In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression. (Cancer Sci 2011; 102: 1501–1508)
Breast cancer is typically hormone-dependent; exposure to estrogen enhances cellular growth and proliferation of breast cancer. However, it is known that estrogen also has suppressive effects against tumor progression; estrogen could suppress invasion and metastasis in the later stages of breast cancer. The effects of estrogen are mediated by the binding of estrogen receptors (ER),(1) ERα and ERβ, members of the nuclear receptor superfamily that function as ligand-induced transcription factors.(1–3)
The transforming growth factor (TGF)-β superfamily is a large, evolutionarily conserved family of secreted multifunctional peptides involved in almost every aspect of cellular behavior.(4) When TGF-β binds to TGF-β receptors termed type I, it triggers the phosphorylation of members of the Smad family.(5,6) Smad2 and Smad3 are receptor-activated Smads (R-Smad) downstream of TGF-β receptor type I activation.(7–9) Smad4 serves as a common partner (Co-Smad) for all R-Smads. Both R-Smads and Co-Smad serve as tumor suppressors; mutation, deletion, or silencing of these genes has been observed in several human cancers.(10,11) Moreover, clinical and preclinical data have shown that the TGF-β/Smad pathway plays a crucial role in suppressing primary tumor formation.(12,13) In contrast, recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for the Smad-dependent promotion of cancer metastasis by TGF-β.(14,15)
In our previous study, we found that ERα forms a protein complex with phosphorylated Smads (pSmad2 and pSmad3) and a ubiquitin ligase for Smad (Smurf) in the nucleus, and induces the Smurf-mediated ubiquitination and degradation of pSmad proteins to inhibit TGF-β/Smad signaling in an estrogen-dependent manner.(16) Thus, estrogen inhibits TGF-β/Smad signaling through a non-genomic function of ERα.
In the present study, we provide evidence that ERα-dependent suppression of breast cancer metastasis is mediated by inhibiting TGF-β signaling. We also show that Smad and pSmad protein levels and TGF-β-dependent transcription were decreased by estrogen, and not by antiestrogens, the pure ER antagonist ICI182,780 (ICI), or the selective ER modulator, tamoxifen (TAM). Consistent with these effects of ligands, the migration and invasion of breast cancer cells were suppressed by estrogen treatment, and not by ICI or TAM. In addition, we identified estrogen-regulated TGF-β target genes whose expressions were associated with breast cancer invasiveness and poor prognosis in patients.
Materials and Methods
Establishment of MCF-7-M5 cells. Parent MCF-7 cells suspended in DMEM containing 10% FBS medium were initially seeded into the top chamber with the non-coated membrane. After incubation for 24 h, migrated cells were harvested, cultured, then reseeded into a migration chamber. This process was repeated five times and highly migratory MCF-7 cell lines, MCF-7-M5 cells, were generated.
Supplemental Materials and Methods can be found in Data S1.
Estrogen receptor α suppresses lung metastasis of breast cancer cells in vivo. We previously reported that ERα regulates TGF-β/Smad signaling through a non-genomic pathway.(16) In addition, we have shown that ERα forms a protein complex with Smad and Smurf, and that Smad ubiquitination and subsequent degradation by ERα are enhanced in an estrogen-dependent manner, resulting in inhibition of TGF-β signaling.(16) To examine the effect of ERα on breast cancer metastasis, we generated the MDA-MB-231 breast cancer cell line expressing ERα (Fig. 1a). No significant difference was observed in cell proliferation, colony formation, or tumor formation between control MDA-MB-231 cells and ERα-expressing MDA-MB-231 cells (Fig. S1). Next, we carried out an in vivo lung metastasis assay using these cells. We discovered several metastatic tumors in the lungs of mice injected with control MDA-MB-231 cells (Fig. 1b,C,D). In contrast, significantly fewer metastatic tumors were observed in mice injected with ERα-expressing MDA-MB-231 cells (Fig. 1b,E,F). We also quantified lung metastasis by real-time RT-PCR and confirmed these observations (Fig. 1c). These results suggest that ERα prevents metastasis of breast cancer cells.
