Apoptosis

, Volume 17, Issue 7, pp 666–678 | Cite as

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance

  • Craig D. Simpson
  • Rose Hurren
  • Dahlia Kasimer
  • Neil MacLean
  • Yanina Eberhard
  • Troy Ketela
  • Jason Moffat
  • Aaron D. Schimmer
Original Paper

Abstract

Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.

Keywords

Anoikis Genome wide shRNA screen α/β Hydrolase domain containing 4 ABHD4 

Notes

Ackonwledgments

This work was supported by the Canadian Cancer Society Research Institute, the Canadian Institutes of Health Research, the Ontario Ministry of Research and Innovation, and the Ministry of Long Term Health and Planning in the Province of Ontario. CDS holds a Banting and Best Doctoral Research Fellowship from the Canadian Institutes of Health Research. ADS is a Leukemia and Lymphoma Society Scholar in Clinical Research.

References

  1. 1.
    Meredith JE Jr, Fazeli B, Schwartz MA (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4(9):953–961PubMedGoogle Scholar
  2. 2.
    Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124(4):619–626PubMedCrossRefGoogle Scholar
  3. 3.
    Aoudjit F, Vuori K (2001) Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis. J Cell Biol 152(3):633–643PubMedCrossRefGoogle Scholar
  4. 4.
    Rytömaa M, Martins LM, Downward J (1999) Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr Biol 9(18):1043–1046PubMedCrossRefGoogle Scholar
  5. 5.
    Stupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA (2001) Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol 155(3):459–470PubMedCrossRefGoogle Scholar
  6. 6.
    Rytömaa M, Lehmann K, Downward J (2000) Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling. Oncogene 19(39):4461–4468PubMedCrossRefGoogle Scholar
  7. 7.
    Jan Y, Matter M, J-t Pai, Chen Y-L, Pilch J, Komatsu M, Ong E, Fukuda M, Ruoslahti E (2004) A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell 116(5):751–762PubMedCrossRefGoogle Scholar
  8. 8.
    Ruoslahti E, Reed JC (1994) Anchorage dependence, integrins, and apoptosis. Cell 77(4):477–478PubMedCrossRefGoogle Scholar
  9. 9.
    Krajewska M, Krajewski S, Banares S, Huang X, Turner B, Bubendorf L, Kallioniemi OP, Shabaik A, Vitiello A, Peehl D, Gao GJ, Reed JC (2003) Elevated expression of inhibitor of apoptosis proteins in prostate cancer. Clin Cancer Res 9(13):4914–4925PubMedGoogle Scholar
  10. 10.
    Mawji IA, Simpson CD, Gronda M, Williams MA, Hurren R, Henderson CJ, Datti A, Wrana JL, Schimmer AD (2007) A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis. Cancer Res 67(17):8307–8315PubMedCrossRefGoogle Scholar
  11. 11.
    Mawji IA, Simpson CD, Hurren R, Gronda M, Williams MA, Filmus J, Jonkman J, Da Costa RS, Wilson BC, Thomas MP, Reed JC, Glinsky GV, Schimmer AD (2007) Critical role for Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein in anoikis resistance and distant tumor formation. J Natl Cancer Inst 99(10):811–822PubMedCrossRefGoogle Scholar
  12. 12.
    Simpson CD, Mawji IA, Anyiwe K, Williams MA, Wang X, Venugopal AL, Gronda M, Hurren R, Cheng S, Serra S, Zavareh RB, Datti A, Wrana JL, Ezzat S, Schimmer AD (2009) Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation. Cancer Res 69(7):2739–2747PubMedCrossRefGoogle Scholar
  13. 13.
    Bello D, Webber MM, Kleinman HK, Wartinger DD, Rhim JS (1997) Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18. Carcinogenesis 18(6):1215–1223PubMedCrossRefGoogle Scholar
  14. 14.
    Tsao SW, Wang X, Liu Y, Cheung YC, Feng H, Zheng Z, Wong N, Yuen PW, Lo AKF, Wong YC, Huang DP (2002) Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes. Biochim Biophys Acta 1590(1–3):150–158PubMedCrossRefGoogle Scholar
  15. 15.
    Carter BZ, Gronda M, Wang Z, Welsh K, Pinilla C, Andreeff M, Schober WD, Nefzi A, Pond GR, Mawji IA, Houghten RA, Ostresh J, Brandwein J, Minden MD, Schuh AC, Wells RA, Messner H, Chun K, Reed JC, Schimmer AD (2005) Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells. Blood 105(10):4043–4050PubMedCrossRefGoogle Scholar
