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. 2013 May 16;32(20):2555-64.
doi: 10.1038/onc.2012.275. Epub 2012 Jul 2.

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

D M Cittelly  1 , J Finlay-Schultz, E N Howe, N S Spoelstra, S D Axlund, P Hendricks, B M Jacobsen, C A Sartorius, J K Richer
Affiliations

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

D M Cittelly et al. Oncogene. .

Abstract

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44(+) cell population. Downregulation of miR-29 members potentiates the expansion of CK5(+) and CD44(+) cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

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Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Promotion of CK5+ cells in ER+PR+ breast cancer cells by progestins
A. Top: Treatment of T47D cells with 100 nM MPA, R5020 or P4 for 24h results in up to 20% increase in CK5+ cells compared to ethanol (OH) treated cells. Bottom: BT474 cells pretreated with estradiol for 48h to upregulate PR and then treated with the same progestins for 24h results in ~10% increase in CK5+ cells. CK5 expression was determined by immunohistochemistry and percentage of CK5+ cells was measured in 3 fields and at least 300 cells. DAPI shows nuclei. B. Left: By quantitative RT-PCR CK5 mRNA levels increase during the first 12h following P4 (T47D cells) or MPA (BT474 cells) treatment. Data represent fold change in CK5 mRNA normalized to β-actin mRNA levels relative to levels of control 0h-time point. Bars are mean ± SEM of biological triplicates. Asterisks represent statistical significance compared to 0h. Right: Western blots shows PR (PRA, PRB isoforms) and CK5 expression in T47D and BT474 cells after 24h treatment with P4. α-tubulin was used as loading control. CK5 antibody may recognize CK6. Since both CK5/6 are basal markers (55), both are P4-regulated, and are usually co-expressed(11), we refer to it as CK5 thereafter. C. By qRT-PCR, miR-29a b and c are decreased in T47D and BT474 cells at 6, and 18h after treatment with 100 nM P4 as compared to ethanol-treated (OH) cells. Data represent miRNA levels normalized to RNU6 relative to miR-29a levels in control cells. Bars = mean± SEM. In all experiments, BT474 cells were pre-treated with 10nM 17-β-estradiol for 48h to induce PR expression prior to progestin stimulation. D. CD44hi cells express significantly less miR-29abc than CD44lo cells. T47D cells were treated with vehicle or 100nM P4 for 24h. Top: Cells were immunolabeled and CD44+ (CD44hi) and CD44− (CD44lo) cells sorted from P4-treated triplicate samples. Bottom: miRNA expression relative to miR-29a expression in CD44lo cells (n=3). Bars =mean± SEM. For all panels, *P<0.05; **P<0.01; ***P<0.0001 ANOVA and Bonferoni post-hoc t-tests. Concentrations of progestins used in this study are within physiological range (i.e P4 ranges from 5.4 to 85.86nM during luteal phase in humans according to NIH clinical test guidelines).
Figure 2
Figure 2. P4 induces transcriptional downregulation of miR-29a via c-Myc
A. P4 decreases pri-miR-29a levels as early as 1h after treatment. T47D cells were treated for 0.5, 1 or 3h with 100nM P4 and pri-miR-29a levels analyzed by qRT-PCR. Data represents pri-miR-29a levels normalized to GAPDH, relative to pri-miR-29a levels at time 0. **P<0.01 compared to 0h. B. si-MYC prevents P4-induced upregulation of c-Myc in T47D cells. T47D cells were plated at 60% confluence, attached overnight and then transfected with either 20nM siNC (−) or siMYC (+). After 24h, cells were treated with 100nM P4 for the indicated times. Total cell lysates were used to measure c-Myc expression by western blot. α-tubulin is used as a loading control. C. c-Myc inhibition abolishes downregulation of mature miR-29 (left) and pri-miR-29a (right) 18h after P4 treatment. T47D cells were transfected with either 20nM siNC or 20nM siMYC and 24h later, treated with vehicle (OH) or 100nM P4 for 18h. Data represents miR-29a or pri-miR-29a levels normalized to RNU6B, relative to siNC-OH-treated cells. *P<0.05. D. P4 inhibitor RU486 prevents P4-mediated upregulation of c-Myc and repression of miR-29a. Left: T47D cells were treated for 18h with either vehicle (OH), 100nM P4 (P4) or 100nM P4 in combination with 100nM RU486 for the indicated times, and c-Myc expression analyzed by western blot. Right. qRT-PCR shows miR-29a levels normalized to RNU6B and relative to OH-treated cells. Bars represent mean± SEM from biological replicates (n=5). *P<0.05.
