title: Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers.
abstract: BACKGROUND
  CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.
  
  
  METHODS
  Quantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.
  
  
  RESULTS
  This study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.
  
  
  CONCLUSIONS
  Our data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. akw582@mail.usask.ca.
authors.forename: Alison K
authors.initials: AK
authors.lastname: Ward
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. pam187@mail.usask.ca.
authors.forename: Paul
authors.initials: P
authors.lastname: Mellor
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. ses747@mail.usask.ca.
authors.forename: Shari E
authors.initials: SE
authors.lastname: Smith
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. scj503@mail.usask.ca.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kendall
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. nat163@mail.usask.ca.
authors.forename: Natasha A
authors.initials: NA
authors.lastname: Just
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. frederick.vizeacoumar@usask.ca.
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Vizeacoumar
authors.affiliation: Epidemiology and Performance Measurement, Saskatchewan Cancer Agency, 4-2105 8th Street, Saskatoon, SK, S7H 0T8, Canada. sabuj.sarker@saskcancer.ca.
authors.forename: Sabuj
authors.initials: S
authors.lastname: Sarker
authors.affiliation: Epidemiology and Performance Measurement, Saskatchewan Cancer Agency, 4-2105 8th Street, Saskatoon, SK, S7H 0T8, Canada. zoe.phillips@saskcancer.ca.
authors.forename: Zoe
authors.initials: Z
authors.lastname: Phillips
authors.affiliation: Epidemiology and Performance Measurement, Saskatchewan Cancer Agency, 4-2105 8th Street, Saskatoon, SK, S7H 0T8, Canada. riaz.alvi@saskcancer.ca.
authors.forename: Riaz
authors.initials: R
authors.lastname: Alvi
authors.affiliation: Department of Pathology and Lab Medicine, Royal University Hospital, 2841 - 103 Hospital Drive, Saskatoon, SK, S7N 0W8, Canada. anurag.saxena@usask.ca.
authors.forename: Anurag
authors.initials: A
authors.lastname: Saxena
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. franco.vizeacoumar@usask.ca.|Cancer Research, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. franco.vizeacoumar@usask.ca.
authors.forename: Franco J
authors.initials: FJ
authors.lastname: Vizeacoumar
authors.affiliation: Cancer Research, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. svein.carlsen@shaw.ca.
authors.forename: Svein A
authors.initials: SA
authors.lastname: Carlsen
authors.affiliation: Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. deborah.anderson@saskcancer.ca.|Cancer Research, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. deborah.anderson@saskcancer.ca.
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Anderson
mesh_terms: D000368:Aged; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D018572:Disease-Free Survival; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D011379:Prognosis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D064726:Triple Negative Breast Neoplasms / Q000145:classification / Q000235:genetics* / Q000473:pathology; D056811:Unfolded Protein Response / Q000235:genetics
chemical_list: C485618:CREB3L1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger
references.citation: Hum Pathol. 2013 Aug;44(8):1577-85
references.pmid: 23517923
references.citation: Breast Cancer Res Treat. 2003 Apr;78(3):323-35
references.pmid: 12755491
references.citation: Cell Host Microbe. 2011 Jul 21;10(1):65-74
references.pmid: 21767813
references.citation: Clin Chem. 2015 Jan;61(1):290-6
references.pmid: 25361948
references.citation: J Biol Chem. 2004 Jun 18;279(25):25935-8
references.pmid: 15070890
references.citation: EMBO J. 2005 Oct 5;24(19):3470-81
references.pmid: 16148948
references.citation: Cell Death Differ. 2012 Dec;19(12):1939-49
references.pmid: 22705851
references.citation: Mol Cancer. 2006;5:8
references.pmid: 16504004
references.citation: Cell. 2007 Nov 16;131(4):660-8
references.pmid: 18022361
references.citation: Int J Biochem Cell Biol. 2015 May;62:1-14
references.pmid: 25724736
