Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells

Accepted Manuscript Title: Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells Author: Guillaume Vares, Sei Sai, Bing Wang, Akira Fujimori, Mitsuru Nenoi, Tetsuo Nakajima PII: DOI: Reference:

S0304-3835(15)00222-0 http://dx.doi.org/doi:10.1016/j.canlet.2015.03.030 CAN 12352

To appear in:

Cancer Letters

Received date: Revised date: Accepted date:

20-2-2015 20-3-2015 20-3-2015

Please cite this article as: Guillaume Vares, Sei Sai, Bing Wang, Akira Fujimori, Mitsuru Nenoi, Tetsuo Nakajima, Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells, Cancer Letters (2015), http://dx.doi.org/doi:10.1016/j.canlet.2015.03.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Progesterone generates cancer stem cells through

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membrane progesterone receptor-triggered signaling in

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basal-like human mammary cells

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Guillaume Vares1*, Sei Sai2, Bing Wang1, Akira Fujimori1, Mitsuru Nenoi1, Tetsuo

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Nakajima1

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Therapy. National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku,

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Research Center for Radiation Protection, 2Research Center for Charged Particle

Chiba 263-8555, Japan

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* Corresponding author. Telephone: +81-43-206-4730. E-mail: [email protected]

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Highlights  In basal-like PR-negative MCF10A cells, progesterone and 1 Gy X-rays generate cancer stem cells (CSCs).  Stimulation of membrane progesterone receptor (mPR) by progesterone activates the PI3K/Akt/NFκB pathway.  The downregulation of miR-29c triggers upregulated levels of KLF4 and is necessary for CSC initiation and maintenance.

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Abstract

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Ionizing radiation and cumulative exposure to steroid hormones are known risk

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factors for breast cancer. There is increasing evidence that breast tumors are driven by

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a subpopulation of tumor-initiating cancer stem cells (CSCs). In MCF10A non-

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cancerous basal-like PR- cells, progesterone treatment and X-rays generated ALDH+

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and CD44+/CD24- CSCs. Here, we report that in irradiated MCF10A cells,

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progesterone activated the PI3k/Akt pathway via membrane progesterone receptor

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(mPR). Inhibition of the PI3k/Akt pathway counteracted the generation of CSCs by

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progesterone and irradiation. The stimulation of PI3K/Akt via mPR resulted in the

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inactivation of FOXO transcriptional activity, the upregulation of snail and slug

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expression and a downregulation of miR-29 expression, which led to increased levels

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of KLF4, a transcription factor required for breast CSC maintenance. Stabilization of

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miR-29 expression impeded the generation of CSCs, while its inhibition alone was

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sufficient to generate CSCs. This study provides a new mechanistic basis for

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progesterone and radiation-induced breast cancer risk in basal cells. In addition, the

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elucidation of new pathways and miRNA regulations involved in CSC generation and

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maintenance may open the door to potential novel anti-CSC strategies.

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Keywords

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progesterone; cancer stem cells; basal breast cancer; membrane progesterone receptor;

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radiation; miRNA

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1. Introduction

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Breast cancer is responsible for 13.7% of all cancer deaths worldwide. Although

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much progress has been made in the understanding and cure of breast cancer,

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significant challenges remain. Among the four main subtypes of breast cancer, basal-

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like breast cancer (BBC) is of particular interest, due to its high frequency, relative

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lack of effective therapies and poor prognosis. BBC represents the predominant

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subtype of triple-negative (TN) breast cancer1, that do not express estrogen receptor

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(ER), progesterone receptor (PR) or epidermal growth factor receptor 2 (Her2).

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Ionizing radiation (for example as treatment for other cancers) and cumulative

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exposure to steroid hormones (as seen in postmenopausal women under hormone

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replacement therapy) are known risk factors for breast cancer2-8. Although basal

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mammary cells do not express hormonal receptors (progesterone and estrogen

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receptors) and TN breast cancer does not respond to hormonal therapy, a new family

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of G-protein coupled membrane progestin receptors (mPRs) has been identified as in

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intermediary of progesterone signaling in PR- cell lines, as well as in normal and

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breast cancer tissue, including TN/BBC9,10. mPRs initiate various signaling pathways

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associated with G-protein activation11-15. Recent investigations have shown that

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progesterone activation of mPR could result in either the activation or the repression

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of the PI3K/Akt signaling pathway16,17.

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A number of cancers appear to be initiated and driven by stem-like cells that are

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capable of self-renewing and of causing the different lineage of cancer cells

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comprising a tumor. Breast CSCs were first described as a subpopulation of

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CD44+CD24lowESA+lineage− human breast cancer cells capable of initiating tumors in

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immune-deficient NOD/SCID mice18. Subsequently, a number of reports have

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observed breast CSCs on the basis of cell-surface markers, such as CD44+CD24−19, or

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by measuring cellular activities, such as the expression of aldehyde dehydrogenase

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(ALDH)20. Breast CSCs were shown to exhibit the capacity for self-renewal and

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multi-lineage differentiation, tumor-initiating properties and resistance to

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radiotherapy21.

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Like breast tumors, breast CSCs are characterized by significant morphological and

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molecular heterogeneity. There is active discussion concerning the role of each CSC

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subset in the initiation of various breast cancers. Basal-derived cell lines generally

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exhibit higher tumor initiation potential and contain more CD44+/CD24- CSCs.

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Although it was suggested that BRCA1 BBCs originate from luminal and not basal

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progenitors22, there is still controversy about the role of basal progenitors in BBC23.

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MCF10A is a spontaneously immortalized non-transformed human mammary cell

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line with a basal-like phenotype24, whose morphogenesis on reconstituted basement

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membrane closely parallels that of normal breast epithelium25. Whereas untreated

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MCF10A cell populations contain almost no CSCs26, MCF10A CSCs can be

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generated after TGFβ/TNFα-induced epithelial to mesenchymal transition (EMT)27,

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Ras transformation28-30 or combined exposure to ionizing radiation and

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progesterone31. MCF10A CSCs and non-CSCs exist in a dynamic equilibrium that can

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be influenced by an inflammatory feedback loop involving NFκB, Lin28, IL6,

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STAT3, PTEN, CYLD and several microRNAs30.

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miRNAs are emerging as playing an essential role in regulating breast CSC

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generation, maintenance and behavior32. Several EMT- and CSC-associated miRNA

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regulations have been reported in breast models33,34. In PR+ T47D breast cancer cells,

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progesterone treatment rapidly downregulated miR-29, resulting in the upregulation

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of Krüppel-like factor 4 (KLF4), a transcription factor required for the maintenance of

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breast CSCs35. Several reports have shown that miR-29 is repressed by NFκB36-38,

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suggesting that miR-29 might be a downstream target of the PI3K/Akt signaling

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pathway.

