Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression

Abstract

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Figure 1

FXR expression and activation in MCF-7 and MCF-7TR1 cells. (a) Western blot analysis of HER2, EGFR, ERα in total protein extracts from MCF-7 and MCF-7TR1 cells; β-Actin was used as loading control. (b) Soft Agar growth assay in MCF-7 and MCF-7TR1 cells plated in 0.35% agarose and treated with EGF 100ng/ml in the presence or absence of Herceptin (10µg/ml). After 14 days of growth colonies > 50 µm diameter were counted. n.s. (nonsignificant); *p<0.05 compared to vehicle or EGF. (c) Total RNA was extracted from MCF-7 and MCF-7TR1 cells, reverse transcribed and cDNA was subjected to PCR using primers specific for FXR or 36B4 (upper panel); NC: negative control, RNA sample without the addition of reverse transcriptase. Nuclear proteins were extracted from MCF-7 and MCF-7TR1 and then western blotting analysis was performed using anti-FXR antibody. Lamin B was used as loading control (lower panel). (d) MCF-7 and MCF-7TR1 cells were transiently transfected with a FXR responsive reporter gene (FXRE-IR1) with either empty vector (e.v.) or FXR-DN (dominant negative) expression plasmid. After transfection cells were treated for 24h with vehicle (−) or increasing doses of CDCA (25–50–100µM) and then luciferase activity was measured. Results represent the mean ± SD of three different experiments each performed in triplicate. *p<0.05 compared to vehicle. Numbers on top of the blots represent the average fold change versus control of MCF-7 cells normalized for β-Actin.

Figure 2

FXR ligands effects on breast cancer cells proliferation. MTT growth assays in MCF-10A, MCF-7 and MCF-7TR1 cells treated with vehicle (−) or increasing doses of CDCA (12,5–25–50–100 µM) (a) or GW4064 (0,3-3–6,5–10 µM) (b) for 7 days. Cell proliferation is expressed as fold change ± SD relative to vehicle treated cells, and is representative of three different experiments each performed in triplicate. (c) MCF-7 and MCF-7TR1 cells, transiently transfected with either empty vector (e.v.) or FXR-DN vector plasmids, were treated with vehicle (−) or CDCA 50µM for 4 days before testing cell viability using MTT assay. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. (d) MTT growth assay in MCF-7 and MCF-7TR1 cells treated with vehicle (−) or CDCA 50µM in the presence or not of Tam 1µM for 4 days. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. (e) Soft Agar growth assay in MCF-7 and MCF-7TR1 cells plated in 0.35% agarose and treated as above indicated. After 14 days of growth colonies > 50 µm diameter were counted. n.s. (nonsignificant); *p<0.05 compared to vehicle or Tam.

Figure 3

Effects of CDCA on HER2 expression and its transduction pathways in MCF-7 and MCF-7TR1 cells. (a) MCF-7 and MCF-7TR1 cells were treated for 24h with vehicle (−) or CDCA 25 and 50µM before lysis. Equal amounts of total cellular extract were analyzed for HER2 levels by Western blotting. β-Actin was used as loading control. (b) Cells were transiently transfected with either empty vector (e.v.) or FXR-DN plasmids and then treated with vehicle (−) or CDCA 50µM for 24h and HER2 levels were evaluated by Western blotting. β-Actin was used as loading control. (c) Immunoblot analysis showing phosphorylated HER2 (pHER2 Tyr¹²⁴⁸) and MAPK (pMAPK Thr²⁰²/Tyr²⁰⁴), total HER2, total MAPK in MCF-7 and MCF-7TR1 cells pretreated for 24h with CDCA 50µM and then treated for 10 min with EGF 100ng/ml. β-Actin was used as loading control. (d) MTT growth assay (upper panel) and soft agar assay (lower panel) in cells treated with CDCA 50µM with or without EGF 100ng/ml for 4 days and 14 days respectively. The MTT assay results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. The soft agar assay values are represented as a mean of colonies number >50 µm diameter counted at the end of assay. Percentages of inhibition induced by CDCA versus EGF treatment alone are showed. (e) Cells were treated for 24h with vehicle (−) or EGF 100ng/ml in the presence or not of CDCA 50µM before lysis and then cellular extracts were analyzed for cyclin D1 levels by Western Blot analysis. β-Actin was used as loading control. Numbers on top of the blots represent the average fold change versus control of MCF-7 cells normalized for β-Actin.

Figure 4

Effects of CDCA on human HER2 promoter activity. (a) mRNA HER2 content, evaluated by real time RT-PCR, after treatment with vehicle or CDCA 50µM as indicated. Each sample was normalized to its GAPDH mRNA content. *p<0.05 and ** p<0.001 compared to vehicle. (b) Schematic map of the human HER2/neu promoter region constructs used in this study. All of the promoter constructs contain the same 3’ boundary. The 5’ boundaries of the promoter fragments varied from −500 (pNeuLite) to −232 (−232 pNeuLite). A mutated NF-κB binding site is present in NF-κB mut construct. HER2 transcriptional activity in MCF-7 (c) and MCF-7TR1 (d) cells transfected with promoter constructs are shown. After transfection, cells were treated in the presence of vehicle (−) or CDCA 50µM for 6h. The values represent the means ± SD of three different experiments each performed in triplicate. *p<0.05 compared to vehicle.

