Chromatin , Gene , and RNA Regulation Towards Sustained Silencing of HER 2 / neu in Cancer By Epigenetic Editing

The human epidermal growth factor receptor-2 (HER2/neu/ERBB2) is overexpressed in several cancer types. Although therapies targeting the HER2/neu protein result in inhibition of cell proliferation, the anticancer effect might be further optimized by limiting HER2/neu expression at the DNA level. Towards this aim, epigenetic editing was performed to suppress HER2/neu expression by inducing epigenetic silencing marks on theHER2/neu promoter.HER2/neu expression and HER2/neu promoter epigenetic modification status were determined in a panel of ovarian and breast cancer cell lines.HER2/neu-overexpressing cancer cells were transduced to express a zinc finger protein (ZFP), targeting the HER2/neu gene, fused to histone methyltransferases (G9a, SUV39-H1)/super KRAB domain (SKD). Epigenetic assessment of the HER2/neu promoter showed that HER2/neu-ZFP fused to G9a efficiently induced the intended silencing histone methylation mark (H3K9me2). Importantly, H3K9me2 induction was associated with a dramatic downregulation of HER2/neu expression in HER2/neuoverexpressing cells. Downregulation by SKD, traditionally considered transient in nature, was associated with removal of the histone acetylation mark (H3ac). The downregulation of HER2/neu by induced H3K9 methylation and/or reduced H3 acetylation was sufficient to effectively inhibit cellular metabolic activity and clonogenicity. Furthermore, genome-wide analysis indicated preferential binding of the ZFP to its target sequence. These results not only show that H3K9 methylation can be induced but also that this epigenetic mark was instructive in promoting downregulation of HER2/neu expression. Implications: Epigenetic editing provides a novel (synergistic) approach tomodulate expression of oncogenes.Mol Cancer Res; 11(9); 1029–39. 2013 AACR.

[1]  L. Bagella,et al.  Histone Deacetylase Inhibitors in the Treatment of Hematological Malignancies and Solid Tumors , 2010, Journal of biomedicine & biotechnology.

[2]  Pilar Blancafort,et al.  Epigenetic reprogramming of cancer cells via targeted DNA methylation , 2012, Epigenetics.

[3]  D. Trono,et al.  The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development. , 2012, Cell reports.

[4]  Jiri Bartek,et al.  Epithelial junction opener JO-1 improves monoclonal antibody therapy of cancer. , 2011, Cancer research.

[5]  J. Martens,et al.  Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. , 2003, Molecular cell.

[6]  Sandip K. Mishra,et al.  Dynamic chromatin remodeling on the HER2 promoter in human breast cancer cells , 2001, FEBS letters.

[7]  C. Pabo,et al.  Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo , 2002, Current Biology.

[8]  M. Rots,et al.  Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. , 2003, Nucleic acids research.

[9]  M. Goldenberg.  Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. , 1999, Clinical therapeutics.

[10]  E. Pujade-Lauraine,et al.  HER-2 overexpression is an independent marker of poor prognosis of advanced primary ovarian carcinoma: a multicenter study of the GINECO group. , 2004, Annals of oncology : official journal of the European Society for Medical Oncology.

[11]  C. Barbas,et al.  Positive and negative regulation of endogenous genes by designed transcription factors. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Baselga,et al.  Primary results from EMILIA, a phase III study of trastuzumab emtansine (T-DM1) versus capecitabine (X) and lapatinib (L) in HER2-positive locally advanced or metastatic breast cancer (MBC) previously treated with trastuzumab (T) and a taxane. , 2012, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  P. Blancafort,et al.  Breaking through an epigenetic wall , 2013, Epigenetics.

[14]  M. Teitell,et al.  Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase , 2005, Nucleic acids research.

[15]  E. D. de Vries,et al.  Trastuzumab pharmacokinetics influenced by extent human epidermal growth factor receptor 2-positive tumor load. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[16]  P. Atadja,et al.  Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. , 2003, Molecular cancer therapeutics.

[17]  R. Nahta,et al.  HER2 therapy: Molecular mechanisms of trastuzumab resistance , 2006, Breast Cancer Research.

