Altered Histone Modifications and Their Regulating Enzymes in Fulvestrant Resistant Breast Cancer Cell Line In Vitro
SE Elsheikh, AR Green, RA McClelland, IR Hutcheson, JM Gee, RI Nicholson, IO Ellis. School of Molecular Medical Science, Nottingham University, Nottingham, United Kingdom; Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom
Background: Clinical studies have demonstrated that Fulvestrant is a valuable treatment in postmenopausal women with advanced breast cancer who have progressed on prior endocrine therapy. However, Fulvestrant resistance commonly emerges, where in vitro data indicate this state can have increased proliferative and invasive capacity. Thus, understanding the mechanisms responsible for Fulvestrant resistance remains a key challenge if we are to improve treatment and survival for breast cancer patients. The purpose of this study was thus to begin to examine aspects of epigenetic regulation in a Fulvestrant resistant model, profiling histone acetylation/methylation and its modifying enzymes. Such studies could reveal new therapeutic avenues to treat endocrine-resistant breast cancer.
Design: A Fulvestrant-resistant breast cancer cell line (FASMCF), developed by continuous culture of the endocrine responsive MCF-7 parental line in Fulvestrant (10-7M)-supplemented medium. Immunocytochemistry was used to compare the level of a series of histone lysine acetylation (H3K18, H4K12, and H4K16), lysine methylation (H3K4, H4K20) and arginine methylation (H4R3) marks between the parental MCF7 and FASMCF cell lines prepared as paraffin cell pellets. Q-PCR was used to compare the level of various histone modifying enzyme genes (e.g. PCAF, NCOA1, HAT1, MYST3, HDAC1, HDAC2, and EZH2) in both cell lines.
Results: The level of histone lysine acetylation (H3K18, H4K16) and methylation (H4K20) was higher in FASMCF cells compared to MCF-7 cells, as revealed by nuclear immunostaining. Furthermore, Q-PCR showed that the mRNA expression of the histone acetyl transferases HAT1 and MYST3 and the histone methyltransferase EZH2 were upregulated in FASMCF cells.
Conclusions: The study identifies differences in the level of histone marks between Fulvestrant-resistant and responsive breast cancer cells, accompanied by upregulation of genes that have been reported to be responsible for such modifications. Such changes may be contributory to the mechanisms underlying the gene profile of Fulvestrant resistance in vitro. The findings suggest combination treatments of Fulvestrant and histone deacetyltransferase inhibitor (HDACI) or demethylating agents, that merit experimental investigation in the context of treating endocrine-resistant breast cancer.
Monday, March 9, 2009 1:00 PM
Poster Session II # 59, Monday Afternoon