Cargando…

New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Paranjpe, Ameya, Bailey, Nathan I., Konduri, Santhi, Bobustuc, George C., Ali-Osman, Francis, Yusuf, Mohd. A., Punganuru, Surendra R., Madala, Hanumantha Rao, Basak, Debasish, Mostofa, AGM, Srivenugopal, Kalkunte S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Editorial Department of Journal of Biomedical Research 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044712/
https://www.ncbi.nlm.nih.gov/pubmed/27845303
http://dx.doi.org/10.7555/JBR.30.20160040
_version_ 1782456991233343488
author Paranjpe, Ameya
Bailey, Nathan I.
Konduri, Santhi
Bobustuc, George C.
Ali-Osman, Francis
Yusuf, Mohd. A.
Punganuru, Surendra R.
Madala, Hanumantha Rao
Basak, Debasish
Mostofa, AGM
Srivenugopal, Kalkunte S.
author_facet Paranjpe, Ameya
Bailey, Nathan I.
Konduri, Santhi
Bobustuc, George C.
Ali-Osman, Francis
Yusuf, Mohd. A.
Punganuru, Surendra R.
Madala, Hanumantha Rao
Basak, Debasish
Mostofa, AGM
Srivenugopal, Kalkunte S.
author_sort Paranjpe, Ameya
collection PubMed
description 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 O(6)-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 O(6)-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 O(6)-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O(6)-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.
format Online
Article
Text
id pubmed-5044712
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Editorial Department of Journal of Biomedical Research
record_format MEDLINE/PubMed
spelling pubmed-50447122016-10-13 New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy Paranjpe, Ameya Bailey, Nathan I. Konduri, Santhi Bobustuc, George C. Ali-Osman, Francis Yusuf, Mohd. A. Punganuru, Surendra R. Madala, Hanumantha Rao Basak, Debasish Mostofa, AGM Srivenugopal, Kalkunte S. J Biomed Res Original Article 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 O(6)-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 O(6)-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 O(6)-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O(6)-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. Editorial Department of Journal of Biomedical Research 2016-09 2016-06-10 /pmc/articles/PMC5044712/ /pubmed/27845303 http://dx.doi.org/10.7555/JBR.30.20160040 Text en © 2016 by the Journal of Biomedical Research. All rights reserved.
spellingShingle Original Article
Paranjpe, Ameya
Bailey, Nathan I.
Konduri, Santhi
Bobustuc, George C.
Ali-Osman, Francis
Yusuf, Mohd. A.
Punganuru, Surendra R.
Madala, Hanumantha Rao
Basak, Debasish
Mostofa, AGM
Srivenugopal, Kalkunte S.
New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title_full New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title_fullStr New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title_full_unstemmed New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title_short New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy
title_sort new insights into estrogenic regulation of o(6)-methylguanine dna-methyltransferase (mgmt) in human breast cancer cells: co-degradation of er-α and mgmt proteins by fulvestrant or o(6)-benzylguanine indicates fresh avenues for therapy
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044712/
https://www.ncbi.nlm.nih.gov/pubmed/27845303
http://dx.doi.org/10.7555/JBR.30.20160040
work_keys_str_mv AT paranjpeameya newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT baileynathani newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT kondurisanthi newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT bobustucgeorgec newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT aliosmanfrancis newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT yusufmohda newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT punganurusurendrar newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT madalahanumantharao newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT basakdebasish newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT mostofaagm newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy
AT srivenugopalkalkuntes newinsightsintoestrogenicregulationofo6methylguaninednamethyltransferasemgmtinhumanbreastcancercellscodegradationoferaandmgmtproteinsbyfulvestrantoro6benzylguanineindicatesfreshavenuesfortherapy