ORIGINAL RESEARCH ARTICLE
Journal of
Non-Genomic Estrogen/Estrogen Receptor a Promotes Cellular Malignancy of Immature Ovarian Teratoma In Vitro
Cellular Physiology
YAO-CHING HUNG,1,2,3 WEI-CHUN CHANG,1,2,3 LU-MIN CHEN,1,2,3 YING-YI CHANG,1,2,4 LING-YU WU,1,2 WEI-MIN CHUNG,1,2,3 TZE-YI LIN,1,2 LIANG-CHI CHEN,1,2 1,2,3 AND WEN-LUNG MA * 1
Sex Hormone Research Center, Department of Obstetric and Gynecology, China Medical University Hospital, Taichung, Taiwan
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Department of Pathology, China Medical University Hospital, Taichung, Taiwan
3
Graduate Institution of Clinical Medical Science, School of Medicine, China Medical University, Taichung, Taiwan
4
Graduate Institution of Public Health Science, School of Public Health, China Medical University, Taichung, Taiwan
Malignant immature ovarian teratomas (IOTs) most often occur in women of reproductive age. It is unclear, however, what roles estrogenic signaling plays in the development of IOT. In this study, we examined whether estrogen receptors (ERa and b) promote the cellular malignancy of IOT. Estradiol (E2), PPT (propylpyrazole), and DPN (diarylpropionitrile) (ERa- and b-specific agonists, respectively), as well as ERa- or ERb-specific short hairpin (sh)RNA were applied to PA-1 cells, a well-characterized IOT cell line. Cellular tumorigenic characteristics, for example, cell migration/invasion, expression of the cancer stem/progenitor cell marker CD133, and evidence for epithelial-mesenchymal transition (EMT) were examined. In PA-1 cells that expressed ERa and ERb, we found that ERa promoted cell migration and invasion. We also found that E2/ERa signaling altered cell behavior through non-classical transactivation function. Our data show non-genomic E2/ERa activations of focal adhesion kinase-Ras homolog gene family member A (FAK-RhoA) and ERK governed cell mobility capacity. Moreover, E2/ERa signaling induces EMT and overexpression of CD133 through upregulation micro-RNA 21 (miR21; IOT stem/progenitor promoter), and ERK phosphorylations. Furthermore, E2/ERa signaling triggers a positive feedback regulatory loop within miR21 and ERK. At last, expression levels of ERa, CD133, and EMT markers in IOT tissue samples were examined by immunohistochemistry. We found that cytosolic ERa was co-expressed with CD133 and mesenchymal cell markers but not epithelial cell markers. In conclusion, estrogenic signals exert malignant transformation capacity of cancer cells, exclusively through non-genomic regulation in female germ cell tumors. J. Cell. Physiol. 229: 752–761, 2014. ß 2013 Wiley Periodicals, Inc. Female Hormones in Immature Ovarian Teratoma
Ovarian cancers are classified according to the type of cells from which they start. Cancerous ovarian tumors can start from four common cell types, namely epithelial cells (ovarian carcinoma), stromal cells (ovarian adenoma), salpinge cells (fallopian tube cancer), and germ cells (ovarian teratoma) (Zhang et al., 2008; Wasim et al., 2009). Ovarian teratoma, a mixed germ cell tumor, comprises approximately 20% of ovarian neoplasms (Koshy et al., 2005; Moniaga and Randall, 2011; Sviracevic et al., 2011); however, the pathogenesis of this type of tumor is poorly understood (Oliveira et al., 2004; Ye et al., 2012). There are two major subtypes of ovarian teratoma, namely immature and mature types (Tewari et al., 2000; Mahdi et al., 2011). Immature ovarian teratoma (IOT) accounts for approximately 50% of all ovarian teratomas and occurs predominantly in women of reproductive age (Ulbright, 2005; Mahdi et al., 2011). It is well known that female hormones such as estrogen and progesterone play pivotal roles in reproductive function throughout reproductive age; however, little is known about their impact on IOT development and progression. Actions of Estrogen/Estrogen Receptors and Their Pathological Function
Female hormones are steroidal hormones that exert their biological functions by binding to specific receptors (e.g., estrogen receptor; ESR1 (Walter et al., 1985) and ESR2 (Mosselman et al., 1996); or, ERa and ERb) (Johnson and ß 2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .
O’Malley, 2012). Estrogen receptors (ERs) translocate to the nucleus where they bind to specific DNA sequences called estrogen response elements (EREs) upon estrogen challenge (Johnson and O’Malley, 2012; Ma et al., 2012a). It is well known that estrogenic actions are confounding factors for breast cancer carcinogenesis and cancer progression (Hammond et al., 2010). However, the functions and expressions of ERa and ERb in IOT are poorly documented (Salonen et al., 2009).
Yao-Ching Hung, Wei-Chun Chang, and Lu-Min Chen having equal contribution to this work. Conflict of interests: All the authors claimed no conflict of interests. Contract grant sponsor: National Science Council of Taiwan; Contract grant numbers: NSC-101-2314-B-039-014, 101-2314-B039-027-MY3. *Correspondence to: Wen-Lung Ma, Sex Hormone Research Center, Department of Obstetric and Gynecology, China Medical University Hospital, Taichung 404, Taiwan. E-mail:
[email protected] Manuscript Received 24 August 2013 Manuscript Accepted 14 October 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 19 October 2013. DOI: 10.1002/jcp.24495
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In addition to E2/ERs transactivation functions, non-genomic rapid actions of estrogens have been documented in a variety of human diseases (Okoh et al., 2011). Graber et al. (1993) reported that the functions of non-genomic E2/ERs influence breast cancer development. Furthermore, Turner et al. (1992) reported that non-genomic E2/ERs functions affect bone formation through osteoblast differentiation. Although several studies have described the existence of non-genomic E2/ERs functions in human diseases, few studies have evaluated the importance. In 2009, Salonen et al. examined ERa and ERb protein expressions in male patients with testicular teratoma and found that most of the patients expressed both receptors. Furthermore, the researchers found that the transactivation activity of ERE in testicular teratoma cells (NCC-IT) was twofold to threefold higher than that of vehicle control cells but that the increased activity could be reversed by an ER antagonist (ICI182780) (Salonen et al., 2009). However, very few changes in cellular behavior were noted after E2 treatment. In the present study, we used the IOT cell line PA1 to examine the effect estrogen has on the cellular malignancy of IOT. Epithelial-mesenchymal transition (EMT) plays important roles in carcinogenesis (Zavesky et al., 2011), the development of malignant neoplasms (De Craene and Berx, 2013), stem/ progenitor characteristics (CSPC) (Yu et al., 2012; Shirkoohi, 2013), and chemotherapy resistance (Ahmed et al., 2010; Sampieri and Fodde, 2012), which qualities have recently been linked to estrogenic functions (Zavesky et al., 2011). Germ cell tumors provide an excellent model for studying CSPC. Moreover, cancer cell stemness is linked to cancer progression and metastatic potential. The human ovarian teratocarcinoma cell line PA-1 comprises cells with multi-pluripotency and high self-renew capacities (McGowanJordan et al., 1994; Sekiya, 1995). In this study, we also examined the roles that estrogen/ER signaling play in EMT and CSPC in IOT cells. Materials and Methods IOT patient data from a single cohort study Patient data were obtained from the Cancer Registry Database at the China Medical University Hospital and comprised data on 14 patients who were treated for IOT at that hospital during the period 1987–2013. Tissue samples from those patients were also obtained. Use of the tissue samples was approved by the Internal Review Board of the China Medical University Hospital, Taiwan (DMR101-IRB2-276). Cell cultures, plasmids, and reagents. PA-1 and HeyA8 cells were the courtesy of Professor Min-Chie Hung of the MD Anderson Cancer Center, Houston, USA. MCF7 cells were presented as a gift from Dr. Yu-Ping Sher of the Graduate Institute of Clinical Medical Science, China Medical University, Taiwan. Cells were maintained in D-MEM phenol red-free medium with 10% FCS (or 10% charchol-dextran treated FCS [CD-FCS] while measuring ligand effects), 1% penicillin–streptomycin, and 1% EAA and were sub-cultured every 2–3 days. ERa and ERb lentiviral-based shRNAs (pLKO-scramble, shESR1, and shESR2; ERa: TRCN0000003300; and ERb: TRCN0000364067) were obtained from the National RNAi Core Facility Platform located at the institute of Molecular Biology/Genome Research Center, Academia Sinica, supported by the National Core Facility Program for Biotechnology (grant NSC100-2319-B-001-002). The knockdown-miR21 construct (miRZip-21) was obtained from Systems Biosciences, SBI; CD133 antibody for flowcytometry (CD133-APC conjugated, MACS#130-090-826), ICI-182780 (I127), Estradiol (E2) (E8875), PPT (propylpyrazole triol; H6036), and DPN (diarylpropionitrile; H5915) were obtained from Sigma– Aldrich. Focal adhesion kinase inhibitor I (1,1,2,4,5benzenetetraamine, 4HCl, NSC 667249) and ROCK (Rho kinase) JOURNAL OF CELLULAR PHYSIOLOGY