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We have shown that combined exposure of MCF10A cells to progesterone and 10 Gy

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X-rays triggered stemness- and cancer-associated miRNA regulations, and the

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initiation of ALDH+ and CD44+/CD24- CSCs31. Here we investigated the role of

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mPR, the PI3K/Akt signaling pathway and miR-29 in the generation of MCF10A

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CSCs.

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2. Material and Methods

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2.1

Cell culture

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Non-tumorigenic basal-like MCF10A breast epithelial cells were maintained in

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DMEM/F12 supplemented with 5% horse serum, 20 ng/mL epidermal growth factor

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(EGF), 10 µg/mL insulin, 100 µg/mL hydrocortisone and 10 ng/mL cholera toxin.

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Cultures were grown in 5% CO2 at 95% humidity.

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2.2

Irradiation, hormonal treatment and oligonuclotide transfection

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MCF10A cells were irradiated in serum-free medium 72 hours after plating, using an

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X-ray generator (ISOVOLT Titan-320, General Electric, Fairfield, CT, USA).

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Irradiation dose was 1 Gy at a dose-rate of 0.9 Gy/min. Starting one day after plating,

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natural progesterone diluted in ethanol (10 µM) was added daily to the culture

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medium until cell collection at a final concentration of 10 nM. MCF10A cells were

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also cotreated with 20 nM wortmannin (diluted in ethanol), 10 µg/mL caffeic acid

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phenethyl ester (CAPE, diluted in ethanol). miRIDIAN miRNA mimic and antagomir

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for miR-29 (GE Dharmacon, Lafayette, CO, USA) were transfected using

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Lipofectamine RNAimax (Life Technologies, Carlsbad, CA, USA) according to the

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manufacturer’s instructions, at a final concentration of 10 nM. For miR-29

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modulation experiments, cells not transfected with mimic or antagomir were treated

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with lipofectamine alone.

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2.3

FOXO reporter assay

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MCF10A cells were transiently co-transfected with a PI3K/Akt pathway-responsive

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FOXO luciferase reporter vector (or with a non-inducible luciferase vector as negative

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control) and with a constitutively-expressing Renilla luciferase vector (SABioscience,

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San Diego, CA, USA). Firefly and Renilla luciferase activities were measured using

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the Dual Luciferase Assay System (Promega, Madison, WI, USA). Normalized

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luciferase activity for the FOXO reporter was obtained by substracting the

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background luminescence and by calculating the ratio of firefly luminescence from

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the FOXO reporter to Renilla luminescence from the control Renilla luciferase vector.

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2.4

Sorting of ALDH+ cells

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As previously31, ALDH activity in the cells was measured by flow cytometry using

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the ALDEFLUOR kit (Stemcell technologies, Vancouver, BC, Canada). Cells with

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low and high levels of ALDH enzymatic activity (respectively ALDH- and ALDH+

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cells) were sorted using a FACSAria cell sorter (BD Biosciences, Franklin Lakes, NJ,

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USA). As a negative control, cells were treated with diethylaminobenzaldehyde

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(DEAB), a specific ALDH inhibitor.

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2.5

Mammosphere culture

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Sorted ALDH- and ALDH+ cells were resuspended in complete mammosphere cell

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culture medium (MammoCult; Stemcell Technologies) supplemented with

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Mammocult proliferation supplement, hydrocortisone and heparin. Then they were

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seeded in ultra-low adherent plates (Corning, Corning, NY, USA) at densities of

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5,000 to 40,000 cells per well and grown for 7 days. Spheres larger than 60 µm in size

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were counted.

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2.6

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profiling

RNA extraction and real-time PCR-based gene expression

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Gene expression levels were measured in MCF10A cells and in sorted CSCs and non-

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CSCs. Measurements were not performed in sorted cells for experimental conditions

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that did not trigger the generation of ALDH+ CSCs (after progesterone treatment

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alone or after administration of miR-29 mimic or wortmannin in irradiated and treated

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cells). Primers sets for SNAI1 (snail), SNAI2 (slug), KLF4, PAQR7 (mPRα), PAQR8

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(mPRβ), PAQR5 (mPRγ), PAQR9 (mPRε), PAQR3 (a related PAQR family member),

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MPR (nuclear PR), MRPL19 (mitochondrial ribosomal protein L19) and 18S rRNA

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were used as previously described39-42. 250 ng RNA per sample was reverse

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transcribed using the RT2 first strand kit (SABiosciences), then real-time PCR

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reactions were performed in triplicate with an Applied Biosystems 7300 Real-Time

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PCR system (Life Technologies), using the RT2 SYBR Green PCR Master Mix

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(SABiosciences). Data analysis was performed using the web-based GeneGlobe

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software from Qiagen

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(http://www.qiagen.com/products/genes%20and%20pathways/data-analysis-center-

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overview-page/). The Ct2 method was used to compute relative gene expression

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levels, normalized against L19 (mPRs and nuclear PR) or 18S ribosomal RNA (snail,

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slug, KLF4). For each gene, fold changes (compared to levels in control cells) were

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calculated.

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2.7

Statistical analysis

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Quantitative data were presented as means of at least three independent experiments ±

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standard deviation. Statistical significance was determined by using the Student’s t-

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test between two groups (p<0.01 was considered significant).

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3. Results 3.1

Generation of CSCs by progesterone and ionizing radiation

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relies on PI3K and NFκB

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We previously reported than progesterone and 10 Gy ionizing radiation could

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generate CSCs in the MCF10A cell line31. Because an irradiation dose of 10 Gy

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resulted in significant cell death (35% of cells died within the three days following

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irradiation), we here exposed cells to 1 Gy X-rays, which caused the death of less than

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5% of the cells within the same time frame (data not shown). As previously observed,

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progesterone treatment alone did not lead to a higher proportion of ALDH+ cells.

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Only when combined with radiation exposure, progesterone treatment triggered

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increased ALDH+ cells levels (Figure 1). Co-treatment with wortmannin (a PI3K

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inhibitor) and caffeic acid phenethyl ester (CAPE, an NFκB inhibitor) prevented

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increased ALDH+ cell frequencies, suggesting that the generation of CSCs by

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progesterone and ionizing radiation requires PI3K and NFκB, respectively.

