Embryonic NANOG Activity Defines Colorectal Cancer Stem Cells and Modulates through AP1‐ and TCF‐dependent Mechanisms

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG‐pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1‐expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear β‐cateninHigh. Moreover, single NANOG1‐CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem‐like Wnt‐associated target genes. Furthermore, we show that NANOG1‐expression is positively regulated by c‐JUN and β‐catenin/TCF4. Ectopic expression of c‐Jun in murine ApcMin/+‐ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c‐JUN binds to the NANOG1‐promoter via the octamer M1 DNA element. Collectively, our data suggest that β‐Catenin/TCF4 and c‐JUN together drive a subpopulation of CRC tumor cells that adopt a stem‐like phenotype via the NANOG1‐promoter. STEM Cells2012;30:2076–2087

[1]  H. Nakagama,et al.  Differential expression of nanog1 and nanogp8 in colon cancer cells. , 2012, Biochemical and biophysical research communications.

[2]  A. Vaiopoulou,et al.  OCT4 spliced variant OCT4B1 is expressed in human colorectal cancer , 2012, Molecular carcinogenesis.

[3]  Chulan Kwon,et al.  Notch Post-Translationally Regulates β-Catenin Protein in Stem and Progenitor Cells , 2011, Nature Cell Biology.

[4]  Chang Gun Kim,et al.  A Tcf/Lef element within the enhancer region of the human NANOG gene plays a role in promoter activation. , 2011, Biochemical and biophysical research communications.

[5]  J. Shen,et al.  NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation , 2011, Oncogene.

[6]  Hanns Lochmüller,et al.  Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines. , 2011, Stem cell research.

[7]  A. Ruiz i Altaba,et al.  Loss of WNT-TCF addiction and enhancement of HH-GLI1 signalling define the metastatic transition of human colon carcinomas , 2010, EMBO molecular medicine.

[8]  D. Galetta,et al.  Atypical expression and distribution of embryonic stem cell marker, OCT4, in human lung adenocarcinoma , 2010, Journal of surgical oncology.

[9]  M. Celeste Simon,et al.  O2 regulates stem cells through Wnt/β-catenin signalling , 2010, Nature Cell Biology.

[10]  S. Lyle,et al.  Quiescent, Slow-Cycling Stem Cell Populations in Cancer: A Review of the Evidence and Discussion of Significance , 2010, Journal of oncology.

[11]  A. Ruiz i Altaba,et al.  NANOG regulates glioma stem cells and is essential in vivo acting in a cross‐functional network with GLI1 and p53 , 2010, The EMBO journal.

[12]  Mauro Biffoni,et al.  Hedgehog controls neural stem cells through p53‐independent regulation of Nanog , 2010, The EMBO journal.

[13]  T. Dingermann,et al.  Transcriptional properties of human NANOG1 and NANOG2 in acute leukemic cells , 2010, Nucleic acids research.

[14]  Louis Vermeulen,et al.  Wnt activity defines colon cancer stem cells and is regulated by the microenvironment , 2010, Nature Cell Biology.

[15]  Jian-ming Li,et al.  Over-expression of Nanog predicts tumor progression and poor prognosis in colorectal cancer , 2010, Cancer biology & therapy.

[16]  K. Mimori,et al.  Defined factors induce reprogramming of gastrointestinal cancer cells , 2009, Proceedings of the National Academy of Sciences.

[17]  A. Ruiz i Altaba,et al.  Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion , 2009, EMBO molecular medicine.

[18]  B. Spencer‐Dene,et al.  The Links between Transcription, β-catenin/JNK Signaling, and Carcinogenesis , 2009, Molecular Cancer Research.

[19]  K. Mimori,et al.  Comprehensive Analysis of the Clinical Significance of Inducing Pluripotent Stemness-Related Gene Expression in Colorectal Cancer Cells , 2009, Annals of Surgical Oncology.

[20]  G. Daley,et al.  Functional Evidence that the Self‐Renewal Gene NANOG Regulates Human Tumor Development , 2009, Stem cells.

[21]  Hans Clevers,et al.  Crypt stem cells as the cells-of-origin of intestinal cancer , 2009, Nature.

[22]  Mike J. Mason,et al.  Role of the Murine Reprogramming Factors in the Induction of Pluripotency , 2009, Cell.

[23]  G. Pan,et al.  NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. , 2008, Cell stem cell.

[24]  M. Krause,et al.  Exploring the role of cancer stem cells in radioresistance , 2008, Nature Reviews Cancer.

[25]  A. Regev,et al.  An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors , 2008, Nature Genetics.

