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

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Tumor Biology

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.

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References

  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.

    Article  CAS  PubMed  Google Scholar 

  2. Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature. 2002;418:935–41.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  4. Fu L, Lee CC. The circadian clock: pacemaker and tumour suppressor. Nat Rev Cancer. 2003;3:350–61.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Gauger MA, Sancar A. Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer. Cancer Res. 2005;65:6828–34.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  21. McCain J. The cancer genome atlas: new weapon in old war? Biotechnol Healthc. 2006;3:46–51B.

    PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kai K, Arima Y, Kamiya T, Saya H. Breast cancer stem cells. Breast Cancer. 2010;17:80–5.

    Article  PubMed  Google Scholar 

  30. Marhaba R, Zoller M. Cd44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol. 2004;35:211–31.

    Article  CAS  PubMed  Google Scholar 

  31. Louderbough JM, Schroeder JA. Understanding the dual nature of cd44 in breast cancer progression. Mol Cancer Res. 2011;9:1573–86.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

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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.

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Author notes

  1. Yingying Mao

    Present address: College of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, China

Authors and Affiliations

  1. School of Public Health, Yale University, New Haven, CT, USA

    Yingying Mao, Alan Fu, Daniel I. Jacobs & Yong Zhu

  2. Department of Epidemiology and Health Statistics, Zhejiang University School of Public Health, Hangzhou, Zhejiang Province, China

    Yingying Mao, Mingjuan Jin & Kun Chen

  3. Department of Epidemiology, Tulane School of Public Health and Tropical Medicine and Tulane Cancer Center, New Orleans, LA, USA

    Aaron E. Hoffman

  4. College of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, China

    Yong Zhu

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  1. Yingying Mao
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Correspondence to Yong Zhu.

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Mao, Y., Fu, A., Hoffman, A.E. et al. The circadian gene CRY2 is associated with breast cancer aggressiveness possibly via epigenomic modifications. Tumor Biol. 36, 3533–3539 (2015). https://doi.org/10.1007/s13277-014-2989-3

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  • DOI: https://doi.org/10.1007/s13277-014-2989-3

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