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3.2

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pathway

Stimulation of mPR by progesterone activates the PI3K/Akt

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We hypothesized that progesterone effects in MCF10A cells rely on membrane

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progestin receptors (mPRs). We first measured the expression of mPR-family and

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nuclear PR transcripts in untreated and progesterone-treated MCF10A cells (Figure

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2). PAQR5 (mpRε) and PGR (nuclear PR) transcripts were not detected. Expression

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of PAQR7 (mPRα), PAQR8 (mPRβ), PAQR9 (mPRε), PAQR3 was observed but those

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transcripts were not significantly modulated after progesterone treatment. Those

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results confirmed the absence of nuclear PR and the presence of mPRs in MCF10A

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cells. To examine the role of PI3K/Akt pathway in mPR-mediated signaling, we

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performed luciferase reporter assays in transfected MCF10A cells (Figure 3). Because

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the activation of Akt phosphorylates FOXOs, resulting in their export to the

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cytoplasm and in the inhibition of FOXO-dependent transcription, PI3K/Akt pathway

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activity is inversely correlated to FOXO transcriptional activity. PI3K/Akt pathway

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was activated after administration of progesterone or Org-OD-02-0, a specific mPR

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agonist43. Co-treatment with the nuclear PR antagonist mifepristone (RU-486) did not

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reduce the activation of the PI3K/Akt pathway by progesterone. Furthermore,

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PI3K/Akt pathway was inhibited by wortmannin, alone or combined with

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progesterone or Org-OD-02-0 treatment. These findings suggest that the activation of

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mPR by progesterone triggers the PI3K/Akt signaling pathway.

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3.3

Downregulation of miR-29c is necessary for CSC initiation and

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maintenance

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To evaluate the role of miR-29c in CSC initiation and maintenance, we treated

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MCF10A cells with miR-29c mimic and antagomir (Figure 4). Co-administration of

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progesterone and miR-29c mimic in irradiated cells counteracted the generation of

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CSCs. On the contrary, administration of miR-29c antagomir alone was sufficient to

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trigger increased CSC levels. In irradiated MCF10A cells treated with progesterone

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and wortmannin, administration of miR-29c antagomir resulted in the generation of

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CSCs. We then measured the ability of ALDH+ cells to grow “tumorspheres” (or

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“mammospheres”) in anchorage-independent conditions during the week following

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irradiation (Figure 5). As observed previously, ALDH+ cells exhibited increased

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tumorsphere-forming abilities, compared to ALDH- cells. Addition of miR-29c mimic

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significantly reduced the numbers of mammospheres formed during the same time

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frame, whereas addition of miR-29c antagomir resulted in high mammosphere

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numbers. Altogether, our results suggest that the down-regulation of miR-29c is

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required for both CSC initiation and maintenance in our model.

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3.4

SNAI1 (Snail), SNAI2 (Slug) and KLF4 are upregulated in CSCs

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We measured by real-time PCR the expression of SNAI1 (snail), SNAI2 (slug) and

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KLF4 genes in MCF10A cells and in sorted CSC and non-CSC populations (Figure

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6). SNAI1, SNAI2 and KLF4 were upregulated after exposure to radiation and

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progesterone treatment. A strong upregulation of these three genes was observed in

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sorted CSCs, but not in non-CSCs. Addition of miR-29 mimic did not prevent SNAI1

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and SNAI2 upregulation, but addition of wortmannin or miR-29 antagomir did. KLF4

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levels were not significantly modulated after the addition of miR-29 mimic or

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wortmannin, but they were after addition of miR-29 antagomir. These findings

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indicate that KLF4 levels are correlated with CSC phenotype. snail and slug are

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upregulated as a consequence of the activation of the PI3K/Akt pathway but are not

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required for CSC phenotype.

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4. Discussion

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Among the main subtypes of breast carcinoma described to date, basal-like tumors are

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of particular interest as they show unfavorable prognosis, lack of established therapy,

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resistance to chemotherapy and a certain propensity to metastasis.

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The question whether the heterogeneity of breast cancer subtypes derives from their

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cellular origin is hotly debated and not yet settled23. Recent evidence suggests that

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luminal progenitors can initiate basal-like breast cancer22 and that basal cells are

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capable of generating both luminal and basal lineages44, reflecting the complexity of

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breast cancer ontogeny. In this study, we have investigated the generation of CSCs in

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basal-like cells. MCF10A cells exhibit a basal molecular signature in gene expression

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profiling studies45 and exhibit some similarities with BBC (such as the lack of

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progesterone and estrogen receptor expression or the overexpression of the MYC

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oncogene)46. For these reasons, MCF10A cells are considered to be an appropriate

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model for BBC initiation studies46,47.

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In the normal breast, PR is expressed in a subset of luminal epithelial cells, and not in

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basal cells; in response to progesterone, PR+ cells communicate with other cells

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through paracrine interactions48,49. However, new reports by us and others have

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shown that PR- MCF10A cells can also directly respond to progesterone

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stimulation31,50-52. Whereas we did not detect nuclear PR expression in MCF10A

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cells, we could measure the expression of mPRα, mPRβ, mPRε and PAQR3

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transcripts. Progesterone treatment did not significantly influence mPR expression

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levels, contrary to earlier evidence suggesting that the abundance of mPRα transcripts

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in the human endometrium is correlated with progesterone levels40.

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We previously have described the generation of CSCs in the MCF10A cell line by

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progesterone treatment combined with 10 Gy ionizing radiation exposure 31. Since a

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dose of 10 Gy X-rays triggered significant cell death, it could be argued that increased

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CSC proportions might partly result from enrichment in radioresistant cells, even

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though there are very few CSCs in untreated MCF10A cells and progesterone

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protected cells against radiation-induced death53. Thus in this study, we evaluated the

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ability of lower irradiation doses to elicit the initiation of CSCs. Three days after

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irradiation, cell death levels were too small to contribute to the enrichment in CSCs.

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Emerging evidence suggests that the PI3K/PTEN/Akt pathway plays an important

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role in the initiation and maintenance of CSCs, as observed in prostate cancer54,

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colorectal cancer55, pancreatic cancer56 or ERα+ breast cancer57. Here we describe a

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new mechanism for CSC induction in mammary basal-like cells, relying on PI3K/Akt

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pathway stimulation by mPR, in agreement with the described role of this pathway in

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CSC biology. Furthermore, our results are consistent with a recent study reporting the

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activation of the PI3K/Akt pathway by mPR as the underlying mechanism of

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progestin-induced sperm hypermotility in the Atlantic croaker fish species16. On the

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contrary, in MDA-MB-468 basal breast cancer cells, progesterone-triggered mPR-

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alpha mediated the repression of EMT through Caveolin 1 (Cav-1), which

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subsequently inactivated the PI3K/Akt pathway17. MDA-MB-468 cells contain a

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mutant pten gene and exhibit sustained PI3K/Akt activation, a common occurrence in

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breast cancer58. However, it was shown that Cav-1 expression is considerably lower in

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MCF10A cells than in cancer cell lines59, which may suggest that the effects resulting

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from mPR activation by progesterone might depend on Cav-1 levels. Those results are

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consistent with the idea that progesterone promotes pre-neoplastic progression in the

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normal breast by stimulating proliferation and promoting tumor-initiating cells, but

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suppresses tumor invasion and metastasis in advanced breast cancer by impeding

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EMT60.

