title: New insights into estrogenic regulation ofO6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant orO6-benzylguanine indicates fresh avenues for therapy.
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 withO6-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 toO6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins andO6-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 ofMGMTgene 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.
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Ameya
authors.initials: A
authors.lastname: Paranjpe
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Nathan I
authors.initials: NI
authors.lastname: Bailey
authors.affiliation: Neuro-Oncology Section, Aurora Advanced Cancer Care, Milwaukee, WI 53215, USA.
authors.forename: Santhi
authors.initials: S
authors.lastname: Konduri
authors.affiliation: Neuro-Oncology Section, Aurora Advanced Cancer Care, Milwaukee, WI 53215, USA.
authors.forename: George C
authors.initials: GC
authors.lastname: Bobustuc
authors.affiliation: Department of Surgery, The Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC 27710, USA.
authors.forename: Francis
authors.initials: F
authors.lastname: Ali-Osman
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Mohd A
authors.initials: MA
authors.lastname: Yusuf
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Surendra R
authors.initials: SR
authors.lastname: Punganuru
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Hanumantha Rao
authors.initials: HR
authors.lastname: Madala
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Debasish
authors.initials: D
authors.lastname: Basak
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Agm
authors.initials: A
authors.lastname: Mostofa
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA; Kalkunte.Srivenugopal@ttuhsc.edu.
authors.forename: Kalkunte S
authors.initials: KS
authors.lastname: Srivenugopal
keywords: MGMT DNA repair; anti-estrogens; breast cancer; estrogen signaling; ubiquitin-proteasome pathway