A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance
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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.
KeywordsAnoikis Genome wide shRNA screen α/β Hydrolase domain containing 4 ABHD4
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.
- 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.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
- 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.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.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
- 29.Simpson C, Anyiwe K, Schimmer A (2008) Anoikis resistance and tumor metastasis. Cancer LettGoogle Scholar
- 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.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
- 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
- 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