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Generation of CSCs required activity of the downstream transcription factor NFκB,

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which blocks the ubiquitination and degradation of snail61, resulting in increased

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levels of snail/slug, in accordance with other reports showing that EMT-inducing snail

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expression is associated with CSC phenotype62,63.

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Evidence in several models pinpoint miR-29 as a downstream target for NFκB with a

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tumor suppressor role: Sp1/NFκB/HDAC/miR-29b-dependent KIT overexpression

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contributes to acute myeloid leukemia growth38; miR-29b/c is also repressed by NFκB

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in rhabdomyosarcoma through YY137 and in cholangiocarcinoma36. Because miR-

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29b-2 and miR-29-c are transcribed together and their target genes are predicted to

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overlap64, it is plausible that they are regulated similarly and their biological functions

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are similar. Here we show that the repression of miR-29c (by NFκB or by using a

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specific antagomir) is necessary and sufficient to generate ALDH+ cells in MCF10A

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cells. KLF4 was recently identified as a direct target of miR-29 in PR+ T47D luminal-

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like breast cancer cells; in response to progestin treatment, miR-29-mediated

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upregulation of KLF4 triggers the dedifferentiation of T47D cells into CK5+ and

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CD44+ stem-like cells. Our results suggest that a similar progesterone/miR-29/KLF4

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pathway is responsible for the generation of CSCs in MCF10A cells, via the

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activation of an mPR-triggered PI3K/Akt/NFκB pathway.

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mPR activation by progesterone and ionizing radiation also resulted in the

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upregulation of EMT-inducing snail and slug transcription factors65. Growing

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evidence indicate that EMT and cancer stem cells phenotypes are closely connected66.

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Immortalized mammary epithelial cells (HMLE) compelled to undergo EMT via

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ectopic expression of snail generated cells with CSC characteristics (increased

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mammosphere formation abilities, acquisition of the CD44+/CD24- phenotype and

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tumor-initiating properties)63. Upregulation of snail and slug in MCF10A cells is thus

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likely to contribute to the CSC phenotype.

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In our system, progesterone stimulation alone is not sufficient to induce CSCs. It

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remains to be seen how ionizing radiation contributes to the CSC initiating signal in

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MCF10A cells. Activation of PI3K by ionizing radiation has been described in a

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variety of models67,68. Ionizing radiation exposure was shown to activate multiple

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signal transduction processes through various means, such as DNA damage-induced

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signaling, the inhibition of protein phosphatase by reactive oxygen species or the

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generation of ceramides68. Additional investigations are required to understand the

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interactions between ionizing radiation- and progesterone-induced signaling in

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MCF10A cells, leading to the initiation of CSCs.

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In summary, we suggested the existence a PR-independent pathway in MCF10A

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basal-like breast cells, that involves the activation of mPR by progesterone, which

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triggers PI3K/Akt/NFκB pathway, resulting in the downregulation of miR-29 and the

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upregulation of KLF4, a protein necessary for CSC maintenance. This new pathway

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requires priming by ionizing radiation via a yet unknown mechanism. These findings

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have important implications for understanding potential cancer risk resulting from the

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stimulation of basal cells and for establishing new anti-CSC strategies.

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Conflict of interest statement

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None

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5. Acknowledgements

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This study was funded by JSPS KAKENHI Grant Number 24710066.

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6. References

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1.Prat, A., Adamo, B., Cheang, M. C. U., Anders, C. K., Carey, L. A. and Perou, C.

334

M. Molecular characterization of basal-like and non-basal-like triple-negative breast

335

cancer. Oncologist 18, 123-33 (2013).doi:10.1634/theoncologist.2012-0397

336

2.Calcagno, A. M., Salcido, C. D., Gillet, J. -P., Wu, C. -P., Fostel, J. M., Mumau, M.

337

D., Gottesman, M. M., Varticovski, L. and Ambudkar, S. V. Prolonged drug selection

338

of breast cancer cells and enrichment of cancer stem cell characteristics. J Natl

339

Cancer Inst 102, 1637-52 (2010).doi:10.1093/jnci/djq361

340

3.Hankinson, S. E., Colditz, G. A. and Willett, W. C. Towards an integrated model for

341

breast cancer etiology: The lifelong interplay of genes, lifestyle, and hormones. Breast

342

Cancer Research 6, 213-218 (2004).doi:10.1186/bcr921

343

4.Hulka, B. S. and Moorman, P. G. Breast cancer: hormones and other risk factors.

344

Maturitas 61, 203-213 (2008).doi:10.1016/S0378-5122(00)00196-1

345

5.John, E. M., Phipps, A. I., Knight, J. A., Milne, R. L., Dite, G. S., Hopper, J. L.,

346

Andrulis, I. L., Southey, M., Giles, G. G., West, D. W. and Whittemore, A. S.

347

Medical radiation exposure and breast cancer risk: findings from the Breast Cancer

348

Family Registry. Int J Cancer 121, 386-94 (2007).doi:10.1002/ijc.22668

349

6.Li, X., Lewis, M. T., Huang, J., Gutierrez, C., Osborne, C. K., Wu, M. -F.,

350

Hilsenbeck, S. G., Pavlick, A., Zhang, X., Chamness, G. C., Wong, H., Rosen, J. and

351

Chang, J. C. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J

352

Natl Cancer Inst 100, 672-9 (2008).doi:10.1093/jnci/djn123

353

7.Oeffinger, K. C., Ford, J. S., Moskowitz, C. S., Diller, L. R., Hudson, M. M., Chou,

354

J. F., Smith, S. M., Mertens, A. C., Henderson, T. O., Friedman, D. L., Leisenring, W.

355

M. and Robison, L. L. Breast cancer surveillance practices among women previously

15

Page 15 of 28

356

treated with chest radiation for a childhood cancer. JAMA 301, 404-14

357

(2009).doi:10.1001/jama.2008.1039

358

8.Tanei, T., Morimoto, K., Shimazu, K., Kim, S. J., Tanji, Y., Taguchi, T., Tamaki, Y.