Figure 5

Electrophoretic mobility shift assay of the NF-κB binding site in the HER2 promoter region. (a) Nuclear extracts from MCF-7 and MCF-7TR1 cells were incubated with a double-stranded NF-κB specific sequence probe labeled with [γ³²P]ATP and subjected to electrophoresis in a 6% polyacrylamide gel (lanes 1 and 5). Competition experiments were performed adding as competitor a 100-fold molar excess of unlabeled probe (lanes 2 and 6) or a 100-fold molar excess of unlabeled oligonucleotide containing a mutated NF-κB RE (lanes 3 and 7). Lanes 4 and 8, nuclear extracts from CDCA (50µM) treated MCF-7 and MCF-7TR1 cells respectively incubated with probe. Lane 9, NF-κB protein. Lane 10, probe alone. (b) Nuclear extracts from MCF-7 and MCF-7TR1 cells were incubated with a double-stranded NF-κB specific sequence probe labeled with [γ³²P]ATP (lanes 1 and 7) or with a 100-fold molar excess of unlabeled probe (lanes 2 and 8). Nuclear extracts incubated with anti-NF-κB (lanes 3 and 9) or IgG (lanes 4 and 10). Lanes 5 and 11, NF-κB protein. Lanes 6 and 12, probe alone. (c) Lane 1, NF-κB protein. Lanes 2, 3 and 4, NF-κB protein incubated with increasing doses (1, 3 and 5 µl) of transcribed and translated in vitro FXR protein. Lane 5, probe alone.

Figure 6

FXR inhibits NF-κB recruitment to HER2 promoter. (a) MCF-7 and MCF-7 TR1 cells were treated with vehicle (−) or CDCA 50µM for 1h before lysis. FXR protein was immunoprecipitated using an anti-FXR polyclonal antibody (IP:FXR) and resolved in SDS-PAGE. Immunoblotting was performed using an anti-NF-κB (p65 subunit) monoclonal antibody and anti-FXR antibody. MCF-7 and MCF-7TR1 cells were treated in the presence of vehicle (−) or CDCA 50 µM for 1h, then cross-linked with formaldehyde, and lysed. The precleared chromatin was immunoprecipitated with anti-NF-κB (b), anti-RNA Pol II (c) and anti-HDCA3 (d) antibodies. 5µl volume of each sample and input were analyzed by real time PCR using specific primers to amplify HER2 promoter sequence including the NF-κB site. Similar results were obtained in multiple independent experiments. * p<0.01 compared to vehicle.

Figure 7

Effects of FXR ligand on SKBR3 breast cancer cells. (a) MTT proliferation assay of SKBR3 cells treated with vehicle (−) or increasing doses of CDCA (12,5–25–50–100 µM) for 7 days. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. (b) Soft Agar growth assay in SKBR3 cells plated in 0.35% agarose and treated with vehicle (−) or CDCA 50 µM. After 14 days of growth colonies > 50 µm diameter were counted. (c) SKBR3 cells were treated as indicated with vehicle (−) or CDCA 50µM before lysis. Equal amounts of total cellular extract were analyzed for HER2 levels by Western blotting. β-Actin was used as loading control. Numbers on top of the blots represent the average fold change relative to control normalized for β-Actin. (d) mRNA HER2 content, evaluated by real time RT-PCR, after treatment with vehicle (−) or CDCA 50µM as indicated. Each sample was normalized to its GAPDH mRNA content. (e) SKBR3 cells were transiently transfected with pNeuLite construct. After transfection cells were treated in the presence of vehicle (−) or CDCA 50 µM for 24h and the promoter activity was evaluated. The values represent the means ± SD of three different experiments each performed in triplicate. * p<0.05 compared to vehicle. (f) MTT growth assay in MCF-7TR1 and MCF-7/HER2-18 cells treated with vehicle (−), CDCA 50µM and GW4064 3µM in the presence or not of Tam 1µM for 4 days. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. * p<0.05 compared to Tam.

Figure 8

Proposed working model of the FXR-mediated regulation of HER2 expression in Tam-resistant breast cancer cells. In the absence of CDCA, HER2 expression is regulated by several serum factors, including NF-κB, acting through a regulatory region in HER2 promoter and enabling gene transcription. Upon CDCA treatment, FXR binds NF-κB inhibiting its recruitment on the response element located in the proximal HER2 promoter, causing displacement of RNA polymerase II with consequent repression of HER2 expression.