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-KB 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.

SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel-Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1alpha dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1alpha in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1alpha ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1alpha at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1alpha ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.

Chip profiling of a p53 temperature-sensitive tumor model identified SAK (Snk/Plk-akin kinase), encoding a new member of polo-like kinases (PLKs), as a gene strongly repressed by wild-type p53. Further characterization revealed that SAK expression was downregulated by wild-type p53 in several tumor cell models. Computer search of a 1.7-kb SAK promoter sequence revealed three putative p53 binding sites, but p53 failed to bind to any of these sites, indicating that SAK repression by p53 was not through a direct p53 binding to the promoter. Transcriptional analysis with luciferase reporters driven by SAK promoter deletion fragments identified SP-1 and CREB binding sites, which together conferred a two-fold SAK repression by p53. However, the repression was not reversed by cotransfection of SP-1 or CREB, suggesting a lack of interference between p53 and SP-1 or CREB. Significantly, p53-mediated SAK repression was largely reversed in a dose-dependent manner by Trichostatin A, a potent histone deacetylase (HDAC) inhibitor, suggesting an involvement of HDAC transcription repressors in SAK repression by p53. Biologically, SAK RNA interference (RNAi) silencing induced apoptosis, whereas SAK overexpression attenuated p53-induced apoptosis. Thus, SAK repression by p53 is likely mediated through the recruitment of HDAC repressors, and SAK repression contributes to p53-induced apoptosis.

No abstract