Abstract 2739: Specific interaction of human MGMT with ER-α in breast cancer cells: Co-degradation of MGMT and ER- α proteins by either fulvestrant or O6-benzylguanine and its therapeutic significance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MGMT (O6-methylguanine-DNA methyltransferase) is an antimutagenic DNA repair protein and an established target for improving chemotherapy with alkylating agents. MGMT is highly expressed in breast cancers. This study investigated whether i) MGMT is modulated by endocrine therapies, ii) MGMT interacts with ER-signaling components and iii) if such findings are exploitable for improved breast cancer treatment. The ER-α positive cell lines MCF-7, T47D and ER- α negative MDAMB 468 cells, all MGMT-proficient, were treated with Fulvestrant (Faslodex, ICI 182, 780) or O6-benzylguanine (BG) in various experiments. Fulvestrant is a pure antiestrogen that binds to ER without eliciting any transcriptional effects, but leads to the receptor degradation through the ubiquitin-proteasome (ub-P) pathway. BG is a specific pseudosubstrate for MGMT currently in clinical trials that inactivate the repair protein resulting in its degradation, again by the ub-P pathway. Fulvestrant (0.1-1 µM) induced a dose- and time-dependent inhibition of MGMT's DNA repair activity with a 75% inhibition after 1 µM treatment at 72 h. Interestingly, western blot analyses showed a progressive loss of both ER-α and MGMT proteins in Fulvestrant -treated cells. The MGMT in ER- α negative MDMAB cells was not affected by the drug. Similarly, BG (20-50 µM) induced a time-dependent significant destruction of the ER- α and MGMT proteins in cells. Immunoprecipitation using antibodies to either MGMT or ER- α followed by western blot analyses showed that the two proteins exist physically associated in breast cancer cells. siRNA and shRNAs specific for MGMT downregulated both the ER- α and MGMT proteins in MCF-7 cells confirming the mutual dependence and maintenance of these two proteins. RT-PCR showed the downregulation of several genes transactivated by ER- α in BG-treated cells. Furthermore, bortezomib, a proteasomal inhibitor stabilized both ER- α and MGMT proteins in cells after Fulvestrant exposure. Temozolomide or BCNU caused a 2-4 fold increased cell killing when combined with Fulvestrant. We postulate that portions of ER- α and MGMT proteins exist as a complex and inhibition of either of them results in co- ubiquitination by the same E3 ligase and marked for elimination. The MGMT-deficiency caused by Fulvestrant warrants the use of alkylating agents with endocrine therapeutics [Supported by grants from CPRIT (RP 130266), NIH RO3 CA125872 and the Association for Research of Childhood Cancer]. Citation Format: Kalkunte S. Srivenugopal, Ameya Paranjpe. Specific interaction of human MGMT with ER-α in breast cancer cells: Co-degradation of MGMT and ER- α proteins by either fulvestrant or O6-benzylguanine and its therapeutic significance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2739. doi:10.1158/1538-7445.AM2014-2739