Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

[1]  G. Dontu,et al.  Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells , 2004, Breast Cancer Research.

[2]  P. Laird,et al.  Epigenetic stem cell signature in cancer , 2007, Nature Genetics.

[3]  P. D’Eustachio,et al.  Essential role of STAT3 for embryonic stem cell pluripotency. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Wei Jiang,et al.  High-throughput DNA methylation profiling using universal bead arrays. , 2006, Genome research.

[5]  M. Katsuki,et al.  STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells , 1999, The EMBO journal.

[6]  A. Cassidy,et al.  Hypomethylation of retrotransposable elements correlates with genomic instability in non‐small cell lung cancer , 2009, International journal of cancer.

[7]  Kentaro Yamashita,et al.  Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. , 2006, Cancer cell.

[8]  Carlos Caldas,et al.  Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis , 2007, Nature Reviews Cancer.

[9]  A. Puisieux,et al.  Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition , 2008, PloS one.

[10]  F. Vesuna,et al.  Interleukin-6 induces an epithelial–mesenchymal transition phenotype in human breast cancer cells , 2009, Oncogene.

[11]  Z. Herceg,et al.  Epigenetic interplay between histone modifications and DNA methylation in gene silencing. , 2008, Mutation research.

[12]  S. Lehnert,et al.  Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. , 1987, Development.

[13]  Cassandra R. Farthing,et al.  Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes , 2008, PLoS genetics.

[14]  R. Weinberg,et al.  Enrichment of a population of mammary gland cells that form mammospheres and have in vivo repopulating activity. , 2007, Cancer research.

[15]  A. Bird.  DNA methylation patterns and epigenetic memory. , 2002, Genes & development.

[16]  Haiyan I. Li,et al.  Purification and unique properties of mammary epithelial stem cells , 2006, Nature.

[17]  Zohar Yakhini,et al.  Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer , 2007, Nature Genetics.

[18]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  L. Hennighausen,et al.  Signaling pathways in mammary gland development. , 2001, Developmental cell.

[20]  Paolo Boffetta,et al.  Quantitative analysis of DNA methylation profiles in lung cancer identifies aberrant DNA methylation of specific genes and its association with gender and cancer risk factors. , 2009, Cancer research.

[21]  M. Wicha,et al.  Regulation of Mammary Stem/Progenitor Cells by PTEN/Akt/β-Catenin Signaling , 2009, PLoS biology.

[22]  Sean V. Tavtigian,et al.  Hepatocellular Carcinoma Displays Distinct DNA Methylation Signatures with Potential as Clinical Predictors , 2010, PloS one.

[23]  I. Weissman,et al.  Stem cells, cancer, and cancer stem cells , 2001, Nature.

[24]  Hua Yu,et al.  Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells , 2001, Oncogene.

[25]  T. Hirano,et al.  Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors , 2000, Oncogene.

[26]  Dongxin Zhao,et al.  WNT/β-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells , 2006 .

[27]  Heike Knüpfer,et al.  Significance of interleukin-6 (IL-6) in breast cancer (review) , 2007, Breast Cancer Research and Treatment.

[28]  R. Beroukhim,et al.  Molecular definition of breast tumor heterogeneity. , 2007, Cancer cell.

[29]  A. Sultan,et al.  Co‐overexpression of Janus kinase 2 and signal transducer and activator of transcription 5a promotes differentiation of mammary cancer cells through reversal of epithelial–mesenchymal transition , 2008, Cancer science.

[30]  S. Thorgeirsson,et al.  Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. , 2007, The Journal of clinical investigation.

[31]  Michael Weber,et al.  Genomic patterns of DNA methylation: targets and function of an epigenetic mark. , 2007, Current opinion in cell biology.

[32]  Hitoshi Niwa,et al.  Open conformation chromatin and pluripotency. , 2007, Genes & development.

[33]  T. Dale,et al.  Wnt Signalling in Mammalian Development and Cancer , 2004, Cancer and Metastasis Reviews.

[34]  Taylor Jensen,et al.  Epigenetic inactivation of the HOXA gene cluster in breast cancer. , 2006, Cancer research.

[35]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[36]  A. Feinberg,et al.  The epigenetic progenitor origin of human cancer , 2006, Nature Reviews Genetics.

[37]  D. Santini,et al.  IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. , 2007, The Journal of clinical investigation.

[38]  E. Lander,et al.  The Mammalian Epigenome , 2007, Cell.

[39]  A. Smith,et al.  Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. , 1998, Genes & development.

[40]  Zena Werb,et al.  Stromal Effects on Mammary Gland Development and Breast Cancer , 2002, Science.

[41]  Taylor Jensen,et al.  Agglomerative epigenetic aberrations are a common event in human breast cancer. , 2008, Cancer research.

[42]  G. Dontu,et al.  In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. , 2003, Genes & development.

[43]  J. Blay,et al.  Prognostic value of serum levels of interleukin 6 and of serum and plasma levels of vascular endothelial growth factor in hormone-refractory metastatic breast cancer patients , 2003, British Journal of Cancer.

[44]  G. Dontu,et al.  Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. , 2006, Cancer research.

[45]  Peter A. Jones,et al.  The fundamental role of epigenetic events in cancer , 2002, Nature Reviews Genetics.

[46]  Kornelia Polyak,et al.  Breast cancer: origins and evolution. , 2007, The Journal of clinical investigation.

[47]  J. Visvader,et al.  Cancer stem cells in solid tumours: accumulating evidence and unresolved questions , 2008, Nature Reviews Cancer.

[48]  A. Bird,et al.  DNA methylation landscapes: provocative insights from epigenomics , 2008, Nature Reviews Genetics.

[49]  Angel Porgador,et al.  Cell type-specific DNA methylation patterns in the human breast , 2008, Proceedings of the National Academy of Sciences.

[50]  J. Margolick,et al.  Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance , 2007, Proceedings of the National Academy of Sciences.

[51]  Kelly M. McGarvey,et al.  A stem cell–like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing , 2007, Nature Genetics.

[52]  A. Feinberg,et al.  Genome-wide methylation analysis of human colon cancer reveals similar hypo- and hypermethylation at conserved tissue-specific CpG island shores , 2008, Nature Genetics.

[53]  Danila Coradini,et al.  Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. , 2005, Cancer research.