Abstract 1867: Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer
Authors: Goyette Sandy , Mafuvadze Benford , Cook Matthew T. , Liang Yayun , Hyder Salman M. 
Published: August 2015

Abstract
Combined hormone replacement therapy (HRT) containing progestins (P) and estrogens (E) has been shown to increase the risk of breast cancer in post-menopausal women (JNCI, 2000, 92:328). We showed that the commonly used P, medroxyprogesterone acetate (MPA), increases production of the potent angiogenic growth factor VEGF in breast cancer cells (Int J Cancer, 2001, 92:469). Furthermore, it has been established that P drives tumor progression in an in-vivo xenograft model (Can Res 2007; 67:9929). CD44 has recently been shown to be expressed by cancer stem cells (CSC) (Nature Reviews Cancer 2011; 11:254), and has therefore been used as a marker for detecting and enriching of tumor initiating cells. CD44 binds potent growth factors such as VEGF and acts as a co-receptor that helps mediate proliferative and migratory signaling for a variety of receptor tyrosine kinases. Thus, we hypothesized that MPA increases CD44 levels in breast cancer cells and promotes the emergence of self-renewing stem-cell-like-cells, which support MPA-accelerated tumors in vivo. Human T47-D human breast cancer cells were treated with MPA for 24h. Subsequent FACS analysis showed that MPA caused a significant increase in CD44 protein; CD44 was induced 10-fold (Control 5.9%, MPA 60.7%). A less robust, but significant increase also occurred in BT474 cells (Control 4.0%, MPA 11.2%). Further studies showed that MPA-induced CD44high cells also had high levels of ALDH enzyme activity, a characteristic of stem cells. CD44high cell induction was completely repressed by the anti-progestin RU-486, indicating involvement of progesterone receptors. Induction of CD44 protein occurred in a time-dependent manner, with a significant increase 6h after MPA treatment and reaching a peak after 24h. Interestingly, CD44high expression remained elevated for at least another 24 hours, even though MPA was removed from the treatment media, indicating that once induced, CD44 may be a stable protein. Importantly, various natural and synthetic P that are used widely in HRT prescriptions (Progesterone, Norethindrone, Norgestrel), brought about a similar increase in CD44high cells. Considering P has previously been linked to increased VEGF secretion by hormone-responsive cancer cells, we tested whether VEGF signaling plays a role in the elevation of CD44high cells. However, this process was found to be VEGF-independent. Our results show that natural and synthetic progestins induce a CD44high cell population which is likely associated with development of MPA-induced tumors. Thus targeted therapies against CD44 may be useful in controlling progression of P-driven tumors.
Supported in part by COR award from the College of Veterinary Medicine, University of Missouri.
Citation Format: Sandy Goyette, Benford Mafuvadze, Matthew T. Cook, Yayun Liang, Salman M. Hyder. Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer. [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 1867. doi:10.1158/1538-7445.AM2015-1867