Upregulation of the orphan nuclear receptor NR4A3 in drug-induced apoptosis of tumor cells and its relationship to mitochondrial VDAC1

3597 Mitochondria play a critical role in the execution of tumor cells following drug exposure. We have previously reported the apoptosis inducing activity of the novel small molecule, C1 (merodantoin), a photoproduct of merocyanine 540. This includes fragmentation of DNA, activation of caspases 2, 3, and 9, translocation of Bax to the mitochondria and the reciprocal egress of cytochrome C to the cytosol. In addition, exposure of tumor cells to C1 results in an early increase in intracellular hydrogen peroxide production and cytosolic acidification, thus creating an environment conducive for death execution. To further understand the signaling circuitry and upstream modulators of C1-induced apoptosis in tumor cells, here we used a high throughput microarray analysis to pick out genes that were up- or down-regulated upon C1 treatment. We identified among other genes, NR4A3, which encodes an orphan nuclear receptor, transcriptionally upregulated within 1-2 hours of treatment. These data were then validated using NR4A3 specific primers by Real time PCR. Furthermore, to identify partners in the signal transduction pathway mediated by NR4A3 we looked at the co-expression neighborhood of NR4A3 in cancer datasets and found that it includes among other genes, voltage dependent anion channel 1 (VDAC1), a mitochondrial outer membrane protein involved in mitochondria-mediated apoptosis. Of note, VDAC1 transcript and total protein levels did not change upon drug treatment, however a significantly higher mitochondrial VDAC1 was detected in the mitochondria following 12 hours incubation with the compound. To further gain insight into the relationship between NR4A3 expression and mitochondrial localization of VDAC1, the expression of NR4A3 was silenced by siRNA. Interestingly, silencing NR4A3 blocked C1-induced mitochondrial translocation to or expression of VDAC1. These data suggest that NR4A3 could modulate the apoptotic response by impacting on translocation of critical proteins to mitochondria, which are involved in apoptotic signaling. In summary, here we report a novel role for NR4A3 in drug-induced apoptosis, indicating specific involvement of mitochondria.