DNA Methyltransferase Inhibitor, Zebularine, Delays Tumor Growth and Induces Apoptosis in a Genetically Engineered Mouse Model of Breast Cancer

Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA methylation. We examined the effect of zebularine on mammary tumor growth in genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were randomized at 46 days of age into control (n = 25) and zebularine (n = 25) treatment groups and monitored for parameters of tumor growth. Zebularine was administered at 5 mg/mL in drinking water. We observed a significant delay in the growth of mammary tumors in zebularine-treated mice with a statistically significant reduction (P = 0.0135) in total tumor burden at 94 days of age when the mice were sacrificed. After 48 days of zebularine treatment, the tumors were predominantly necrotic compared with untreated animals. In addition, a high apoptotic index by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay was observed as early as 13 days following treatment. Immunoblot analysis showed depletion of DNMT1 and partial depletion of DNMT3b after zebularine treatment. Microarray analyses of global gene expression identified upregulation of twelve methylation-regulated genes as well as a set of candidate cancer genes that participate in cell growth and apoptosis. In summary, zebularine inhibits the growth of spontaneous mammary tumors and causes early onset of tumor cell necrosis and apoptosis in a genetically engineered mouse model of breast cancer. Defining the parameters of zebularine-mediated tumor inhibition may advance the future development of DNA methyltransferase inhibitors as an effective cancer treatment. Mol Cancer Ther; 11(2); 370–82. ©2011 AACR.

[1]  I. Bièche,et al.  Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[2]  A. Lindblom,et al.  Cyclin D2 expression in familial and sporadic breast cancer. , 2002, Oncology reports.

[3]  Min Chen,et al.  Enhanced growth inhibition by combined DNA methylation/HDAC inhibitors in lung tumor cells with silenced CDKN2A. , 2010, International journal of oncology.

[4]  R. Sutherland,et al.  Cyclin D2 activates Cdk2 in preference to Cdk4 in human breast epithelial cells , 1997, Oncogene.

[5]  S. Sanche,et al.  Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression. , 2007, Experimental hematology.

[6]  K. Camphausen,et al.  Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine , 2005, Clinical Cancer Research.

[7]  R. Nagler,et al.  Prognostic Role of Apoptotic, Bcl-2, c-erbB-2 and p53 Tumor Markers in Salivary Gland Malignancies , 2003, Oncology.

[8]  S. Naber,et al.  Akt1 ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice. , 2007, Cancer research.

[9]  Pedram Argani,et al.  Quantitative Multiplex Methylation-Specific PCR Assay for the Detection of Promoter Hypermethylation in Multiple Genes in Breast Cancer , 2004, Cancer Research.

[10]  R. Cardiff,et al.  Induction of mammary epithelial hyperplasias and mammary tumors in transgenic mice expressing a murine mammary tumor virus/activated c-src fusion gene. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  K. Kinzler,et al.  p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. , 1995, Genes & development.

[12]  Gangning Liang,et al.  Preferential response of cancer cells to zebularine. , 2004, Cancer cell.

[13]  B. Nielsen,et al.  Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas. , 2005, Carcinogenesis.

[14]  B. Komm,et al.  The Wnt antagonist secreted frizzled‐related protein‐1 controls osteoblast and osteocyte apoptosis , 2005, Journal of cellular biochemistry.

[15]  John N. Hutchinson,et al.  Requirement for Both Shc and Phosphatidylinositol 3′ Kinase Signaling Pathways in Polyomavirus Middle T-Mediated Mammary Tumorigenesis , 1998, Molecular and Cellular Biology.

[16]  J. Beisler.  Isolation, characterization, and properties of a labile hydrolysis product of the antitumor nucleoside, 5-azacytidine. , 1978, Journal of medicinal chemistry.

[17]  J. Herman,et al.  Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. , 1995, Cancer research.

[18]  F. Šorm,et al.  Effect of 5-aza-2'-deoxycytidine against leukemic and hemopoietic tissues in AKR mice. , 1968, Neoplasma.

[19]  E. Liu,et al.  Global Expression Profiling Identifies Signatures of Tumor Virulence in MMTV-PyMT-Transgenic Mice , 2004, Cancer Research.

[20]  Peter A. Jones,et al.  Cellular differentiation, cytidine analogs and DNA methylation , 1980, Cell.

[21]  Matthew Tudor,et al.  Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation , 2001, Nature Genetics.

