Molecular sequelae of proteasome inhibition in human multiple myeloma cells

Abstract

The proteasome inhibitor PS-341 inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM.

Fig 1.

Transcriptional profile of PS-341-treated MM-1S cells by oligonucleotide-microarray analysis. Representative functional clusters of transcriptional changes induced by PS-341 (100 nM, 1–8 h) include molecules implicated in caspase-mediated apoptosis, growth factor signaling and cell cycle control, heat shock proteins, and proteasome subunits. Color saturation is proportional to magnitude of the difference from the respective control.

Fig 2.

Protective effect of the IGF/Akt pathway against apoptosis induced by proteasome inhibition. (A) IGF-1 (200 ng/ml) lowers the sensitivity to PS-341 (10 nM). Cells were serum-starved overnight and then incubated with or without PS-341 in serum-free medium for additional 18 h. (B) PS-341 (5 and 10 nM)-induced cell death (quantified by MTT, mean ± SD) in MM.1S cells transfected with a vector expressing constitutively active Akt (open bars), or control vector (filled bars). After overnight serum starvation, cells were incubated with or without PS-341 in serum-free medium for additional 18 h.

Fig 3.

Increased expression of stress-response proteins in apoptosis induced by proteasome inhibition. (A) Immunoblotting confirms PS-341 induces time-dependent increase in levels of hsp90, hsp70, and hsp27. (B) The hsp90 inhibitor NSC683664 (100 nM) sensitizes MM.1S cells to a subtoxic concentration of PS-341 (5 nM).

Fig 4.

Schematic diagram of apoptotic pathways activated by PS-341 inhibition.