inhibitor (Y-27632; (R)-(þ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide, 2HCl) were obtained from MERCK, and ERK inhibitor (PD98059) was obtained from Sigma– Aldrich. Cell migration/invasion and growth assays. For the wound healing cell migration assay, 2 105 cells were seeded onto 12-well plates and allowed to adhere to the wells for 18 h. A 200-ml pipette tip was then used to create a linear wound area. Photographs were taken under a light microscope at 0 h. We observed wound closure for 8 h and then took photographs. Migration activity was defined by subtracting the wound area (mm2) at 0 h from that at 8 h. The photographic images were analyzed by NIS Elements BR3.1v software (Nikon). The protocol for the transwell invasion assay followed that reported previously with minor modifications (Ma et al., 2012b). Briefly, 5 104 cells were seeded in a transwell (Corning, NY) that had been precoated with matrigel (Invitrogen) and allowed to invade for 24 h. After incubation, the cells in the inner layer of the chambers were scrapped out and the cells in the outer chamber were stained with trypan blue and counted under a light microscope (Nikon, Eclipse 80i). Expression assays (RNA, protein, and promoter assays). The expression levels of miR-21, miR-99a, CD133 mRNA (Hubbard et al., 2009), SCF (stem cell factor) mRNA, BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) mRNA, and Nanog (Homeobox protein NANOG) mRNA were determined using quantitative RT-PCR (Q-PCR) as previously described (Ma et al., 2008). Complementary DNAs (cDNAs) were subjected to Q-PCR assay using a C1000 Cycler CFX96 Real-time System (BioRad Laboratories, Inc., Hercules, CA) with iQ SYBR Green Supermix (Bio-Rad). A real-time detection system (Bio-Rad) and the KAPATM SYBR FAST One-Step qRT-PCR Kit (KAPABIOSYSTEMS, US) were used according to the manufacturers’ instructions. Relative gene expressions were obtained by normalization to the expression levels of housekeeping genes (U6 or actin). Threshold value (Ct) dynamics were used (2DDCt) for quantitation of gene expression. The following qRT-PCR primer sequences were used: CD133 forward 50 -TCT CTA TGT GGT ACA GCC G-30 , reverse 50 -TGA TCC GGG TTC TTA CCT G-30 ; Nanog forward 50 -GGG CCT GAA GAA AAC TAT CCA TCC30 , reverse 50 -TGC TAT TCT TCG GCC AGT TGT TTT-30 ; SCF forward 50 -ACT GAC TCT GGA ATC TTT CTC AGG-30 , reverse 50 -GAT GTT TTG CCA AGT CAT TGT TGG-30 ; miR-21 50 -TAGCTTATCAGACTGATGTTGA-30 ; and miR-99a forward 50 -AAC CCG TAG ATC CGA TCT TGT G-30 . Protein expression was analyzed using an immunoblotting assay (Yeh et al., 1999; Ma et al., 2008). The primary antibodies used were: ERa (SC-543, Santa Cruz), ERb (#ab3577, Abcam), ERK/pERK (#4695/#9101, Cell Signaling), FAK/pFAK (#3285/#3283, Cell Signaling), RhoA/pRhoA (#2117/sc32954, Cell Signaling/Santa Cruz), ROCK (sc-5560, Santa Cruz), E-cadherin (#3195, Cell Signaling), Vimentin (#GTX62264, Gene Tex), N-Cadherin (#610921, Cell Signaling), Slug (#9627528, Abcam), Integrin-b1 (sc-9936, Santa Cruz). Reporter gene assays were performed as previously described (Yeh et al., 1999). The promoter constructs were ERE- and MMTV-luciferase. Thymidine kinase-driven Renilla reniformis luciferase (pRL-TK) served as a transfection efficiency control. Flowcytometry for cell cycle, apoptosis assays, and CD133 populations. We used flowcytometry to determine cell cycle and apoptosis. Cells (106) were harvested and fixed in 4% paraformaldehyde, treated with RNase (1 mM; Sigma–Aldrich), washed, stained with propidium iodide (P.I.; 5 mM; Sigma–Aldrich) for 5 min, and then injected into a flow cytometer (FACS, BD FACAria) to evaluate cell cycle. To investigate apoptosis, cells (106) were harvested, stained with P.I. for 5 min, and then directly injected into the flow cytometer (FACS, BD FACAria). To verify CD133þ populations, cells were detached, incubated in 5% BSA for 30 min at room temperature to block non-specific binding of antibodies to the cell membrane, incubated with antibody CD133-
