During tumor progression, cancer cells undergo dynamic transformations, including tumor growth, angiogenesis, and metastatic dissemination. There is a crucial need in cancer therapeutics to develop novel approaches that target critical, ideally multiple steps, during tumor progression. In this proposal, we have designed artificial transcription factors (ATFs) made of zinc finger (ZF) domains as novel therapeutic strategy to inhibit multiple processes during tumor progression. We have targeted the mammary serine protease inhibitor (maspin) gene (SERPINB5), Maspin is an important therapeutic target since its overexpression is associated with tumor suppression, decreased angiogenesis, motility and metastasis in breast and prostate tumor models. Metastatic cells have developed several mechanisms to down-regulate maspin. Maspin is rarely mutated in aggressive tumors. Instead, silencing of maspin involves both, transcriptional regulation and aberrant promoter methylation. Our objective is to construct ATFs able to specifically re-activate maspin in metastatic breast cell lines, overcoming epigenetic silencing. Our hypothesis is that ATFs up-regulating maspin, by themselves or in combination with drugs that increase chromatin accessibility (methyltransferase and histone deacetylase inhibitors), will be able to re-activate maspin functions in tumor cells, reduce tumor growth and metastatic spread in nude mice. First, we have engineered highly specific ATFs made of 6ZF domains targeting 18-base pairs (bp) sites in the maspin promoter. The ZFs are linked to a potent transcriptional activator domain and expressed in tumor cells using retroviral vectors. We will investigate if the ATFs are able to specifically reactivate maspin in several breast cancer cells comprising a methylated and silenced maspin promoter. We will study if the ATFs synergize with methyltransferase and histone deacetylase inhibitors, to up-regulate maspin. We will assess if these ATFs are able to down-regulate cell invasion and to induce apoptosis in metastatic cell lines. Finally, the ATFs will be expressed using Adeno- Associated Viruses (AAVs) and we will study their capability to down-regulate tumor growth and metastasis in an orthotopic xenograft model of breast cancer in nude mice. Metastatic lesions will be monitored in real time using Bioluminescence Imaging (BLI). This work should lead to the characterization of novel anti-cancer regulators, able to reprogram the aberrant epigenetic silencing of tumor suppressors

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA125273-05
Application #
8026864
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Snyderwine, Elizabeth G
Project Start
2007-05-01
Project End
2013-02-28
Budget Start
2011-03-01
Budget End
2013-02-28
Support Year
5
Fiscal Year
2011
Total Cost
$269,078
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Beltran, A S; Graves, L M; Blancafort, P (2014) Novel role of Engrailed 1 as a prosurvival transcription factor in basal-like breast cancer and engineering of interference peptides block its oncogenic function. Oncogene 33:4767-77
Wang, Yuhua; Su, Hsing-Hao; Yang, Yang et al. (2013) Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. Mol Ther 21:358-67
Juárez-Moreno, Karla; Erices, Rafaela; Beltran, Adriana S et al. (2013) Breaking through an epigenetic wall: re-activation of Oct4 by KRAB-containing designer zinc finger transcription factors. Epigenetics 8:164-76
Hu, Yunxia; Haynes, Matthew T; Wang, Yuhua et al. (2013) A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo. ACS Nano 7:5376-84
Rivenbark, Ashley G; Stolzenburg, Sabine; Beltran, Adriana S et al. (2012) Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics 7:350-60
Lara, Haydee; Wang, Yuhua; Beltran, Adriana S et al. (2012) Targeting serous epithelial ovarian cancer with designer zinc finger transcription factors. J Biol Chem 287:29873-86
Hassiotou, Foteini; Beltran, Adriana; Chetwynd, Ellen et al. (2012) Breastmilk is a novel source of stem cells with multilineage differentiation potential. Stem Cells 30:2164-74
Stolzenburg, Sabine; Rots, Marianne G; Beltran, Adriana S et al. (2012) Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer. Nucleic Acids Res 40:6725-40
Beltran, Adriana S; Blancafort, Pilar (2011) Reactivation of MASPIN in non-small cell lung carcinoma (NSCLC) cells by artificial transcription factors (ATFs). Epigenetics 6:224-35

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