[18]  H. Hurst.  Update on HER-2 as a target for cancer therapy: The ERBB2 promoter and its exploitation for cancer treatment , 2001, Breast Cancer Research.

[19]  Albert Jeltsch,et al.  Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity. , 2013, Journal of molecular biology.

[20]  M. Rots,et al.  Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer , 2012, Nucleic acids research.

[21]  N. Hynes,et al.  ErbB receptors and signaling pathways in cancer. , 2009, Current opinion in cell biology.

[22]  L. Tse,et al.  Survival benefits from lapatinib therapy in women with HER2-overexpressing breast cancer: a systematic review , 2010, Anti-cancer drugs.

[23]  R. Schüle,et al.  Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology. , 2010, Carcinogenesis.

[24]  D J Segal,et al.  Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Pernette J. Verschure,et al.  Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes , 2012, Nucleic acids research.

[26]  Yuhua Wang,et al.  Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors* , 2012, The Journal of Biological Chemistry.

[27]  Pilar Blancafort,et al.  Promoter-targeted phage display selections with preassembled synthetic zinc finger libraries for endogenous gene regulation. , 2004, Journal of molecular biology.

[28]  Monilola A. Olayioye,et al.  Update on HER-2 as a target for cancer therapy: Intracellular signaling pathways of ErbB2/HER-2 and family members , 2001, Breast Cancer Research.

[29]  M. Rots,et al.  Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer , 2013, British Journal of Cancer.

[30]  M. Rots,et al.  Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer , 2011, British Journal of Cancer.

[31]  S. Berger,et al.  Epigenetic Drugs Can Stimulate Metastasis through Enhanced Expression of the Pro-Metastatic Ezrin Gene , 2010, PloS one.

[32]  T. Younis,et al.  Trastuzumab for HER2-Positive Metastatic Breast Cancer: Clinical and Economic Considerations , 2012, Clinical Medicine Insights. Oncology.

[33]  T. Sera.  Zinc-finger-based artificial transcription factors and their applications. , 2009, Advanced drug delivery reviews.

[34]  Peter A. Jones,et al.  Epigenetic Modifications as Therapeutic Targets , 2010, Nature Biotechnology.

[35]  C. Barbas,et al.  Zinc Finger Transcription Factors Designed for Bispecific Coregulation of ErbB2 and ErbB3 Receptors: Insights into ErbB Receptor Biology , 2005, Molecular and Cellular Biology.

[36]  Werner Scheuer,et al.  Strongly enhanced antitumor activity of trastuzumab and pertuzumab combination treatment on HER2-positive human xenograft tumor models. , 2009, Cancer research.

[37]  D. Cunningham,et al.  Targeted Therapy for Gastric Cancer , 2012, Current Treatment Options in Oncology.

[38]  M. Rots,et al.  Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors , 2013, Molecular oncology.

[39]  J. Herman,et al.  Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. , 2011, Cancer discovery.

[40]  J. Hechtman,et al.  HER2/neu gene amplification and protein overexpression in gastric and gastroesophageal junction adenocarcinoma: a review of histopathology, diagnostic testing, and clinical implications. , 2012, Archives of pathology & laboratory medicine.

[41]  M. Hung,et al.  The HER-2-Targeting Antibodies Trastuzumab and Pertuzumab Synergistically Inhibit the Survival of Breast Cancer Cells , 2004, Cancer Research.

[42]  J. Baselga,et al.  Randomized study of Lapatinib alone or in combination with trastuzumab in women with ErbB2-positive, trastuzumab-refractory metastatic breast cancer. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[43]  D. Trono,et al.  The Krüppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo * , 2012, The Journal of Biological Chemistry.

[44]  Inti Zlobec,et al.  HER2 gene status in primary breast cancers and matched distant metastases , 2007, Breast Cancer Research.

[45]  G. Sauter,et al.  Close association between HER-2 amplification and overexpression in human tumors of non-breast origin , 2007, Modern Pathology.

[46]  M. Moasser,et al.  A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer , 2011, British Journal of Cancer.

[47]  M. Rots,et al.  Step into the groove: engineered transcription factors as modulators of gene expression. , 2006, Advances in genetics.