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APC (MACS, Miltenyi Biotech) or isotype antibody for 30 min, and then subjected to flow cytometric analysis (FACS, BD FACSAria). Prediction of transcription factor binding and transcription regulatory module prediction by PReMOD. The genes of interest were subjected to PReMod (Ferretti et al., 2007) (http://genomequebec.mcgill.ca/PReMod/search/ module) and TFSEARCH: Searching Transcription Factor Binding Sites (ver 1.3) (http://www.cbrc.jp/research/db/TFSEARCH.html) to predict potential ER binding sites and other transcription factors. A 1.8 kbp sequence (57917986–57919801) covering the 50 -promoter and 30 -untranslated region of the miR-21 gene was included for prediction. Statistical analysis. All assays were performed at least three times. Values were calculated and compared using the Student’s ttest. A P-value 85 (Fig. 5H) (HREF¼http://www.cbrc.jp/ htbin/bget_tfmatrix?> M00173; M00199; M00074). The three putative ERK downstream transcription factors included one
NON-GENOMIC ERa PROMOTES IOT
Fig. 4. Estrogenic signals promote EMT and CSPC in PA1 cells. A: Estrogenic signals have little influence on cell cycle. PA1 cells were treated with Veh, E2 (upper left panel), or PPT, DPN (upper left panel) for 4 days and then we evaluated the cell cycle. Quantitation data were plotted to show G0/G1, S, and G2/M phases among different treatment groups (lower panel). B: Estrogenic signals have borderline effect on cell death. PA1 cells were treated with Veh, E2 (upper left panel), or PPT, DPN (upper left panel) for 4 days and then we evaluated cell death by P. I staining and analysis by flowcytometry. Quantitation data are plotted to show percentage of positive staining among treatments. C: Estrogen/ERa signals promote CD133 populations in PA1 cells. PA1 cells were treated with Veh, E2, or PPT for 7 days and then we measured CD133þ populations by flowcytometry. The values of CD133þ/total cells were plotted on graph. D: Estrogen/ERa signals promote cellular EMT in PA1 cells. PA1 cells were treated with Veh, E2, or PPT for 24 h and then we measured E-cadherin (E-Cad), N-Cadherin (N-Cad), Slug, and b1-integrin (b1-ITG) expressions (left panels). Expression of EMT markers was also examined in PA1 cells with scramble (sc) RNA (scER) or ERa-specific small hairpin (sh) RNA (shERa) stable clones (right panels). Representative images were shown, and actin expressions served as a loading control. All data were from at least three independent experiments where indicates P-values less than 0.05.
ETS binding module (chr17: 57918477; binding power 85.8) and two AP1 binding modules (chr 17: 57919095, binding power 86.6; chr 17: 57919114, binding power 94.8). Taken together, the data in Figures 4 and 5 show that nongenomic E2/ERa promotes IOT EMT and CSPC and that this function is mediated by ERK activation and miR-21 upregulation. The findings from our study indicate that E2/ERa triggers a positive feedback loop within ERK and miR-21 to facilitate IOT cancer EMT and stemness. Cytosolic ERa is co-expressed with CD133 and mesenchymal markers in human IOT tissue
The tissue samples evaluated in this study were obtained from 14 patients with IOT. The mean age at diagnosis was 27.5 years (Fig. 6A). As shown in Figure 6B, H&E staining revealed a typical immature ovarian tumor of undifferentiated cell morphology. We then performed immunohistochemical staining on normal JOURNAL OF CELLULAR PHYSIOLOGY
and tumor lesions of the same patients and compared the locations and levels of expression of ERa, E-cadherin, Vimentin, and CD133. We found that E-cadherin was expressed in normal epithelial tissue (Fig. 6C, 1st row left) but was almost undetectable in tumor tissue (Fig. 6C, 1st row right). Vimentin expression was absent in normal epithelial tissue but was abundant in tumor lesions (Fig. 6C, 2nd row). In healthy tissue, expression of ERa (Fig. 6C, 3rd row) was mostly localized in the nucleus and in cytosol whereas in tumor tissue, expression was localized almost exclusively in cytosol. We also found that CD133 expression (Fig. 6C, 4th row) was less abundant in normal epithelial tissue than in tumor tissue. These results support our hypothesis that ERa promotes the progression of malignant IOT by facilitating EMT and CSPC. In summary (Fig. 7), non-genomic E2/ERa signaling promotes IOT cellular malignancy by increasing cell invasion, migration, EMT, and CSPC. ERa-mediated IOT progression might be governed by RhoA/ROCK and ERK activation, which