359

and Noguchi, S. Association of breast cancer stem cells identified by aldehyde

360

dehydrogenase 1 expression with resistance to sequential Paclitaxel and epirubicin-

361

based chemotherapy for breast cancers. Clin Cancer Res 15, 4234-41

362

(2009).doi:10.1158/1078-0432.CCR-08-1479

363

9.Dressing, G. E., Alyea, R., Pang, Y. and Thomas, P. Membrane Progesterone

364

Receptors (mPRs) Mediate Progestin Induced Antimorbidity in Breast Cancer Cells

365

and Are Expressed in Human Breast Tumors. Hormones and Cancer 3, 101-112

366

(2012).doi:10.1007/s12672-012-0106-x

367

10.Dressing, G. E. and Thomas, P. Identification of membrane progestin receptors in

368

human breast cancer cell lines and biopsies and their potential involvement in breast

369

cancer. Steroids 72, 111-6 (2007).doi:10.1016/j.steroids.2006.10.006

370

11.Karteris, E., Zervou, S., Pang, Y., Dong, J., Hillhouse, E. W., Randeva, H. S. and

371

Thomas, P. Progesterone signaling in human myometrium through two novel

372

membrane G protein-coupled receptors: potential role in functional progesterone

373

withdrawal at term. Mol Endocrinol 20, 1519-34 (2006).doi:10.1210/me.2005-0243

374

12.Pang, Y. and Thomas, P. Progesterone signals through membrane progesterone

375

receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast

376

cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear

377

progesterone receptor. Steroids 76, 921-8 (2011).doi:10.1016/j.steroids.2011.01.008

378

13.Pang, Y., Dong, J. and Thomas, P. Characterization, neurosteroid binding and

379

brain distribution of human membrane progesterone receptors δ and ε (mPRδ and

16

Page 16 of 28

380

mPRε) and mPRδ involvement in neurosteroid inhibition of apoptosis. Endocrinology

381

154, 283-95 (2013).doi:10.1210/en.2012-1772

382

14.Thomas, P., Pang, Y., Dong, J., Groenen, P., Kelder, J., de Vlieg, J., Zhu, Y. and

383

Tubbs, C. Steroid and G protein binding characteristics of the seatrout and human

384

progestin membrane receptor alpha subtypes and their evolutionary origins.

385

Endocrinology 148, 705-18 (2007).doi:10.1210/en.2006-0974

386

15.Zhu, Y., Rice, C. D., Pang, Y., Pace, M. and Thomas, P. Cloning, expression, and

387

characterization of a membrane progestin receptor and evidence it is an intermediary

388

in meiotic maturation of fish oocytes. Proc Natl Acad Sci U S A 100, 2231-6

389

(2003).doi:10.1073/pnas.0336132100

390

16.Tan, W. and Thomas, P. Activation of the Pi3k/Akt Pathway and Modulation of

391

Phosphodiesterase Activity via Membrane Progestin Receptor-Alpha (mPRalpha)

392

Regulate Progestin-Initiated Sperm Hypermotility in Atlantic Croaker. Biology of

393

Reproduction biolreprod.113.112896 (2014).doi:10.1095/biolreprod.113.112896

394

17.Zuo, L., Li, W. and You, S. Progesterone reverses the mesenchymal phenotypes of

395

basal phenotype breast cancer cells via a membrane progesterone receptor mediated

396

pathway. Breast Cancer Research 12, R34 (2010).doi:10.1186/bcr2588

397

18.Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. and Clarke, M.

398

F. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U

399

S A 100, 3983-8 (2003).doi:10.1073/pnas.0530291100

400

19.Sheridan, C., Kishimoto, H., Fuchs, R. K., Mehrotra, S., Bhat-Nakshatri, P.,

401

Turner, C. H., Goulet, R., Badve, S. and Nakshatri, H. CD44+/CD24- breast cancer

402

cells exhibit enhanced invasive properties: an early step necessary for metastasis.

403

Breast Cancer Research 8, R59 (2006).doi:10.1186/bcr1610

17

Page 17 of 28

404

20.Ginestier, C., Hur, M. H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown,

405

M., Jacquemier, J., Viens, P., Kleer, C. G., Liu, S., Schott, A., Hayes, D., Birnbaum,

406

D., Wicha, M. S. and Dontu, G. ALDH1 is a marker of normal and malignant human

407

mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555-

408

67 (2007).doi:10.1016/j.stem.2007.08.014

409

21.Ablett, M. P., Singh, J. K. and Clarke, R. B. Stem cells in breast tumours: are they

410

ready for the clinic? Eur J Cancer 48, 2104-16 (2012).doi:10.1016/j.ejca.2012.03.019

411

22.Molyneux, G., Geyer, F. C., Magnay, F. -A., McCarthy, A., Kendrick, H.,

412

Natrajan, R., MacKay, A., Grigoriadis, A., Tutt, A., Ashworth, A., Reis-Filho, J. S.

413

and Smalley, M. J. BRCA1 Basal-like Breast Cancers Originate from Luminal

414

Epithelial Progenitors and Not from Basal Stem Cells. Cell Stem Cell 7, 403-417

415

(2010).doi:10.1016/j.stem.2010.07.010

416

23.Blanpain, C. Tracing the cellular origin of cancer. Nature Cell Biology 15, 126-134

417

(2013).doi:10.1038/ncb2657

418

24.DiRenzo, J., Signoretti, S., Nakamura, N., Rivera-Gonzalez, R., Sellers, W., Loda,

419

M. and Brown, M. Growth factor requirements and basal phenotype of an

420

immortalized mammary epithelial cell line. Cancer Res 62, 89-98 (2002).

421

25.Debnath, J., Muthuswamy, S. K. and Brugge, J. S. Morphogenesis and

422

oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional

423

basement membrane cultures. Methods 30, 256-68 (2003).

424

26.Charafe-Jauffret, E., Ginestier, C., Iovino, F., Wicinski, J., Cervera, N., Finetti, P.,

425

Hur, M. -H., Diebel, M. E., Monville, F., Dutcher, J., Brown, M., Viens, P., Xerri, L.,

426

Bertucci, F., Stassi, G., Dontu, G., Birnbaum, D. and Wicha, M. S. Breast cancer cell

427

lines contain functional cancer stem cells with metastatic capacity and a distinct

18

Page 18 of 28

428

molecular signature. Cancer Res 69, 1302-13 (2009).doi:10.1158/0008-5472.CAN-

429

08-2741

430

27.Asiedu, M. K., Ingle, J. N., Behrens, M. D., Radisky, D. C. and Knutson, K. L.

431

TGFβ/TNFα-Mediated Epithelial–Mesenchymal Transition Generates Breast Cancer

432

Stem Cells with a Claudin-Low Phenotype. Cancer Research 71, 4707-4719

433

(2011).doi:10.1158/0008-5472.CAN-10-4554

434

28.Morel, A. -P., Lièvre, M., Thomas, C., Hinkal, G., Ansieau, S. and Puisieux, A.

