Transforming Growth Factor-B1, Transforming Growth Factor-B2, and Transforming Growth Factor-B3 Enhance Ovarian Cancer Metastatic Potential by Inducing a Smad3-Dependent Epithelial-to-Mesenchymal Transition

Transforming growth factor-B (TGF-B) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-B/ Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-B1, TGF-B2, and TGF-B3 induced pro–matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-tomesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-B–mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-B1, TGF-B2, and TGF-B3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-B–stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-B/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential. (Mol Cancer Res 2008;6(5):695–705) Introduction More than 80% of ovarian cancers are thought to originate from the single layer of epithelia surrounding the ovary, called the ovarian surface epithelia (OSE). OSE cells are distinct from other epithelia in that they possess a relatively uncommitted phenotype, expressing both epithelial and mesenchymal characteristics (1). The coelemic epithelium gives rise to OSE cells and cells that form the Müllerian duct, which subsequently differentiate into the endocervix, endometrium, and Fallopian tube (2). Ovarian cancer is unique from other cancer models, because cancer cells acquire more specialized epithelial markers, such as E-cadherin, when compared with their hypothesized tissue of origin, the OSE (3). Ovarian cancer is thought to arise from neoplastic changes that result in the pathologic differentiation of OSE into the established histologic subtypes, which include serous (fallopian tube– like), endometrioid (endometrium-like), and mucinous (endocervical-like) adenocarcinoma among others. Although OSE have a more uncommitted phenotype than malignant epithelia, we hypothesize that cancer cells undergo an epithelialto-mesenchymal transition (EMT) to disseminate within the i.p. cavity or metastasize to a distant site. EMTs occur as a part of developmental and physiologic programs that orchestrate events, such as neural crest formation, palatal fusion, and wound healing (4). Cancer cells in advanced stages become aggressive and invasive due to the pathologic initiation of an EMT that often recapitulates developmental signaling cascades. EMTs involve coordination of a complex genetic program that results in the loss of epithelial markers (e.g., E-cadherin, occludin) and the acquisition of mesenchymal markers (e.g., N-cadherin, vimentin). Cancer cells that undergo an EMT change from a compact, cuboidal (epithelial) shape to a spindle-like, fibroblastic (mesenchymal) morphology. The transition causes cells to lose cell-cell contacts and cell polarity, become motile, and secrete proteases that degrade extracellular matrix (5). Transforming growth factor-h (TGF-h) is thought to contribute to oncogenesis by inducing an EMT that promotes tumor metastasis (6). Received 6/22/07; revised 12/28/07; accepted 1/14/08. Grant support: NIH grant RO1 HD044464 (T.K. Woodruff). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Teresa K. Woodruff, Northwestern University, 2205 Tech Drive, Hogan 4-150, Evanston, IL 60208. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0294 Mol Cancer Res 2008;6(5). May 2008 695 on June 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from The TGF-h superfamily participates in a diverse array of reproductive, differentiation, and developmental programs (7). Family members are subdivided into the TGF-h/activin/nodal branch and the bone morphogenetic protein/growth and differentiation factor branch because of the specific receptorregulated Smads they activate (8). TGF-h1, TGF-h2, and TGFh3 exert biological effects by binding to type I (ThRI) and type II (ThRII) receptors. Upon ligand binding to ThRII, ThRI is