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

et al. (2011) Suppression of Breast Tumor Growth and Metastasis by an Engineered Transcription Factor. PLoS ONE 6(9): e24595. doi:10.1371/journal.pone.0024595 Suppression of Breast Tumor Growth and Metastasis by an Engineered Transcription Factor Adriana S. Beltran 0 Angela Russo 0 Haydee Lara 0 Cheng Fan 0 Paul M. Lizardi 0 Pilar Blancafort 0 Austin John Cooney, Baylor College of Medicine, United States of America 0 1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Department of Pharmacology, University of Illinois at Chicago , Chicago , Illinois, United States of America, 4 Department of Marine Biotechnology, CICESE Research Institute, Ensenada, Mexico, 5 Department of Pathology, Yale University School of Medicine , New Haven, Connecticut , United States of America 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. - Funding: This work was supported from National Cancer Institute/National Institutes of Health grants 1R01CA125273, 3R01CA125273-03S1 and Department of Defense (DoD) W81XWH-10-1-0265 (PB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) is a multifunctional protein possessing tumor and metastasis suppressive functions [1,2]. Additionally, Maspin over-expression inhibits in vivo angiogenesis [3]. The multifaceted nature of Maspin affecting many molecular mechanisms during neoplastic disease progression makes it a very attractive target in cancer biology. Importantly, clinical data shows that high Maspin levels are associated with better prognosis in breast, lung and prostate carcinomas [4,5,6]. As a class II tumor suppressor gene, Maspin is not mutated, rearranged or deleted in tumor cells. Instead, its expression is regulated by means of transcription factors [7] and epigenetic modifiers [8,9]. While Maspin is expressed at high levels by epithelial cells, it is down-regulated in mesenchymal cells, such as stromal fibroblasts. In breast cancer cell lines and cancer specimens, silencing of Maspin correlates with acquisition of invasive and metastatic behavior. Epigenetic mechanisms controlling Maspin silencing include both, DNA [9] and H3K9 histone methylation [10]. Hence epigenetic mechanisms are reversible yet inherited during cell division, blockade of Maspin promoter silencing offers a potent strategy to reactivate tumor suppressor function. To this end, we have previously described the construction of Artificial Transcription Factors (ATFs) made of sequence-specific six Zinc Finger (ZF) domains[11] designed to bind unique 18-base pair recognition sites in the Maspin proximal promoter [12]. The ZFs were linked to a VP64 transactivator domain, which mediates a strong promoter up-regulation by recruitment of the polII transcriptional complex. In cell systems, both in lung and breast cancer cell lines, retroviral transduction of one of the ATFs, ATF-126, led to a potent induction of apoptosis and inhibition of cell invasion [12,13]. Furthermore, these ATFs were able to directionally demethylate the Maspin promoter and this effect depended on upon the orientation of the ATF along the DNA [13]. Consistently, we found that ATFs synergized with both methyltransferase and histone deacetylase inhibitors to reactivate silenced Maspin [13,14,15]. These previous observations suggested that ATF-126 was able to partially reprogram or revert the epigenetic state of the Maspin promoter, resulting in a re-activation of the endogenous gene. However, the impact of ATF-126 in inhibiting tumor progression in preexisting tumors and/or metastases in vivo has never been addressed. Herein, we have taken advantage of an inducible viral vector system to control the expression of ATF-126 in pre-existing breast tumor growths and experimental metastases in immunodeficient mice. Chemical induction of ATF-126 in vivo resulted in tumor suppression as well as in inhibition of breast tumor cell colonization. Furthermore, genome-wide DNA microarrays of MDA-MB-231 cells induced with ATF-126 revealed that breast tumor cells acquired a 550-gene signature that was found overrepresented in estrogen receptor positive (ER+) breast cancer cell lines and in the normal-like intrinsic subtype of breast cancer. Our data indicates that ATF-126 up-regulates novel Maspin-dependent targets possessing tumor and metastasis suppressive functions, which are found epigenetically silenced in aggressive tumors. Our results outline a possible mechanism by which ATF-126 reprograms aggressive tumor cells towards a more normal-like, more benign, and more differ (...truncated)