• ISSN 1674-8301
  • CN 32-1810/R
Volume 30 Issue 5
Sep.  2016
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Article Contents
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. 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[J]. The Journal of Biomedical Research, 2016, 30(5): 393-410. doi: 10.7555/JBR.30.20160040
Citation: 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. 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[J]. The Journal of Biomedical Research, 2016, 30(5): 393-410. doi: 10.7555/JBR.30.20160040

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

doi: 10.7555/JBR.30.20160040
Funds:

This work was supported by grants from the Cancer

  • Received: 2016-04-02
  • Revised: 2016-04-10
  • Issue Date: 2016-09-28
  • 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 ER–negative 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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