activated and phosphorylates the receptor-regulated Smads, Smad2 and Smad3. Phosphorylated receptor-regulated Smads then bind to the co-Smad, Smad4, and translocate to the nucleus to modulate gene expression. TGF-h also initiates Smadindependent pathways, including those mediated by the mitogen-activated protein kinase family members (TAK1, extracellular signal-regulated kinase, p38, c-Jun-NH2-kinase) and phosphatidylinositol 3-kinase (9). TGF-h can function as a tumor suppressor by inhibiting cell proliferation, but can also promote metastasis in various cancer models (10, 11). Although TGF-h plays an important role in ovarian physiology, much remains to be elucidated about how this hormone contributes to ovarian cancer progression, particularly in the regulation of an EMT. TGF-h1 elevates matrix metalloproteinase (MMP) secretion and invasion through Matrigel in ovarian cancer cells (12, 13). Another study reports that TGF-h1 induces an EMT in two ovarian adenocarcinoma cell lines, derived from a rare variant of aMüllerian mixed tumor (14). Several clinical studies suggest that TGF-h is involved in ovarian tumor progression. TGF-h1, TGF-h2, and TGF-h3 are FIGURE 1. TGF-h1, TGF-h2, and TGF-h3 mediate an EMT and MMP secretion in ovarian cancer cells. OVCA429 cells were serum-starved for 24 h and then treated with vehicle (Veh ), 10 ng/mL TGF-h1, TGF-h2, TGF-h3, or activin A for 72 h. A. The TGF-h ligands stimulate an EMT in cancer cells. Top, brightfield images of cells showing that TGF-h initiates the loss of cell-cell junctions and the change from an epithelial to a fibroblastic morphology (bar, 20 Am); bottom, OVCA429 cells immunostained with anti –E-cadherin, anti-occludin, anti – N-cadherin, or anti-vimentin (FITC, white arrowheads ) and counterstained with 4¶,6-diamidino-2-phenylindole (blue ) to show nuclei (bar, 50 Am).B. Anti –E-cadherin and anti – N-cadherin immunoblots of quiescent cells treated with vehicle, activin A, TGF-h1, TGF-h2, or TGF-h3. The same blot was stripped and reprobed with anti –h-actin as a loading control. C. TGF-h1, TGF-h2, and TGF-h3 trigger pro–MMP-2 and pro–MMP-9 secretion in cancer cells, but not in the normal cell line IOSE80. Conditioned media from quiescent OVCA429 and IOSE80 cells treated with vehicle, TGF-h1, TGF-h2, or TGF-h3 were subjected to gelatin zymography to analyze MMP secretion. Do et al. Mol Cancer Res 2008;6(5). May 2008 696 on June 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from overexpressed in 44%, 66%, and 66% of malignant ovarian tumors, respectively (15). Notably, TGF-h1 expression is less frequent than TGF-h2 and TGF-h3, underscoring the relevance of studying the TGF-h2 and TGF-h3 isoforms. Furthermore, TGF-b1 and latent TGF-b1-binding protein mRNAs are upregulated in ovarian carcinoma tissue relative to benign tissue (16), and TGF-h1 levels are elevated in the plasma and peritoneal fluid of ovarian cancer patients (17). Although the role of TGF-h1 in oncogenesis has been studied extensively, the function of TGF-h2 and TGF-h3 in ovarian cancer and other cancers remains relatively unexplored. In the current study, we investigated the potential of TGFh1, TGF-h2, and TGF-h3 to initiate an EMT in ovarian cancer cells. All three isoforms were able to efficiently induce an EMT in ovarian cancer cells, disrupting cell-cell junctions and stimulating MMP secretion. Furthermore, Smad3 is required for the EMT mediated by all three TGF-h family members, and down-regulation of Smad3 expression inhibits the loss of cellcell contacts and transition to a fibroblastic morphology. Within the context of a three-dimensional collagen matrix, Smad3 is required for TGF-h–regulated secretion of pro-MMP and active MMP and invasion. Consistent with the in vitro findings, analysis of serous cystadenocarcinoma revealed that higher Smad3 nuclear expression correlates with poor outcome. Taken