435

Generation of breast cancer stem cells through epithelial-mesenchymal transition.

436

PLoS One 3, e2888 (2008).doi:10.1371/journal.pone.0002888

437

29.Hirsch, H. A., Iliopoulos, D., Tsichlis, P. N. and Struhl, K. Metformin selectively

438

targets cancer stem cells, and acts together with chemotherapy to block tumor growth

439

and prolong remission. Cancer Res 69, 7507-11 (2009).doi:10.1158/0008-5472.CAN-

440

09-2994

441

30.Iliopoulos, D., Hirsch, H. A., Wang, G. and Struhl, K. Inducible formation of

442

breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via

443

IL6 secretion. Proceedings of the National Academy of Sciences 108, 1397-1402

444

(2011).doi:10.1073/pnas.1018898108

445

31.Vares, G., Cui, X., Wang, B., Nakajima, T. and Nenoi, M. Generation of breast

446

cancer stem cells by steroid hormones in irradiated human mammary cell lines. PLoS

447

One 8, e77124 (2013).doi:10.1371/journal.pone.0077124

448

32.Takahashi, R. -U., Miyazaki, H. and Ochiya, T. The role of microRNAs in the

449

regulation of cancer stem cells. Front. Genet 4, (2014).doi:10.3389/fgene.2013.00295

450

33.Iliopoulos, D., Lindahl-Allen, M., Polytarchou, C., Hirsch, H. A., Tsichlis, P. N.

451

and Struhl, K. Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated

19

Page 19 of 28

452

repression required for the formation and maintenance of cancer stem cells. Mol Cell

453

39, 761-72 (2010).doi:10.1016/j.molcel.2010.08.013

454

34.Ibarra, I., Erlich, Y., Muthuswamy, S. K., Sachidanandam, R. and Hannon, G. J. A

455

role for microRNAs in maintenance of mouse mammary epithelial progenitor cells.

456

Genes Dev 21, 3238-43 (2007).doi:10.1101/gad.1616307

457

35.Cittelly, D. M., Finlay-Schultz, J., Howe, E. N., Spoelstra, N. S., Axlund, S. D.,

458

Hendricks, P., Jacobsen, B. M., Sartorius, C. A. and Richer, J. K. Progestin

459

suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4.

460

Oncogene 32, 2555-2564 (2012).doi:10.1038/onc.2012.275

461

36.Mott, J. L., Kurita, S., Cazanave, S. C., Bronk, S. F., Werneburg, N. W. and

462

Fernandez-Zapico, M. E. Transcriptional suppression of mir-29b-1/mir-29a promoter

463

by c-Myc, hedgehog, and NF-kappaB. J Cell Biochem 110, 1155-64

464

(2010).doi:10.1002/jcb.22630

465

37.Wang, H., Garzon, R., Sun, H., Ladner, K. J., Singh, R., Dahlman, J., Cheng, A.,

466

Hall, B. M., Qualman, S. J., Chandler, D. S., Croce, C. M. and Guttridge, D. C. NF-

467

kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and

468

rhabdomyosarcoma. Cancer Cell 14, 369-81 (2008).doi:10.1016/j.ccr.2008.10.006

469

38.Liu, S., Wu, L. -C., Pang, J., Santhanam, R., Schwind, S., Wu, Y. -Z., Hickey, C.

470

J., Yu, J., Becker, H., Maharry, K., Radmacher, M. D., Li, C., Whitman, S. P., Mishra,

471

A., Stauffer, N., Eiring, A. M., Briesewitz, R., Baiocchi, R. A., Chan, K. K., Paschka,

472

P., Caligiuri, M. A., Byrd, J. C., Croce, C. M., Bloomfield, C. D., Perrotti, D., Garzon,

473

R. and Marcucci, G. Sp1/NFkappaB/HDAC/miR-29b regulatory network in KIT-

474

driven myeloid leukemia. Cancer Cell 17, 333-47

475

(2010).doi:10.1016/j.ccr.2010.03.008

20

Page 20 of 28

476

39.Akaogi, K., Nakajima, Y., Ito, I., Kawasaki, S., Oie, S. -H., Murayama, A.,

477

Kimura, K. and Yanagisawa, J. KLF4 suppresses estrogen-dependent breast cancer

478

growth by inhibiting the transcriptional activity of ERα. Oncogene 28, 2894-2902

479

(2009).doi:10.1038/onc.2009.151

480

40.Fernandes, M. S., Pierron, V., Michalovich, D., Astle, S., Thornton, S., Peltoketo,

481

H., Lam, E. W., Gellersen, B., Huhtaniemi, I., Allen, J. and Brosens, J. J. Regulated

482

expression of putative membrane progestin receptor homologues in human

483

endometrium and gestational tissues. J Endocrinol 187, 89-101

484

(2005).doi:10.1677/joe.1.06242

485

41.Herfs, M., Hubert, P., Kholod, N., Caberg, J. H., Gilles, C., Berx, G., Savagner, P.,

486

Boniver, J. and Delvenne, P. Transforming Growth Factor-β1-Mediated Slug and

487

Snail Transcription Factor Up-Regulation Reduces the Density of Langerhans Cells in

488

Epithelial Metaplasia by Affecting E-Cadherin Expression. The American Journal of

489

Pathology 172, 1391-1402 (2008).doi:10.2353/ajpath.2008.071004

490

42.Li, H., Sun, L., Xu, Y., Li, Z., Luo, W., Tang, Z., Qiu, X. and Wang, E.

491

Overexpression of MTA3 Correlates with Tumor Progression in Non-Small Cell Lung

492

Cancer. PLoS ONE 8, e66679 (2013).doi:10.1371/journal.pone.0066679at

493



494

43.Kelder, J., Azevedo, R., Pang, Y., de Vlieg, J., Dong, J. and Thomas, P.

495

Comparison between steroid binding to membrane progesterone receptor alpha

496

(mPRalpha) and to nuclear progesterone receptor: correlation with physicochemical

497

properties assessed by comparative molecular field analysis and identification of

498

mPRalpha-specific agonists. Steroids 75, 314-22

499

(2010).doi:10.1016/j.steroids.2010.01.010

21

Page 21 of 28

500

44.Keymeulen, A. V., Rocha, A. S., Ousset, M., Beck, B., Bouvencourt, G., Rock, J.,

501

Sharma, N., Dekoninck, S. and Blanpain, C. Distinct stem cells contribute to

502

mammary gland development and maintenance. Nature 479, 189

503

(2011).doi:doi:10.1038/nature10573

504

45.Charafe-Jauffret, E., Ginestier, C., Monville, F., Finetti, P., Adélaïde, J., Cervera,

505

N., Fekairi, S., Xerri, L., Jacquemier, J., Birnbaum, D. and Bertucci, F. Gene

506

expression profiling of breast cell lines identifies potential new basal markers.

