Epigenetic reversion of breast carcinoma phenotype is accompanied by DNA sequestration

The importance of microenvironment and context in regulation of tissue-specific genes is finally well established. DNA exposure to, or sequestration from, nucleases can be used to detect differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we utilized an established 3-D assay where normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like vs tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion where the malignant cells are resistant to digestion relative to non-malignant cells. Reversion of the T4-2 breast cancer cells by either cAMP analogs, or a phospatidylinositol 3-kinase (P13K) inhibitor not only reverted the phenotype, but also the chromatin sensitivity to AluI. By using different cAMP-analogs, we show that the cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependentprotein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin also reverted the malignant phenotype, polarized the acini, and changed chromatin sequestration. These experiments show not only that modifying the tumor microenvironment can alter the organization of tumor cells but also that architecture of the tissues and the global chromatin organization are coupled and yet highly plastic. Introduction We have shown previously that the degree of malignancy, the organization of cytoskeleton, and the composition of the extracellular matrix (ECM) influence chromatin structure (Maniotis et al., 2005). We found that the DNA of cultured cell lines from malignant tumors, transformed fibroblasts harboring 3 oncogenes, and cells collected from human tumors were more resistant to nucleases compared to DNA from normal or nonmalignant and weakly malignant cells. In addition, cells with the same genotype exhibit different degrees of DNA sequestration and exposure when cytoskeletal components were selectively disrupted, or when they were cultured on different ECM components (Maniotis et al., 2005). Without viral insertion, additional deletion, or mutation of genes, it is possible to revert malignant tumor cells into cells those behave phenotypically normal. On 2-dimensional (2-D) surfaces, malignant cells can be induced by addition of cAMP to cease blebbing, form an organized cytoskeleton, and develop contact-inhibited monolayers (Krystosek et al., 1990; Puck et al., 2002). In a 3-dimensional (3-D) tissue culture system, it is possible to induce breast cancer cells to form normal tissue structures resembling breast acini (Wang et al., 2002; Weaver et al., 2002; Weaver et al., 1997). In experimental animal models, teratocarcinoma cells placed into mammalian embryos (Mintz and Illmensee, 1975), avian embryos transformed with Rous sarcoma virus (Dolberg and Bissell, 1984), and metastatic, aneuploid melanoma cells placed in chick embryos (Kulesa et al., 2006) develop into normal structures and tissues, and do not form tumors as they would in the adult organisms. These findings demonstrate that the phenotypes of malignant, metastatic, or transformed cells are highly plastic and regulated by the environment in which they are placed. The mechanisms underlying these phenotypic plasticities are not understood. In addition, it is not known if changes in cell phenotype are accompanied by epigenetic changes in DNA organization. Here we have employed a well-characterized, phenotypically breast tumor model system in 3-D (Bissell and Labarge, 2005; Petersen et al., 1992; Weaver et al., 1997) to determine whether the transition of a tumor cells from disorganized clusters to organized, polar acinus-like structures is accompanied by global epigenetic changes in chromatin structure that could be quantified using the degree of resistance to DNAdegrading enzymes. We show that the organization of DNA in a malignant, mammary epithelial cell line follows tissue architecture. Moreover, tissue phenotype, and DNA organization are plastic, and reversible. We take advantage of these observations to test the following question: Through manipulation of single molecules in the microenvironment, is it possible to reversibly control DNA exposure/sequestration, cell polarity, tumor morphology, and ultimately, tumor behavior? Materials and Methods Cell lines and Cell culture MCF10A, a nonmalignant human breast epithelial cells was obtained from American Type Culture Collection (Rockville, MD); the spontaneously transformed and malignant human breast epithelial line, HMT-3522 T4-2, was isolated by (Briand et al., 1996) and was from the laboratory of Mina J Bissell at Lawrence Berkley National Lab. MCF10A cells were maintained in DMEM/F12 (Biowhittaker, Inc. Walkersville, MD) containing 20 ng/mL EGF (Calbiochem, Corp, San Diego, CA), 1.4 x 10 M hydrocortisone (BD Bioscience, San Jose, CA), 0.1 ng/mL Cholera toxin (Sigma, St Louis, MO), 10 X 10 g/mL human Insulin (Calbiochem Corp.), 2 mM Glutamine L, 5% horse serum (Fisher, Ontario, Canada) and penicillin/streptomycin. HMT-3522 T4-2 cells were routinely grown in H14 medium without 10 ng/ml EGF on Vitrogen-coated plates (Biowhittaker, Inc. Walkersville, MD) as described (Weaver et al., 2002). 3-D Cultures Three-dimensional cultures were prepared based on previously described protocols (Debnath et al., 2003; Weaver et al., 1997) with some modifications: Cover slips (18 x 18 mm) were coated with 120 μl of Reduced growth factor Matrigel (BD Bioscience, San Jose, CA). Single cell suspensions (0.5 1.0 X 10 cells per slip) were seeded on top of polymerized Matrigel, incubated for 30 min and over-laid with 2.5 ml of culture medium containing no EGF or serum. Cells were overlaid with medium containing 2% Matrigel. Cultures were grown for days indicated in figure legends, adding new medium every