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A Phenotype-Based RNAi Screening for Ras-ERK/MAPK Signaling-Associated Stem Cell Regulators in C. elegans

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RNAi and Small Regulatory RNAs in Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1622))

Abstract

Stem cells have the ability to self-renew and to generate differentiated cell types. A regulatory network that controls this balance is critical for stem cell homeostasis and normal animal development. Particularly, Ras-ERK/MAPK signaling pathway is critical for stem cell self-renewal and differentiation in mammals, including humans. Aberrant regulation of Ras-ERK/MAPK signaling pathway results in either stem cell or overproliferation. Therefore, the identification of Ras-ERK/MAPK signaling pathway-associated regulators is critical to understand the mechanism of stem cell (possibly cancer stem cell) control. In this report, using the nematode C. elegans mutants, we developed a methodology for a phenotype-based RNAi screening that identifies stem cell regulator genes associated with Ras-ERK/MAPK signaling within the context of a whole organism. Importantly, this phenotype-based RNAi screening can be applied for other stem cell-associated signaling pathways such as Wnt/β-catenin and Notch using the C. elegans.

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References

  1. Morrison SJ, Kimble J (2006) Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441(7097):1068–1074. doi:10.1038/nature04956

    Article  CAS  PubMed  Google Scholar 

  2. Huang G, Ye S, Zhou X, Liu D, Ying QL (2015) Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network. Cell Mol Life Sci 72(9):1741–1757. doi:10.1007/s00018-015-1833-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111. doi:10.1038/35102167

    Article  CAS  PubMed  Google Scholar 

  4. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453(7194):519–523. doi:10.1038/nature06968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Li J, Wang G, Wang C, Zhao Y, Zhang H, Tan Z, Song Z, Ding M, Deng H (2007) MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation 75(4):299–307. doi:10.1111/j.1432-0436.2006.00143.x

    Article  CAS  PubMed  Google Scholar 

  6. Burdon T, Stracey C, Chambers I, Nichols J, Smith A (1999) Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol 210(1):30–43. doi:10.1006/dbio.1999.9265

    Article  CAS  PubMed  Google Scholar 

  7. Salasznyk RM, Klees RF, Hughlock MK, Plopper GE (2004) ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin. Cell Commun Adhes 11(5–6):137–153. doi:10.1080/15419060500242836

    Article  CAS  PubMed  Google Scholar 

  8. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460(7251):118–122. doi:10.1038/nature08113

    Article  CAS  PubMed  Google Scholar 

  9. Hamilton WB, Brickman JM (2014) Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm. Cell Rep 9(6):2056–2070. doi:10.1016/j.celrep.2014.11.032

    Article  CAS  PubMed  Google Scholar 

  10. Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, Lin X, Hahm HS, Hao E, Hayek A, Ding S (2009) A chemical platform for improved induction of human iPSCs. Nat Methods 6(11):805–808. doi:10.1038/nmeth.1393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sundaram MV (2006) RTK/Ras/MAPK signaling. WormBook:1–19. doi:10.1895/wormbook.1.80.1

  12. Whelan JT, Hollis SE, Cha DS, Asch AS, Lee MH (2012) Post-transcriptional regulation of the Ras-ERK/MAPK signaling pathway. J Cell Physiol 227(3):1235–1241. doi:10.1002/jcp.22899

    Article  CAS  PubMed  Google Scholar 

  13. Cha DS, Datla US, Hollis SE, Kimble J, Lee MH (2012) The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline. Biochim Biophys Acta 1823(10):1847–1855. doi:10.1016/j.bbamcr.2012.07.006

    Article  CAS  PubMed  Google Scholar 

  14. Lee MH, Hook B, Lamont LB, Wickens M, Kimble J (2006) LIP-1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans. EMBO J 25(1):88–96. doi:10.1038/sj.emboj.7600901

    Article  CAS  PubMed  Google Scholar 

  15. Vaid S, Ariz M, Chaturbedi A, Kumar GA, Subramaniam K (2013) PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells. Development 140(8):1645–1654. doi:10.1242/dev.088013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Morgan CT, Lee MH, Kimble J (2010) Chemical reprogramming of Caenorhabditis elegans germ cell fate. Nat Chem Biol 6(2):102–104. doi:10.1038/nchembio.282. nchembio.282 [pii

  17. Sell S (1993) Cellular origin of cancer: dedifferentiation or stem cell maturation arrest? Environ Health Perspect 101(Suppl 5):15–26

    Article  PubMed  PubMed Central  Google Scholar 

  18. Friedmann-Morvinski D, Verma IM (2014) Dedifferentiation and reprogramming: origins of cancer stem cells. EMBO Rep 15(3):244–253. doi:10.1002/embr.201338254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lewis TS, Hunt JB, Aveline LD, Jonscher KR, Louie DF, Yeh JM, Nahreini TS, Resing KA, Ahn NG (2000) Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell 6(6):1343–1354

    Article  CAS  PubMed  Google Scholar 

  20. Arur S, Ohmachi M, Nayak S, Hayes M, Miranda A, Hay A, Golden A, Schedl T (2009) Multiple ERK substrates execute single biological processes in Caenorhabditis elegans germ-line development. Proc Natl Acad Sci U S A 106(12):4776–4781. doi:10.1073/pnas.0812285106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tullai JW, Schaffer ME, Mullenbrock S, Kasif S, Cooper GM (2004) Identification of transcription factor binding sites upstream of human genes regulated by the phosphatidylinositol 3-kinase and MEK/ERK signaling pathways. J Biol Chem 279(19):20167–20177. doi:10.1074/jbc.M309260200

