Med1 plays a critical role in the development of tamoxifen resistance

Abstract

Understanding the molecular pathways that contribute to the development of tamoxifen resistance is a critical research priority as acquired tamoxifen resistance is the principal cause of poor prognosis and death of patients with originally good prognosis hormone-responsive breast tumors. In this report, we provide evidence that Med1, an important subunit of mediator coactivator complex, is spontaneously upregulated during acquired tamoxifen-resistance development potentiating agonist activities of tamoxifen. Phosphorylated Med1 and estrogen receptor (ER) are abundant in tamoxifen-resistant breast cancer cells due to persistent activation of extracellular signal-regulated kinases. Mechanistically, phosphorylated Med1 exhibits nuclear accumulation, increased interaction with ER and higher tamoxifen-induced recruitment to ER-responsive promoters, which is abrogated by inhibition of Med1 phosphorylation. Stable knockdown of Med1 in tamoxifen-resistant cells not only reverses tamoxifen resistance in vitro but also in vivo. Finally, higher expression levels of Med1 in the tumor significantly correlated with tamoxifen resistance in ER-positive breast cancer patients on adjuvant tamoxifen monotherapy. In silico analysis of breast cancer, utilizing published profiling studies showed that Med1 is overexpressed in aggressive subsets. These findings provide what we believe is the first evidence for a critical role for Med1 in tamoxifen resistance and identify this coactivator protein as an essential effector of the tamoxifen-induced breast cancer growth.

Fig. 1.

Tamoxifen promotes tumor growth, clonogenicity and augments ER-responsive gene expression and ER-mediated transactivation in tamoxifen-resistant cells. (A) Tumor growth curves of MCF7-P and MCF7-OHT cells implanted subcutaneous in athymic mice in the presence of estrogen and tamoxifen treatment. (B) Anchorage-dependent growth of MCF7-P and MCF7-OHT cells in the presence of various doses of 17β-estradiol (E2) or 4-OHT. Colonies containing >50 normal-appearing cells were counted. *, P < 0.001, compared with untreated controls (MCF7-P and MCF7-OHT); #, P < 0.001, compared with untreated controls (MCF-P and MCF7-OHT). (C) Immunoblot analysis of ER protein in MCF7-P and MCF7-OHT cells. (D) Semi-quantitative reverse transcription–PCR analysis of messenger RNA (mRNA) expression levels of cathepsin-D and c-Myc mRNA in MCF7-P and MCF7-OHT cells treated with 1 μM of 4-OHT for 2 h. (E) Real-time quantitative PCR analysis of EBAG9, pS2 and IGF1 expression in MCF7-P and MCF7-OHT cells treated with 1 μM of 4-OHT for 2 h. *, P < 0.005, compared with untreated controls (MCF7-P); #, P < 0.001, compared with untreated controls (MCF7-OHT). (F) Luciferase activity of pERE-Luc in MCF7-P and MCF7-OHT cells treated with either ethanol vehicle alone or 100 nM E₂ or 1 μM OHT for 24 h. *, P < 0.001, compared with untreated controls (MCF7-P and MCF7-OHT); **, P < 0.02, compared with untreated controls (MCF7-P); #, P < 0.001, compared with untreated controls (MCF7-OHT).

Fig. 2.

Increased expression and phosphorylation of Med1 in tamoxifen-resistant cells due to persistent activation of ERK. (A) Immunoblot analysis of Med1, ERK, phospho-Med1 and phospho-ERK in MCF7-P and MCF7-OHT. (B) Immunoblot analysis of pMed1, pERK and ERK in MCF7-P and MCF7-OHT cells treated with EGF and U1026. (C) Immunoblot analysis of ER, p-ER (S-167) and p-ER (S-118) in MCF7-P and MCF7-OHT cells. (D) Immunoblot analysis of ER, p-ER (S-167) and p-ER (S-118) in MCF7-P and MCF7-OHT cells treated with EGF and of U1026.

Fig. 3.

Med1 associates with ER and tamoxifen increases recruitment of Med1 to ER-responsive gene promoters. (A) Immunoprecipitation of Med1 protein from MCF7-P and MCF7-OHT cells followed by immunoblot analysis of ER. (B) ChIP was performed using antibodies specific for ER and Med1 from MCF7-P and MCF7-OHT cells untreated and treated with 4-OHT (1 μM for 2 h). The purified DNA was analyzed by PCR using specific primers spanning the EREs of cathepsin D and c-myc gene promoters. (C) ChIP was performed as in (B). The purified DNA was analyzed by real-time quantitative PCR analysis using specific primers spanning the EREs of EBAG9, pS2 and IGF1 gene promoters. **, P < 0.001, compared with MCF-P treated with OHT.

Fig. 4.

Med1 phosphorylation is important for its nuclear accumulation and tamoxifen-induced recruitment to ER-responsive gene promoters. (A) Immunoblot analysis of p-Med1 and Med1 in cytoplasmic and nuclear fractions of MCF7-OHT cells treated with EGF or U0126. (B) Immunofluorescence analysis of p-Med1 in MCF7-OHT cells treated as in A. (C) ChIP analysis of ER and Med1 in MCF7-OHT cells treated with 4-OHT alone or in combination with U0126. The purified DNA was analyzed by PCR using specific primers spanning the EREs of cathepsin D, c-myc and pS2 gene promoters. (D) ChIP was performed as in (C). The purified DNA was analyzed by real-time quantitative PCR analysis using specific primers spanning the EREs of EBAG9, pS2 and IGF1 gene promoters. *, P < 0.001, compared with cells treated with OHT alone (ER ChIP). **, P < 0.001, compared with cells treated with OHT alone (Med1 ChIP).

