Dual effects of TGF-beta on ERalpha-mediated estrogenic transcriptional activity in breast cancer

Abstract

Background: TGF-beta 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-beta regulates ERalpha-induced gene transcription and potential mechanisms of frequent TGF-beta resistance in breast cancer.

Methods: Effect of TGF-beta on ERalpha-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 ERalpha-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays.

Results: A complex of Smad3/4 mediates TGF-beta inhibition of ERalpha-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-beta from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERalpha-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma.

Conclusion: These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-beta suppression of ERalpha transactivity in breast cancer cells.

Figure 1

The Smad3/4 complex confers TGF-β repression on ERα-mediated transcription. Smads (Smad2, Smad3, and Smad4 alone or combinations of Smad2/4 or Smad3/4) or empty vector was co-transfected into MCF-7 cells (a) or MDA-MB-468 cells (b). The cells were treated with (+) or without (-) E₂ and/or TGF-β as indicated for 16 hours prior to lysis, and analyzed for luciferase activity. (c-d) MCF-7 cells were transfected with Smad2/4 or Smad3/4 and treated with (+) or without (-) E2 and/or TGF-β as indicated for 8 hours. Total mRNA was isolated and the mRNA levels of PS2 and C-MYC were detected and quantified by reverse transcription and semi-quantitative real time PCR.

Figure 2

The EREaccommodates Smad3/4 and ERα complexes in the nucleus. (a) MCF-7 cells were treated with E₂, E₂ + TGF-β1 or vehicle (as a negative control) for 1 hr as indicated. Lysates were subjected to immunoprecipitation with anti-Smad4, anti-Smad2/Smad3 or anti-ERα antibody. The Smad-associated endogenous ERα was detected by immunoblot analysis. The expression levels of endogenous proteins were monitored by immunoblotting of total cell protein. (b) COS-1 cells were transfected with HA-ERα, FLAG-Smad3 and FLAG-Smad4 expression plasmids. Cells were treated with (+) or without (-) E₂ and TGF-β1 and subjected to sequential immunoprecipitation, DNA pull-down (DNAP), and immunoblotting. Wild-type (WT) and mutant (mt) biotinylated ERE oligonucleotides were used in the DNAP. The presence of HA-ERα, FLAG-Smad3 and FLAG-Smad4 in the DNAP was detected with anti-FLAG and anti-HA antibodies.

Figure 3

Smad4 is essential for the inhibitory effect of TGF-β in ERα-mediated transcriptional activation. (a) RNAi was performed in MCF-7 cells using Smad4 siRNA (see materials and methods). (b) MCF-7 cells were transfected with either siRNA targeting Smad4 or scrambled siRNA (as a control). Sixty hours after transfection, the cells were treated with (+) or without (-) E₂ or TGF-β for 6 hrs. Total RNA was isolated and the mRNA levels of PS2 and C-MYC were quantitated by RT-PCR.

Figure 4

Expression of Smad4 in breast cancer cell lines and ERα-positive infiltrating beast carcinoma. (a and b) MCF-7 and MDA-MB-231 cells were treated with TGF-β for 1 hr and then subjected to immunofluorescence analysis with anti-Smad2/3 and anti-Smad4 antibodies to localize endogenous Smad2/3 and Smad4. Nuclei were visualized by DNA staining with DAPI. (c-g) Immunohistochemical staining of Smad4 in human breast tissue: (c) Benign breast ductal epithelial (1000×). (d) ERα-positive infiltrating breast carcinoma (1000×). (e) Residual benign ductal epithelial (arrow) and surrounding ERα-positive infiltrating breast carcinoma (200×). (f) Residual benign breast ductal epithelial in panel e (1000×). (g) Surrounding infiltrating breast carcinoma in panel e (1000×).