Progesterone (P4) has emerged as an important hormone-regulating mammary stem cell (MaSC) populations. In breast cancer, P4 and synthetic analogs increase the number of stem-like cells within luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancers. These cells gain expression of de-differentiated cell markers CD44 and cytokeratin 5 (CK5), lose luminal markers ER and PR, and are more therapy resistant. We previously described that P4 downregulation of microRNA (miR)-29a contributes to the expansion of CD44high and CK5+ cells. Here we investigated P4 downregulation of miR-141, a member of the miR-200 family of tumor suppressors, in facilitating an increase in stem-like breast cancer cells. miR-141 was the sole member of the miR-200 family P4-downregulated at the mature miRNA level in luminal breast cancer cell lines. Stable inhibition of miR-141 alone increased the CD44high population, and potentiated P4-mediated increases in both CD44high and CK5+ cells. Loss of miR-141 enhanced both mammosphere formation and tumor initiation. miR-141 directly targeted both PR and signal transducer and activator of transcription 5A (Stat5a), transcription factors important for MaSC expansion. miR-141 depletion increased PR protein levels, even in cell lines where PR expression is estrogen dependent. Stat5a suppression via small interfering RNA or a small-molecule inhibitor reduced the P4-dependent increase in CK5+ and CD44high cells. These data support a mechanism by which P4-triggered loss of miR-141 facilitates breast cancer cell de-differentiation through deregulation of PR and Stat5a, two transcription factors important for controlling mammary cell fate.
A major role of progesterone (P4) in the mammary gland is to promote lobulo-alveolar development during specific stages, such as puberty, pregnancy and lactation.1 In murine models, this process involves the P4-dependent expansion of mammary stem cells (MaSCs) that in turn rapidly generate additional lobular epithelial cells.2,3 This occurs through a paracrine mechanism whereby progesterone receptor (PR)+ cells in the luminal compartment signal through receptor activator of nuclear factor kappa-B ligand (RANKL) to expand basal-located stem cells.4,5 In humans, P4 increases progenitor cells in recapitulated breast epithelial acini structures.6 RANKL is P4 regulated in human breast,7 and thus this expansion could also occur through inter-cellular signaling. Expansion of pre-malignant stem cell populations may explain increased breast cancer incidence among women taking hormone therapies containing progestin.8 We previously demonstrated in estrogen receptor (ER)+ and PR+ breast cancer cells that P4 treatment leads to the emergence of cells expressing cytokeratin 5 (CK5),9,10 a marker of stem and progenitor cells in the normal breast and breast cancer.11, 12, 13 CK5+, compared with the bulk CK5−, tumor cells are endocrine and chemotherapy resistant, and have enhanced mammosphere-forming potential.11,14 Progestin exposure of existing breast cancers may thus negatively affect tumor progression.
MicroRNAs (miRNAs/miRs) are small regulatory RNA molecules that regulate expression of specific target genes by base-pairing to their mRNAs and interfering with their translation and/or inducing their degradation. miRNA maturation is regulated by several processing steps. The primary miRNA transcript (pri-miRNA) is usually RNA Pol II generated and can include multiple miR sequences from a cluster. The pri-miRNA is then processed to its precursor hairpin form (pre-miRNA) by the ribonuclease III protein Drosha, exported out of the nucleus and cleaved into its mature form by the ribonuclease III enzyme Dicer.15 miRNA species are in general downregulated in cancer compared with normal adult tissue.16 Loss of the miR-200 family (miR-200abc, miR-141, miR-429) in particular leads to epithelial–mesenchymal transition in normal and cancer cells.17 Restoration of miR-200c is sufficient to revert mesenchymal breast cancer cells into a more epithelial phenotype.17, 18, 19, 20, 21 Progestins regulate multiple miRNAs in breast cancer cells, the majority (~70%) of which are downregulated22, 23, 24 providing a mechanism to indirectly control cellular differentiation. We previously demonstrated that progestin suppression of miR-29 family members facilitates induction of the transcription factor KLF4 and potentiates the expansion of CD44+ and CK5+ breast cancer cells.24 miR-141 is also progestin downregulated in breast cancer cells,22 and we therefore speculated that it may contribute to expansion of de-differentiated breast cancer cells.
