Anti-ESR1 monoclonal antibody, clone UF222.6E22

Topotecan is a clinically active anticancer agent for the management of various human tumors. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I and formation of DNA double-strand breaks, recent studies suggest that mechanisms involving generation of reactive free radicals and induction of oxidative stress may play a significant role in topotecan-dependent tumor cell death. We have shown that topotecan generates a topotecan radical following one-electron oxidation by a peroxidase-hydrogen peroxide system which reacts with reduced glutathione and cysteine, forming the glutathiyl and cysteinyl radicals, respectively. While little is known how these events are involved in topotecan-induced tumor cell death, we have now examined the effects of topotecan short (1 h) and long (24 h) exposure on global gene expression patterns using gene expression microarray analysis in human breast MCF-7 cancer cells, a wild-type p53 containing cell line. We show here that topotecan treatment significantly down-regulated estrogen receptor alpha (ER alpha/ESR1) and antiapoptotic BCL2 genes in addition to many other p53-regulated genes. Furthermore, 8-oxoguanine DNA glycosylase (OGG1), ferredoxin reductase (FDXR), methionine sulfoxide reductase (MSR), glutathione peroxidases (GPx), and glutathione reductase (GSR) genes were also differentially expressed by topotecan treatment. The differential expression of these genes was observed in a wild-type p53-containing breast ZR-75-1 tumor cell line following topotecan treatment. The involvement of reactive oxygen free radical sensor genes, the oxidative DNA damage (OGG1) repair gene and induction of pro-apoptotic genes suggest that reactive free radical species play a role in topotecan-induced tumor cell death.

Purpose Although tamoxifen remains the frontline treatment for ER alpha-positive breast cancers, resistance to this drug limits its clinical efficacy. Most tamoxifen-resistant patients retain ER alpha expression which may support growth and progression of breast cancers. Therefore, we investigated epigenetic regulation of ER alpha that may provide a rationale for targeting ER alpha in these patients. Methods Expression levels of the mixed-lineage leukemia (MLL) family of proteins in tamoxifen-resistant breast cancer cells and publicly available breast cancer patient data sets were analyzed. Histone methylation levels in ER alpha promoter regions were assessed using chromatin immunoprecipitation. Expression levels of ER alpha and its target gene were analyzed using western blotting and real-time qPCR. Cell-cycle was analyzed by flow cytometry. Results The expression of MLL3 and SET-domain-containing 1A (SET1A) were increased in tamoxifen-resistant breast cancers. An MLL3 chromatin immunoprecipitation-sequencing data analysis and chromatin immunoprecipitation experiments for MLL3 and SET1A suggested that these proteins bound to enhancer or intron regions of the ESR1 gene and regulated histone H3K4 methylation status. Depletion of MLL3 or SET1A downregulated the expression level of ER alpha and inhibited the growth of tamoxifen-resistant breast cancer cells. Additional treatment with fulvestrant resulted in a synergistic reduction of ER alpha levels and the growth of the cells. Conclusions The enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported the ER alpha-dependent growth of these cells by increasing ER alpha expression. Our results suggest that targeting these histone methyltransferases might provide an attractive strategy to overcome endocrine resistance.