Figure 3
Figure 3. Stable inhibition of miR-29 increases the CK5+ and CD44+ populations in response to P4
ER+PR+ breast cancer cells stably expressing inhibitors of miR-29a, b or c (29aZIP, 29bZIP or 29cZIP lentiviral vectors) were compared to SCR-control cells for expression of basal markers and stem-like properties. A. T47D 29aZIP cells express more CD44+ cells in response to P4 compared to SCR-ZIP cells. Cells were plated at 60% confluence, attached overnight and then treated with ethanol (OH) or 100nM P4 for 24h. Cells were labeled and CD44 expression measured by flow cytometry. Data represents percentage of CD44+ cells from at least 3 independent experiments. Bars = mean± SEM. B. T47D 29aZIP cells express more CK5+ cells in response to P4 compared to SCRZIP cells. Left: CK5 expression (green) was determined by immunofluorescence in vehicle or P4 treated T47D SCRZIP and 29aZIP cells. Representative image shows DAPI (blue) and CK5+ expression (20X objective). Right: Percentage of CK5+ cells (green) was measured in 3 fields and at least 300 cells per condition in biological triplicates. Bars = mean± SEM. C. T47D and BT474 cells expressing 29aZIPs form significantly more colonies in 3D-culture compared to SCRZIP control cells. 1000 cells were plated in matrigel and colonies counted 14 days later. Colonies and their diameter were digitally measured and a the number of colonies > or <60μm diameter (representing colonies of at least 10 cells) were counted. Bars represent mean± SEM from biological triplicates. For all panels, *P<0.05; **P<0.01; ***P<0.0001 ANOVA followed by Bonferoni post-hoc t-tests.
Figure 4
Figure 4. Stable inhibition of miR-29a increases tumor growth
Ovariectomized female NOD/SCID mice were injected with 1x 106 T47D-SCRZIP or T47D-29aZIP cells in the left and right 4th mammary fat pad respectively. Pellets containing either estrogen alone (E2) or in combination with MPA (E2+MPA) were implanted subcutaneously at time of tumor cell injection (n=8 per group) and tumor size measured using a caliper weekly after injection. A. Tumor growth in E2 treated mice. B. Tumor growth in E2+MPA treated mice. For A and B, data represent mean± SEM. *P<0.05 paired t-test compared to SCR control. Images show representative tumors for each group at the end of the experiment. C. Representative immunohistochemistry showing Ki67 staining SCRZIP and 29aZIP tumors. Magnification 20X. Tumors grown in MPA show decreased cellularity compared to E2-treated tumors, but no differences in the percentage of Ki67 positive cells was observed among SCR-ZIP and 29aZIP tumors.
Figure 5
Figure 5. KLF4, a transcription factor required for maintenance of breast cancer stem-cells, is upregulated by progesterone prior to the increase in CK5+ cells
A. Time course of progestin-induced increases in KLF4 and CK5 protein level. Western blot of KLF4 and CK5 protein expression at 6, 18 and 24h following treatment of T47D cells with 100 nM P4. α-tubulin was used as loading control. KLF4 expression normalized to α-tubulin and relative to OH-treated control for each time point is shown in the graph on the right (n=2). B. Levels of KLF4 and CK5 mRNA were measured following treatment with 100 nM P4 in T47D cells relative to β-actin mRNA levels and normalized to 0h. Bars =mean± SEM from biological triplicates. Asterisks indicate statistically significant data compared to 0h. *P<0.05 (ANOVA followed by Bonferoni post-hoc t-tests). C. Cells were treated with 100 nM P4 for 24h, immunolabeled, then sorted for expression of CD44hi and CD44lo populations. CK5 and KLF4 mRNA levels were normalized to β-actin mRNA and expressed relative to levels in CD44lo cells. ***P<0.0001 paired t-test.
Figure 6
Figure 6. Progesterone-mediated downregulation of miR-29 relieves repression of KLF4