The cancer stem cell (CSC) theory posits that a subset of pre-existing CSCs, the putative origins of malignancies, perpetuate tumors through indefinite self-renewal and replicative potentials.25 Breast CSCs, compared with bulk tumor cells, are relatively resistant to conventional drugs and are mostly ER−PR−, thus avoiding endocrine therapies.14,25,26 Notably, a CSC state may be acquired either spontaneously or through environmental signals during tumor progression.27,28 Progesterone is one such factor that controls MaSC plasticity; this could be partially facilitated through miR regulation. We therefore tested whether miR-141 suppression could contribute to P4-mediated cellular de-differentiation. Here we demonstrate that miR-141 inhibition enhanced the P4-mediated increase in CD44high and CK5+ cells. miR-141 directly targeted PR and signal transducer and activator of transcription 5A (Stat5a), a transcription factor and P4-regulated gene29,30 that regulates the mammary luminal progenitor population.31 Stat5a inhibition reduced the P4-mediated increase in CK5+ and CD44high populations. These findings emphasize that hormonal alteration of miR levels contributes to the regulation of breast CSCs via transcription factors that promote MaSC populations.
P4 downregulates mature miR-141 in luminal breast cancer cell lines
The miR-200 family is organized on two different chromosomes; the miR-141 gene is downstream of miR-200c on chr12p13, with the remaining members (miR-200b-200a-429) clustered on chr1p36 (Figure 1a). To determine whether miR-141 expression correlates with a particular breast cancer subtype, we assessed miR-141 expression in multiple breast cancer cell lines. miR-141 was expressed at detectable levels in luminal and basal-like triple negative cell lines, and was absent in mesenchymal-like triple negative cell lines (Supplementary Figure 1), where the miR-200c/141 cluster is silenced by CpG methylation.32
Global miRNA profiling of T47D breast cancer cells treated with vehicle or the synthetic progestin medroxyprogesterone acetate (MPA) identified miR-141 downregulation at 6 h post treatment.22 To confirm P4-mediated regulation of miR-141, luminal breast cancer cell lines T47D, BT474 and ZR75-1 were treated with vehicle (ethanol) or 100 nM P4 for 6 or 24 h, and mature miR-141 levels measured by quantitative PCR. BT474 and ZR75-1 cells were pre-treated with 10 nM 17β-estradiol (E2) for 48 h to induce PR levels. In all three cell lines, mature miR-141 levels significantly decreased at 6 h post P4 treatment, and returned to pre-treatment levels at 24 h (Figure 1b). Co-treatment with the progestin antagonist RU486 for 6 h blocked miR-141 downregulation, suggesting a PR-dependent mechanism. E2 alone had no effect on miR-141 levels (Supplementary Figure 2). In contrast, mature miR-200a, miR-200b and miR-200c levels did not significantly change with 6 h P4 treatment (Figure 1c). An expanded time course confirmed 6 h as the maximal miR-141 reduction point, with no changes in the other family members (Supplementary Figure 3).
As miR-141 is located downstream of miR-200c on the miR-200c/-141 cluster,32 but P4 decreases only mature miR-141, we sought to determine if P4 regulates the pri-miRNA transcript. Quantitative PCR with primers specific to pri-miR-141 or pri-miR-200c (provided in Supplementary Figure 4a) found that both transcripts decreased at 6 h post P4 treatment, and were blocked by RU486 (Figure 1d). An extended time course (0–12 h) confirmed maximal downregulation of pri-miR-141 and pri-miR-200 at 6 h (Supplementary Figure 4b). There is emerging evidence for differential processing of miRs located in the same cluster,33 with separate transcription units observed for miR-141 and miR-200c.32 These data suggest differential biogenesis of miR-141 and miR-200c following P4 treatment; selective underexpression of miR-141 has been previously observed in prostate cancer.34
Stable inhibition of miR-141 potentiates the P4-mediated expansion of CD44high and CK5+ cells
We evaluated CD44high and CK5+ populations as both are reported markers of CSCs and are P4-regulated.9,24,35 Although there is considerable overlap between the two populations, a distinct CD44highCK5− population exists.9 As miR-141 is underexpressed in CD44highCD24−/low breast CSCs isolated from patient tumors,36 we sought to determine whether miR-141 levels are changed in the P4-induced CD44high population. T47D cells were treated with vehicle or P4 for 24 h then fluorescence-activate cell sorting performed to isolate the CD44high and CD44low populations. Expression of mature miR-141 was significantly reduced in both the endogenous and P4-induced CD44high populations (Figure 2a). Although this could indicate direct expansion of the CD44high population, conversion is more probable given a cell doubling time of 36 h and maximal downregulation of miR-141 at 6 h.
