5

A screen for small molecule inhibitors of ErbB2 transcription identified two drug classes, histone deacetylase (HDAC) and proteasome inhibitors, that accelerate decay of mature ErbB2 mRNA. We have also shown that ErbB2 transcript stability is dependent on a conserved hairpin-forming U-rich element within its endogenous 3’ UTR, thought to bind to the transcript-stabilizing UTR-binding protein, HuR. To identify transcript specific stability mechanisms sensitive to HDAC and/or proteasome inhibitors, we used an expression microarray strategy analyzing total RNA purified from replicate SKBr3 cultures treated (5 hr) with either a transcript-destabilizing dose of HDAC inhibitor (LAQ824 or trichostatin/TSA), a proteasome inhibitor (PS-341), or a dose of actinomycin D (ActD) designed to establish normal transcript decay. Microarrays with 13K UniGene annotated sequences included 45% also found in a novel database (Gorospe et al., 2004; 13,560 total entries) of human cDNAs with evolutionarily conserved U-rich/HuR-binding motifs within their 3’UTRs. Over 60% (307/486) of the ActD-inhibited short-lived SKBr3 transcripts corresponded to genes containing these U-rich/HuR-binding elements. Unsupervised clustering of the 5,789 arrayed U-rich genes unambiguously distinguished ActD from HDAC and proteasome inhibitor treatments. A subset of 346 U-rich transcripts showed HDAC inhibitor induced destabilization comparable to or greater than that observed for ErbB2. Another subset of 444 U-rich transcripts showed proteasome inhibitor induced destabilization comparable to or greater than that observed for ErbB2; and most of these genes (416) were destabilized moreso by proteasome inhibition than by HDAC inhibition. Experiments looking at treatment effects on the HuR and AUF proteins that compete for binding to U-rich elements in these breast cancer transcripts are expected to elucidate how HDAC and proteasome inhibitors differentially inhibit cancer growth by destabilizing critical transcripts.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]