Evidence that mitogen-activated protein kinase phosphatase-1 induction by proteasome inhibitors plays an anti-apoptotic role

ABSTRACT Inhibitors of the proteasome, a multicatalytic proteinase complex responsible for intracellular proteolysis, activate programmed cell death in part through the c-Jun-N-terminal kinase (JNK). Proteasome inhibitors also induce mitogenactivated protein kinase phosphatase-1 (MKP-1), however, which can inactivate JNK, and we therefore considered the hypothesis that MKP-1 induction may be antiapoptotic. Overexpression of MKP-1 in A1N4-myc human mammary epithelial and BT-474 breast carcinoma cells decreased proteasome inhibitor-mediated apoptosis. On the other hand, BT-474 cells stably expressing an MKP-1 small interfering RNA (siMKP-1) and MKP-1 knockout mouse embryo fibroblasts underwent enhanced apoptosis compared with their respective controls. MKP-1-mediated inhibition of apoptosis was associated with decreased phospho-JNK levels, whereas MKP-1 suppression or inactivation enhanced phospho-JNK. Anthracyclines repress MKP-1 transcription, suggesting that they could enhance proteasome inhibitor-mediated apoptosis. Such combinations induced increased cell death in association with enhanced phospho-JNK and decreased MKP-1 levels. Inhibition of JNK signaling decreased the proapoptotic activity of the anthracycline/proteasome inhibitor regimen. Xenograft studies showed the combination was more effective at inducing tumor growth delay, associated with suppression of MKP-1 and enhancement of apoptosis and phospho-JNK. Infection of anthracycline/proteasome inhibitor-treated A1N4-myc cells with Adenoviral-MKP-1 suppressed apoptosis and phospho-JNK. Finally, the anthracycline/proteasome inhibitor regimen activated apoptosis and phospho-JNK to a greater extent in BT-474/ siMKP-1 cells than controls. These findings for the first time demonstrate that proteasome inhibitor-mediated induction of MKP-1 is antiapoptotic through inhibition of JNK. Furthermore, they suggest that a proteasome inhibitor/anthracycline regimen holds potential for enhanced antitumor activity in part through repression of MKP-1, supporting clinical evaluation of such combinations. The majority of regulated intracellular eukaryotic protein turnover occurs through the ubiquitin-proteasome pathway ABBREVIATIONS: NF, nuclear factor; JNK, c-Jun-N-terminal kinase; MAPK, mitogen-activated protein kinase; MKP, mitogen-activated protein kinase phosphatase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; PS-341, bortezomib; Z-LLF-CHO, N-benzyloxycarbonyl-leucyl-leucyl-phenylalanyl-aldehyde; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; MEF, mouse embryo fibroblast; DP, dominant-positive; ERK, extracellular signal-regulated kinase; CMV, cytomegalovirus; ss, scrambled sequence; GFP, green fluorescent protein; DN, dominant-negative; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; HSC, heat shock cognate protein; siMKP-1, small interfering RNA targeting MKP-1; ssMKP-1, small interfering RNA with a scrambled sequence that does not target MKP-1; HSP, heat shock protein