507

Oncogene 25, 2273-2284 (2006).doi:10.1038/sj.onc.1209254

508

46.Pires, M. M., Hopkins, B. D., Saal, L. H. and Parsons, R. E. Alterations of EGFR,

509

p53 and PTEN that mimic changes found in basal-like breast cancer promote

510

transformation of human mammary epithelial cells. Cancer Biol Ther 14, 246-53

511

(2013).doi:10.4161/cbt.23297

512

47.Croucher, D. R., Hochgräfe, F., Zhang, L., Liu, L., Lyons, R. J., Rickwood, D.,

513

Tactacan, C. M., Browne, B. C., Ali, N., Chan, H., Shearer, R., Gallego-Ortega, D.,

514

Saunders, D. N., Swarbrick, A. and Daly, R. J. Involvement of Lyn and the Atypical

515

Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway. Cancer

516

Research 73, 1969-1980 (2013).doi:10.1158/0008-5472.CAN-12-1472

517

48.Clarke, R. B., Howell, A., Potten, C. S. and Anderson, E. Dissociation between

518

steroid receptor expression and cell proliferation in the human breast. Cancer Res 57,

519

4987-91 (1997).

520

49.Clarke, M. F., Dick, J. E., Dirks, P. B., Eaves, C. J., Jamieson, C. H. M., Jones, D.

521

L., Visvader, J., Weissman, I. L. and Wahl, G. M. Cancer stem cells--perspectives on

522

current status and future directions: AACR Workshop on cancer stem cells. Cancer

523

Res 66, 9339-44 (2006).doi:10.1158/0008-5472.CAN-06-3126

22

Page 22 of 28

524

50.Behera, M. A., Dai, Q., Garde, R., Saner, C., Jungheim, E. and Price, T. M.

525

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis

526

in MCF-10A benign breast epithelial cells. Am J Physiol Endocrinol Metab 297,

527

E1089-96 (2009).doi:10.1152/ajpendo.00209.2009

528

51.Krämer, E. A., Seeger, H., Krämer, B., Wallwiener, D. and Mueck, A. O. The

529

effect of progesterone, testosterone and synthetic progestogens on growth factor- and

530

estradiol-treated human cancerous and benign breast cells. Eur J Obstet Gynecol

531

Reprod Biol 129, 77-83 (2006).doi:10.1016/j.ejogrb.2005.12.004

532

52.Krämer, E. A., Seeger, H., Krämer, B., Wallwiener, D. and Mueck, A. O.

533

Characterization of the stimulatory effect of medroxyprogesterone acetate and

534

chlormadinone acetate on growth factor treated normal human breast epithelial cells. J

535

Steroid Biochem Mol Biol 98, 174-8 (2006).doi:10.1016/j.jsbmb.2005.11.002

536

53.Vares, G., Ory, K., Lectard, B., Levalois, C., Altmeyer-Morel, S., Chevillard, S.

537

and Lebeau, J. Progesterone prevents radiation-induced apoptosis in breast cancer

538

cells. Oncogene 23, 4603-13 (2004).doi:10.1038/sj.onc.1207601

539

54.Dubrovska, A., Kim, S., Salamone, R. J., Walker, J. R., Maira, S. -M., García-

540

Echeverría, C., Schultz, P. G. and Reddy, V. A. The role of PTEN/Akt/PI3K signaling

541

in the maintenance and viability of prostate cancer stem-like cell populations.

542

Proceedings of the National Academy of Sciences 106, 268-273

543

(2009).doi:10.1073/pnas.0810956106

544

55.Fang, D. D., Zhang, C. C., Gu, Y., Jani, J. P., Cao, J., Tsaparikos, K., Yuan, J.,

545

Thiel, M., Jackson-Fisher, A., Zong, Q., Lappin, P. B., Hayashi, T., Schwab, R. B.,

546

Wong, A., John-Baptiste, A., Bagrodia, S., Los, G., Bender, S., Christensen, J. and

547

VanArsdale, T. Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502

548

in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells

23

Page 23 of 28

549

Harboring a PIK3CA Mutation. PLoS ONE 8, e67258

550

(2013).doi:10.1371/journal.pone.0067258

551

56.Mueller, M. -T., Hermann, P. C., Witthauer, J., Rubio-Viqueira, B., Leicht, S. F.,

552

Huber, S., Ellwart, J. W., Mustafa, M., Bartenstein, P., D'Haese, J. G., Schoenberg,

553

M. H., Berger, F., Jauch, K. -W., Hidalgo, M. and Heeschen, C. Combined targeted

554

treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.

555

Gastroenterology 137, 1102-13 (2009).doi:10.1053/j.gastro.2009.05.053

556

57.Hardt, O., Wild, S., Oerlecke, I., Hofmann, K., Luo, S., Wiencek, Y., Kantelhardt,

557

E., Vess, C., Smith, G. P., Schroth, G. P., Bosio, A. and Dittmer, J. Highly sensitive

558

profiling of CD44+/CD24− breast cancer stem cells by combining global mRNA

559

amplification and next generation sequencing: Evidence for a hyperactive PI3K

560

pathway. Cancer Letters 325, 165-174 (2012).doi:10.1016/j.canlet.2012.06.010

561

58.Pandolfi, P. P. Breast Cancer — Loss of PTEN Predicts Resistance to Treatment.

562

N Engl J Med 351, 2337-2338 (2004).doi:10.1056/NEJMcibr043143

563

59.Joshi, B., Strugnell, S. S., Goetz, J. G., Kojic, L. D., Cox, M. E., Griffith, O. L.,

564

Chan, S. K., Jones, S. J., Leung, S. -P., Masoudi, H., Leung, S., Wiseman, S. M. and

565

Nabi, I. R. Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal

566

Adhesion Dynamics and Tumor Cell Migration and Invasion. Cancer Research 68,

567

8210-8220 (2008).doi:10.1158/0008-5472.CAN-08-0343

568

60.Obr, A. E. and Edwards, D. P. The biology of progesterone receptor in the normal

569

mammary gland and in breast cancer. Molecular and Cellular Endocrinology 357, 4-

570

17 (2012).doi:10.1016/j.mce.2011.10.030

571

61.Wu, Y., Deng, J., Rychahou, P. G., Qiu, S., Evers, B. M. and Zhou, B. P.

