Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

Abstract

The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

“…The identified transcription factor binding sites in this region experimentally elucidated by previous studies include the sequences in the negative regulator site HRE and the positive regulator binding sites, ETS, AP1, ATF-126 and ATF-2 sites (Maekawa et al, 2007;Zhang et al, 1997 a and b;Beltran et al, 2007). These sites are 60-83% identical with human suggesting these transcription factors may be active across the different species (Fig 9B).…”

Reprogramming epigenetic silencing: artificial transcription factors synergize with chromatin remodeling drugs to reactivate the tumor suppressor mammary serine protease inhibitor

“…2C). When retrovirally transduced in MDA-MB-231 cells, only the ATF-126 was able to strongly reactivate the promoter (70-fold relative to controls, in the absence of drugs), whereas ATF-97 and ATF-452 alone had a much weaker activity (27).…”

“…In a previous report, we have described the construction of three ATFs designed to bind 18-bp sites in the maspin proximal promoter (27). The ATFs were constructed by linkage of six sequence-specific ZF domains with the VP64 transactivator domain (Fig.…”

“…Real-time PCR Expression Assays ATF-transduced cells and control cells, drug treated or nontreated, were collected, and the RNA was extracted and 2.5 Ag were used for reverse transcription. Quantification of maspin and VP64 activator domain was obtained by realtime quantitative PCR using fluorescent Taqman assays (Applied Biosystems) as described (27). The primers and probes for the VP64 activator domain were the following: 5 ¶-AAGCGACGCATTGGATGAC-3 ¶ (forward primer), 5 ¶-GGAACGTCGTACGGGTAGTTAATT3 ¶ (reverse primer), and 5 ¶-6FAM-TCGGCTCCGATGCT-MGBNFQ-3 ¶ (probe).…”

“…Our laboratory has recently applied a new strategy to specifically reactivate tumor suppressor genes silenced by epigenetic mechanisms in aggressive tumors (27). We have targeted the tumor suppressor gene maspin using three rationally designed artificial transcription factors (ATFs).…”