ABSTRACT
To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.
Subject(s)
Cell Cycle Proteins/genetics , Microtubule-Associated Proteins/genetics , Neoplasm Proteins/genetics , Neoplasms/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , RNA, Small Interfering/genetics , Stearoyl-CoA Desaturase/genetics , Xenopus Proteins/genetics , ran GTP-Binding Protein/genetics , Cell Cycle/genetics , Cell Death/genetics , Cell Line, Tumor , Cell Survival/genetics , Gene Library , Genes, ras , Humans , Neoplasms/pathology , RNA InterferenceABSTRACT
To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting approximately 4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G(2)-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein.
