Molecular and Cellular Pathobiology Epigenetic Regulation by Z-DNA Silencer Function Controls Cancer-Associated ADAM-12 Expression in Breast Cancer: Cross-talk between MeCP2 and NF1 Transcription Factor Family Bimal K. Ray 1 , Srijita Dhar 1 , Carolyn Henry 2 , Alexander Rich 3 , and Alpana Ray 1 Abstract A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA–mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells. Cancer Res; 73(2); 1–9. Ó2012 AACR. Introduction Metastatic spread of cancer is regarded as the greatest hurdle to cancer cure. In the metastatic cascade, multiple interrelated pathways are activated, which include proteolytic breakdown of the tumor membrane and spreading of cancer cells into the surrounding tissues, migration, and successful attachment of the escaped cancer cells at new sites and colonization and proliferation of cancer cells at secondary locations. Advances in cancer research indicate that genetic mutations along with epigenetic alterations also contribute to metastasis-related gene expression. In many human cancers, markedly high-level expression of a multifunctional protein, ADAM-12, is detected (1–7). In patients with breast and bladder cancer, increase of ADAM-12 is shown to correlate with disease progression and tumor stage (2, 5, 8) and in animal models, ADAM-12 is found to be required for aggressive tumor progression (3, 9). ADAM-12 is capable of supporting several steps of the cancer-metastasis cascade. It proteolytically degrades several components of extracellular matrix (2), facilitates cell–cell and cell–extracellular matrix (ECM) attachments (10), and pro- motes cell proliferation by increasing bioavailability of growth factors (2, 5). Incidentally, 3 somatic mutations in ADAM-12 gene are frequently seen in breast cancers (11). These muta- tions cause mutant ADAM-12 proteins to be retained in the endoplasmic reticulum (ER) rather than at the cell surface (12). It is speculated that increased accumulation of ADAM-12 in the ER may be linked to tumor growth, which is yet to be exper- imentally determined. Normal cellular expression of ADAM-12 usually is very low and highly regulated. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) that acts as transcriptional silencer of ADAM-12 expression (13). Here, we provide evidence that marked increase of ADAM- 12 level in breast cancer cells is, at least in part, due to loss of NRE-silencer function. The results reveal a novel mode of epigenetic regulation, which involves cross-talk between MeCP2, a prominent epigenetic regulator, and 2 members of Authors' Affiliations: 1 Departments of Veterinary Pathobiology and 2 Vet- erinary Medicine and Surgery, University of Missouri, Columbia, Missouri; and 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Alpana Ray, Department of Veterinary Pathobi- ology, University of Missouri, 126A Connaway Hall, Columbia, MO 65211; Phone: 573-882-6728; Fax: 573-884-5414; E-mail: rayal@missouri.edu, and Bimal K. Ray, E-mail: rayb@missouri.edu doi: 10.1158/0008-5472.CAN-12-2601 Ó2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1