A loss-of-function RNA interference screen for molecular targets in cancer.
The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair 'bar code', allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-kappaB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IkappaB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes.
Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Vu N
VN
Ngo
R Eric
RE
Davis
Laurence
L
Lamy
Xin
X
Yu
Hong
H
Zhao
Georg
G
Lenz
Lloyd T
LT
Lam
Sandeep
S
Dave
Liming
L
Yang
John
J
Powell
Louis M
LM
Staudt
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000074018:B-Cell CLL-Lymphoma 10 Protein; D053468:CARD Signaling Adaptor Proteins; D020169:Caspases; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D015195:Drug Design; D006162:Guanylate Cyclase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018442:Lymphoma, B-Cell, Marginal Zone / Q000235:genetics / Q000378:metabolism; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D034622:RNA Interference; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000074018:B-Cell CLL-Lymphoma 10 Protein; C118591:BCL10 protein, human; D053468:CARD Signaling Adaptor Proteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D020169:Caspases; C400109:MALT1 protein, human; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; C426242:CARD11 protein, human; D006162:Guanylate Cyclase