To determine whether miR-141 suppression exacerbates the P4-induced expansion of CD44high and CK5+ populations, we generated stable control (SCR-ZIP) and miR-141-inhibited (141-ZIP) cell lines. SCR-ZIP and 141-ZIP T47D cells were treated with vehicle or P4 for 24 h and the CD44high and CK5+ populations measured by flow cytometry, or immunofluorescence and western blot, respectively. P4 treatment led to a fivefold increase in the CD44high population (5%) in SCR-ZIP cells (Figure 2b), as previously described.24 Vehicle-treated 141-ZIP compared with SCR-ZIP cells had a 12-fold increase in the CD44high population (12%). P4-treated 141-ZIP compared with SCR-ZIP had a eightfold increase in the CD44high population (40%; Figure 2b). CK5+ cells were absent in vehicle-treated SCR-ZIP and 141-ZIP T47D and BT474 cells. The observed P4-induced CK5+ cells in SCR-ZIP cell lines were potentiated approximately twofold in 141-ZIP cell lines (Figure 2c). By western blot analysis, total PR protein levels increased by 1.5-fold in 141-ZIP cells, and still underwent ligand-dependent downregulation. CK5 protein levels increased by >2-fold in P4-treated 141-ZIP compared with SCR-ZIP cells (Figure 2d). These results indicate that downregulation of miR-141 alone contributes to expanding the CD44high population, and potentiates the P4-mediated increases in the CD44high and CK5+ populations.
Stable inhibition of miR-141 increases the mammosphere and tumor-initiating capacity of breast cancer cells
To test whether miR-141 inhibition affects breast cancer cell self-renewal, we performed mammosphere formation assays using T47D SCR-ZIP and 141-ZIP cells. P4 treatment alone increased mammosphere formation of SCR-ZIP cells (Figure 3a). 141-ZIP compared with SCR-ZIP cells had increased mammosphere formation in both control and P4-treated samples. Thus, inhibition of miR-141 alone increases the sphere-forming capability of luminal breast cancer cells, potentially due to increasing the CD44high population, and enhances the P4-mediated increase in mammosphere formation, potentially due to increased CD44high and CK5+ cells.
We next evaluated the ability of SCR-ZIP and 141-ZIP cells to initiate tumors; cells were pre-treated for 24 h with P4 in vitro then injected bilaterally into the fourth mammary fat pads of female nude mice at dilutions of 102–104. Measured at 5 and 6 weeks post implantation, 141-ZIP cells initiated tumors more efficiently compared with SCR-ZIP cells (Table 1). These data show that loss of miR-141 enhances tumor-initiating ability, likely due to amplified CD44high and CK5+ populations.
To determine whether the observed differences in sphere and tumor formation could be due to differential cell growth rate, we measured proliferation of SCR-ZIP and 141-ZIP in vitro and in vivo. For in vitro experiments, SCR-ZIP, 141-ZIP T47D and BT474 cells were plated in sextuplicate in 96-well plates, treated with vehicle or 100 nM P4 (T47D), or E2 and E2+P4 (BT474) and proliferation was measured via the Incucyte kinetic live-cell imaging system over 4 days. In two luminal breast cancer lines, 141-ZIP compared with SCR-ZIP cells had significantly reduced proliferation in the absence or presence of P4 (Figure 3b).
