Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

Keywords: endoplasmic reticulum; hepatocellular carcinoma; proteasome inhibitor; stress; unfolded protein response.

Figure 1. Antiproliferative and pro-apoptotic effects of oprozomib in monotherapy or in combination with modulators of ER stress in human hepatoma HepG2 cells

(A) MTT assay (B) BrdU incorporation (C) Caspase-3/7 activity. OZ: oprozomib. *p < 0.05, **p < 0.01, ***p < 0.001 compared to oprozomib 0 nM; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to the respective concentration of oprozomib alone. Results are representative of 2 independent experiments.

Figure 2. Oprozomib modulates the UPR pattern in HepG2 cells

(A) Real-time PCR analysis of relative mRNA levels of UPR-mediated genes after 48 hours of incubation with the indicated treatments. (B) Immunoblotting of UPR-mediated proteins. (C) To measure the half-life of CHOP protein in HepG2 cells, cells were pre-treated for 1 hour with 1 μg/ml tunicamycin and a time-course in the presence of 50 μg/ml cycloheximide, which blocks protein synthesis, was performed. Band intensities were quantified using ImageJ software. (D) Half-life of CHOP protein was determined by plotting optical density (arbitrary unit) calculated from densitometric analysis of bands in panel C versus hours of treatment. Data represent the mean ± SD of three independent experiments. (E) BrdU incorporation of HepG2 cells incubated with indicated treatments for 48 hours. OZ: 400 nM oprozomib. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Impact of oprozomib in monotherapy or in combination with ER stress modulators on an orthotopic and a xenograft model for HCC

(A) Photographs of representative livers after different treatments. (B) Reticulin staining. Scale bar: 100 μm. (C) Assessment of tumor burden in the carcinogen-induced mouse model in randomly selected high-power fields. (D) Hepatic caspase-3/7 activity ex vivo. Values represent the mean ± SD. (E) Real-time PCR analysis and (F) Immunoblotting of UPR targets in isolated DEN-induced tumors following the indicated treatments. Densitometric analysis relative to tubulin is indicated below. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Oprozomib and UPR modulation in experimental HCC

(A) Evolution of tumor volume in mice bearing HepG2-derived xenograft tumors. (B) Final tumor weights. (C) TUNEL immunofluorescence in HepG2 xenografts and (D) quantification of TUNEL-positive index (n = 6). OZ: oprozomib. Values represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Schematic model outlining the mechanisms of oprozomib with indication of the point of action of the applied products

Persistent ER stress activates the tripartite UPR-mediated transcriptional program followed by translation of these UPR proteins, which leads to proteotoxicity-mediated tumor cell death. Although oprozomib did not induce the UPR and even inhibited ATF6-mediated transcription, it increased the UPR-mediated protein levels by prolonging their half-life. This UPR dysregulation allows for enhanced proteotoxicity through supplementary boosting PERK activity by tunicamycin, nelfinavir (also inhibits ATF6) or salubrinal.