factor antibody arrays to assay the cell culture media, we found that insulin-like growth factor 2 (IGF2) was selectively up-regulated in response to steroid hormone treatment only in those cases which also demonstrated increased CSC self-renewal. Changes in gene expression in response to treatment are also being examined by next generation RNA sequencing. RNA from vehicle and steroid-treated DCIS cells was isolated from cases that either did or did not respond to E+P treatment. Differential expression analysis was used to identify genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing mammosphere-forming efficiency. Ingenuity Pathway Analysis was used to characterize the biological functions and disease states associated with gene changes observed. The top results included cellular movement, cancer, and reproductive system disease. Many of the gene changes observed were associated with changes that appear during epithelial mesenchymal transition (EMT). This supports our hypothesis that the cases which respond to steroid hormone treatment by increasing stem cell self- renewal may have greater invasive potential. Additionally, MIND xenografts in ovariectomized mice are being created and treated with steroid hormones using cells from these same DCIS cases to determine if these cases also have greater growth and invasion potential in vivo. Differences in gene expression of Wnt4, RankL, FGF9, IGF2, and amphiregulin will also be examined, as these have been shown to be involved in the signaling pathways regulating steroid hormone-induced stem cell proliferation. Conclusions: Our results indicate that steroid hormone treatment can enrich the stem cell population by increasing MS efficiency in some but not all DCIS cases, and that IGF2 may be involved in this selective ability. Based on these data, we expect that DCIS cases with higher MS efficiency in response to steroid hormone treatment will also show higher invasion potential in vivo and in vitro. 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. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Therese Cusick, Lisa May, William Smith, Fariba Behbod. Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C41.