To evaluate tumor growth in vivo, we injected 1 × 106 T47D SCR-ZIP and 141-ZIP cells in the fourth mammary fat pad of NOD/SCID mice supplemented with either E2 alone or E2+MPA. SCR-ZIP and 141-ZIP cells were implanted on opposing mammary glands for internal comparison. There was no statistical size difference between SCR-ZIP and 141-ZIP tumors in the E2-treated group (Figure 3c). Tumors in E2+MPA-supplemented mice were overall smaller as previously observed.24 141-ZIP compared with SCR-ZIP tumors were not statistically different in volume or mass, except for one data point at 69 days post implantation (Figure 3c). We conclude that miR-141 inhibition decreases short-term cell proliferation, and has no overall effect on long-term tumor growth. Therefore, its enhancement of sphere and tumor initiation is not a function of increased growth capacity.
PR is a direct target of miR-141 and increases with miR-141 inhibition
To determine relevant targets of miR-141 that may be involved in promoting the expansion of stem-like cells, we focused on transcription factors important in mammary gland differentiation. As miR-141 is predicted to target the PGR gene, which encodes both isoforms of PR (PR-B, PR-A), we first analyzed the effect of miR-141 manipulation on PR expression. PR protein expression significantly increased in three different luminal breast cancer cell lines (T47D, BT474 and ZR75-1) with miR-141 inhibition (141-ZIP; Figures 2d and 4a). Conversely, PR expression was decreased in the same three cell lines when miR-141 was overexpressed using a lentiviral vector carrying its precursor sequence (Pre-141) or a scrambled control (Pre-SCR; Figure 4b).
To test if miR-141 directly targets the PR transcript, we analyzed four predicted miR-141-binding sites (Figure 4c); three within the 3′ untranslated region (UTR) as identified through Targetscan (http://www.targetscan.org/) and one in the last exon predicted based on Argonaute HITS-CLIP analysis and corresponding seed match with prediction algorithms.37 These sequences were each placed separately downstream of a luciferase reporter gene and luciferase activity measured in the presence of control or miR-141 mimics. MiR-141 mimic significantly decreased luciferase activity with the coding site (PGR EXON), but not the 3′UTR sites, and mutation of the predicted coding miR-141-binding site rescued the decrease (Figure 4c; hatched bars). These results indicate direct targeting of PR through a miR-141 site in the last exon, which is present in transcripts for both PR-A and PR-B isoforms.38
To further test this in context, PR-negative HEK293 cells were transiently transfected with plasmids expressing PR-A and PR-B that contain ~998 bp of the 3′UTR.38 These contain the exonic-binding site but none of the three predicted 3′UTR-binding sites. Expression of both PRA and PRB protein was reduced by miR-141 mimic but not control (Figure 4d). These data confirm that PR expression levels can be directly altered by miR-141 targeting of PR transcripts. The heightened PR expression observed with miR-141 inhibition may be a contributing mechanism that helps potentiate the P4-mediated expansion of CK5+ and CD44high populations.
Stat5a is a direct target of miR-141 and is important for the P4-mediated increase in CK5+ cells
Stat5a is a progestin-regulated gene in the normal mammary gland that dictates luminal cell fate and is necessary for full lobular-alveolar development.39,40 Stat5a is also progestin regulated in luminal breast cancer,30,41 and is a predicted target of miR-141. We therefore investigated miR-141 regulation of Stat5a expression and its involvement in the P4 expansion of stem-like cells. Treatment with a miR-141 mimic blocked P4-mediated Stat5 upregulation (Figure 5a). To determine if miR-141 directly targets Stat5a, we placed the predicted miR-141-binding site sequence downstream of a luciferase reporter gene and measured luciferase activity in the presence of control or miR-141 mimics. miR-141 mimic significantly decreased luciferase activity for the Stat5a miR-141 site construct in both T47D and BT474 cells; mutation of the predicted miR-141-binding site blocked this decrease (Figure 5b). We also analyzed a predicted miR-141 site in Stat5b and found no significant regulation (Supplementary Figure 5). These data support that miR-141 directly and specifically regulates Stat5a in breast cancer cells.
