Suppression of Breast Tumor Growth and Metastasis by an Engineered Transcription Factor

Abstract

Maspin is a tumor and metastasis suppressor playing an essential role as gatekeeper of tumor progression. It is highly expressed in epithelial cells but is silenced in the onset of metastatic disease by epigenetic mechanisms. Reprogramming of Maspin epigenetic silencing offers a therapeutic potential to lock metastatic progression. Herein we have investigated the ability of the Artificial Transcription Factor 126 (ATF-126) designed to upregulate the Maspin promoter to inhibit tumor progression in pre-established breast tumors in immunodeficient mice. ATF-126 was transduced in the aggressive, mesenchymal-like and triple negative breast cancer line, MDA-MB-231. Induction of ATF expression in vivo by Doxycycline resulted in 50% reduction in tumor growth and totally abolished tumor cell colonization. Genome-wide transcriptional profiles of ATF-induced cells revealed a gene signature that was found over-represented in estrogen receptor positive (ER+) “Normal-like” intrinsic subtype of breast cancer and in poorly aggressive, ER+ luminal A breast cancer cell lines. The comparison transcriptional profiles of ATF-126 and Maspin cDNA defined an overlapping 19-gene signature, comprising novel targets downstream the Maspin signaling cascade. Our data suggest that Maspin up-regulates downstream tumor and metastasis suppressor genes that are silenced in breast cancers, and are normally expressed in the neural system, including CARNS1, SLC8A2 and DACT3. In addition, ATF-126 and Maspin cDNA induction led to the re-activation of tumor suppressive miRNAs also expressed in neural cells, such as miR-1 and miR-34, and to the down-regulation of potential oncogenic miRNAs, such as miR-10b, miR-124, and miR-363. As expected from its over-representation in ER+ tumors, the ATF-126-gene signature predicted favorable prognosis for breast cancer patients. Our results describe for the first time an ATF able to reduce tumor growth and metastatic colonization by epigenetic reactivation of a dormant, normal-like, and more differentiated gene program.

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

“…Engineering binding specificity is achieved by grafting the ␣-helical domain of each ZF known to interact with the target DNA triplet (13). 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).…”

“…Although most epithelial cells express high levels of Maspin, the promoter is down-regulated by epigenetic mechanisms in several cancers, such as breast (28,29) and ovarian cancer (30). Endogenous Maspin reactivation by ATF-126 was associated with decreased tumor growth by enhancement of apoptosis (10), cell invasion (10), and suppression of metastatic colonization in breast (18) and lung (31) cancer cells. Although most studies have been focused on breast, prostate, and lung cancer, the functional role of Maspin as tumor and metastasis suppressor in EOC has not been investigated.…”

Prolonged re-expression of the hypermethylated geneEPB41L3using artificial transcription factors and epigenetic drugs

“…Artificial transcription factor (ATF)-mediated expressional changes, however, have shown to be only transient, as targeted promoters are incompletely reprogrammed and genes tend to return to the 'normal' state after the ATF is removed. 7,8 Recently, a number of TSGs were successfully reactivated using ZFPs linked to VP64, including maspin, 7,9,10 CDKN2A, 11 and C13ORF18, 12 and re-activation of these 3 TSGs was associated with site-specific DNA demethylation, while re-expression of the latter two was also shown to be accompanied with a decreased repressive histone methylation status. Despite these epigenetic changes, ATF-induced effects are transient, and more stable epigenetic reprogramming is required to prolong the effects.…”

Writing and Rewriting the Epigenetic Code of Cancer Cells: From Engineered Proteins to Small Molecules

“…We have characterized an activator ATF able to reactivate silenced maspin in breast and lung cancer lines having high degree of promoter methylation. When delivered in these cell lines, the ATF suppressed breast cancer growth and totally abolished metastatic colonization (Lund et al, 2004;Beltran, 2011;Beltran et al, 2011b). Moreover, the effect of the ATF in reactivating endogenous maspin was highly synergistic with small molecule inhibitors of DNA methyltransferases and histone deacetylases (Beltran et al, 2008).…”

“…Highly methylated promoters, for example tumor suppressors, are associated with condensed chromatin, which could impede TFs to find their targets in chromatin. In the case of the maspin tumor suppressor, for example, the degree or extent of transactivation by ZF proteins is highly dependent on the targeted region of the promoter and in the DNA methylation level of the cell line examined (Beltran et al, 2011b). We have characterized an activator ATF able to reactivate silenced maspin in breast and lung cancer lines having high degree of promoter methylation.…”