references.citation: Brain Res Mol Brain Res. 1999 May 21;69(1):93-103
references.pmid: 10350641
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7690-4
references.pmid: 8755537
references.citation: Endocr Relat Cancer. 2006 Dec;13(4):1109-20
references.pmid: 17158757
references.citation: Sci Signal. 2013 Apr 2;6(269):pl1
references.pmid: 23550210
references.citation: Genome Res. 2002 Jun;12(6):996-1006
references.pmid: 12045153
references.citation: Mol Cell. 2000 Nov;6(5):1099-108
references.pmid: 11106749
references.citation: Nat Cell Biol. 2005 Feb;7(2):186-94
references.pmid: 15665855
references.citation: Epigenetics. 2014 Dec;9(12):1626-40
references.pmid: 25625847
references.citation: Ann Surg Oncol. 2010 Jul;17(7):1862-9
references.pmid: 20162457
references.citation: Cancer Res. 2005 Jul 1;65(13):5785-91
references.pmid: 15994954
references.citation: Am J Physiol Cell Physiol. 2014 Oct 1;307(7):C582-94
references.pmid: 25143348
references.citation: BMC Mol Biol. 2010;11:79
references.pmid: 21050451
references.citation: Nat Cell Biol. 2011 Mar;13(3):184-90
references.pmid: 21364565
references.citation: Sci Rep. 2014;4:7097
references.pmid: 25403569
references.citation: Histopathology. 1991 Nov;19(5):403-10
references.pmid: 1757079
references.citation: Cancer Res. 2004 Sep 1;64(17):5943-7
references.pmid: 15342372
references.citation: Br J Cancer. 1997;76(9):1234-40
references.pmid: 9365176
references.citation: Genes Dev. 1999 Jun 15;13(12):1501-12
references.pmid: 10385618
references.citation: Curr Protoc Hum Genet. 2014;82:7.24.1-13
references.pmid: 25042719
references.citation: PLoS One. 2015;10(6):e0129233
references.pmid: 26110425
references.citation: Front Pharmacol. 2013 Feb 11;4:10
references.pmid: 23403503
references.citation: Biochim Biophys Acta. 2012 Jun;1819(6):604-15
references.pmid: 22182827
references.citation: Cancer Res. 2008 Apr 1;68(7):2132-44
references.pmid: 18381418
references.citation: J Mol Med (Berl). 2007 Apr;85(4):331-41
references.pmid: 17216204
references.citation: J Natl Cancer Inst. 2005 Oct 19;97(20):1498-506
references.pmid: 16234563
references.citation: Mod Pathol. 1998 Feb;11(2):155-68
references.pmid: 9504686
references.citation: Cancer Metastasis Rev. 2009 Jun;28(1-2):219-23
references.pmid: 19172382
references.citation: Am J Pathol. 1998 Jul;153(1):5-9
references.pmid: 9665458
references.citation: FASEB J. 2007 Dec;21(14):4013-27
references.pmid: 17660348
references.citation: Nature. 2014 Apr 3;508(7494):103-7
references.pmid: 24670641
references.citation: Elife. 2012;1:e00090
references.pmid: 23256041
references.citation: Exp Mol Pathol. 2013 Dec;95(3):276-87
references.pmid: 24045095
references.citation: Methods Mol Biol. 2000;132:365-86
references.pmid: 10547847
references.citation: J Cell Biol. 2012 Jun 25;197(7):857-67
references.pmid: 22733998
references.citation: Oncogene. 2010 Jul 8;29(27):3881-95
references.pmid: 20453876
references.citation: Breast Cancer Res. 2008;10(5):R81
references.pmid: 18823530
references.citation: Int J Oncol. 2012 Aug;41(2):721-32
references.pmid: 22664488
references.citation: BMC Cancer. 2015;15:639
references.pmid: 26391005
references.citation: Methods Mol Biol. 2012;825:223-35
references.pmid: 22144248
references.citation: J Neurochem. 2006 Feb;96(4):1090-100
references.pmid: 16417584
references.citation: Cancer Discov. 2012 May;2(5):401-4
references.pmid: 22588877
references.citation: Breast Cancer Res. 2008;10(5):R82
references.pmid: 18826564
references.citation: J Clin Oncol. 1999 May;17(5):1442-8
references.pmid: 10334529
references.citation: Mol Cell Biol. 2013 Dec;33(24):4985-95
references.pmid: 24126059
references.citation: Int J Cancer. 2008 Jul 1;123(1):85-8
references.pmid: 18386815
references.citation: Oncogene. 1998 May 7;16(18):2321-31
references.pmid: 9620549
references.citation: Biochem Cell Biol. 2007 Dec;85(6):751-8
references.pmid: 18059533
references.citation: Pediatr Res. 2015 Dec;78(6):609-17
references.pmid: 26309238
references.citation: Proc Natl Acad Sci U S A. 1984 Nov;81(22):6993-7
references.pmid: 6209710
references.citation: Breast Cancer Res Treat. 2010 Oct;123(3):725-31
references.pmid: 20020197