To determine whether Stat5a upregulation has a functional role in the P4-induced expansion of CK5+ cells, we employed two methods to block Stat5a. First, we used small interfering RNA to reduce Stat5a protein levels; P4-mediated increases in Stat5 and CK5 expression were both blocked in the presence of siSTAT5A (Figure 5c). We then analyzed the effects of siSTAT5A on CK5 promoter activity using T47D cells stably expressing a luciferase reporter gene driven by the human KRT5 (CK5) promoter.11 The P4-mediated increase in CK5 expression was significantly inhibited in the presence of siSTAT5A (Figure 5d). Second, we analyzed the effects of the Stat5 inhibitor Pimozide on the P4-mediated increase in CK5 expression. Pimozide is a Stat5 inhibitor that acts through inhibition of phospho-Stat5 production, but has no effect on nuclear factor-κB, Stat3 or Stat1.42 Treatment of T47D cells with 500 nM Pimozide for 24 h inhibited P4 induction of CK5+ cells, measured by immunofluorescence for CK5 (Figure 6a) and CK5-promoter-luciferase activity (Figure 6b). Likewise, Pimozide reduced the P4 induction of CD44high cells by half (Figure 6c). Taken together, these results suggest that loss of miR-141 contributes to positive regulation of Stat5a, which in turn contributes to the P4-dependent expansion of CK5+ and CD44high breast cancer cells.
Progestins have emerged as crucial regulators of stem cells in the normal mammary gland and in breast cancer. In normal tissue, this regulation occurs both temporally during peak P4 levels, and spatially, as luminal-located cells signal to basally located MaSCs to expand.2,3 Our group was first to describe that progestins increase a population of CK5+ stem-like cells in breast cancer.9,10 Luminal breast cancer cells appear to directly convert from CK5− to CK5+ based on tracing studies using a CK5 promoter reporter.11,43 Interestingly, transformed human mammary epithelial cells can convert to a CD44+CD24−/low phenotype spontaneously or via cytokine signals.27,28 The P4-mediated expansion of stem-like cells, although necessary for normal breast function, is detrimental in breast cancer and may cause increased treatment resistance and recurrence. Sustained progestin use in hormone replacement therapy is hypothesized to increase breast tumorigenesis through expansion of pre-malignant stem cells.8 As breast cancer cells lose compartmentalization and become more reliant on autocrine signaling,44 we focused on intracellular P4 signaling events that could potentially influence cell phenotype. In this article, we describe a mechanism by which P4 suppression of miR-200 family member miR-141 cooperates to increase stem-like breast cancer cells.
We demonstrate that miR-141 is the only miR-200 family member temporally downregulated at the mature level in response to progestins in breast cancer cells. Although the primary transcript for the miR-141/200c cluster is downregulated, only mature miR-141 levels decrease (Figure 1). There is emerging evidence supporting differential processing, through multiple mechanisms including differential processing, maintenance of miRNA stability and direct or indirect degradation.45, 46, 47, 48, 49 A prime example is context-dependent processing of miR-18a within the miR-17 cluster of intronic miRNAs through the RNA-binding protein hnRNP A1.33 miR-200c is sometimes more abundant than miR-141 because of differential splicing that creates independent transcription units.32 This could also explain why it is less sensitive to transient downregulation of the primary transcript. Notably, miR-141, but not miR-200c, is underexpressed in CD44highCD24−/low prostate CSCs, further supporting differential regulation of miRNAs located in the same cluster.34 miR-141 is constitutively underexpressed in CD44high cells, as previously reported,36 and is transiently downregulated by P4 in the total cell population. This indicates its loss helps set in motion the gain of CD44high and CK5+ cells (Figure 2). This is similar to a feedback loop described in hepatocarcinoma cells; several miRNAs and HNF4a set oncogenesis into motion that cannot be reversed once initiated, even when miRNAs are restored to normal levels.50