Fig. 5.

Stable knockdown of Med1 reverses tamoxifen resistance in tamoxifen-resistant cells. (A) Immunoblot analysis of Med1 in stable pools of Med1-depleted (Med1^shRNA) and vector control (pLKO.1) MCF7-P and MCF7-OHT cells. (B) Clonogenicity of MCF7-P-pLKO.1, MCF7-P-pMed1^shRNA, MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA cells in the presence of 4-OHT (1 μM). *, P < 0.005, compared with untreated controls (MCF7-P-pLKO.1); **, P < 0.001, compared with untreated controls (MCF7-P-pMed1^shRNA); #, P < 0.001, compared with untreated MCF7-OHT-pLKO.1; #*, P < 0.001, compared with untreated MCF7-OHT-pMed1^shRNA. (C) Soft agar colony formation in MCF7-P-pLKO.1, MCF7-P-pMed1^shRNA, MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA cells in the presence of 1 μM 4- OHT. *, P < 0.005, compared with untreated controls (MCF7-OHT-pLKO.1); #, P < 0.001, compared with tamoxifen-treated MCF7-OHT-pLKO.1. (D) Real-time quantitative PCR analysis of EBAG9, pS2 and IGF1 expression in MCF7-P-pLKO.1, MCF7-P-pMed1^shRNA, MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA cells treated with 1 μM of 4-OHT for 2 h. *, P < 0.005, compared with untreated controls (MCF7-P-pLKO.1); #, P < 0.001, compared with untreated controls (MCF7-P-pMed1^shRNA); **, P < 0.001, compared with untreated controls (MCF7-OHT-pLKO.1); ***, P < 0.005, compared with untreated controls (MCF7-OHT-pMed1^shRNA).

Fig. 6.

Stable knockdown of Med1 reverses tamoxifen resistance in LCC2 and MCF7-5/23, tamoxifen-resistant cells. (A) Immunoblot analysis of Med1 in MCF7 and LCC2 cells. (B) Clonogenicity of MCF7-pLKO.1, MCF7-pMed1^shRNA, LCC2-pLKO.1 and LCC2-pMed1^shRNA cells in the presence of 4-OHT (1 μM). *, P < 0.005, compared with untreated controls; **, P < 0.001, compared with untreated controls; #, P < 0.001, compared with untreated LCC2-pLKO.1; #*, P < 0.001, compared with tamoxifen-treated LCC2-pLKO.1. (C) Immunoblot analysis of MCF7-5/21 and MCF7-5/23 cells. (D) Clonogenicity of MCF7-5/21-pLKO.1, MCF7-5/21-pMed1^shRNA, MCF7-5/23-pLKO.1 and MCF7-5/23- pMed1^shRNA cells in the presence of 4-OHT (1 μM). *, P < 0.005, compared with untreated controls; **, P < 0.001, compared with untreated controls; #, P < 0.001, compared with untreated MCF7-5/23-pLKO.1; #*, P < 0.001, compared with tamoxifen-treated MCF7-5/23-pLKO.1.

Fig. 7.

Stable knockdown of Med1 reverses tamoxifen resistance in tamoxifen-resistant tumors in vivo. MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA cells derived tumors were developed in nude mice and treated with vehicle and 4-OHT. (A) Tumor growth #, P < 0.001, comparing tamoxifen-treated MCF7-OHT-pLKO.1 with tamoxifen-treated MCF7-OHT-pMed1^shRNA. (B) Representative tumor images and tumor weight. (C) Immunohistochemical analysis using anti-Ki-67 antibody. Bar diagram shows quantitation of Ki-67 expression in tumors. Columns, mean (n = 6); *, P < 0.01, compared with vehicle controls; **, P < 0.001, compared with tamoxifen-treated MCF7-OHT-pLKO.1. (D) Semi-quantitative reverse transcription–PCR analysis of messenger RNA expression levels of c-Myc and cathepsin D in tamoxifen-treated and untreated MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA tumors. (E) Immunohistochemical analysis of PR expression in MCF7-OHT-pLKO.1 and MCF7-OHT-pMed1^shRNA untreated and treated with tamoxifen. Bar diagram shows quantitation of PR expression in tumors. Columns, mean (n = 6); *, P < 0.005, compared with vehicle controls; **, P < 0.001, compared with tamoxifen-treated MCF7-OHT-pLKO.1.

Fig. 8.

Increased Med1 expression associates with higher tumor grade and recurrence after tamoxifen treatment. (A) Examples of immunohistochemical Med1 staining of breast cancer. Breast cancer with a) low cytoplasmic and nuclear Med1 expression, b) high cytoplasmic but low nuclear Med1, c) high nuclear but low cytoplasmic and d) both high cytoplasmic and nuclear Med1. (B) Kaplan–Meier plots of breast cancer recurrences in relation to total Med1 status (high versus low) and treatment. Recurrence-free survival of patients was assessed among those who had been randomly assigned to tamoxifen or to no adjuvant systemic tamoxifen (control) treatment.