Abstract
The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1Ahigh) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1Alow) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1Alow cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.
Highlights
Recent studies have established that increased protein synthesis is crucial for neoplastic transformation
(253J B-V, SW780, UM-UC10, and UM-UC13) for characterization bortezomib for 24 h and plasma membrane integrity was measured by trypan blue uptake. (The presence of RFP in the expression construct prevented our use of the PI/FACS cell death assay.) Mean 6 SEM, n = 3. *, P,0.02
Bortezomib induced strong upregulation of mRNA encoding the major inducible isoform of Hsp72 (HSPA1A) in the most bortezomib-resistant cell line (253J B-V) but not in the most drug-sensitive line (UM-UC13) (Fig. S1). We confirmed these results using quantitative real-time RT-PCR, demonstrating that HSPA1A mRNA was strongly induced by bortezomib in 253JB-V and SW780 cells (,25–60 fold over untreated levels), whereas expression increased only slightly induced (,2–4 fold over untreated levels) in UM-UC10 and UM-UC13 cells (Fig. 1B)
Summary
Recent studies have established that increased protein synthesis (translation) is crucial for neoplastic transformation. As a consequence of this increase, cancer cells appear to be vulnerable to agents inhibiting the elimination of aggregated or misfolded proteins produced as a normal byproduct of protein synthesis. The proteasome plays a central role in the clearance of damaged proteins, and proteasome inhibitors induce tumor cell death in large part via protein aggregation and proteotoxicity. Cytoprotective mechanisms are upregulated by proteasome inhibition, limiting the impact on cancer cell death [1,2,3]. It is possible that tumors that possess defect(s) in these cytoprotective mechanisms will be especially sensitive to proteasome inhibitors. It may be possible to develop therapeutic approaches that disrupt these cytoprotective mechanisms thereby promoting proteasome inhibitor sensitivity in tumors that would otherwise be resistant to this class of drugs
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