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Fig. 5. Estrogenic signals promote cell EMT and CSPC through an ERK-miR21 positive regulatory feedback loop. A: CSPC marker gene expression in PA1 cells upon E2 treatments. The mRNA expression of CD133, SCF, BMI-1, and Nanog were measured in PA1 cells that had been treated with either Veh or 10 nM E2 for 7 days. B: miR21 and miR-99a mRNA expression in PA1 cells treated with Veh or E2 for 7 days. C, D: Estrogen/ERa-induced CSPC is ERK mediated. PA1 cells were pre-treated with PD98059 for 2 h, and then treated with Veh, E2 (C), or PPT (D) for 7 days. Then we measured CD133þ populations. E: Estrogen/ERa-induced CSPC is mediated by miR-21. The PA1 cells were infected with control miRZip vector or miR21 knockdown constructs and then treated with Veh or E2 for 7 days before measuring CD133 populations. F: Estrogen/ERa-induced EMT is mediated by miR-21. The PA1 cells were infected with control miRZip vector or miR21 knockdown constructs and then treated with Veh or E2 for 24 h. We then measured E-Cad, N-Cad, Vimentin, Slug, and b1-ITG protein expressions. G: Estrogen/ERa-induced ERK phosphorylation is mediated by miR-21. The PA1 cells were infected with control miRZip vector or miR21 knockdown constructs and then treated with Veh or E2 for 2 h. We then measured pERK, ERK, and actin protein expressions. Representative images are in the upper panels and quantitation results are shown in the lower panels. All data were from at least three independent experiments where indicates P-values less than 0.05. H: Computational simulation of ERK downstream ETS/AP1 putative binding modules. The pre-miR-21 (has-miR-21) is located on chromosome 17 (57918627–57918698). Around 1.2 kb (57917986–57919801), including 50 and 30 of has-miR-21 region, was subjected to PReMod and TFSEARCH, which revealed three putative ETS/AP1 binding modules.
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NON-GENOMIC ERa PROMOTES IOT
remains a threat for women of reproductive age. Estrogenic signals are suspected to participate in the development of IOT; however, these signals have never been studied. Herein, we have demonstrated a strong association between non-genomic E2/ERa actions and IOT development. The conclusion was based on the following findings: (1) PA-1 cell invasion and migration were enhanced by E2 and PPT treatments. (2) E2/ERs transactivation function was absent in the reporter gene assay. (3) E2/PPT had little effect on overall population cell cycle, but exerted a minor effect on cell death. Furthermore, E2/PPT increased the expression of CD133 and cellular EMT. (4) Knockdown of ERa expression resulted in a reduction in E2- or PPT-altered cell mobility, EMT formation, and CSPC stemness. (5) Cytosolic ERa expression was higher in IOT lesions and was accompanied by Vimentin and CD133 expression. However, nuclear ERa staining was dominant in healthy tissue with E-cadherin co-expression. The non-genomic E2/ERa actions have been reported previously (Migliaccio et al., 1996). The major concept of the non-genomic action in Migliaccio et al. paper is that ovarian hormones could go through rapid activation of cellular signal molecules either alone through ER, or interaction with progesterone receptor (PR) (Migliaccio et al., 1998). Those findings provided evidence that co-existence of rapid E2 actions through Src/p21/Erk pathway and/or E2/ERa transactivation function could promote breast cancer progression. In this report, we revealed the novel non-genomic E2/ERa action, bypass transactivation function, to influence on cancer development. It is of great interest to know if lost of E2/ERa transactivation function promote cancer. Results in this paper demonstrated that E2/ERa signals promote IOT cancer mobility, EMT, and CSPC. We found that the ERa in patients with IOT is not activated in the nucleus but in cytosol as a result of interaction with phosphorylated FAK, ERK, ROCK, as well as interaction with miR21. E2/ERa triggers a positive feedback loop among ERK phosphorylation and miR21 expression to alter EMT and CSPC populations
Fig. 6. ERa was co-expressed with mesenchymal and CSPC markers, but not epithelial markers in IOT lesions. A: Age distribution of IOT patients of the single cohort study. B: H&E stains of a representative IOT lesion. C: Immunohistochemistry staining of E-cadherin, Vimentin, ERa, and CD133 were performed healthy (left-hand side) and IOT (right-hand side) tissues from the same patient.
