Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) is a promising agent in selectively killing
tumor cells. However, TRAIL monotherapy has not been successful as many
cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as
doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput
small interfering RNA screening and genome-wide gene expression profiling on
doxorubicin-treated U1690 cells to explore novel mechanisms underlying
doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and
dihydroorotate dehydrogenase (
DHODH) was identified as a potential candidate.
DHODH is the rate-limiting
enzyme in the
pyrimidine synthesis pathway, and its expression was downregulated by
doxorubicin. We demonstrated that silencing of
DHODH or inhibition of
DHODH activity by
brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition,
uridine, an end product of the
pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both
brequinar and
doxorubicin. Furthermore, several other
cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by
brequinar. Taken together, our findings have identified a novel
protein target and its inhibitor,
brequinar, as a potential agent in TRAIL-based combinatorial
cancer therapy and highlighted for the first time the importance of mitochondrial
DHODH enzyme and
pyrimidine pathway in mediating TRAIL sensitization in
cancer cells.