title: A kinome-wide siRNA screen identifies multiple roles for protein kinases in hypoxic stress adaptation, including roles for IRAK4 and GAK in protection against apoptosis in VHL-/- renal carcinoma cells, despite activation of the NF-κB pathway.
abstract: Hypoxia induces changes to cancer cells that make them more resistant to treatment. We have looked at signaling pathways that facilitate these changes by screening the human kinome for effects on hypoxic responses in SW480 colon cancer cells. Hits identified in the screen were examined for effects on multiple molecular responses to hypoxia, including the endoplasmic reticulum stress and DNA damage responses in colon, melanoma, and renal cancer lines. To validate the hits from the small interfering RNA studies, we developed cell lines expressing stable short hairpin RNAs (shRNAs) in the A498 renal carcinoma cell line. Several lines, including those expressing shRNAs against DYRK1B, GAK, IHPK2, IRAK4, and MATK, showed an inability to form spheroid cultures. In addition, shRNAs targeting IRAK4 and GAK were incapable of 2D growth under anoxia. In the GAK shRNA-expressing line, nuclear factor-κB (NF-κB) was localized to the nucleus, but in the IRAK4 shRNA line, NF-κB levels were increased but the extent of nuclear localization was unchanged. Dominant negative mutants of IRAK4 and GAK also showed strong apoptotic effects in A498 cells under anoxia, supporting a direct link between these kinases and survival of the VHL(-/-) RCC line, which is typically highly resistant to hypoxic stress as a result of high and constitutive levels of Hif-1α.
authors.affiliation: Applied Genomics, Department of Biological Technologies, Wyeth Research, Cambridge, MA, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Pan
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Andrew
authors.initials: A
authors.lastname: Hill
authors.forename: Peter
authors.initials: P
authors.lastname: Lapan
authors.forename: Steve
authors.initials: S
authors.lastname: Berasi
authors.forename: Brian
authors.initials: B
authors.lastname: Bates
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
authors.forename: Steven
authors.initials: S
authors.lastname: Haney
mesh_terms: D017209:Apoptosis; D002292:Carcinoma, Renal Cell; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D005799:Genes, Dominant; D057166:High-Throughput Screening Assays; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D016328:NF-kappa B / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000502:physiology; D013312:Stress, Physiological; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C486325:GAK protein, human; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D017346:Protein Serine-Threonine Kinases; C497446:VHL protein, human
keywords: RNA interference; RNAi; cancer and cancer drugs; image analysis; phenotypic drug discovery; shRNA