Tumor Biology

, Volume 36, Issue 5, pp 3533–3539 | Cite as

The circadian gene CRY2 is associated with breast cancer aggressiveness possibly via epigenomic modifications

  • Yingying Mao
  • Alan Fu
  • Aaron E. Hoffman
  • Daniel I. Jacobs
  • Mingjuan Jin
  • Kun Chen
  • Yong Zhu
Research Article

Abstract

Although the role of core circadian gene cryptochrome 2 (CRY2) in breast tumorigenesis has been demonstrated, the correlations of CRY2 with clinical parameters in breast cancer patients and its involvement in epigenetic processes such as DNA methylation remain relatively unexplored. In the current study, we first queried the Oncomine database and the Gene Expression-Based Outcome for Breast Cancer Online (GOBO) database to identify associations between CRY2 expression levels and clinical parameters in breast cancer patients. We then silenced CRY2 in vitro and performed a genome-wide methylation array to determine the epigenetic impact of CRY2 silencing. The Ingenuity Pathway Analysis software was used to further explore the genes exhibiting altered methylation identified using the array. We found that CRY2 was frequently down-regulated in breast cancer tissue compared to adjacent normal tissue or breast tissue from healthy controls. Lower CRY2 expression was associated with estrogen receptor (ER)-negativity (P < 0.0001), higher tumor grade (P < 0.0001), and shorter overall survival time in breast cancer patients (HR = 1.44, 95 % confidence interval (CI) 1.09–1.91). Genome-wide methylation analysis showed that a total of 515 CpG sites were hypermethylated following CRY2 knockdown, while 730 sites were hypomethylated. The pathway analysis revealed several cancer-relevant networks with genes exhibiting significantly altered methylation following CRY2 silencing. These findings suggest that the core circadian gene CRY2 is associated with breast cancer progression and prognosis, and that knockdown of CRY2 causes the epigenetic dysregulation of genes involved in cancer-relevant pathways, which provide further evidence supporting a role of the circadian system in breast tumorigenesis.

Keywords

CRY2 Breast cancer Biomarker DNA methylation Network analysis 

Notes

Acknowledgments

This work was supported by the National Institutes of Environmental Health Sciences (NIEHS) grant ES018915.

Conflicts of interest

The authors declare that they have no competing interests.

Authors’ contributions

YM carried out the DNA methylation assay, public data search and analysis, and drafted the manuscript. AF and DIJ participated in the data analysis and manuscript preparation. AEH constructed the expression vector and helped with the DNA methylation analysis. MJ and KC participated in the design of the study and helped the statistical analysis. YZ conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Supplementary material

13277_2014_2989_MOESM1_ESM.doc (206 kb)
Supplementary Table 1 and 2 (DOC 206 kb)