    Article  CAS  PubMed  Google Scholar 

  22. Kosako H, Yamaguchi N, Aranami C, Ushiyama M, Kose S, Imamoto N, Taniguchi H, Nishida E, Hattori S (2009) Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport. Nat Struct Mol Biol 16(10):1026–1035. doi:10.1038/nsmb.1656

    Article  CAS  PubMed  Google Scholar 

  23. Bachorik JL, Kimble J (2005) Redundant control of the Caenorhabditis elegans sperm/oocyte switch by PUF-8 and FBF-1, two distinct PUF RNA-binding proteins. Proc Natl Acad Sci U S A 102(31):10893–10897. doi:10.1073/pnas.0504593102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hajnal A, Berset T (2002) The C. elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes. EMBO J 21(16):4317–4326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Benson JA, Cummings EE, O'Reilly LP, Lee MH, Pak SC (2014) A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate. Methods 68(3):529–535. doi:10.1016/j.ymeth.2014.05.011

    Article  CAS  PubMed  Google Scholar 

  26. Lackner MR, Kim SK (1998) Genetic analysis of the Caenorhabditis elegans MAP kinase gene mpk-1. Genetics 150(1):103–117

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Eisenmann DM, Kim SK (1997) Mechanism of activation of the Caenorhabditis elegans ras homologue let-60 by a novel, temperature-sensitive, gain-of-function mutation. Genetics 146(2):553–565

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77(1):71–94

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Subramaniam K, Seydoux G (2003) Dedifferentiation of primary spermatocytes into germ cell tumors in C. elegans lacking the pumilio-like protein PUF-8. Curr Biol 13(2):134–139. doi:S0960982203000058 [pii]

    Article  CAS  PubMed  Google Scholar 

  30. O'Reilly LP, Long OS, Cobanoglu MC, Benson JA, Luke CJ, Miedel MT, Hale P, Perlmutter DH, Bahar I, Silverman GA, Pak SC (2014) A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of alpha1-antitrypsin deficiency. Hum Mol Genet 23(19):5123–5132. doi:10.1093/hmg/ddu236

    Article  PubMed  PubMed Central  Google Scholar 

  31. Lee MH, Ohmachi M, Arur S, Nayak S, Francis R, Church D, Lambie E, Schedl T (2007) Multiple functions and dynamic activation of MPK-1 extracellular signal-regulated kinase signaling in Caenorhabditis elegans germline development. Genetics 177(4):2039–2062. doi:10.1534/genetics.107.081356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Grant B, Hirsh D (1999) Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 10(12):4311–4326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ward S, Roberts TM, Strome S, Pavalko FM, Hogan E (1986) Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins. J Cell Biol 102(5):1778–1786

    Article  CAS  PubMed  Google Scholar 

  34. Yoon DS, Pendergrass DL, Lee MH (2016) A simple and rapid method for combining fluorescent in situ RNA hybridization (FISH) and immunofluorescence in the C. elegans germline. MethodsX. 3:378–85. PMID: 27257608

    Google Scholar 

  35. Kobet RA, Pan X, Zhang B, Pak SC, Asch AS, Lee MH (2014) Caenorhabditis elegans: a model system for anti-cancer drug discovery and therapeutic target identification. Biomol Ther 22(5):371–383. doi:10.4062/biomolther.2014.084

    Article  CAS  Google Scholar 

  36. Morgan CT, Noble D, Kimble J (2013) Mitosis-meiosis and sperm-oocyte fate decisions are separable regulatory events. Proc Natl Acad Sci U S A 110(9):3411–3416. doi:10.1073/pnas.1300928110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgment

We thank the members of the Lee’s laboratory (especially Eunsuk Kim) and Faten A. Taki (Biology, ECU) as well as Jiwoo Lee and Jiah Lee (St. Peter School, Greenville, NC) for critical reading. This work was supported in part by the Vidant Medical Center Cancer Research and Education Fund, Brody Brothers Grant, Oncology Internal Grant, Brody Seed/Bridge Grant, and NIGMS (1R15GM112174-01A1) grant to MHL. C. elegans strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

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Authors and Affiliations

  1. Division of Hematology/Oncology, Department of Medicine, Brody School of Medicine at East Carolina University, Greenville, NC, 27834, USA

    Myon-Hee Lee Ph.D. & Dong Suk Yoon

  2. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA

    Myon-Hee Lee Ph.D.

Authors
  1. Myon-Hee Lee Ph.D.
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  2. Dong Suk Yoon
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Correspondence to Myon-Hee Lee Ph.D. .

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Editors and Affiliations

  1. Department of Biology, East Carolina University, Greenville, North Carolina, USA

    Baohong Zhang

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Lee, MH., Yoon, D.S. (2017). A Phenotype-Based RNAi Screening for Ras-ERK/MAPK Signaling-Associated Stem Cell Regulators in C. elegans . In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7108-4_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7106-0

  • Online ISBN: 978-1-4939-7108-4

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