To examine whether the effects of ERα on cancer metastasis are mediated by its non-genomic function, we generated MDA-MB-231 cells expressing a mutated ERα, ERα(mC), which cannot bind to estrogen response elements (ERE). The expression of ERα(mC) did not affect cell proliferation, colony formation, or tumor formation (Fig. S1). However, lung metastasis of MDA-MB-231 cells was decreased by the expression of ERα(mC) (Fig. 1b,G,H,c). These results suggest that suppression of breast cancer metastasis by ERα is mediated by its non-genomic function.
Estrogen and ERα suppress migration and invasion of breast cancer cells through inhibition of TGF-β signaling. When TGF-β binds to its cell surface receptors, the activated receptors phosphorylate specific Smad proteins, Smad2 and Smad3, which are key transducers of TGF-β signaling. To verify whether ERα suppresses breast cancer metastasis by inhibiting TGF-β/Smad signaling, we generated a MDA-MB-231 cell line expressing both ERα and a constitutively active form of Smad2, Smad2SE (Fig. 2a).(17) Transforming growth factor-β-induced transcription of 9×CAGA-Luc(18) and p800-Luc(19) was inhibited by ERα expression and further suppressed by estrogen (Fig. 2b,c, lanes 4, 5). Smad2SE expression abolished the ERα and estrogen-dependent reduction of TGF-β-dependent transcription (Fig. 2b,c, lanes 6, 7). In a Transwell migration assay, TGF-β-induced migration was inhibited by estrogen in ERα-expressing MDA-MB-231 cells (Fig. 2d, middle). However, the inhibitory effect of estrogen on TGF-β-induced migration was not observed in MDA-MB-231 cells expressing both ERα and Smad2SE (Fig. 2d, right). In addition, invasive activity was reduced by estrogen in ERα-expressing MDA-MB-231 cells (Fig. 2e, middle), but not in MDA-MB-231 cells expressing both ERα and Smad2SE (Fig. 2e, right). Taken together, these results indicate that estrogen and ERα suppress TGF-β-induced migration and invasion by inhibiting TGF-β signaling.
Estrogen and antiestrogens regulate Smad and pSmad protein levels. Estrogen is known to promote breast cancer tumorigenesis.(20) However, our results indicate that estrogen suppresses breast cancer invasion and metastasis by inhibiting TGF-β signaling.(16) In contrast, previous studies have indicated that antiestrogens such as ICI and TAM have suppressive effects on breast cancer tumorigenesis.(20,21) However, the effects of these antiestrogens on breast cancer metastasis remain unclear. Therefore, we investigated the effects of antiestrogens on TGF-β signaling. Total Smad and pSmad protein levels were reduced by ERα expression and further decreased by estrogen (Fig. 3a, lanes 3, 4). However, total Smad and pSmad protein levels were unaltered by TAM (Fig. 3a, lane 6) and slightly increased by ICI compared with no treatment (Fig. 3a, lanes 3, 5). Because ICI acts as a pure ER antagonist by facilitating the rapid degradation of ER,(21) ERα levels were significantly reduced by ICI treatment. Thus, it is possible that ICI increases total Smad and pSmad protein levels by abrogating Smad degradation by ERα. These data suggest that estrogen and antiestrogens regulate pSmad2 and pSmad3 protein levels through ERα.
Estrogen and antiestrogens regulate Smad-dependent transcription and TGF-β target gene expression. Next, we examined whether the regulation of Smad protein levels by estrogen and antiestrogens affected Smad-dependent transcription and TGF-β target gene expression. Consistent with previous reports, estrogen, unlike antiestrogens, increased ERE-mediated transcriptional activity (Fig. 3b). A constitutively active TGF-β type I receptor (ALK5 TD) expression induced TGF-β-dependent transcription of 9×CAGA-Luc or p800-Luc, which was inhibited by ERα expression and further suppressed by estrogen (Fig. 3c,d, lanes 3, 4). In contrast, ALK5 TD-induced transcription was enhanced by ICI compared with no treatment (Fig. 3c,d, lanes 3, 5) and unchanged by TAM (Fig. 3c,d, lanes 3, 6). We also examined whether estrogen and antiestrogens affect the mRNA levels of the ERα target gene pS2 and TGF-β/Smad target genes plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-9 (MMP-9). Estrogen, but not antiestrogens, increased the expression of pS2 (Fig. 3e). Transforming growth factor-β-induced expression of PAI-1 and MMP-9 was decreased by estrogen (Fig. 3f,g, lane 3). In contrast, ICI, unlike TAM, slightly increased TGF-β-induced expression of these genes (Fig. 3f,g, lanes 4, 5). These results indicate that antiestrogens do not inhibit TGF-β-dependent transcription. Taken together, these results suggest that estrogen and antiestrogens regulate TGF-β signaling by controlling Smad protein levels through ERα.
Estrogen, unlike antiestrogens, abrogates migration and invasion of highly migratory MCF-7 cells. We investigated whether the regulation of TGF-β signaling by estrogen and antiestrogens affected the migratory and invasive potentials of MCF-7 cells that endogenously expressed ERα and Smads. It is known that the migratory and invasive potentials of MCF-7 cells are very low. Therefore, we generated highly migratory MCF-7 cells, MCF-7-M5 cells, using the Transwell migration assay system. We first compared the effects of ERα ligands on MCF-7 and MCF-7-M5 cells. The expression levels and transcriptional activity of ERα in MCF-7-M5 cells were comparable to those in parental MCF-7 cells (Fig. S2a,b). In addition, the effects of ERα ligands or TGF-β on proliferation of MCF-7-M5 cells were similar to those on proliferation of MCF-7 cells (Fig. S2c,d).
Next, we examined the migratory and invasive potential of MCF-7-M5 cells. MCF-7-M5 cells have significantly higher migratory potential than parental MCF-7 cells (Fig. 4a). We also observed that migration of MCF-7-M5 cells was suppressed by a specific inhibitor of TGF-β receptor kinase, SB-505124 (Fig. 4a). Moreover, the invasive potential of MCF-7-M5 cells was higher than that of parental MCF-7 cells and suppressed by SB-505124 (Fig. 4b). We also investigated the expression levels of E-cadherin and N-cadherin as epithelial–mesenchymal transition (EMT) markers using MCF-7 and MCF-7-M5 cells. E-cadherin expression was decreased and N-cadherin expression was increased in MCF-7-M5 cells compared with those in MCF-7 cells (Fig. 4c, lanes 1, 2). In addition, SB-505124 treatment upregulated E-cadherin expression and downregulated N-cadherin expression (Fig. 4c, lanes 2, 3). These results suggest that the enhancement of the invasive potential and EMT of MCF-7-M5 cells is partly due to facilitation of TGF-β signaling.
Next, we examined the effects of estrogen and antiestrogens on EMT in MCF-7-M5 cells. E-cadherin expression was increased and N-cadherin expression was decreased by estrogen, but not by antiestrogens (Fig. 4c, lanes 4–6). These results suggest that estrogen suppresses TGF-β-induced EMT in breast cancer cells, whereas antiestrogens do not suppress it. We next examined the effects of estrogen and antiestrogens on the migratory and invasive potential of MCF-7-M5 cells. Both migratory and invasive potential was suppressed by estrogen, slightly enhanced by ICI, and unaffected by TAM (Fig. 4d,e). Taken together, these observations indicate that invasion and EMT of MCF-7-M5 cells were suppressed by estrogen, but not by ICI or TAM.
Estrogen counteracts the expression of TGF-β target genes associated with poor prognosis in breast cancer patients. As mentioned before, TGF-β/Smad signaling is reported to facilitate metastasis.(14,15) As shown in Figure 4(d,e), migration and invasion of breast cancer cells were suppressed by estrogen. Therefore, we identified the TGF-β target genes that were downregulated by estrogen in MCF-7-M5 cells by DNA microarray analysis. We found that the expression of 18 target genes, which are reported to be upregulated by TGF-β signaling,(22,23) was downregulated more than twofold by estrogen in MCF-7-M5 cells. To investigate the relationship between these target genes and breast cancer invasiveness, we used the Oncomine cancer profiling database (http://www.oncomine.org), which provides publicly available datasets on cancer gene expression. Of 12 genes, we focused on six, fibroblast growth factor 2 (FGF2), latent transforming growth factor beta binding protein 2 (LTBP2), dynamin 3 (DNM3), T-box-3 (TBX3), hect domain and RCC1 (CHC1)-like domain 1 (HERC1), and integrin alpha 4 (ITGA4), whose expression was downregulated more than fourfold by estrogen (Table 1). The Oncomine database revealed that the expression of these six genes was upregulated in invasive breast carcinoma tissues compared to their expression in normal breast tissues (Fig. 5a).(24–26) The relationship between the expression of these genes and prognosis in breast cancer patients was examined using the PrognoScan database (http://gibk21.bio.kyutech.ac.jp/PrognoScan/index.html). PrognoScan is a large collection of publicly available cancer microarray datasets with clinical annotation and a tool for assessing the biological relationship between gene expression and prognosis.(27) As shown in Figure 5(b), the expression of LTBP2, DNM3, TBX3, and HERC1 inversely correlated with distant metastasis-free survival or overall survival in breast cancer patients.(22–27)
Table 1. Identification of transforming growth factor-β target genes regulated by estrogen and correlated with breast cancer invasiveness
Gene expression with E2 treatment compared to DMSO†
Gene expression in invasive breast carcinoma tissues compared to normal breast tissues‡
†Results of DNA microarray analysis using MCF-7-M5 cells with estrogen (E2) treatment compared to no treatment. ‡Data and statistics obtained from the Oncomine database.
Fibroblast growth factor 2
Up (P = 2.00E−3)
Latent transforming growth factor beta binding protein 2
Up (P = 1.30E−6)
Up (P = 1.92E−7)
Up (P = 3.00E−3)
Hect domain and RCC1 (CHC1)-like domain (RLD) 1
Up (P = 2.65E−7)
Integrin alpha 4
Up (P = 9.87E−4)
Up (P = 5.84E−5)
Runt-related transcription factor 1
Up (P = 6.75E−10)
Protein tyrosine phosphatase, non-receptor type 21
Up (P = 9.63E−5)
MOB1, Mps one binder kinase activator-like 2B (yeast)
Up (P = 6.73E−8)
Transcription factor 12
Up (P = 7.10E−14)
BTG family, member 2
Up (P = 2.24E−10)
Therefore, we examined whether estrogen and antiestrogens regulated the expression of these genes using MCF-7-M5 cells. The mRNA levels of these four genes were decreased by estrogen, but not by ICI or TAM (Fig. 6a, lanes 2–4). Next, to confirm whether these four genes were regulated by TGF-β signaling, we treated MCF-7-M5 cells with TGF-β. The expression of LTBP2, DNM3, TBX3, and HERC1 was increased by TGF-β treatment (Fig. 6a, lane 5). Transforming growth factor-β-induced expression of these genes was decreased by estrogen, but not by antiestrogens (Fig. 6a, lanes 6–8). Next, we applied the same combined treatment to ERα-expressing MDA-MB-231 cells. The expression of LTBP2, DNM3, TBX3, and HERC1 was stimulated by TGF-β treatment in ERα-expressing MDA-MB-231 cells (Fig. 6b, lane 2). Transforming growth factor-β-induced expression of these genes was decreased by estrogen, but not by antiestrogens (Fig. 6b, lanes 3–5).
