Genetic reprogramming of tumor cells by zinc finger transcription factors

Abstract

Cancer arises by the accumulation of genetic alterations in DNA leading to aberrant gene transcription. Expression-profiling studies have correlated genomewide expression signatures with malignancy. However, functional analysis elucidating the contribution and synergy of genes in specific cancer cell phenotypes remains a formidable obstacle. Herein, we describe an alternative genetic approach for identification of genes involved in tumor progression by using a library of zinc finger artificial transcription factors (ATFs) and functional screening of tumor cells as a source of genetic plasticity and clonal selection. We isolated a six-zinc finger transcriptional activator (TF 20-VP, TF 20 containing the VP64 activator domain) that acts to reprogram a drug-sensitive, poorly invasive, and nonmetastatic cell line into a cell line with a drugresistant, highly invasive, and metastatic phenotype. Differential expression profiles of cells expressing TF 20-VP followed by functional studies, both in vitro and in animal models, revealed that invasion and metastasis requires coregulation of multiple target genes. Significantly, the E48 antigen, associated with poor metastasis-free survival in head and neck cancer, was identified as one specific target of TF 20-VP. We have shown phenotypic modulation of tumor cell behavior by E48 expression, including enhanced cell migration in vitro and tumor cell dissemination in vivo. This study demonstrates the use of ATFs to identify the group of genes that cooperate during tumor progression. By coregulating multiple targets, ATFs can be used as master genetic switches to reprogram and modulate complex neoplastic phenotypes. drug resistance ͉ metastasis ͉ invasion ͉ transcriptional regulation ͉ RNA inteference

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

“…We have constructed multimodular 6ZF proteins referred as artificial transcription factors (ATFs), recognizing sequences in targeted promoters with dissociation constants in the picomolar range (10,(13)(14)(15)(16)(17)(18). In an ATF, the 6ZF scaffold can be linked to a variety of protein modules to promote transcriptional activation (19 -23), repression (24,25), and more recently, epigenetic editing (26).…”

Targeting the absence: Homozygous DNA deletions as immutable signposts for cancer therapy

“…The DST strategy brings together both existing and new methodologies, such as the Ub fusion technique (22,23); the split-Ub assay (24); ZF DNA-recognizing proteins (43)(44)(45); restriction nucleases that are derived either from engineered ZF proteins (37)(38)(39)(40)(41) or from homing endonucleases (35,36) and that are configured (in DST designs) as split nucleases; DNA sequence-enabled assembly of a split protein (31); a new arrangement of split Ub-type domains in a polypeptide chain that enables a double proteolytic cleavage once two chains associate in vivo; and a new feedback mechanism that receives input from a circuit operating as a Boolean OR gate and involves the activation of split nucleases, which destroy the DST vector in normal (nontarget) cells (Figs. 1-3).…”

“…6. The fourth and last domain of DST-f1 is ZF1-1, a ZF protein domain, similar to the previously described and extensively characterized ZF proteins that recognize specific DNA sequences (43)(44)(45)(46). The ZF domains ZF1-1 and ZF1-2, of the fusions DST-f1 and DST-f2, respectively ( Fig.…”

Genome-Editing Technologies: Principles and Applications

“…Early work on the use of engineered zinc-finger transcription factors revealed that synthetic transcriptional modulators are effective tools for a broad range of applications, enabling such tasks as inhibiting viral replication (Papworth et al 2003;Reynolds et al 2003;Segal et al 2004;Eberhardy et al 2006), modulating the expression of disease-associated loci (Graslund et al 2005;Wilber et al 2010), inducing angiogenesis for accelerated wound healing (Rebar et al 2002), and genomic screening of cellular targets for cancer progression and drug resistance Blancafort et al 2005Blancafort et al , 2008. Facilitated by many of the insights gained from zinc-finger transcription factor technology, both TALEs and CRISPR-Cas9 have now further expanded the possibilities of engineered transcriptional activators and repressors.…”