A. Left: Predicted binding sites for miR-29a and b in the KLF4 3′UTR. The KLF4 3′UTR containing the putative miR-29 and miR-200c binding sites cloned downstream of luciferase (pMIRGLO-KLF4) was transfected into T47D cells with either 50 nM negative control (NC), miR-29a (29a), miR29b (29b), miR-200c or miR-29a in combination with miR-29a inhibitor (29a +29a inh) and luciferase activity measured 24h after transfection. Data represents relative luciferase activity normalized to renilla-luciferase present in the pmiRGLO vector. Bars are mean ± SEM. *P<0.05, **P<0.01: ***P<0.001 Right: T47D cells transfected with negative control (NC) or miR-29 mimics (miR-29) in combination with pMIRGLO-KLF4 were treated with either ethanol (OH) or 100 nM P4 for 48h. P4 alone can relieve repression of the 3′UTR-KLF4 and miR-29a partially blocks this effect. B. T47D and BT474 cells stably expressing SCR-ZIPs or 29a-ZIPs were treated for 24h with vehicle or 100nM P4. Western blot of endogenous KLF4 protein in 29aZIP cells compared to SCR-ZIPs (quantified on the right) in T47D and BT474 cells with KLF4 expression normalized to α-tubulin and relative to expression of SCR-ZIP cells treated with vehicle in biological triplicate samples. *P<0.05. C. Stable overexpression of miR29a (pre-miR-29a) decreases P4 induced KLF4 in T47D and BT474 cells.
Figure 7
Figure 7. miR-29 repression and KLF4 upregulation contribute to maximal promotion of CK5+ and CD44+ cells
A. Transient miR-29 and siKLF4 transfection decreases P4 induced activation of CK5 promoter. T47D cells stably expressing a luciferase reporter driven by the KRT5-promoter were plated at 10000 cells/well in 96-well plates. After 24h cells were transfected with 50 nM miR-29a (29a) or miR-29b mimics or negative control (NC), 5 nM on-target pool siKLF4 or siNC in combination with a renilla-luciferase plasmid (pRL-SV40) using Dual-transfection reagent. Cells were treated for additional 24h with ethanol (OH) or 100 nM P4 and luciferase activity measured 24h after transfection and normalized to renilla-luciferase control. *P<0.05 B. T47D cells were transfected with 5 or 10 nM siKLF4-smart pool or si-NC control in combination with pGFP plasmid to control for transfection efficiency. After 24h, cells were treated with either ethanol (OH) or 100 nM P4 for 24h and western blotting was performed to detect KLF4 and CK5. C. T47D were transfected with 10nM siNC or si-KLF4. After 24h, cells were treated with vehicle (OH) or 100nM P4 for 24h and CD44+ expression was measured by flow cytometry. Left: Representative flow-charts. Right. Percentage of CD44+ cells from biological triplicates *P<0.05.
Figure 8
Figure 8. Current model
Progestins acting through PR induce transcriptional activation of c-MYC and KLF4. C-MYC is rapidly upregulated to repress miR-29a/b1, thus relieving post-transcriptional repression of KLF4 mRNA. Downregulation of miR-29, and upregulation of KLF4 contribute to maximal expansion of CK5 and CD44+ cells, which in turns increase tumor initiating capability.
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References

    1. Mulac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proceedings of the National Academy of Sciences. 2003;100(17):9744–9749. - PMC - PubMed
    1. Ismail PM, Amato P, Soyal SM, DeMayo FJ, Conneely OM, O’Malley BW, et al. Progesterone involvement in breast development and tumorigenesis—as revealed by progesterone receptor “knockout” and “knockin” mouse models. Steroids. 2003 Nov;68(10–13):779–87. - PubMed
    1. Lange CA. Challenges to defining a role for progesterone in breast cancer. Steroids. 2008 Oct;73(9–10):914–21. - PMC - PubMed
    1. Lydon JP, Ge G, Kittrell FS, Medina D, O’Malley BW. Murine Mammary Gland Carcinogenesis Is Critically Dependent on Progesterone Receptor Function. Cancer Research. 1999;59(17):4276–4284. - PubMed
    1. Magnusson C, Baron JA, Correia N, Bergström R, Adami HO, Persson I. Breast-cancer risk following long-term oestrogen- and oestrogen-progestin-replacement therapy. Int J Cancer. 1999 May 5;81(3):339–44. - PubMed

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