Ubiquitin B: An Essential Mediator Of HDACi-Induced Tumor-Selective Killing In Human Cancer Cells

Background Histone deacetylase inhibitors (HDACis) are now emerging as a new class of anticancer agents with potent activity in the inhibition of proliferation and induction of apoptosis and differentiation in a wide spectrum of tumors. At least 12 HDACis are under evaluation in over 100 clinical trials and have produced encouraging therapeutic responses with surprisingly good safety profiles. The clinical potential of HDACis has been well exemplified by the successful development of Vorinostat which was recently approved by the US Food and Drug Administration for treatment of cutaneous T-cell lymphoma. Despite the rapid clinical progress achieved, the mechanism of action of HDACis is not yet well understood. One of the central questions is how these agents selectively kill tumor cells while sparing normal cells. Identification of the critical intracellular targets responsible for the tumor selectivity of HDACis will further improve the design of optimized clinical protocols. More attractively, unraveling the potential "death programs" selectively activated in tumor but not in normal cells will have broader implications for the understanding of tumorigenesis and the design of targeted therapies.Objective To screen some novel functional proapoptotic genes associated with HDAC inhibitors by suppression of mortality by antisense rescue technique and to explore the possible mechanisms of tumor-selective killing by HDAC inhibitors.Methods MCF-7 cells treated with 250 nmol/L Trichostatin A for different time points were harvested to construct an antisense cDNA library. HeLa cells were transfected with the library, and were selscted with Trichostatin A and Hygromycin B for 2 weeks. Then surviving colonies were amplified and Hirt DNA were extracted for sequencing.Results Twenty-four genes were identified, the most significant of which was ubiquitin B (UbB). The expression of UbB was selectively up-regulated by TSA in tumor cells, but not non-malignant cells. Further observation indicated that TSA induced a substantial dissipation of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and proteolytic cleavage of caspases 3/9 in HeLa cells which was apparently mediated by ubiquitylation and the subsequent degradation of mitochondrial membrane proteins including BCL-2 and MCL-1. In contrast, knockdown of UbB expression inhibited the TSA-induced apoptotic cascade by abolishing TSA-induced ubiquitylation and the subsequent degradation of mitochondrial membrane proteins. Furthermore, apicidine, another HDACi, exhibited activity similar to that of TSA. Interestingly, TSA induced UbB-dependent proteasomal degradation of BCR-ABL fusion protein in K562 leukemic cells.Conclusion Ubiquitin B and Ubb-dependent proteasomal protein degradation play an essential role in HDACi-induced tumor selectivity. The mechanism provides a novel starting point for dissecting the molecular mechanism underlying the tumor selectivity of HDACi.