572

Stabilization of snail by NF-kappaB is required for inflammation-induced cell

573

migration and invasion. Cancer Cell 15, 416-28 (2009).doi:10.1016/j.ccr.2009.03.016

24

Page 24 of 28

574

62.Heiden, K. B., Williamson, A. J., Doscas, M. E., Ye, J., Wang, Y., Liu, D., Xing,

575

M., Prinz, R. A. and Xu, X. The sonic hedgehog signaling pathway maintains the

576

cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail

577

expression. J Clin Endocrinol Metab 99, E2178-87 (2014).doi:10.1210/jc.2014-1844

578

63.Mani, S. A., Guo, W., Liao, M. -J., Eaton, E. N., Ayyanan, A., Zhou, A. Y.,

579

Brooks, M., Reinhard, F., Zhang, C. C., Shipitsin, M., Campbell, L. L., Polyak, K.,

580

Brisken, C., Yang, J. and Weinberg, R. A. The Epithelial-Mesenchymal Transition

581

Generates Cells with Properties of Stem Cells. Cell 133, 704-715

582

(2008).doi:10.1016/j.cell.2008.03.027

583

64.Kriegel, A. J., Liu, Y., Fang, Y., Ding, X. and Liang, M. The miR-29 family:

584

genomics, cell biology, and relevance to renal and cardiovascular injury. Physiol

585

Genomics 44, 237-44 (2012).doi:10.1152/physiolgenomics.00141.2011

586

65.Wang, Y. and Zhou, B. P. Epithelial-mesenchymal transition in breast cancer

587

progression and metastasis. Chin J Cancer 30, 603-11

588

(2011).doi:10.5732/cjc.011.10226

589

66.Scheel, C. and Weinberg, R. A. Cancer stem cells and epithelial–mesenchymal

590

transition: Concepts and molecular links. Seminars in Cancer Biology 22, 396-403

591

(2012).doi:10.1016/j.semcancer.2012.04.001

592

67.Li, H. -F., Kim, J. -S. and Waldman, T. Radiation-induced Akt activation

593

modulates radioresistance in human glioblastoma cells. Radiation Oncology 4, 43

594

(2009).doi:10.1186/1748-717X-4-43

595

68.Valerie, K., Yacoub, A., Hagan, M. P., Curiel, D. T., Fisher, P. B., Grant, S. and

596

Dent, P. Radiation-induced cell signaling: inside-out and outside-in. Molecular

597

Cancer Therapeutics 6, 789-801 (2007).doi:10.1158/1535-7163.MCT-06-0596

598 599 25

Page 25 of 28

600

7. Figure legends

601

Figure 1. Generation of CSCs by progesterone and ionizing radiation relies on

602

PI3K and NFκB. The percentages of ALDH+ MCF10A cells were evaluated by flow

603

cytometry three days after irradiation (1 Gy X-rays). Hormonal treatment was

604

performed two days before irradiation and every day afterwards. Pg: progesterone,

605

CAPE: caffeic acid phenethyl ester (NFκB inhibitor), W: wortmannin (PI3K

606

inhibitor). Results are the average of at least three independent experiments. Error

607

bars represent standard deviation. Asterisks denote significant differences (t-test, *

608

p<0.01).

609 610

Figure 2. MCF10A cells express membrane progestin receptors but not nuclear

611

progesterone receptor. PAQR7 (mPRα), PAQR8 (mPRβ), PAQR5 (mPRγ), PAQR9

612

(mPRε), PAQR3 and MPR (nuclear PR) gene expression levels, normalized against

613

MRPL19 levels (mitochondrial ribosomal protein L19), were measured by real-time

614

PCR in MCF10A cells, untreated (Control) or 24 hours after progesterone treatment

615

(Pg). Lower delta Ct values (Ct value of RNA – Ct value of L19) correspond to

616

higher gene expression levels. N.D.: not detected. Results are the average of at least

617

three independent experiments. Error bars represent standard deviation.

618 619

Figure 3. Stimulation of mPR by progestins activates the PI3K/Akt pathway. In

620

order to monitor the activity of PI3K/Akt pathway, we measured FOXO

621

transcriptional activity in MCF10A cells transiently co-transfected with a FOXO

622

luciferase reporter vector (or with a non-inducible luciferase vector as negative

623

control) and with a constitutively-expressing Renilla luciferase vector. Normalized

26

Page 26 of 28

624

luciferase activity for the FOXO reporter was obtained by substracting the

625

background luminescence and by calculating the ratio of firefly luminescence from

626

the FOXO reporter to Renilla luminescence from the control Renilla luciferase vector.

627

Pg: progesterone, RU: mifepristone (RU-486, PR antagonist), OD: Org OD 02-0

628

(mPR agonist), W: wortmannin (PI3K inhibitor). Results are the average of at least

629

three independent experiments. Error bars represent standard deviation. Asterisks

630

denote significant differences (t-test, * p<0.01).

631 632

Figure 4. Downregulation of miR-29c is necessary for CSC initiation and

633

maintenance. The percentages of ALDH+ MCF10A cells were evaluated by flow

634

cytometry three days after irradiation (1 Gy X-rays). Hormonal treatment was

635

performed two days before irradiation and every day afterwards. Antagomir for miR-

636

29 was transfected two days before irradiation and every other day afterwards. miR-

637

29 mimic was transfected on the day of irradiation and two days later. Pg:

638

progesterone, W: wortmannin (PI3K inhibitor), mimic-29: miR-29 mimic, antagomir-

639

29: miR-29 antagomir. Results are the average of at least three independent

640

experiments. Error bars represent standard deviation. Asterisks denote significant

641

differences (t-test, * p<0.01).

642 643

Figure 5. miR-29 downregulation is associated with increased mammmosphere-

644

forming abilities. Sorted ALDH- and ALDH+ MCF10A cells were plated in ultra-low

645

adherence plates and the number of mammospheres formed after 7 days was counted.

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ALDH+ cells were transfected with miR-29 mimic or antagomir every other day until

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counting. Results are the average of at least three independent experiments. Error bars

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represent standard deviation. Asterisks denote significant differences (t-test, *

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p<0.01).

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Figure 6. SNAI1 (snail), SNAI2 (slug) and KLF4 are upregulated in CSCs. Gene

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expression levels, normalized against 18S rRNA, were measured by real-time PCR in

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whole cell populations and in sorted CSCs and non-CSCs. Pg: progesterone, W:

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wortmannin (PI3K inhibitor), mimic-29: miR-29 mimic, antagomir-29: miR-29

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antagomir. Results are the average of at least three independent experiments. Error

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bars represent standard deviation. Asterisks denote significant differences (t-test, *

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p<0.01).

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