Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy

Abstract

Background Functional disturbances of the endoplasmic reticulum (ER) lead to activation of the unfolded protein response (UPR), which is involved in the consecutive steps of carcinogenesis. In human hepatocellular carcinoma (HCC), the UPR is shown to be activated; however, little is known about the UPR kinetics and effects of UPR modulation in HCC. Methods We sequentially monitored the UPR over time in an orthotopic mouse model for HCC and explored the effects of UPR modulation on cell viability and proliferation in vitro and in the mouse model. Results The expression of ER-resident chaperones peaked during tumor initiation and increased further during tumor progression, predominantly within the nodules. A peak in Ire1 signaling was observed during tumor initiation. The Perk pathway was activated during tumor progression, and the proapoptotic target Chop was upregulated from week 5 and continued to rise, especially in the tumors. The Atf6 pathway was modestly activated only after tumor initiation. Consistent with the UPR activation, electron microscopy demonstrated ER expansion and reorganization in HCC cells in vivo. Strikingly, under ER stress or hypoxia, the Perk inhibitor and not the Ire1 inhibitor reduced cell viability and proliferation via escalating proteotoxic stress in vitro. Notably, the Perk inhibitor significantly decreased tumor burden in the mouse model. Conclusion We provide the first evaluation of the UPR dynamics in a long-term cancer model and identified a small molecule inhibitor of Perk as a promising strategy for HCC therapy.

Next-Generation Proteasome Inhibitor Oprozomib Synergizes with Modulators of the Unfolded Protein Response to Suppress Hepatocellular Carcinoma

“…Again, addition of tunicamycin or salubrinal further increased caspase-3/7 activity (p < 0.001). Although the IRE1 and PERK inhibitors were previously validated [15], these compounds did not affect the sensitivity of HCC cells to 100-400 nM OZ in HepG2 cells. Since tunicamycin increased the sensitivity but is not clinically applicable because of its toxicity, the HIV protease inhibitor nelfinavir, which represents one of the few clinically applicable ER stress-inducing agents [16], was tested.…”

“…Of note, human HCC, in contrast to unaffected adjacent liver tissue, is characterized by increased CHOP staining [29], suggesting that tumor-selective effects can be achieved. Additionally, we previously reported the high expression of Chop in the HCC nodules compared to the non-HCC tissue of the DEN-induced mouse model [15].…”

“…Combination with the chemical ER stress inducer tunicamycin, which inhibits N-linked protein glycosylation, or with the recently developed small-molecules selectively inhibiting the IRE1 or PERK pathway or with salubrinal, which inhibits eIF2α dephosphorylation, was evaluated [15]. In HepG2 cells, 48 hours of incubation with 100-400 nM OZ dose-dependently reduced cell viability, as shown by a tetrazolium MTT spectrophotometric assay (p < 0.001; Figure 1A and Table S1).…”

“…However, identification of sensitivity enhancers by UPR modulation could allow for a reduction of dose and dose-related toxicity. Because ER stress potentiates the antitumor efficacy of OZ, we speculate that OZ exerts a stronger effect on hypoxic ER-stressed tumor cells compared to the normal liver tissue [15]. Consequently, OZ could be more efficacious in combination with antiangiogenic treatments promoting tumor hypoxiainduced UPR [11,31].…”

“…Since we previously reported the UPR pattern in the DEN-induced mouse model [15], we examined the impact of OZ administration on the expression of UPR markers in HCC. OZ administration reduced the levels of Grp78 (p < 0.01) and Chop (p < 0.05) mRNA in the isolated tumors ( Figure 3E).…”