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

Vandewynckel YP, Laukens D, Bogaerts E, Paridaens A, Van den Bussche A, Verhelst X, Van Steenkiste C, Descamps B, Vanhove C, Libbrecht L, De Rycke R, Lambrecht BN, Geerts A, Janssens S, Van Vlierberghe H - Hepatol Int (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatology and Gastroenterology, Ghent University, De Pintelaan 185, 1K12 IE, 9000 Ghent, Belgium.

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.

No MeSH data available.


Related in: MedlinePlus

Temporal dynamics of genes regulated by Perk or Atf6 in the HCC model. a Real-time PCR analysis of Perk-regulated genes. b Immunoblotting and immunostaining for Perk-regulated genes in livers treated for 30 W with DEN or saline. Positive control received tunicamycin for 72 h. Arrows indicate tumors. Scale bar 100 µm. c Caspase-3 activity of liver lysates of the indicated tissue after 30 weeks of saline or DEN administration. d Real-time PCR analysis of Atf6 target genes. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. Data are presented as the mean ± SD. One-way ANOVA was applied for statistical analysis. *p < 0.05, **p < 0.01
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Fig2: Temporal dynamics of genes regulated by Perk or Atf6 in the HCC model. a Real-time PCR analysis of Perk-regulated genes. b Immunoblotting and immunostaining for Perk-regulated genes in livers treated for 30 W with DEN or saline. Positive control received tunicamycin for 72 h. Arrows indicate tumors. Scale bar 100 µm. c Caspase-3 activity of liver lysates of the indicated tissue after 30 weeks of saline or DEN administration. d Real-time PCR analysis of Atf6 target genes. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. Data are presented as the mean ± SD. One-way ANOVA was applied for statistical analysis. *p < 0.05, **p < 0.01

Mentions: At W25, during tumor progression, Atf4 mRNA upregulation was observed and limited to the nodules (Fig. 2a). At W30, expression expanded to the surrounding tissues (Fig. 2a, b). Phosphorylation of eIf2α was increased in both surrounding and tumor tissue at W30 (Fig. 2b). Immunostaining for phospho-eIf2α showed a diffuse distribution in the surrounding tissue, intensifying toward the core of the nodules at W30.Fig. 2


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

Vandewynckel YP, Laukens D, Bogaerts E, Paridaens A, Van den Bussche A, Verhelst X, Van Steenkiste C, Descamps B, Vanhove C, Libbrecht L, De Rycke R, Lambrecht BN, Geerts A, Janssens S, Van Vlierberghe H - Hepatol Int (2014)

Temporal dynamics of genes regulated by Perk or Atf6 in the HCC model. a Real-time PCR analysis of Perk-regulated genes. b Immunoblotting and immunostaining for Perk-regulated genes in livers treated for 30 W with DEN or saline. Positive control received tunicamycin for 72 h. Arrows indicate tumors. Scale bar 100 µm. c Caspase-3 activity of liver lysates of the indicated tissue after 30 weeks of saline or DEN administration. d Real-time PCR analysis of Atf6 target genes. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. Data are presented as the mean ± SD. One-way ANOVA was applied for statistical analysis. *p < 0.05, **p < 0.01
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4289530&req=5

Fig2: Temporal dynamics of genes regulated by Perk or Atf6 in the HCC model. a Real-time PCR analysis of Perk-regulated genes. b Immunoblotting and immunostaining for Perk-regulated genes in livers treated for 30 W with DEN or saline. Positive control received tunicamycin for 72 h. Arrows indicate tumors. Scale bar 100 µm. c Caspase-3 activity of liver lysates of the indicated tissue after 30 weeks of saline or DEN administration. d Real-time PCR analysis of Atf6 target genes. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. Data are presented as the mean ± SD. One-way ANOVA was applied for statistical analysis. *p < 0.05, **p < 0.01
Mentions: At W25, during tumor progression, Atf4 mRNA upregulation was observed and limited to the nodules (Fig. 2a). At W30, expression expanded to the surrounding tissues (Fig. 2a, b). Phosphorylation of eIf2α was increased in both surrounding and tumor tissue at W30 (Fig. 2b). Immunostaining for phospho-eIf2α showed a diffuse distribution in the surrounding tissue, intensifying toward the core of the nodules at W30.Fig. 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatology and Gastroenterology, Ghent University, De Pintelaan 185, 1K12 IE, 9000 Ghent, Belgium.

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.

No MeSH data available.


Related in: MedlinePlus