Abstract
Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.
Highlights
Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a powerful tool for inducing and understanding important cellular phenotypes [1,2]
Several groups have created artificial ZF-TF libraries by randomly assembling combinations of individual zincfinger domains with pre-characterized DNA-binding specificities into libraries of either three, four, or six-finger proteins [1,2]. Each member of such a library has the potential to alter the expression of a spectrum of different genes in a cell, if fused to a transcriptional regulatory domain (e.g.—an activation or a repression domain) [1,2] Using 25 different zinc-finger domains (Table 1), we used a ‘‘mix and match’’ approach to create a combinatorial ZF-TF library consisting of as many as 390,625 (254) different four-finger proteins each fused to a NF -KB p65 activation domain encoded in a retroviral vector that can confer puromycin-resistance (Figure 1B)
We undertook a different approach in which we identified six members of a combinatorial zinc finger transcription factor library capable of inducing stable anti-endocrine drug resistance in a clonal breast cancer cell line
Summary
Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a powerful tool for inducing and understanding important cellular phenotypes [1,2]. By ‘‘mixing and matching’’ collections of individual zinc fingers with various preselected DNA-binding specificities, large collections (or ‘‘libraries’’) of multi-finger arrays, each predicted to bind a different spectrum of target DNA sequences, can be assembled [1,2]. These multi-finger arrays can in turn be fused to transcriptional regulatory domains to create libraries of artificial ZF-TFs capable of activating or repressing expression of specific genes. We reasoned that pertubation of molecular gene expression patterns in cells could lead to anti-endocrine resistance and that identification of these gene expression alterations could potentially lead to identification of novel and more effective therapeutic markers and targets
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