Transcription factors involved in controlling cell fate decisions are common miRNA targets. In breast cancer, these include miR-200 or miR-205 targeting of epithelial–mesenchymal transition and stem cell-promoting transcription factors Zeb1/2, Suz12, TWIST1 and BMI1.17, 18, 19, 20, 21,51,52 Here we demonstrate miR-141 directly targets two transcription factors involved in regulating mammary cell differentiation: PR and Stat5a. P4 repression of miR-141 may allow for increased PR translation to recover from its ligand-dependent downregulation. The enhanced number of P4-induced CD44high and CK5+ cells with miR-141 inhibition could be partially due to more robust PR signaling. Stable inhibition of miR-141 was in fact sufficient to induce PR expression in breast cancer cell lines that typically require E2/ER-dependent PR expression (Figure 4). We also demonstrate that miR-141 targets both PR-A and PR-B at a site within the last coding exon mapped by genome-wide analysis of miR-binding sites in luminal breast cancer cells.37
The two highly conserved Stat5 transcription factors (transcribed from two separate genes, STAT5A and STAT5B) are both positively regulated by progestins in breast cancer.30 We demonstrate that Stat5a is specifically targeted by miR-141 in breast cancer cells. Stat5a is also targeted by miR-141 in the bovine mammary gland.53 Selective deletion of Stat5 genes determined that Stat5a, but not Stat5b, is required for mammary development.39 Furthermore, Stat5a-null mammary epithelial cells show impaired differentiation, and are unable to expand the luminal progenitor population, which produces mature ER+PR+ alveolar cells.40,54 Notably, CK5 is a major marker of luminal progenitor cells, the putative origin of BRCA1 breast cancers.12 We demonstrate that knockdown and inhibition of Stat5a reduces P4-mediated expansion of CK5+ and CD44high breast cancer cells (Figures 5 and 6). Stat5 is activated by another P4-regulated protein, prolactin receptor (PRLR). Prolactin (PRL) has been shown to reduce the P4 increase in CK5+ breast cancer cells, potentially though blocking induction of the transcriptional repressor BCL6.55 Other reports found PRL antagonism could reduce clonogenicity and improve chemotherapy treatment of breast cancer cells and primary tumor xenografts,56 and could block PRL/Stat5 induction of basal stem-like populations in prostate cancer.57 Thus, interplay between PR-Stat5a-PRLR signaling in regulating the breast cancer CK5+ population requires further investigation, and could be a source for therapeutic intervention.
Both PR and Stat5a are positive prognostic indicators in breast cancer, and predict better response to tamoxifen-based endocrine therapy.58,59 Loss of Stat5 signaling measured by nuclear phopho-Stat5 correlates with worse prognosis.60 Conversely, both PR- and Stat5a-null animals exhibit reduced mammary tumorigenesis in murine models;1,39 this could be due to reduced stem/progenitor cell populations. In breast cancer cells, P4 upregulates Stat5a transcriptionally through its promoter and post transcriptionally by depleting its repressor miR-141. Progestin regulation of stem-like cancer cell populations is complex; our data demonstrate that miRs are utilized to modulate expression of mammary cell fate transcription factors. Downregulation of miR-141 is sufficient for increasing the CD44high population, as we previously demonstrated with miR-29,24 and serves to amplify the progestin signal in increasing the CD44high/CK5+ populations. This luminal to basal/stem-like cell switch likely involves convergence of multiple signaling factors, including miRs as we demonstrate here. Ultimately, there may be opportunities for small molecule manipulation of breast cancers to maintain cells in a more treatment-vulnerable state.
Materials and Methods
Luminal breast cancer cell lines (T47D, BT474 and ZR75-1) were obtained from the University of Colorado Cancer Center Tissue Culture core. T47D cells were maintained in minimal Eagle’s medium, 5% fetal bovine serum, 1 × non-essential amino acids (NEAA), 1 × 10−9 M insulin, 0.1 mg/ml sodium pyruvate and 2 mM L-glutamine. BT474 and ZR75-1 cells were maintained in RPMI, 10% fetal bovine serum and 2.05 mM L-glutamine.