together, these results suggest that the TGF-h/Smad3 pathway plays an important role in ovarian tumor progression by promoting the dissemination of cancer cells. Results TGF-b1, TGF-b2, and TGF-b3 Induce an EMT and MMP Secretion in Ovarian Cancer Cells The role of TGF-h, particularly that of TGF-h2 and TGF-h3, in regulating EMTs in ovarian cancer remains relatively unexplored. Therefore, we treated the human cancer cell line, OVCA429, with TGF-h1, TGF-h2, and TGF-h3 to determine if any of these family members can initiate an EMT, causing cells to lose cell-cell contacts and to change from a compact, epithelial morphology to a spindle-shaped fibroblastic morphology. Notably, all three ligands efficiently induced a fibroblastic morphology in OVCA429 cells after 72 h (Fig. 1A, brightfield images). We further investigated the effects of TGF-h1, TGF-h2, and TGF-h3 on the epithelial markers, E-cadherin and occludin, and the mesenchymal markers, vimentin and N-cadherin, by indirect immunofluorescent staining. All three ligands elicited a fibroblastic, undifferentiated phenotype in OVCA429 cells, disrupting adherens junctions and tight junctions, as shown by E-cadherin (Fig. 1A, i-iv) and occludin immunofluorescence (Fig. 1A, v-viii), respectively. E-cadherin staining was present at adherens junctions in vehicle-treated cells (Fig. 1A, i, white arrowhead), but was markedly decreased in TGF-h1–treated, TGF-h2–treated, and TGF-h3–treated cells (Fig. 1A, ii-iv). Similarly, occludin staining was present at tight junctions in vehicle-treated cells (Fig. 1A, v, white arrowhead), but was severely diminished in TGF-h–treated cells (Fig. 1A, vi-viii). Furthermore, treatment with all three ligands increased the immunostaining of the mesenchymal marker vimentin, an intermediate filament protein (Fig. 1A, x-xii, white arrowheads), and N-cadherin (Fig. 1A, xiv-xvi, white arrowheads) when compared with vehicle-treated cells (Fig. 1A, ix and xiii). Consistent with these observations, TGF-h1, TGF-h2, and TGF-h3 up-regulated N-cadherin and down-regulated E-cadherin protein expression (Fig. 1B). Cells were treated with activin A, for 72 h, as a negative control, because activin A did not mediate an EMT in OVCA429 cells (data not shown). Treatment with activin A did not affect E-cadherin or Ncadherin expression (Fig. 1B). FIGURE 2. TGF-h1, TGF-h2, and TGF-h3 stimulate Smad3 phosphorylation and nuclear localization. OVCA429 cells were made quiescent and then treated with vehicle, 10 ng/mL TGF-h1, TGF-h2, or TGF-h3. A. Anti –phosphorylated Smad3 immunoblot. OVCA429 cells were treated with TGF-h ligands for 0, 10, 20, 30, 45, and 60 min. The same blot was stripped and reprobed with anti-Smad3 as a loading control. B. Anti –phosphorylated Smad2 immunoblot. OVCA429 cells were treated with TGF-h ligands for 0, 10, 20, 30, 45, and 60 min. The same blot was reprobed with anti-Smad2 as a loading control. Lysates from LhT2 cells treated with activin A were used as a control for anti –phosphorylated Smad2 immunoreactivity. C. Fluorescent micrograph of cells transfected with Smad3-GFP for 24 h before treatment with vehicle, TGF-h1, TGF-h2, or TGF-h3 for 30 min. Bar, 50 Am. Smad3 Mediates an EMT in Ovarian Cancer Cells Mol Cancer Res 2008;6(5). May 2008 697 on June 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Because the TGF-h ligands caused ovarian cancer cells to undergo dramatic changes in cell morphology suggestive of an EMT, the effect of these ligands on MMP secretion was also evaluated. Conditioned media was collected from cells treated with TGF-h and subjected to gelatin zymography. TGF-h treatment had no effect on MMP-2 and MMP-9 secretion in the cell line, IOSE80 (normal human OSE cells transfected with SV40 large T antigen to allow for longer passage times in vitro ; Fig. 1C, top), but augmented both pro–MMP-2 and pro–MMP9 secretion in