Original Articles Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells Guillaume Vares a, *, Sei Sai b , Bing Wang a , Akira Fujimori a , Mitsuru Nenoi a , Tetsuo Nakajima a a Research Center for Radiation Protection, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan b Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan ARTICLE INFO Article history: Received 20 February 2015 Received in revised form 20 March 2015 Accepted 20 March 2015 Keywords: Progesterone Cancer stem cells Basal breast cancer Membrane progesterone receptor Radiation miRNA A B ST R AC T Ionizing radiation and cumulative exposure to steroid hormones are known risk factors for breast cancer. There is increasing evidence that breast tumors are driven by a subpopulation of tumor-initiating cancer stem cells (CSCs). In MCF10A non-cancerous basal-like PR - cells, progesterone treatment and X-rays generated ALDH + and CD44 + /CD24 - CSCs. Here, we report that in irradiated MCF10A cells, progesterone activated the PI3K/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the PI3K/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of PI3K/Akt via mPR resulted in the inactivation of FOXO transcriptional activity, the upregulation of snail and slug expression and a downregulation of miR-29 expression, which led to increased levels of KLF4, a transcription factor required for breast CSC maintenance. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk in basal cells. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential novel anti-CSC strategies. © 2015 Elsevier Ireland Ltd. All rights reserved. Introduction Breast cancer is responsible for 13.7% of all cancer deaths worldwide. Although much progress has been made in the understanding and cure of breast cancer, significant challenges remain. Among the four main subtypes of breast cancer, basal-like breast cancer (BBC) is of particular interest, due to its high frequency, relative lack of effective therapies and poor prognosis. BBC represents the predominant subtype of triple-negative (TN) breast cancer [1] that does not express estrogen receptor (ER), progesterone receptor (PR) or epidermal growth factor receptor 2 (Her2). Ionizing radiation (for example as treatment for other cancers) and cumulative exposure to steroid hormones (as seen in postmenopausal women under hormone replacement therapy) are known risk factors for breast cancer [2–8]. Although basal mammary cells do not express hormonal receptors (progesterone and estrogen receptors) and TN breast cancer does not respond to hormonal therapy, a new family of G-protein coupled membrane progestin receptors (mPRs) has been identified as in intermediary of progesterone signaling in PR - cell lines, as well as in normal and breast cancer tissue, including TN/BBC [9,10]. mPRs initiate various signaling pathways associated with G-protein activation [11–15]. Recent investigations have shown that progesterone activation of mPR could result in either the activation or the repression of the PI3K/Akt signaling pathway [16,17]. A number of cancers appear to be initiated and driven by stem- like cells that are capable of self-renewing and of causing the different lineage of cancer cells comprising a tumor. Breast CSCs were first described as a subpopulation of CD44 + CD24 low ESA + lineage - human breast cancer cells capable of initiating tumors in immune- deficient NOD/SCID mice [18]. Subsequently, a number of reports have observed breast CSCs on the basis of cell-surface markers, such as CD44 + CD24 - [19], or by measuring cellular activities, such as the expression of aldehyde dehydrogenase (ALDH) [20]. Breast CSCs were shown to exhibit the capacity for self-renewal and multi- lineage differentiation, tumor-initiating properties and resistance to radiotherapy [21]. Like breast tumors, breast CSCs are characterized by significant morphological and molecular heterogeneity. There is active discussion concerning the role of each CSC subset in the initiation of various breast cancers. Basal-derived cell lines generally exhibit higher tumor initiation potential and contain more CD44 + /CD24 - CSCs. Although * Corresponding author. Tel.: +81 43 206 4730; fax: +81 43 255 6497. E-mail address: vares@nirs.go.jp (G. Vares). http://dx.doi.org/10.1016/j.canlet.2015.03.030 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved. Cancer Letters 362 (2015) 167–173 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet