Dual effects of TGF-β on ERα-mediated estrogenic transcriptional activity in breast cancer
© Ren et al; licensee BioMed Central Ltd. 2009
Received: 22 July 2009
Accepted: 27 November 2009
Published: 27 November 2009
TGF-β resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-β regulates ERα-induced gene transcription and potential mechanisms of frequent TGF-β resistance in breast cancer.
Effect of TGF-β on ERα-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERα-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays.
A complex of Smad3/4 mediates TGF-β inhibition of ERα-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-β from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERα-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma.
These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-β suppression of ERα transactivity in breast cancer cells.
Estrogens act as mitogens to promote cell proliferation in both normal breast tissue and breast carcinomas through their binding to estrogen receptors (ER). The ERα is a transcriptional activator and regulates gene transcription either by directly binding to the estrogen-responsive element (ERE) or by interacting with other transcription factors [1, 2]. Gene amplification or overexpression of ERα was found in some breast cancer [3, 4]. Approximately 70% of breast cancers are ERα positive and estrogen dependent. ERα has become an important prognostic marker and a therapeutic target in breast cancer [5, 6].
In contrast to estrogens, which induce proliferation of breast cancer cells, transforming growth factor-β (TGF-β) inhibits the growth of human breast cancer cells in culture [7, 8]. TGF-β is the prototypic inhibitor of cell cycle progression and appears to directly antagonize the effects of many different mitogenic growth factors. A well-characterized TGF-β signaling pathway is initiated by the association between TGF-β and its two cell surface receptors, resulting in the formation of the receptor heterocomplex and activation of the type I receptor, which in turn activates the cytoplasmic receptor regulated-Smad (R-Smad: Smad2 and Smad3) proteins via phosphorylation . Phosphorylated R-Smad associates with Smad4. The resulting heteromeric Smad complexes then translocate into the nucleus, where they regulate gene transcription in collaboration with other factors. The importance of the TGF-β signaling pathway in cancer development is underscored by the presence of downregulation or inactivating mutations in genes encoding TGF-β receptors and Smads in human carcinomas [10–12].
While the role of TGF-β in breast cancer is ambiguous, as it was shown to display both tumor-suppressing and -enhancing effects, loss of responsiveness to TGF-β is believed to be a major factor in tumor formation [13–15]. Activation of TGF-β represents one of the physiological countermeasures that are invoked to protect transformed cells against ERα excessive mitogenic stimulation. Additionally, inhibition of some breast cancer cell growth by tamoxifen appears to be mediated by TGF-β signaling pathway . Inhibition of Tβ RII expression abolished antiestrogen-dependent growth inhibition [17, 18]. It has been shown that Smad2, Smad3 and Smad4 all have physical interactions with ERα and that Smad4 acts as a transcriptional co-repressor for ERα and inhibits tumor growth by inducing apoptosis in ERα-positive cells [19–22]. Although the regulated gene targets of Smads/ERα have not been identified, these findings imply that Smads-mediated cross-talk with the estrogen receptor plays an important role in development and/or progression of breast cancer.
In this study, we investigated how TGF-β regulates ERα-induced gene transcription and potential mechanisms of frequent TGF-β resistance in breast cancer. We demonstrated that Smad4 is essential for TGF-β-mediated inhibition of ERα estrogenic transcription activity. Either overexpression of Smad3 or inhibition of Smad4 expression switches TGF-β to an activator for ERα transactivation in breast cancer cells. In addition, we found that the expression of Smad4 was downregulated with increased cytoplasmic localization in ERα-positive human infiltrating breast cancer tissue.
Cell Culture, Transient Transfection and Reporter Assays
MCF-7 cells were purchased from the American Type Culture Collection and maintained according to the manufacturer's instructions. MDA-MB-231 and MDA-MB-468 cells were a gift from Dr. Joseph Messina (Department of Pathology, University of Alabama at Birmingham, Birmingham, AL). These two cell lines were incubated in Leibovitz's L-15 medium with 2 mM L-glutamine (ATCC) supplemented with antibiotics and 10% fetal bovine serum (Cellgro) at 37°C in 5% CO2. COS-1 cells were incubated in Dulbecco's modified Eagle's medium (Cellgro) supplemented with antibiotics and 10% fetal bovine serum at 37% in 5% CO2. The methods used for transient transfection and luciferase assay have been described in Wu L. et al. . Similarly, 2× ERE-TATA reporter plasmid was used to examine the function of TGF-β/Smads on the estrogen response element. The breast cancer cell line (MCF-7, MDA-MB-231 or MDA-MB-468) cells were split and plated at 5 × 104 cells/24-well plate. The cells were starved with Dulbecco's modified Eagle's phenol red-free medium supplemented with 10% charcoal-stripped bovine serum (Cellgro) for 24 hours. Transient transfections were performed using LipofectAMINE plus reagent (Invitrogen) with 0.2 μg of luciferase reporter and 2-20 ng (2 ng for MCF-7 cells; 20 ng for MDA-MB-231/468 cells) of the hERα expression plasmid. The amount of co-transfected Smad expression plasmid is indicated in the figures and text. Aliquots of cells were treated with E2, 1 nM (Sigma), TGF-β1, 100 pM (R&D), or a combination of E2 + TGF-β1. Sixteen hours after the treatment, luciferase activity was assayed in each cell line using the Dual-luciferase™ assay kit (Promega) according to the manufacturer's instructions.
HA-tagged human ERα plasmid was cloned into a pCDNA3 vector as described in previous studies . The expression vectors pFLAG-Smad2, pFLAG-Smad3 and pFLAG-Smad4 were gifts from Dr. Rik Derynck (University of California at San Francisco, San Francisco, CA). The 2× ERE-TATA reporter was a gift from Dr. Valerie Clark (Duke University Medical Center, Durham, NC).
Semi Quantitative Real Time PCR
The target cDNA sequence was evaluated using Primer3 http://frodo.wi.mit.edu/primer3. The sequences of primers used in RT-PCR assays are listed as follows: β-actin upper primer, 5'-AGACTTCGAGCAGGAGCTGG-3'; β-actin lower primer, 5'-CGGATGTCAACGTCACACTT-3'; PS2 upper primer, 5'-TTGTGGTTTTCCTGGTGTCA-3'; PS2 lower primer, 5'-CCGAGCTCTGGGACTAATCA-3'; C-MYC upper primer, 5'-CTCCTGGCAAAAGGTCAGAG-3'; C-MYC lower primer, 5'-TCGGTTGTTGCTGATCTGTC-3'. Total RNA was extracted from each breast cancer cell line using STAT-60 (TEL-TEST Inc.). The detailed procedures for mRNA purification, reverse transcription and semi quantitative real time PCR have been described previously .
Co-immunoprecipitation and Immunoblotting assays
To detect endogenous-endogenous, endogenous-exogenous or exogenous-exogenous protein complexes, breast cancer cells or COS-1 cells were transfected with expression plasmid(s) encoding the indicated proteins or left untransfected. Thirty-six hours post-transfection, aliquots of cells were incubated for 1 hr in the presence or absence of β-estradiol (E2) and/or TGF-β1. Cells were lysed with radioimmune precipitation assay buffer , precleared with purified mouse or rabbit IgG , and centrifuged at 13,000 × g for 30 mins. Supernatants were incubated with antibodies and protein A/G beads for 3 hr at 4°C on a rotating platform. Immunoprecipitates were separated by 7%-12% SDS-PAGE and transferred to PVDF membranes (BioRad). Immunoblotting was carried out as previously described . The antibodies used for immunoprecipitation and immunoblotting are listed as follows: anti-FLAG M2 (F3165, Sigma), anti-HA (rabbit polyclonal, H9658, Sigma), anti-Smad2/3 , anti-Smad4 (sc-7154, and sc-7966, Santa Cruz), and anti-ERα (sc-8002, Santa Cruz).
The procedure for immunofluorescence staining was adapted from the online protocol http://www.bdbiosciences.com/pharmingen/protocols/Immunofluorescence_Microscopy.shtml of BD Biosciences: Pharmingen: Protocols. The primary antibodies used were: monoclonal mouse anti-Smad2/3 (S66220, BD Transduction Laboratories) and polyclonal rabbit anti-Smad4 (sc-7154, Santa Cruz). The secondary Texas-red™-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse antibodies were used for immunofluorescence detection. The DNA dye 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (D21490, "FluoroPureTM" grade, Molecular Probes) was used after the secondary antibodies to visualize the nucleus of the cells. Images were captured with an Olympus IX70 inverted fluorescence microscope and MagnaFire SP (Optronics) digital camera and visualized using Adobe Photoshop 7.0 and Image-pro® Express 4.5 (MediaCybernetics Inc.) software.
Immunohistochemical staining was performed on serial paraffin-embedded sections of human breast tissue using UltraVision One detection system (HRP polymer & DAB Plus chromogen, Thermo Fisher Scientific, Fremont, CA). The mouse monoclonal antibody against Smad4 was used at a dilution of 1:250. Sections of 4 μm were deparaffinized and boiled in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH9.0) for 15 minutes. They were incubated with Ultra V Block for 5 min to block nonspecific binding. The sections were then incubated overnight with anti-Smad4 antibody at 4°C. After washing in TBS 0.025% Triton X-100, the slides were incubated in Hydrogen Peroxide Block for 10 minutes. After washing in TBS buffer, they were incubated with UltraVison One HRP Polymer (second antibodies) for 30 minutes, followed by staining with DAB Plus Chromogen and Substrate. Counterstaining was performed with hematoxylin.
Sequential DNA Precipitation Assay
To study the ERα-associated ERE complex, COS-1 cells were co-transfected with HA-tagged ERα, FLAG-tagged Smad3 and FLAG-tagged Smad4. The ERα-containing protein complexes were first immunoprecipitated with an anti-HA antibody and eluted with HA-tagged peptide (I2149, Sigma). The eluates were then subjected to DNA precipitation assays as described by Chen et al  and Bonni et al . The sequences of biotin-labeled wild-type and mutant ERE oligonucleotides are listed as follows: Biotin-ERE (forward): 5'-GATCTCGAGTCAGGTCACAGTGACCTGA-3'; Biotin-ERE (reverse): 5'-TCAGGTCACTGTGACCTGACTCGAGATC-3'; Biotin-mutantERE (forward): 5'-GATCTCGAGTCACCGCACAGTGAAATGA-3'; Biotin-mutantERE (reverse): 5'-TCATTTCACTGTGCGGTGACTCGAGATC-3'. Finally, the precipitated protein complexes which contain ERE-bound ERα were examined by immunoblotting assays with an anti-FLAG antibody.
Small Interfering RNA (siRNA)
siRNA targeting Smad4 was prepared by cloning between the ApaI and EcoRI site of the Multi Cloning Sites of the pBS/U6 vector. The specific sequence for Smad4 was determined with assistance from siRNA Target Finder (Ambion®, Inc.). DNA oligonucleotides with the following sequences, along with their complementary strands, were synthesized by Operon (QIAGEN): siRNA for Smad4:
Forward: 5'-CATTGGATGGGAGGCTTCATTCAAGCTTTGAAGCCTCCCATCCAA TGTTTTTT-3'; Reverse: 5'-AATTAAAAAACATTGGATGGGAGGCTTCAAAGCTT GAATGAAGCCTCCCATCCAATGGGCC-3'. Similar oligonucleotides with the following scrambled sequences were designed as negative controls:
Forward: 5'-TAGTCTAGGAGGTCGAGTCTTCAAGCTTGACTGACCTCCTAGACT ATTTTTT-3'; Reverse: 5'-AATTAAAAAATAGTCTAGGAGGTCGAGTCAAGCTTG AAGACTCGACCTCCTAGACTAGGCC-3'. Each pair of DNA oligos was annealed and cloned into the pBS/U6 vector between ApaI and EcoRI site. MCF-7 cells were transfected with siRNA using LipofectAMINE plus reagent. Sixty hours after transfection, cells were treated with or without E2 and/or TGF-β for 8 hours. Total RNA and cell lysates were prepared and subjected to RT-PCR or western blotting analysis respectively.
The Smad3/4 Complex Confers TGF-β Repression of ERα-Mediated Gene Transcription
ERα regulates gene transcription either by binding directly to the promoter of target genes (such as PS2) or by binding indirectly through a mechanism involving other transcription factors such as Sp1 and AP1 (such as C-MYC). To determine whether Smad4 and R-Smads also regulate ERα downstream genes, we measured the endogenous mRNA levels of PS2 and C-MYC, using endogenous β-actin mRNA as a control. The MCF-7 hormone-dependent breast cancer cell line was transiently transfected with Smad2/4 or Smad3/4 and treated with TGF-β1 for 8 hours. Total mRNA was extracted followed by reverse transcription and semi-quantitative real time PCR. As shown in Figures 1c and 1d, TGF-β downregulated the expression levels of PS2 and C-MYC mRNA stimulated by E2. Transient expression of Smad3/4 repressed the transcription of these two genes, while overexpression of Smad2/4 did not significantly alter the transcriptional activity. These results clearly indicate that, in breast cancer cell lines, Smad4 can either function as an ERα transcriptional co-repressor on its own or mediate TGF-β suppression by forming a complex with Smad3.
Smad4 Is Essential For TGF-β-Mediated Repression of ERα Transactivation
Abnormal Expression of Smad4 is Detected Some ERα-positive Human Infiltrating Breast Carcinoma
Previous investigations have demonstrated that ER status is a very important factor in the management of breast cancer, and that suppression of ER mitogenic activity is a viable strategy for treatment and prevention of breast cancer. TGF-β is a natural negative growth regulator of epithelial cells during both development and tumorigenesis of the mammary gland. Most breast carcinomas are refractory to the suppressive effect of TGF-β with elevated TGF-β expression, suggesting an important role of TGF-β in breast cancer tumorigenesis.
In previous studies, we have demonstrated that Smad4 can act as a transcriptional corepressor for ERα . Smad3, on the other hand, has been shown to act as a coactivator for ERα . Since TGF-β activation leads to the formation of an active Smad3/4 complex which directly binds to the promoter of many TGF-β responsive genes, it is conceivable that the Smad3/4 complex, rather than Smad3 or Smad4 alone, plays a major role in the crosstalk between TGF-β and estrogen. Our data clearly indicate that the Smad3/4 complex plays the same role as Smad4 alone, i.e., inhibiting ERα-mediated gene transcription. This observation is not surprising since TGF-β treatment alone suppresses estrogen-induced ER target gene expression. However, it adds to the complexity of the question why Smad3 and Smad4 act in opposite ways in the regulation of estrogen signaling, while they have the same effect on TGF-β signaling. One possible explanation is that, in the context of ERα-mediated transcription, Smad3 and Smad4 per se may recruit functionally different cofactors such as histone acetylases and histone deacetylases. The protein complex(es) recruited by Smad3 or Smad4 might determine the role of Smad3 or Smad4 as a transcriptional cofactor in estrogen signaling. In breast epithelial cells that have intact TGF-β/Smad and estrogen signaling networks, the "net effect" of TGF-β may be determined by the protein complex that incorporated both Smad3 and Smad4. In breast carcinoma cells that lack one component of the Smad3/4 complex, Smad3 or Smad4 may act as a coactivator or corepressor for ERα that confers a TGF-β activating or suppressing signal on the estrogen signaling pathway. The high frequency of Smad4 mutations in human tumors [32–35] suggests a role for Smad4 as a tumor suppressor independent of TGF-β signaling. Smad4 mutations in breast carcinoma have also been reported [36, 37]. In addition, the work on Smad4 conditional knockout mice also indicates that Smad4 is required for the suppression function of TGF-β in the proliferation of mammary epithelial cells .
We have shown that, in the presence of Smad4, TGF-β inhibits ERα-mediated transcriptional activity. A decrease in Smad4 expression partially or completely abrogates TGF-β suppression of ERα-target genes, and TGF-β can further activate ER-target genes since Smad3 acts as a transcriptional coactivator for ERα (Figure 1a). It seems that TGF-β can regulate ERα estrogenic transcriptional activity in both a negative and positive manner, and apparently, that the absence of Smad4 can switch TGF-β from a suppressor to an activator of ER signaling. TGF-β dual regulation of ERα transcriptional activity may also explain the biphasic effect in tumors: suppression of tumor growth at the early stages and promotion of tumor spreading during the later stages. Further investigations are needed to identify the target genes that are specifically regulated by R-Smads and/or Smad4, especially in the context of tumor cells. Hopefully, the results from future studies will lead to a better understanding of the dual role of TGF-β in cancer development.
Loss of TGF-β inhibition can be due to mutations or deletions of TGF-β signaling components. Indeed, mutations of TGF-β receptor or Smads-encoding genes have been reported in different forms of cancers, such as colon cancer and pancreatic cancer. Abnormal expression level and cellular distribution of biologically active proteins have been implicated in the tumorigenesis. Aberrant cytoplasmic localization of nucleophosmin in primary acute myeloid leukemia has been implicated in disrupting ARF-MDM2-p53 signal pathway and contributed to leukemogenesis [39, 40]. Significant reduced expression of Smad4 protein was found in some human carcinoma including breast cancer [41–44]. Our data demonstrate that, in TGF-β-treated MDA-MB-231 cells, Smad4 failed to translocate into the nucleus with Smad2/3, therefore losing the function of Smad4 as a repressor in the nucleus and probably contributing to decreased growth inhibition of TGF-β in this cell line. In addition, the expression of Smad4 are decreased and largely restricted in the cytoplasm of some of ERα-positive infiltrating human breast cancer cells in contrast to the benign breast tissue in which Smad4 was strongly expressed in both cytoplasm and nucleus of ductal epithelial cells. Moreover, the marked difference of Smad4 expression is noted between carcinoma cells and surrounding residual normal ductal tissue from same specimen (Fig. 4e-g). These data indicate that the dysregulation of Smad4 protein expression may play a role in the development and progression of ERα-positive breast carcinoma. The fact that aberrant expression of Smad4 is only seen some of ERα-positive infiltrating breast cancer is consistent with the heterogeneity of human breast carcinoma in biological features, development and progression, and therapy response. It is likely that in an ERα-positive breast carcinoma with aberrant expression of Smad4, TGF-β not only is unable to inhibit tumor growth, but also promote tumor progression through enhancing the estrogen-ERα mediated cell proliferation. The mechanism for dysregulation of Smad4 expression in ERα-positive infiltrating breast cancer is still unknown. It could be due to mutations or deletion of Smad4, or aberrant expression of certain oncoproteins. Our findings may provide a basic mechanism to explain the lack of TGF-β responsiveness and anti-estrogen-dependent growth inhibition in some of ERα-positive infiltrating breast carcinoma, support an idea of both tumor suppressing and enhancing effects of TGF-β in the development and/or progression of breast cancer, and open a path to further investigate the cause of aberrant expression of Smad4 in ERα-positive infiltrating breast carcinoma.
We have shown that TGF-β can regulate ERα estrogenic transcriptional activity in both negative and positive manners. Smad4 is essential for TGF-β-mediated inhibition of ERα estrogenic transcription activity, and the inhibition of Smad4 expression switches TGF-β from a repressor to an activator for ERα transactivation in breast cancer cells. We have demonstrated that down-regulation and relatively increased cytoplasmic localization of Smad4 were associated with some ERα-positive infiltrating human breast carcinoma. These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity be one of mechanisms for loss of TGF-β negative regulation on ERα transcriptional activity in breast cancer.
This work was supported by National Aeronautics and Space Administration/University of Alabama in Huntsville Grant NCC8-132 and National Institutes of Health Grant DK60913.
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