[22]  P. Jones,et al.  Zebularine: a new drug for epigenetic therapy. , 2004, Biochemical Society transactions.

[23]  S. Ethier,et al.  Methylation‐associated silencing of SFRP1 with an 8p11‐12 amplification inhibits canonical and non‐canonical WNT pathways in breast cancers , 2009, International journal of cancer.

[24]  H. Hibshoosh,et al.  Induction of autophagy and inhibition of tumorigenesis by beclin 1 , 1999, Nature.

[25]  H. Gómez,et al.  The phosphatidyl inositol 3-kinase/AKT signaling pathway in breast cancer , 2010, Cancer and Metastasis Reviews.

[26]  M. Brattain,et al.  Repression of transforming growth factor-β receptor type I promoter expression by Sp1 deficiency , 2000, Oncogene.

[27]  A. Schönthal,et al.  Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Wei Ye,et al.  Inhibition of DNA methylation and reactivation of silenced genes by zebularine. , 2003, Journal of the National Cancer Institute.

[29]  D. Kwiatkowski,et al.  Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[30]  L. Cantley,et al.  Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation , 1985, Nature.

[31]  K. Kinzler,et al.  p21 is necessary for the p53-mediated G1 arrest in human cancer cells. , 1995, Cancer research.

[32]  Mallika Singh,et al.  Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models , 2010, Nature Biotechnology.

[33]  B. Honoré,et al.  Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells , 1990, The Journal of cell biology.

[34]  C. Bradbury,et al.  Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors , 2005, Leukemia.

[35]  S. Weitzman,et al.  Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation. , 2001, Cancer research.

[36]  David A. Tuveson,et al.  The Use of Targeted Mouse Models for Preclinical Testing of Novel Cancer Therapeutics , 2006, Clinical Cancer Research.

[37]  R. Cardiff,et al.  Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease , 1992, Molecular and cellular biology.

[38]  A. Dobrovic,et al.  Methylation of the BRCA1 gene in sporadic breast cancer. , 1997, Cancer research.

[39]  D. Tuveson,et al.  The use of GEM models for experimental cancer therapeutics , 2008, Disease Models & Mechanisms.

[40]  M. Robert,et al.  An Essential Role for DNA Methyltransferase DNMT3B in Cancer Cell Survival* , 2002, The Journal of Biological Chemistry.

[41]  Wei Dong Chen,et al.  Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer , 2004, Nature Genetics.

[42]  J. Connolly,et al.  Widespread loss of gelsolin in breast cancers of humans, mice, and rats. , 1996, Cancer research.

[43]  Peter A. Jones,et al.  The Epigenomics of Cancer , 2007, Cell.

[44]  D. Ahlquist,et al.  Aberrant methylation of secreted frizzled‐related protein genes in esophageal adenocarcinoma and Barrett's esophagus , 2005, International journal of cancer.

[45]  Michele D. Sobolewski,et al.  Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells , 2010, Breast Cancer Research and Treatment.

[46]  Peter A. Jones,et al.  Long-term Epigenetic Therapy with Oral Zebularine Has Minimal Side Effects and Prevents Intestinal Tumors in Mice , 2008, Cancer Prevention Research.

[47]  Z. Khatib,et al.  Expression profiles of apoptotic genes induced by curcumin in human breast cancer and mammary epithelial cell lines. , 2005, Anticancer research.

[48]  Yi-Song Wang,et al.  WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. , 1994, Cancer research.

[49]  H. Hondermarck,et al.  Synergistic induction of apoptosis in breast cancer cells by cotreatment with butyrate and TNF-alpha, TRAIL, or anti-Fas agonist antibody involves enhancement of death receptors' signaling and requires P21(waf1). , 2004, Experimental cell research.

[50]  Pang-Kuo Lo,et al.  Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer , 2006, Cancer biology & therapy.

[51]  Q. Tao,et al.  Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer. , 2006, Carcinogenesis.

[52]  Gun-Hee Kim,et al.  Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells. , 2009, The Journal of nutritional biochemistry.

[53]  Steffen Zopf,et al.  Apoptosis, proliferation and differentiation patterns are influenced by Zebularine and SAHA in pancreatic cancer models , 2007, Scandinavian journal of gastroenterology.

[54]  M. Mahadevappa,et al.  SARPs: a family of secreted apoptosis-related proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[55]  M. Toyota,et al.  Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer , 2007, Oncogene.

[56]  Peter A. Jones,et al.  Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells , 2004, Molecular and Cellular Biology.