  16. 16.
    Ketela T, Heisler LE, Brown KR, Ammar R, Kasimer D, Surendra A, Ericson E, Blakely K, Karamboulas D, Smith AM, Durbic T, Arnoldo A, Cheung-Ong K, Koh JL, Gopal S, Cowley GS, Yang X, Grenier JK, Giaever G, Root DE, Moffat J, Nislow C (2011) A comprehensive platform for highly multiplexed mammalian functional genetic screens. BMC genomics 12(1):213PubMedCrossRefGoogle Scholar
  17. 17.
    Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK, Carpenter AE, Foo SY, Stewart SA, Stockwell BR, Hacohen N, Hahn WC, Lander ES, Sabatini DM, Root DE (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124(6):1283–1298PubMedCrossRefGoogle Scholar
  18. 18.
    Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK, Kaufman PD (2009) A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4(8):e6529PubMedCrossRefGoogle Scholar
  19. 19.
    Schimmer A, Thomas M, Hurren R, Gronda M, Pellecchia M, Pond G, Konopleva M, Gurfinkel D, Mawji I, Brown E, Reed J (2006) Identification of small molecules that sensitize resistant tumor cells to tumor necrosis factor-family death receptors. Cancer Res 66(4):2367–2375PubMedCrossRefGoogle Scholar
  20. 20.
    Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY (1996) Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134(3):793–799PubMedCrossRefGoogle Scholar
  21. 21.
    Attwell S, Roskelley C, Dedhar S (2000) The integrin-linked kinase (ILK) suppresses anoikis. Oncogene 19(33):3811–3815PubMedCrossRefGoogle Scholar
  22. 22.
    Duxbury M, Ito H, Zinner M, Ashley S, Whang E (2004) Focal adhesion kinase gene silencing promotes anoikis and suppresses metastasis of human pancreatic adenocarcinoma cells. Surgery 135(5):555–562PubMedCrossRefGoogle Scholar
  23. 23.
    Zoppi N, Barlati S, Colombi M (2008) FAK-independent alphavbeta3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts. Biochim Biophys Acta 1783(6):1177–1188PubMedCrossRefGoogle Scholar
  24. 24.
    Simon GM, Cravatt BF (2006) Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway. J Biol Chem 281(36):26465–26472PubMedCrossRefGoogle Scholar
  25. 25.
    Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258(5090):1946–1949PubMedCrossRefGoogle Scholar
  26. 26.
    Grimaldi C, Pisanti S, Laezza C, Malfitano AM, Santoro A, Vitale M, Caruso MG, Notarnicola M, Iacuzzo I, Portella G, Di Marzo V, Bifulco M (2006) Anandamide inhibits adhesion and migration of breast cancer cells. Exp Cell Res 312(4):363–373PubMedCrossRefGoogle Scholar
  27. 27.
    Vallorosi CJ, Day KC, Zhao X, Rashid MG, Rubin MA, Johnson KR, Wheelock MJ, Day ML (2000) Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution. J Biol Chem 275(5):3328–3334PubMedCrossRefGoogle Scholar
  28. 28.
    Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS (2002) The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111(1):29–40PubMedCrossRefGoogle Scholar
  29. 29.
    Simpson C, Anyiwe K, Schimmer A (2008) Anoikis resistance and tumor metastasis. Cancer LettGoogle Scholar
  30. 30.
    Douma S, Van Laar T, Zevenhoven J, Meuwissen R, Van Garderen E, Peeper DS (2004) Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB. Nature 430(7003):1034–1039PubMedCrossRefGoogle Scholar
  31. 31.
    Berezovskaya O, Schimmer AD, Glinskii AB, Pinilla C, Hoffman RM, Reed JC, Glinsky GV (2005) Increased expression of apoptosis inhibitor protein XIAP contributes to anoikis resistance of circulating human prostate cancer metastasis precursor cells. Cancer Res 65(6):2378–2386PubMedCrossRefGoogle Scholar
  32. 32.
    Derksen P, Liu X, Saridin F, van der Gulden H, Zevenhoven J, Evers B, van Beijnum J, Griffioen A, Vink J, Krimpenfort P, Peterse J, Cardiff R, Berns A, Jonkers J (2006) Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 10(5):437–449PubMedCrossRefGoogle Scholar
  33. 33.