References

  1. 1.
    Kondratov RV, Gorbacheva VY, Antoch MP. The role of mammalian circadian proteins in normal physiology and genotoxic stress responses. Curr Top Dev Biol. 2007;78:173–216.CrossRefPubMedGoogle Scholar
  2. 2.
    Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature. 2002;418:935–41.CrossRefPubMedGoogle Scholar
  3. 3.
    Duffield GE, Best JD, Meurers BH, Bittner A, Loros JJ, Dunlap JC. Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells. Curr Biol. 2002;12:551–7.CrossRefPubMedGoogle Scholar
  4. 4.
    Fu L, Lee CC. The circadian clock: pacemaker and tumour suppressor. Nat Rev Cancer. 2003;3:350–61.CrossRefPubMedGoogle Scholar
  5. 5.
    Thresher RJ, Vitaterna MH, Miyamoto Y, Kazantsev A, Hsu DS, Petit C, et al. Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses. Science. 1998;282:1490–4.CrossRefPubMedGoogle Scholar
  6. 6.
    van der Spek PJ, Kobayashi K, Bootsma D, Takao M, Eker AP, Yasui A. Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors. Genomics. 1996;37:177–82.CrossRefPubMedGoogle Scholar
  7. 7.
    Unsal-Kacmaz K, Mullen TE, Kaufmann WK, Sancar A. Coupling of human circadian and cell cycles by the timeless protein. Mol Cell Biol. 2005;25:3109–16.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gauger MA, Sancar A. Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer. Cancer Res. 2005;65:6828–34.CrossRefPubMedGoogle Scholar
  9. 9.
    Zhu Y, Stevens RG, Hoffman AE, Fitzgerald LM, Kwon EM, Ostrander EA, et al. Testing the circadian gene hypothesis in prostate cancer: a population-based case-control study. Cancer Res. 2009;69:9315–22.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hoffman AE, Zheng T, Stevens RG, Ba Y, Zhang Y, Leaderer D, et al. Clock-cancer connection in non-Hodgkin’s lymphoma: a genetic association study and pathway analysis of the circadian gene cryptochrome 2. Cancer Res. 2009;69:3605–13.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hoffman AE, Zheng T, Yi CH, Stevens RG, Ba Y, Zhang Y, et al. The core circadian gene cryptochrome 2 influences breast cancer risk, possibly by mediating hormone signaling. Cancer Prevent Res (Philadelphia, Pa). 2010;3:539–48.CrossRefGoogle Scholar
  12. 12.
    Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Varambally R, Yu J, Briggs BB, et al. Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia. 2007;9:166–80.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Turashvili G, Bouchal J, Baumforth K, Wei W, Dziechciarkova M, Ehrmann J, et al. Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis. BMC Cancer. 2007;7:55.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557–63.CrossRefPubMedGoogle Scholar
  15. 15.
    Richardson AL, Wang ZC, De Nicolo A, Lu X, Brown M, Miron A, et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell. 2006;9:121–32.CrossRefPubMedGoogle Scholar
  16. 16.
    Gluck S, Ross JS, Royce M, McKenna Jr EF, Perou CM, Avisar E, et al. Tp53 genomics predict higher clinical and pathologic tumor response in operable early-stage breast cancer treated with docetaxel-capecitabine +/− trastuzumab. Breast Cancer Res Treat. 2012;132:781–91.CrossRefPubMedGoogle Scholar
  17. 17.
    Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC. Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res. 2009;11:R7.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Radvanyi L, Singh-Sandhu D, Gallichan S, Lovitt C, Pedyczak A, Mallo G, et al. The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer. Proc Natl Acad Sci U S A. 2005;102:11005–10.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Zhao H, Langerod A, Ji Y, Nowels KW, Nesland JM, Tibshirani R, et al. Different gene expression patterns in invasive lobular and ductal carcinomas of the breast. Mol Biol Cell. 2004;15:2523–36.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008;14:518–27.CrossRefPubMedGoogle Scholar
  21. 21.
    McCain J. The cancer genome atlas: new weapon in old war? Biotechnol Healthc. 2006;3:46–51B.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Ringner M, Fredlund E, Hakkinen J, Borg A, Staaf J. GOBO: Gene Expression-Based Outcome for Breast Cancer Online. PLoS One. 2011;6:e17911.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Calvano SE, Xiao W, Richards DR, Felciano RM, Baker HV, Cho RJ, et al. A network-based analysis of systemic inflammation in humans. Nature. 2005;437:1032–7.CrossRefPubMedGoogle Scholar
  24. 24.
    Hoffman AE, Zheng T, Ba Y, Stevens RG, Yi CH, Leaderer D, et al. Phenotypic effects of the circadian gene cryptochrome 2 on cancer-related pathways. BMC Cancer. 2010;10:110.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Xiang S, Coffelt SB, Mao L, Yuan L, Cheng Q, Hill SM. Period-2: A tumor suppressor gene in breast cancer. J Circadian Rhythms. 2008;6:4.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Bourguignon LY, Singleton PA, Zhu H, Diedrich F. Hyaluronan-mediated cd44 interaction with rhogef and rho kinase promotes grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression. J Biol Chem. 2003;278:29420–34.CrossRefPubMedGoogle Scholar
  27. 27.
    Orian-Rousseau V, Morrison H, Matzke A, Kastilan T, Pace G, Herrlich P, et al. Hepatocyte growth factor-induced ras activation requires erm proteins linked to both cd44v6 and f-actin. Mol Biol Cell. 2007;18:76–83.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Bourguignon LY, Wong G, Earle C, Krueger K, Spevak CC. Hyaluronan-cd44 interaction promotes c-src-mediated twist signaling, microrna-10b expression, and rhoa/rhoc up-regulation, leading to rho-kinase-associated cytoskeleton activation and breast tumor cell invasion. J Biol Chem. 2010;285:36721–35.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kai K, Arima Y, Kamiya T, Saya H. Breast cancer stem cells. Breast Cancer. 2010;17:80–5.CrossRefPubMedGoogle Scholar
  30. 30.
    Marhaba R, Zoller M. Cd44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol. 2004;35:211–31.CrossRefPubMedGoogle Scholar
  31. 31.
    Louderbough JM, Schroeder JA. Understanding the dual nature of cd44 in breast cancer progression. Mol Cancer Res. 2011;9:1573–86.CrossRefPubMedGoogle Scholar
  32. 32.
    Vaillant F, Merino D, Lee L, Breslin K, Pal B, Ritchie ME, et al. Targeting bcl-2 with the bh3 mimetic abt-199 in estrogen receptor-positive breast cancer. Cancer Cell. 2013;24:120–9.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Yingying Mao
    • 1
    • 2
  • Alan Fu
    • 1
  • Aaron E. Hoffman
    • 3
  • Daniel I. Jacobs
    • 1
  • Mingjuan Jin
    • 2
  • Kun Chen
    • 2
  • Yong Zhu
    • 1
    • 4
  1. 1.School of Public HealthYale UniversityNew HavenUSA
  2. 2.Department of Epidemiology and Health StatisticsZhejiang University School of Public HealthHangzhouChina
  3. 3.Department of EpidemiologyTulane School of Public Health and Tropical Medicine and Tulane Cancer CenterNew OrleansUSA
  4. 4.College of Basic Medical ScienceZhejiang Chinese Medical UniversityHangzhouChina

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