[26]  Eran Segal,et al.  Module map of stem cell genes guides creation of epithelial cancer stem cells. , 2008, Cell stem cell.

[27]  P. Dröge,et al.  A UTF1-based selection system for stable homogeneously pluripotent human embryonic stem cell cultures , 2007, Nucleic acids research.

[28]  M. Katoh.  Networking of WNT, FGF, Notch, BMP, and Hedgehog Signaling Pathways during Carcinogenesis , 2007, Stem Cell Reviews.

[29]  B. Doble,et al.  Functional redundancy of GSK-3α and GSK-3β in wnt/β-catenin signaling shown by using an allelic series of embryonic stem cell lines , 2007 .

[30]  Riccardo Fodde,et al.  Wnt/β-catenin signaling in cancer stemness and malignant behavior , 2007 .

[31]  T. Yokota,et al.  β-Catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells , 2007 .

[32]  Mala Sinha,et al.  Suppressing Wnt Signaling by the Hedgehog Pathway through sFRP-1* , 2006, Journal of Biological Chemistry.

[33]  F. Yi,et al.  Repression of Nanog Gene Transcription by Tcf3 Limits Embryonic Stem Cell Self-Renewal , 2006, Molecular and Cellular Biology.

[34]  Jin Han,et al.  NANOGP8 is a retrogene expressed in cancers , 2006, The FEBS journal.

[35]  M. Tsuneyoshi,et al.  Gli1, downregulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation , 2005, Gut.

[36]  A. Hart,et al.  The pluripotency homeobox gene NANOG is expressed in human germ cell tumors , 2005, Cancer.

[37]  A. Clark,et al.  Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma , 2005, Cancer.

[38]  Axel Behrens,et al.  Interaction of phosphorylated c-Jun with TCF4 regulates intestinal cancer development , 2005, Nature.

[39]  L. Hyslop,et al.  Downregulation of NANOG Induces Differentiation of Human Embryonic Stem Cells to Extraembryonic Lineages , 2005, Stem cells.

[40]  P. Robson,et al.  Transcriptional Regulation of Nanog by OCT4 and SOX2* , 2005, Journal of Biological Chemistry.

[41]  Zhenhua Yao,et al.  Isolation and characterization of the murine Nanog gene promoter , 2005, Cell Research.

[42]  H. Clevers,et al.  Wnt signalling in stem cells and cancer , 2005, Nature.

[43]  M. Tada,et al.  Octamer and Sox Elements Are Required for Transcriptional cis Regulation of Nanog Gene Expression , 2005, Molecular and Cellular Biology.

[44]  P. Holland,et al.  Eleven daughters of NANOG. , 2004, Genomics.

[45]  Chad A. Cowan,et al.  The Src Family of Tyrosine Kinases Is Important for Embryonic Stem Cell Self-renewal* , 2004, Journal of Biological Chemistry.

[46]  A. Behrens,et al.  The Ubiquitin Ligase SCFFbw7 Antagonizes Apoptotic JNK Signaling , 2004, Science.

[47]  J. Nichols,et al.  Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells , 2003, Cell.

[48]  I. Weissman,et al.  A role for Wnt signalling in self-renewal of haematopoietic stem cells , 2003, Nature.

[49]  R. Fodde,et al.  Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling , 2002, Nature Genetics.

[50]  Bert Vogelstein,et al.  DNMT1 and DNMT3b cooperate to silence genes in human cancer cells , 2002, Nature.

[51]  W F Bodmer,et al.  Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[52]  N. LeLeiko,et al.  Characterization of DNA-protein interactions within a distal regulatory element upstream of a mammalian housekeeping gene promoter. , 1992, The Journal of biological chemistry.

[53]  O. Jänne,et al.  Nucleotide sequence of the 5'-flanking region of the murine ornithine decarboxylase gene. , 1989, Nucleic acids research.

[54]  A. Eb,et al.  Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. , 1985, The EMBO journal.

[55]  G. Martin,et al.  Teratocarcinomas and mammalian embryogenesis. , 1980, Science.

[56]  R. Arceci.  PML targeting eradicates quiescent leukaemia-initiating cells , 2009 .

[57]  Guangjin Pan,et al.  Nanog and transcriptional networks in embryonic stem cell pluripotency , 2007, Cell Research.

[58]  T. Yokota,et al.  Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. , 2007, Biochemical and biophysical research communications.

[59]  R. Fodde,et al.  Wnt/beta-catenin signaling in cancer stemness and malignant behavior. , 2007, Current opinion in cell biology.

[60]  K. Bomsztyk,et al.  Protocol for the fast chromatin immunoprecipitation (ChIP) method , 2006, Nature Protocols.