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Radiotherapy combined with chemotherapy is the treatment of choice for glioblastoma and locally advanced lung cancer, but radioresistance of these two types of cancer remains a significant therapeutic hindrance. To identify molecular target(s) for radiosensitization, we screened a small interfering RNA (siRNA) library targeting all protein kinases and E3 ubiquitin ligases in the human genome and identified tumor necrosis factor receptor-associated factor 2 (TRAF2). Silencing of TRAF2 using siRNA caused a significant growth suppression of glioblastoma U251 cells and moderately sensitized these radioresistant cells to radiation. Overexpression of a really interesting new gene (RING)-deleted dominant-negative TRAF2 mutant also conferred radiosensitivity, whereas overexpression of wild-type (WT) TRAF2 significantly protected cells from radiation-induced killing. Likewise, siRNA silencing of TRAF2 in radioresistant lung cancer H1299 cells caused growth suppression and radiosensitization, whereas overexpression of WT TRAF2 enhanced radioresistance in a RING ligase-dependent manner. Moreover, siRNA silencing of TRAF2 in UM-SCC-1 head and neck cancer cells also conferred radiosensitization. Further support for the role of TRAF2 in cancer comes from the observations that TRAF2 is overexpressed in both lung adenocarcinoma tissues and multiple lung cancer cell lines. Importantly, TRAF2 expression was very low in normal bronchial epithelial NL20 cells, and TRAF2 silencing had a minimal effect on NL20 growth and radiation sensitivity. Mechanistically, TRAF2 silencing blocks the activation of the nuclear factor-kappaB signaling pathway and down-regulates several G(2)-M cell cycle control proteins, resulting in enhanced G(2)-M arrest, growth suppression, and radiosensitization. Our studies suggest that TRAF2 is an attractive drug target for anticancer therapy and radiosensitization.

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.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

Validating potential targets is an important step in the drug discovery process. In this study, we tested the feasibility of using inducible RNA interference (RNAi) in vivo to obtain an unbiased evaluation on the efficacy of inhibiting hypoxia-inducible factor-1alpha (HIF-1alpha) in established tumors. We showed that HIF-1alpha inhibition resulted in transient tumor stasis or tumor regression, and inhibiting HIF-1alpha in early-stage tumors was found to be more efficacious than inhibiting HIF-1alpha in more established tumors. A differential requirement of HIF-1alpha for tumor growth was also observed among different tumor types. Examination of tumors resistant to HIF-1alpha inhibition suggested that the resistance might result from a less hypoxic tumor environment and the level of HIF-1alpha expression in tumors may be a useful marker for predicting tumor response to HIF-1 inhibition. This study shows that inducible RNAi is a versatile tool for evaluating cancer targets in vivo. In addition to broad implications on in vivo validation of cancer targets, results from this study will also be instructive for practical applications of HIF-1-based cancer therapeutics.