title: Pharmacologic reversion of epigenetic silencing of the PRKD1 promoter blocks breast tumor cell invasion and metastasis.
abstract: BACKGROUND
  DNA methylation-induced silencing of genes encoding tumor suppressors is common in many types of cancer, but little is known about how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes protein kinase D1 (PKD1), a serine/threonine kinase that is expressed in cells of the normal mammary gland, where it maintains the epithelial phenotype by preventing epithelial-to-mesenchymal transition.
  
  
  METHODS
  The status of PRKD1 promoter methylation was analyzed by reduced representation bisulfite deep sequencing, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR in invasive and noninvasive breast cancer lines, as well as in humans in 34 cases of "normal" tissue, 22 cases of ductal carcinoma in situ, 22 cases of estrogen receptor positive, HER2-negative (ER+/HER2-) invasive lobular carcinoma, 43 cases of ER+/HER2- invasive ductal carcinoma (IDC), 93 cases of HER2+ IDC and 96 cases of triple-negative IDC. A reexpression strategy using the DNA methyltransferase inhibitor decitabine was used in vitro in MDA-MB-231 cells as well as in vivo in a tumor xenograft model and measured by RT-PCR, immunoblotting and immunohistochemistry. The effect of PKD1 reexpression on cell invasion was analyzed in vitro by transwell invasion assay. Tumor growth and metastasis were monitored in vivo using the IVIS Spectrum Pre-clinical In Vivo Imaging System.
  
  
  RESULTS
  Herein we show that the gene promoter of PRKD1 is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, we show that reversion of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion.
  
  
  CONCLUSIONS
  Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells.
authors: Sahra
authors: S
authors: Borges
authors: Heike
authors: H
authors: Döppler
authors: Edith A
authors: EA
authors: Perez
authors: Cathy A
authors: CA
authors: Andorfer
authors: Zhifu
authors: Z
authors: Sun
authors: Panos Z
authors: PZ
authors: Anastasiadis
authors: E
authors: E
authors: Thompson
authors: Xochiquetzal J
authors: XJ
authors: Geiger
authors: Peter
authors: P
authors: Storz
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000188:drug therapy / Q000235:genetics / Q000556:secondary; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000188:drug therapy / Q000235:genetics / Q000556:secondary; D018275:Carcinoma, Lobular / Q000188:drug therapy / Q000235:genetics / Q000556:secondary; D002465:Cell Movement; D049109:Cell Proliferation; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009361:Neoplasm Invasiveness; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046888:Tissue Array Analysis; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D000077209:Decitabine; C089212:protein kinase D; D011493:Protein Kinase C; D001374:Azacitidine
references: Oncogene. 2007 May 31;26(26):3789-96
references: 17213821
references: Breast Cancer Res. 2009;11(1):R13
references: 19243594
references: Int J Oncol. 2012 Jun;40(6):1839-48
references: 22426824
references: J Pathol. 2003 Oct;201(2):213-20
references: 14517838
references: Nature. 1982 Feb 25;295(5851):667-71
references: 6173772
references: Physiology (Bethesda). 2011 Feb;26(1):23-33
references: 21357900
references: Mol Oncol. 2010 Jun;4(3):242-54
references: 20627830
references: N Engl J Med. 2002 Dec 19;347(25):1999-2009
references: 12490681
references: Science. 2003 Nov 14;302(5648):1153; author reply 1153
references: 14615517
references: J Biol Chem. 2012 Sep 21;287(39):32367-80
references: 22791710
references: Nature. 2012 May 16;486(7403):400-4
references: 22722201
references: Nat Rev Cancer. 2011 Sep 23;11(10):726-34
references: 21941284
references: FEBS Lett. 2007 Sep 4;581(22):4279-87
references: 17707375
references: Oncogene. 2005 Jul 7;24(29):4660-71
references: 15897907
references: J Biol Chem. 2011 Sep 30;286(39):34254-61
references: 21832093
references: Cancer Res. 2011 Feb 15;71(4):1292-301
references: 21285253
references: J Biol Chem. 2004 Apr 9;279(15):15161-6
references: 14722112
references: Nat Rev Cancer. 2006 Feb;6(2):107-16
references: 16491070
references: Nature. 2008 Apr 3;452(7187):564-70
references: 18385730
references: Cancer Res. 1998 Jan 1;58(1):95-101
references: 9426064
references: Nat Cell Biol. 2009 May;11(5):545-56
references: 19329994
references: Nature. 2005 Jul 28;436(7050):518-24
references: 16049480
references: Nat Cell Biol. 2002 Jul;4(7):540-4
references: 12080349
references: Am J Hum Genet. 2002 Jun;70(6):1389-97
references: 11992247
references: Carcinogenesis. 2008 Mar;29(3):629-37
references: 18283041
references: Nature. 2000 Aug 17;406(6797):747-52
references: 10963602
references: J Biol Chem. 2010 Jun 11;285(24):18672-83
references: 20363754
references: Science. 2006 Oct 13;314(5797):268-74
references: 16959974
references: Anticancer Res. 2013 Feb;33(2):393-9
references: 23393329
references: CA Cancer J Clin. 2011 Mar-Apr;61(2):69-90
references: 21296855
references: Biochem Biophys Res Commun. 2003 Jul 25;307(2):254-60
references: 12859948
references: Clin Exp Metastasis. 2007;24(8):647-56
references: 17968664
references: Cancer Res. 2009 Jul 15;69(14):5634-8
references: 19567672
references: Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12754-9
references: 10535995
references: J Biol Chem. 2012 Mar 16;287(12):9473-83
references: 22228765
references: Cancer Cell. 2012 Nov 13;22(5):601-14
references: 23153534
references: Int J Oncol. 2012 Sep;41(3):910-8
references: 22767021
references: PLoS One. 2012;7(1):e30459
references: 22276203
references: N Engl J Med. 2003 Nov 20;349(21):2042-54
references: 14627790
references: Nucleic Acids Res. 2011 Aug;39(15):e100
references: 21622959
references: Cancer Res. 2002 Feb 15;62(4):961-6
references: 11861364
references: Nature. 2009 Jun 18;459(7249):1005-9
references: 19421193
references: Nat Rev Cancer. 2002 Mar;2(3):161-74
references: 11990853
references: Cell. 1980 May;20(1):85-93
references: 6156004
references: J Clin Invest. 2009 Sep;119(9):2678-90
references: 19726885
references: Int J Cancer. 2003 Dec 20;107(6):970-5
references: 14601057
references: Cancer Res. 2002 Nov 15;62(22):6634-8
references: 12438260
references: Cell Signal. 2009 Feb;21(2):253-63
references: 19038333
references: PLoS One. 2011 Feb 25;6(2):e17490
references: 21364760
references: J Clin Invest. 2005 Jan;115(1):44-55
references: 15630443
references: Cancer Res. 2010 Oct 15;70(20):7810-9
references: 20940406
references: J Mammary Gland Biol Neoplasia. 2010 Jun;15(2):117-34
references: 20490631