MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

George C Bobustuc,Sreeram Maddipatla,Santhi D Konduri,Maria-Lourdes M Caparas,Sheila Jeudy,Susan M Constantino,Nikita Shah,Kalkunte S Srivenugopal,Joshua S Smith,Beth Isley,Cheryl H Baker,Arati Limaye,Said Baidas

doi:10.2119/molmed.2012.00010

Abstract

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.

Highlights

Targeting the estrogen signaling pathway dramatically improves the longterm disease-free and overall survival in women with estrogen receptor-positive breast cancer
Ethics Statement Human tissue samples for methylguanine DNA methyl transferase (MGMT) immunoassays were used with the approval of MD Anderson Cancer Center Orlando Oncology Institutional Review Board (IRB) after each patient was consented under IRB protocol no 10.003.01, titled “O6 Methylguanine DNA Methyl Transferase (MGMT) and Tamoxifen Resistance in Patients with Breast Cancer: Study of correlation between clinical resistance and tissue expression of MGMT and serum markers.”
In another set of experiments, tamoxifen-resistant MCF7 breast cancer cells were treated with BG for 48 h before TAM (1 μmol/L) or ICI (1 μmol/L) were added for 24 h prior to ATP assay. In another set of experiments, tamoxifenresistant MCF7 breast cancer cells were transfected with NT small interfering RNA (siRNA) and MGMT siRNA (100 nmol/L) and 72 h after transfection, ATP assay was performed. In another set of experiments we investigated the effect of MGMT siRNA in the presence or absence of BG (50 or 140 μmol/L) on tamoxifen-resistant MCF7 breast cancer cells

Summary

Introduction

Targeting the estrogen signaling pathway dramatically improves the longterm disease-free and overall survival in women with estrogen receptor-positive breast cancer. 70% of human breast tumors express significant levels of estrogen receptor alpha (ERα) [1], making antiestrogen therapy an important therapeutic modality in breast cancer treatment [2,3,4]. Ous correlations between ERα phosphorylation patterns and sensitivity to antiestrogen therapy and clinical indicators of treatment response (delayed disease progression/improved survival) [6,7,8,9,10]. There are different degrees of Ser118 phosphorylation which correlate with different ERα functional states. Functional ERα correlates with low Ser118 phosphorylation similar to Ser118 phosphorylation pattern encountered with estradiol binding which is mitogenactivated protein kinase (MAPK) independent [11]. Endocrine therapy resistant ERα is associated with high Ser 118 phosphorylation which is estradiol independent and has been linked to MAPK

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