Increased proteasome-dependent degradation of estrogen receptor-alpha by TGF-beta1 in breast cancer cell lines

Abstract

Normal mammary epithelial cells are rapidly induced to G(1) arrest by the widely expressed cytokine, transforming growth factor beta (TGF-beta1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERalpha) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-beta1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-beta1 as determined by flow cytometry analysis and 5'-bromo-3'-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-beta1 after 6 h. This decrease in ERalpha reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERalpha transcripts suggested that this decrease was primarily the result of altered ERalpha protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERalpha by TGF-beta1. Collectively, this data supports a role for TGF-beta1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERalpha stability.

Fig. 1

Inducible cell growth by E2 in breast cancer cell lines. Breast cancer cell lines were cultured in the presence or absence of 10 nM E₂ for 24 h on defined media. At 18 h, BrdU was added to the media and at 24 h absorbencies were taken to assess DNA synthesis. All tested cell lines exhibited increased DNA synthesis in the presence of E₂; however, those effects were most dramatic in cell line BT474. This data is the average of six independent treatments (*P < 0.05).

Fig. 2

Effects of TGF-β1 on E2-induced cell proliferation. BT474 cells were cultured in the presence or absence of indicated dose for 24 h in defined media. At 18 h, BrdU was added to the conditioned media and at 24 h absorbencies were taken to assess DNA synthesis. The presence of TGF-β1 appeared to abrogate E₂-induced DNA synthesis. Addition of 10 μM tamoxifen blocked E2-induced DNA synthesis and all observed effects by TGF-β1. This data is the average of six independent treatments (*P < 0.05).

Fig. 3

TGFβ modulation of cell cycle. Standard propidium iodide uptake was used to characterize TGF-β1 induced G₁ phase. A: Control non-transformed MCF-10F cells treated with or without 100 pM TGF-β for 18 h show 50% reduction in S phase and 10% induction into G₁ phase. B: Example of a TGF-β1 sensitive, tumorigenic cell line, MDA-MB231, treated with or without 100 pM TGF-β for 18 h also resulting in a 10% induction into G₁ phase. C: BT474 breast epithelial cell line treated with either 10 nM E₂, 100 pM TGF-β, or a combination of both for 18 h. The results indicate no increase of cells in G₁ phase following TGF-β1 treatment. Furthermore, a 50% reduction in E₂-stimulated S phase was observed, consistent with BrdU studies.

Fig. 4

ERα protein levels in BT474 cells. Protein from BT474 cells treated with or without TGF-β1 in defined media for 24 h was isolated and probed for relative ERα expression. Total protein was isolated from BT474 cells treated for 24 h with experimental agent. Blots were probed for ERα using standard ECL detection. A: Up to a 50% reduction in ERα levels was observed at a concentration of 100 pM TGF-β1. B: An expected 70% reduction in ERα was observed in E₂ only treated samples. ERα levels were not further reduced in TGF-β1 + E₂ samples indicating that TGF-β1 does not act in a concerted additive fashion with E₂ (*P < 0.05).

Fig. 5

Time-course effects of TGF-β1 on ERα levels. Total protein was isolated from TGF-β1 treated and untreated BT474 cell cultures at multiple time points spanning 40 h in defined media. A 50 μg quantity of total protein was resolved by 8% SDS–PAGE and the resulting PVDF membrane probed with antibody to ERα. Untreated samples showed increase in ERα levels with time. TGF-β1 treated samples showed effects occurring between 6 and 12 h that maintain suppressed ERα levels.

Fig. 6

Regulation of ERα transcript levels by TGF-β1. A: BT474 cells were treated for 24 h with 0.01 or 0.10 nM TGF-β1. Total RNA was isolated and RT-PCR was performed as described in Materials and Methods for ERα and 36B4. Combined PCR products were resolved by 0.9% agarose in 1 × TAE. Bands were visualized following ethidium bromide staining and were quantified using ImageQuant software (*P < 0.05). B: Northern analysis was carried out using a cloned fragment of human ERα labeled with α-32P-dCTP as a probe. Low endogenous levels of ERα in the BT474 cell line exceeded the level of sensitivity of this assay; however, slight increases in ERα mRNA levels over untreated samples were apparent. The ERα signal was normalized to the internal control gene 36B4, which remained constant throughout. Qualitative binding was detected by phosphorimager analysis (n = 3 for media; n = 2 for TGF-β1).

Fig. 7

Abrogation of TGF-β1 mediated effects on ERα. Sub-confluent cultures of BT474 cells were treated with or without 100 pM TGF-β1 in the presence or absence of 300 nM MG132 for 24 h. A: A 50% suppression in ERα levels was observed in TGF-β1 treated samples compared with media alone (n = 3). B: No suppression in ERα levels by TGF-β1 was observed relative to control samples in the presence of the proteasome inhibitor MG132 (n = 3).