On-Target pooled small interfering RNAs, miR mimics/inhibitors and transfection reagents were obtained from Thermo-Fisher Scientific (Pittsburgh, PA, USA); pmiR-GLO luciferase vector and Dual Luciferase Reporter assay were obtained from Promega (Madison, WI, USA). Small hairpin RNA vectors were from the University of Colorado Functional Genomics Facility (Boulder, CO, USA). Primary antibodies used included: Stat5 (SC-835, SC-836; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CK5 (mouse NCL-L-CK5 and rabbit 2290-1; Vector Laboratories/Epitomics, Burlingame, CA, USA), PR (Dako, Carpinteria, CA, USA), α-tubulin and β-actin (Sigma-Aldrich, St Louis, MO, USA). For western blot analysis, secondary antibodies included were IRDye 800CW Goat-Anti-Mouse IgG (926-32210) and IRDye 680LT Goat-Anti-Rabbit IgG (926-68021; Li-Cor Biosciences, Lincoln, NE, USA). For western blot imaging, the Odyssey Infrared Imaging System (Li-Cor Biosciences) was used. For immunocytochemistry, secondary antibodies included were AlexaFluor488 goat anti-mouse (A11029) and goat anti-rabbit (A11008), AlexaFluor594 goat anti-mouse (A11032) and goat anti-rabbit (A11037; Invitrogen/Life Technologies, Grand Island, NY, USA). Hormones (17β-estradiol, progesterone and MPA) were purchased from Sigma-Aldrich. Pimozide (573110) was purchased from EMD Millipore (Billerica, MA, USA).
RNA extraction and quantitative reverse-transcriptase–PCR
Total RNA from cultured cell lines was isolated using Trizol; total RNA from flow-sorted cells was isolated using the RNAqueous-Micro kit (Ambion, Austin, TX, USA) following manufacturer’s instructions. 10 000–50 000 cells were collected in provided lysis buffer. Analysis of mature miR-141, miR-200abc and RNU6B (used for normalization) used TaqMan MicroRNA Assays, TaqMan MicroRNA RT kit and TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Carlsbad, CA, USA). Primers for pri-miR-141 and pri-200c were designed to flank their respective stem-loop, and analysis was performed using the Verso cDNA Synthesis kit and ABsolute Blue Sybr Green with Fluorescein (Thermo-Fisher Scientific). β-Actin was used for normalization. The relative mRNA or miR levels were calculated using the Pfaffl method.61
Cells were labeled with antibodies CD44-APC, CD44-FITC, CD24-PE (BD Biosciences, Franklin Lakes, NJ, USA) at a concentration of 1 × 106 cells per ml in PBS+0.5% BSA, and were subjected to either flow cytometry analysis on a Gallios or fluorescence-activated cell sorting analysis on a Moflo XDP 100 (Beckman-Coulter, Indianapolis, IN, USA). Analysis was performed using Kaluza Analysis Software (Beckman-Coulter).
Stable cell lines
Lentiviral vectors were used to stably inhibit miR-141 by expressing complementary sequences to the mature miR (pMIRZIPs, System Biosciences, Mountain View, CA, USA). Lentiviral vectors containing the precursor sequence for miR-141 were used for overexpression (pMIRNAs, System Biosciences). A scrambled non-silencing vector was used as the negative control. Stably expressing cells were selected using green fluorescent protein-based cell sorting (for pMIRNA vectors) or puromycin (miR-ZIP vectors).
Cells were seeded on non-adherent six-well plates at 1000 cells/well in 2 ml of complete Mammocult medium (Stemcell Technologies, Vancouver, BC, Canada). After 24 h, cells were treated with EtOH or 100 nM P4 for 10 days. Mammospheres were photographed, four fields/well and mammospheres larger than 75 μm in size were counted.
Real-time imaging (IncuCyte, ESSEN BioScience Inc, Ann Arbor, MI, USA) was used to measure cell proliferation using non-labeled phase confluence. BT474, T47D SCR-ZIP and 141-ZIP cells were plated at 5000 cells/well in 96-well plates. BT474 cells were pre-treated with E2 for 48 h. Cells were treated in sextuplicate with EtOH or P4. Confluence measurements were started immediately after treatment, and taken every 4 h for a total of 96 h.
Tumor growth and limiting dilution formation in vivo
Tumor xenografts were developed by injecting the indicated amount of cells in 100% Matrigel Basement Membrane Matrix (BD Biosciences) into the fourth mammary fad pad of ovariectomized female NOD/SCID mice (for tumor growth) or nu/nu mice (for limiting dilution experiments; Jackson Labs, Bar Harbor, ME, USA). Silastic pellets containing either 17β-estradiol alone (1 mg) or in combination with MPA (10 mg) were implanted subcutaneously at time of tumor cell injection. Tumors were measured weekly using a digital caliper, and tumor volume estimated using the formula (lw^2)/2. At termination of the experiment, mice were euthanized and tumors were excised and weighed. These experiments were performed under an approved University of Colorado Institutional Animal Care and Use Committee protocol.
