Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer.
In the present study, we have taken the novel approach of using an in vitro model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.
Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff, Wales, United Kingdom. a.stone@garvan.org.au
Andrew
A
Stone
Fatima
F
Valdés-Mora
Julia M W
JM
Gee
Lynne
L
Farrow
Richard A
RA
McClelland
Heidi
H
Fiegl
Carol
C
Dutkowski
Rachael A
RA
McCloy
Robert L
RL
Sutherland
Elizabeth A
EA
Musgrove
Robert I
RI
Nicholson
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D066246:ErbB Receptors / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects*; D052138:Genes, Neoplasm / Q000235:genetics; D055436:Growth Differentiation Factor 15 / Q000494:pharmacology; D006801:Humans; D061986:MCF-7 Cells; D011799:Quinazolines / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology*; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D004967:Estrogens; D055436:Growth Differentiation Factor 15; D011799:Quinazolines; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; D066246:ErbB Receptors; D001374:Azacitidine; D000077156:Gefitinib