Identification of new therapeutic targets in breast cancer using chromatin engineering

Abstract

Oestrogen receptor (ER) α is an oestrogen induced transcription factor that binds to oestrogen responsive elements (ERE) which consequently drives the transcription of oestrogen responsive genes. Almost 70-80% of breast tumors overexpress ERα in which gold standard treatment involves the use of anti-oestrogen in blocking ERα activity. Unfortunately, 30% of ERα-positive tumours fail to respond to anti-oestrogen therapy and a substantial proportion of patients with initial response eventually become resistant. Alternative treatment using aromatase inhibitors involves blocking oestrogen biosynthetic pathway. Nonetheless, the effective use of these inhibitors is still hampered by the problem of resistance. Response to a different endocrine therapy despite resistance to the previous one is suggestive of a continued role for ERα responsive genes in breast cancer progression. Hence, inhibiting the expression of ERα responsive genes as opposed to inhibiting ERα activity, is likely to be of significant therapeutic value in the treatment of breast cancer that becomes refractory to endocrine therapies. The aim of this study was to identify the oestrogen-regulated genes and related molecular pathways in breast cancer, by comparing transcriptomic and proteomic profiles between parental and chromatin engineered MCF-7 breast cancer cell lines. Using chromatin engineering, oestrogen-responsive MCF-7 breast cancer cell line was modified, such that the expression of the oestrogen-regulated genes and MCF-7 cell growth were conditionally inhibited. Quantitive PCR (QPCR) and immunoblotting showed expression of known oestrogen responsive genes, PR, PS2 and Cathepsin D were inhibited by this repressor activity in the clones at 16-hour time point. Microarray gene expression analysis using Illumina HumanRef-8 v3.0 Expression BeadChips on the parental MCF7-TO had identified 51 upregulated genes and 27 downregulated genes in the presence of oestrogen (FDR ≤0.01, fold change ≥ 2). Pathway analysis (Ariadne) showed 29 genes were directly related. Transcriptomic profiles of the clone with repressor activity showed 127 genes were downregulated (NME1/NM23H1,GREB1,MYC) and 89 genes (BCAS1, CTGF,TP53INPI) were upregulated and not affected by the repressor activity. Of these, 78 genes were found to be directly related (FDR ≤0.01, fold change≥ 2). Comparing these two transcriptomic profiles, 62 genes were found to be oestrogen responsive and repressed by the repressor activity (NM23-H1/NME1, MYC and TPD52L1). A novel transcriptional repressor ZNF367 was found to be significantly repressed (Mann-Whitney, p-value ≤ 0.0001). QPCR and RNA interference (RNAi) confirmed overexpression of ZNF367 in MCF7 breast cancer cells and its key function in cell growth respectively. Gene Set Enrichment Analysis (GSEA) identified DNA replication and mitotic cell cycle to be negatively enriched in the clone with repressor activity. Proteomic analysis identified two proteins, SET and NM23-H1/NME1 which act in a novel complex in mediating a caspase alternative apoptosis pathway. In conclusion, this study had identified oestrogen-regulated genes which are growth associated using chromatin engineering that are potential novel therapeutic targets for breast cancer.