    Zhang J, Clatterbuck RE, Rigamonti D, Chang DD, Dietz HC (2001) Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation. Hum Mol Genet 10(25):2953–2960PubMedCrossRefGoogle Scholar
  34. 34.
    Shattil SJ, O’Toole T, Eigenthaler M, Thon V, Williams M, Babior BM, Ginsberg MH (1995) Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit. J Cell Biol 131(3):807–816PubMedCrossRefGoogle Scholar
  35. 35.
    Giannoni E, Buricchi F, Grimaldi G, Parri M, Cialdai F, Taddei ML, Raugei G, Ramponi G, Chiarugi P (2008) Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival. Cell Death Differ 15(5):867–878PubMedCrossRefGoogle Scholar
  36. 36.
    Giannoni E, Fiaschi T, Ramponi G, Chiarugi P (2009) Redox regulation of anoikis resistance of metastatic prostate cancer cells: key role for Src and EGFR-mediated pro-survival signals. Oncogene 28(20):2074–2086PubMedCrossRefGoogle Scholar
  37. 37.
    Bouchard V, Harnois C, Demers M-J, Thibodeau S, Laquerre V, Gauthier R, Vézina A, Noël D, Fujita N, Tsuruo T, Arguin M, Vachon PH (2008) B1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms. Apoptosis 13(4):531–542PubMedCrossRefGoogle Scholar
  38. 38.
    Coll ML, Rosen K, Ladeda V, Filmus J (2002) Increased Bcl-xL expression mediates v-Src-induced resistance to anoikis in intestinal epithelial cells. Oncogene 21(18):2908–2913PubMedCrossRefGoogle Scholar
  39. 39.
    Hisano C, Tanaka R, Fujishima H, Ariyama H, Tsuchiya T, Tatsumoto T, Mitsugi K, Nakamura M, Nakano S (2003) Suppression of anoikis by v-Src but not by activated c-H-ras in human gallbladder epithelial cells. Cell Biol Int 27(5):415–421PubMedCrossRefGoogle Scholar
  40. 40.
    Liu G, Meng X, Jin Y, Bai J, Zhao Y, Cui X, Chen F, Fu S (2008) Inhibitory role of focal adhesion kinase on anoikis in the lung cancer cell A549. Cell Biol Int 32(6):663–670PubMedCrossRefGoogle Scholar
  41. 41.
    Wei L, Yang Y, Zhang X, Yu Q (2004) Altered regulation of Src upon cell detachment protects human lung adenocarcinoma cells from anoikis. Oncogene 23(56):9052–9061PubMedCrossRefGoogle Scholar
  42. 42.
    Simon GM, Cravatt BF (2008) Anandamide biosynthesis catalyzed by the phosphodiesterase GDE1 and detection of glycerophospho-N-acyl ethanolamine precursors in mouse brain. J Biol Chem 283(14):9341–9349PubMedCrossRefGoogle Scholar
  43. 43.
    Simon GM, Cravatt BF (2010) Characterization of mice lacking candidate N-acyl ethanolamine biosynthetic enzymes provides evidence for multiple pathways that contribute to endocannabinoid production in vivo. Mol BioSyst 6(8):1411–1418PubMedCrossRefGoogle Scholar
  44. 44.
    De Petrocellis L, Melck D, Palmisano A, Bisogno T, Laezza C, Bifulco M, Di Marzo V (1998) The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation. Proc Natl Acad Sci USA 95(14):8375–8380PubMedCrossRefGoogle Scholar
  45. 45.
    Melck D, De Petrocellis L, Orlando P, Bisogno T, Laezza C, Bifulco M, Di Marzo V (2000) Suppression of nerve growth factor Trk receptors and prolactin receptors by endocannabinoids leads to inhibition of human breast and prostate cancer cell proliferation. Endocrinology 141(1):118–126PubMedCrossRefGoogle Scholar
  46. 46.
    Mimeault M, Pommery N, Wattez N, Bailly C, Henichart JP (2003) Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production. Prostate 56(1):1–12PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Craig D. Simpson
    • 1
    • 2
  • Rose Hurren
    • 1
  • Dahlia Kasimer
    • 3
  • Neil MacLean
    • 1
  • Yanina Eberhard
    • 1
  • Troy Ketela
    • 3
  • Jason Moffat
    • 3
  • Aaron D. Schimmer
    • 1
    • 2
    • 4
  1. 1.Ontario Cancer InstitutePrincess Margaret HospitalTorontoCanada
  2. 2.Department of Medical BiophysicsUniversity of TorontoTorontoCanada
  3. 3.Donnelly Center and Banting & Best Department of Medical ResearchUniversity of TorontoTorontoCanada
  4. 4.TorontoCanada

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