further triggers an onco-miR (miR-21)-ERK positive feedback loop that regulates EMT and CSPC in IOT. Discussion The roles of E2/ERa non-genomic actions in IOT
The etiology of ovarian teratoma is not clear. Ovarian teratoma predominantly occurs in women of reproductive age whereas ovarian carcinoma tends to occur in peri- to-postmenopausal women. Therefore, anti-estrogen prevention for ovarian carcinoma is not practical for teratoma. Although most teratomas are benign and can be treated surgically, IOT JOURNAL OF CELLULAR PHYSIOLOGY
Our findings indicate that there is a positive regulatory feedback loop between ERK and miR-21 that promotes EMT and CSPC in IOT. Previous studies have shown that ERK phosphorylation is an important marker of cancer EMT transformation (Tse and Kalluri, 2007; Kalluri and Weinberg, 2009) and CSPC maintenance (Rybak et al., 2013). Furthermore, studies have shown that miR-21 expression promotes EMT through Programmed Cell Death 4 (PCD4) and Sprouty-1 (Bronnum et al., 2013), and that it also enriches CD133 populations in IOT (Chung et al., 2013). These two parallel factors may be linked by E2/ERa stimulation. In this study, we have demonstrated consistent with literatures that both ERK activation and miR-21 expression promote CD133 expression and EMT formation, which both activated by E2/ ERa signals. We also found that knock down of miR-21 resulted in suppression of E2-induced ERK phosphorylation. Therefore, we speculated a high possibility of ERK downstream ETS/AP1 would regulation on pre-miR-21 expression. And our computer simulation data support this hypothesis. In addition, the human IOT immunohistological study supported the hypothesis of this study that cytosolic ERa co-expressed with CD133 in IOT lesions. These findings indicate that E2/ERa signaling might be able to trigger the ERK-miR21 positive feedback loop. However, the mechanism explaining how E2/ ERa mediates ERK-miR21 pathway needs to be investigated in a future study. In conclusion, non-genomic E2/ERa signaling governs cancer cell mobility through FAK, ERK, and ROCK activations. Furthermore, non-genomic E2/ERa signals promote cellular
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Fig. 7. Illustration showing how potential non-genomic ERa signaling regulates IOT cell mobility, EMT, and CSPC. Cytosolic ERa is activated by estrogens, which then activate FAK, ROCK, and ERK phosphorylation to facilitate cell migration and invasion. Furthermore, ERa activates ERK, which triggers an ERK-miR21 positive regulatory feedback loop that enhances miR-21 expression and consequently promotes cellular EMT and CSPC enrichment.
EMT and CSPC. Interestingly, this cancer cell stemness promoting effect involves a positive feedback loop between ERK phosphorylation and miR-21 expression. The results of this study provide a molecular insight into the development of malignant IOT. Acknowledgments
We thank Jeffery Conrad for language enhancement. Y.-C. Hung, W.-C. Chang, and L.-M. Chen executed the experiment, data analyses/interpretations, and drafted the manuscript. Y.-Y. Chang participated in experimental designs and translational research suggestion. L.-Y. Wu and W.-M. Chung provided experimental technical assistance. L.-C. Chen and T.-Y. Lin provided professional advice on histology studies. W.-L. Ma designed, supervised, and supported the entire project and was responsible for manuscript editing. Literature Cited Ahmed N, Abubaker K, Findlay J, Quinn M. 2010. Epithelial mesenchymal transition and cancer stem cell-like phenotypes facilitate chemoresistance in recurrent ovarian cancer. Curr Cancer Drug Targets 10:268–278. Bronnum H, Andersen DC, Schneider M, Sandberg MB, Eskildsen T, Nielsen SB, Kalluri R, Sheikh SP. 2013. miR-21 promotes fibrogenic epithelial-to-mesenchymal transition of epicardial mesothelial cells involving programmed cell death 4 and Sprouty-1. PLoS ONE 8:e56280. Carragher NO, Frame MC. 2004. Focal adhesion and actin dynamics: A place where kinases and proteases meet to promote invasion. Trends Cell Biol 14:241–249. Cheng W, Chen L, Yang S, Han J, Zhai D, Ni J, Yu C, Cai Z. 2012. Puerarin suppresses proliferation of endometriotic stromal cells partly via the MAPK signaling pathway induced by 17ss-estradiol-BSA. PLoS ONE 7:e45529.
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