Next, to investigate the functional roles of LTBP2, DNM3, TBX3, and HERC1 in breast cancer migration and invasion, we examined the effects of siRNA-mediated knockdown of LTBP2, DNM3, TBX3, or HERC1 on migration and invasion of MCF-7-M5 cells (Fig. S3 and Fig. 6c,d). Migration and invasion of MCF-7-M5 cells were suppressed by knocking down these genes (Fig. 6c,d). These results confirm that LTBP2, DNM3, TBX3, and HERC1 are involved in migration and invasion of breast cancer cells. Taken together, these results suggest that estrogen inhibits TGF-β target genes associated with breast cancer invasiveness and poor prognosis in breast cancer.
In breast cancer, ERα levels are elevated in premalignant and malignant breast lesions compared to their levels in normal tissues. A fraction of breast cancers that were initially ERα-positive lose ERα expression during tumor progression. Such ERα-negative tumors are more likely to be of higher histological grade than more differentiated ERα-positive tumors. Patients with these tumors show decreased overall survival rates, whereas those with ERα-positive tumors have better prognoses. These results raise the possibility that estrogen has biphasic effects on tumor progression; it may stimulate tumor growth in the early stages of breast cancer and suppress invasion and metastasis at later stages.
In addition, TGF-β has biphasic effects on tumor progression that are opposite to those of estrogen.(28,29) With the identification of inactivating mutations in genes encoding components of the TGF-β signaling pathway in cancer cells, it has become clear that TGF-β contributes to tumor suppression in the early stages of cancer development. Genetic manipulation of the TGF-β signaling pathway in tumor cell lines and experimental animal models has validated the promotive role of TGF-β in metastasis during late cancer progression. Exogenous TGF-β increases the invasiveness and metastatic behavior of breast cancer cells in vivo,(14) although it inhibits the proliferation of these cells in vitro. Transforming growth factor-β normally stimulates production of ECM proteins and induces the proteolytic activity of cancer cells by stimulating matrix-degrading enzyme expression.(30) Increased TGF-β levels aid in generating more invasive cancer cells by decreasing cellular adhesion and increasing cancer cell motility and proteolytic activity.
In the present study, we showed that estrogen suppresses breast cancer invasion through the inhibition of TGF-β signaling (Fig. 7). We also showed that, in contrast to estrogen, ICI slightly enhances TGF-β signaling and breast cancer invasiveness, unlike TAM (Fig. 7). Our data suggest that breast tumor invasiveness and metastasis are suppressed by estrogen, but not by antiestrogens.
Several studies have reported the controversial effects of antiestrogens on cancer invasiveness. In MCF-7 cells, TAM inhibited cell proliferation but increased their invasiveness by upregulating collagenase IV expression(31) and promoted an invasive phenotype through the association of the Src kinase pathway and pro-invasive gene expression.(32) However, exposure to estrogen induces endothelial progenitor cell migration and proliferation through ERs and the PI3K pathway, which are blocked by pretreatment with the pharmacological PI3K blockers or ICI.(33) In contrast, it is also reported that ICI increases P-cadherin expression and promotes invasion of breast cancer cells.(34) Taken together, our observations and previous studies suggest that clinical treatment with TAM or ICI suppresses breast cancer tumorigenesis, but cannot suppress breast cancer metastasis.
We also identified the estrogen-regulated TGF-β target genes, LTBP2, DNM3, TBX3, and HERC1, whose expressions are associated with breast cancer invasiveness and poor prognosis in breast cancer patients. LTBP2 is reported to support melanoma cell migration.(35) It is also reported that increased levels of TBX3 enhance melanoma invasiveness by inhibiting E-cadherin expression.(36) In addition, upregulation of TBX3 contributes to breast cancer cell migration, and its expression is significantly higher in metastatic breast cancer than in non-metastatic tumors.(37) It has been also reported that HERC1 is involved in membrane trafficking, cell proliferation.(38–41) In addition, the expression of HERC1 has been reported to be increased in human tumor cells.(42)DNM3 shows tissue-selective expression; however, the relationship between DNM3 and cancer has not been reported.
Taken together, our results suggest that estrogen regulates breast cancer invasiveness through the suppression of TGF-β target genes such as LTBP2, DNM3, TBX3, and HERC1. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.
This work was supported by the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors have no conflict of interest to report.