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| Ameya Paranjpe,Nathan I. Bailey,Santhi Konduri,George C. Bobustuc,Francis Ali-Osman,Mohd. A. Yusuf,Surendra R. Punganuru,Hanumantha Rao Madala,Debasish Basak,AGM Mostofa,Kalkunte S. Srivenugopal.Journal of Biomedical Research,2016,30(5):/span> |
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| New insights into estrogenic regulation of O6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O6-benzylguanine indicates fresh avenues for therapy |
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Received:April 02, 2016Revised:April 10, 2016
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DOI：10.7555/JBR.30.20160040
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Keywords：estrogen signaling, MGMT DNA repair, ubiquitin-proteasome pathway, breast cancer, anti-estrogens
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Grant Program：
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| Author |
Institution |
| Ameya Paranjpe |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Nathan I. Bailey |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Santhi Konduri |
Neuro-Oncology Section, Aurora Advanced Cancer Care, Milwaukee, WI 53215, USA |
| George C. Bobustuc |
Neuro-Oncology Section, Aurora Advanced Cancer Care, Milwaukee, WI 53215, USA |
| Francis Ali-Osman |
Department of Surgery, The Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC 27710, USA |
| Mohd. A. Yusuf |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Surendra R. Punganuru |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Hanumantha Rao Madala |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Debasish Basak |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| AGM Mostofa |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA |
| Kalkunte S. Srivenugopal |
Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA
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| Abstract ： |
| Endocrine therapy using estrogen receptor-α (ER-α) antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O6-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines (MCF-7, T47D) and ER- negative cell lines (MDAMB-468, MDAMB-231), and established inhibitors of ER-α and MGMT, namely, ICI-182,780 (Faslodex) and O6-benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements (EREs) and two antioxidant-responsive elements (AREs). MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 (nuclear factor-erythroid 2-related factor-2) increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this drug-resistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O6-benzylguanine also induced a specific loss of ER-α and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-α and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-α proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ERCnegative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.
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