OVCA429 carcinoma cells (Fig. 1C, bottom). Collectively, these results suggest that all three TGF-h ligands exert prometastatic effects on ovarian cancer cells by inducing an undifferentiated cell morphology and MMP secretion. Smad3 Regulates TGF-b–Mediated Changes in Cell Morphology, but not MMP Secretion in Cells Cultured on Plastic To determine the relevant Smad pathway activated downstream of TGF-h signaling, the phosphorylation of Smad2 and Smad3 was investigated. TGF-h1, TGF-h2, and TGF-h3 stimulated phosphorylation of Smad3 (Fig. 2A), but only low level or no phosphorylation of Smad2 was detected (Fig. 2B). Lysates from the mouse gonadotrope cell line LhT2, treated with activin A, were used as a positive control for the phosphorylated Smad2 immunoblot (Fig. 2B). The effects of the TGF-h ligands on Smad3 nucleocytoplasmic shuttling were also assessed using a Smad3-GFP construct, because Smad3GFP has been shown to behave similarly to endogenous, FIGURE 3. Smad3 is required for TGF-h– mediated EMT, but not MMP secretion in ovarian cancer cells cultured on plastic. Cells were cultured on plastic dishes and transfected with 50 nmol/L Smad3 siRNA or control (nontargeting) siRNA and then treated with vehicle, 10 ng/mL TGF-h1, TGF-h2, or TGF-h3 for 72 h. A. AntiSmad3 immunoblot showing down-regulation of Smad3 protein in Smad3 -siRNA– transfected cells. The same blot was also reprobed with anti –h-actin as a loading control. B. Brightfield images of quiescent cells transfected with control or Smad3 siRNAs and then treated with vehicle and TGF-h ligands (top ); Anti –E-cadherin immunofluorescence (white arrowheads ) showing that Smad3 -siRNA– transfected cells do not undergo an EMT in response to TGF-h1, TGF-h2, or TGFh3, in contrast to control-siRNA– transfected cells (bottom ); bar, 20 Am. C. Gelatin zymography of conditioned media from cells transfected with 50 nmol/L Smad3 or control (nontargeting) siRNA showing effects on MMP secretion. Do et al. Mol Cancer Res 2008;6(5). May 2008 698 on June 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from wild-type Smad3 (18). Treatment of OVCA429 cells with TGF-h ligands for 30 minutes resulted in predominantly nuclear localization of Smad3-GFP (Fig. 2C) in contrast to the diffuse cytoplasmic and weak nuclear Smad3-GFP localization in vehicle-treated cells (Fig. 2C). Taken together, these results suggest that Smad3 is activated as a result of TGF-h stimulation. We next analyzed the effects of inhibition of Smad3 expression on TGF-h–coordinated changes in cell morphology and MMP secretion. To perform this analysis, cells were transfected with Smad3 -specific small interfering RNA (siRNA) to repress Smad3 expression. Cells were also transfected with nontargeting siRNA as a negative control. Smad3 siRNA transfection dramatically decreased Smad3 expression (Fig. 3A, lanes 5-8), whereas control siRNA treatment displayed minimal effect on Smad3 protein expression (Fig. 3A, lanes 1-4). Transfection of control siRNA did not dramatically alter TGF-h–induced loss of cell-cell junctions as evidenced by the diminution of E-cadherin staining in TGF-h– treated cells when compared with the robust E-cadherin staining in vehicle-treated cells (Fig. 3B, arrowhead; control siRNA). However, attenuation of Smad3 expression abrogated TGF-h– regulated cell scattering and loss of cell-cell junctions, as indicated by the presence of anti–E-cadherin immunofluorescence in both vehicle-treated and TGF-h–treated cells (Fig. 3B, top and bottom, arrowheads ; Smad3 siRNA). In addition, TGF-h–stimulated pro–MMP-2 and pro–MMP-9 secretion were equivalent in both Smad3 and control siRNA-transfected cells (Fig. 3C, compare lanes 2-4 and lanes 6-8). These data indicate that Smad3 is required for TGF-h– mediated loss of cell-cell adhesion and cell shape changes, but does not substantially alter TGF-h–triggered MMP