Immunocytochemistry was performed as described,62 with additions of EtOH/10 nM P4 and/or dimethyl sulfoxide/500 nM Pimozide for 24 h. Images were collected using a Nikon TiE microscope (Nikon, Melville, NY, USA) equipped with a digital camera and NIS Elements software. Adobe Photoshop CS5 was used to perform linear adjustments to brightness/contrast, assemble pictures into multipanel figures and convert images from red-green-blue (RGB) to cyan-magenta-yellow-black (CMYK).
For 3′UTR-targeting experiments, portions of the 3′UTRs of the progesterone receptor gene (PGR) and STAT5A/B were cloned into the pmiR-GLO vector (Promega). Sites cloned from PGR are as follows (numbering based off +1 as first base of the 3′UTR): PGR_EXON, −357–+1; PGR_UTR_1, 2997–3961; PGR_UTR_2, 5764–6710; PGR_UTR_3, 8258–9073. Mutagenesis was performed by altering each predicted miR-141 site to 5′-IndexTermGTCACCA-3′. The site cloned from STAT5A is contained within bases 990–1255; mutagenesis altered the miR-141-binding site to 5′-IndexTermCAGTGTT-3′. Sites cloned from STAT5B are as follows: STAT5B_1, 1-379; STAT5B_2, 1329-1604. Mutagenesis altered STAT5B_1 to 5′-IndexTermTCACAAT; STAT5B_2 was altered to 5′-IndexTermGTCACAA-3′. Cells were plated into 96-well plates at 104 cells/well. Cells were transfected in octuplicate with dual transfection reagent (Invitrogen) using 10 ng of pmiR-GLO empty vector, or vectors containing most common sequence or mutated predicted miR-141-binding sites. Cells were lysed after 24 h and luciferase assays performed using the Dual Luciferase Reporter luciferase assay (Promega).
For K5P-Luc, T47D cells11 were plated at 104 cells/well and transfected with negative control pooled siRNA (siNC) or siSTAT5A. After 24 h, EtOH or P4 was added for an additional 24 h, cells were lysed and luciferase activity was measured. For Pimozide experiments, 500 nM Pimozide or vehicle (dimethyl sulfoxide) was added in combination with EtOH or P4 for 24 h. pRL-SV40 Renilla vector (Promega) was the transfection efficiency control.
Statistics were done using Graphpad Prism version 5.0 for Windows 7 (Graphpad, La Jolla, CA, USA). Two-tailed Student’s t-tests or analysis of variance followed by Bonferroni post-hoc tests were used, as well as paired t-tests for tumor growth. P<0.05 were considered significant.
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We thank the University of Colorado Cancer Center Flow Cytometry and Tissue Culture Cores supported by P30CA046934 and the University of Colorado, Department of Pathology Sequencing Core for their technical assistance and services. This work was supported by Department of Defense BCRP grants W81XWH-11-1-0210 (CAS, JKR) and W81XWH-11-1-0101 (DMC) and NIH R01 CA140985 (CAS). PK was supported by NIH K08 CA164048.
JF-S and PH performed most of the studies. DMC performed experiments in Figures 2a, b and 5c, d. PP performed experiments in Figure 3a. PK provided the PR exonic-binding site for miR-141 (Figure 4c) and technical advice. JF-S, DMC, BMJ, JKR and CAS contributed intellectual design and interpretation of results. JFS wrote the manuscript. BMJ, JKR and CAS provided editorial assistance. All authors read and approved the final manuscript.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Finlay-Schultz, J., Cittelly, D., Hendricks, P. et al. Progesterone downregulation of miR-141 contributes to expansion of stem-like breast cancer cells through maintenance of progesterone receptor and Stat5a. Oncogene 34, 3676–3687 (2015). https://doi.org/10.1038/onc.2014.298
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