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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 chaperones and the Ire1 pathway in the HCC model. a Real-time PCR of Grp78, Grp94 and P58IPK during hepatocarcinogenesis. Orange line compares DEN-treated groups at different time points. From W25, different tissue compartments were isolated: green, non-HCC; blue, surrounding; red, tumors. Dashed lines compare these compartments at W30. Relative fold change was calculated using the ΔΔCT method. b Immunoblotting for UPR-mediated proteins. Results are representative of two independent experiments. Densitometric analysis of the p-Ire1:Ire1 ratio is indicated. c Immunostaining for Grp78 in livers treated for 30 W. Arrows indicate tumor. Positive control received a single injection with tunicamycin. Scale bar 100 µm. d Real-time PCR of Ire1-mediated splicing activity, Erdj4, Canx and Edem1. The Ire1-mediated splicing of Xbp1 mRNA is calculated as the relative ratio of spliced Xbp1 mRNA over total Xbp1 mRNA. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. *p < 0.05, **p < 0.01. (Color figure online)
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Fig1: Temporal dynamics of chaperones and the Ire1 pathway in the HCC model. a Real-time PCR of Grp78, Grp94 and P58IPK during hepatocarcinogenesis. Orange line compares DEN-treated groups at different time points. From W25, different tissue compartments were isolated: green, non-HCC; blue, surrounding; red, tumors. Dashed lines compare these compartments at W30. Relative fold change was calculated using the ΔΔCT method. b Immunoblotting for UPR-mediated proteins. Results are representative of two independent experiments. Densitometric analysis of the p-Ire1:Ire1 ratio is indicated. c Immunostaining for Grp78 in livers treated for 30 W. Arrows indicate tumor. Positive control received a single injection with tunicamycin. Scale bar 100 µm. d Real-time PCR of Ire1-mediated splicing activity, Erdj4, Canx and Edem1. The Ire1-mediated splicing of Xbp1 mRNA is calculated as the relative ratio of spliced Xbp1 mRNA over total Xbp1 mRNA. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. *p < 0.05, **p < 0.01. (Color figure online)

Mentions: Every 5 weeks (W) after DEN administration, mice were killed for the analysis of tumor progression and the expression of the (co-)chaperones Grp78, Grp94 and P58IPK (Fig. 1a). At W25, tumor nodules were observed in a background of fibrosis (Fig. S1). The expression of Grp78 was upregulated at W10 (mRNA: p < 0.05; protein: Fig. 1b) but reduced again at W15 (mRNA: p < 0.05), and once tumors were established, Grp78 mRNA was elevated in the nodules compared to the surrounding (p < 0.05) and non-HCC tissue (p < 0.01). By immunohistochemistry, we demonstrated an inhomogeneous pattern of Grp78-positive HCC cells within the nodules, but only a few Grp78-positive cells in the surrounding tissue (Fig. 1c). Grp94 mRNA followed a similar temporal pattern, i.e., increased from W25 only in the surrounding and tumor tissue (p < 0.05, Fig. 1a). Accordingly, in addition to a tendency to increase at W10, co-chaperone P58IPK exhibited upregulation in the surrounding tissue and nodules from W25 (Fig. 1a).Fig. 1


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 chaperones and the Ire1 pathway in the HCC model. a Real-time PCR of Grp78, Grp94 and P58IPK during hepatocarcinogenesis. Orange line compares DEN-treated groups at different time points. From W25, different tissue compartments were isolated: green, non-HCC; blue, surrounding; red, tumors. Dashed lines compare these compartments at W30. Relative fold change was calculated using the ΔΔCT method. b Immunoblotting for UPR-mediated proteins. Results are representative of two independent experiments. Densitometric analysis of the p-Ire1:Ire1 ratio is indicated. c Immunostaining for Grp78 in livers treated for 30 W. Arrows indicate tumor. Positive control received a single injection with tunicamycin. Scale bar 100 µm. d Real-time PCR of Ire1-mediated splicing activity, Erdj4, Canx and Edem1. The Ire1-mediated splicing of Xbp1 mRNA is calculated as the relative ratio of spliced Xbp1 mRNA over total Xbp1 mRNA. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. *p < 0.05, **p < 0.01. (Color figure online)
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Fig1: Temporal dynamics of chaperones and the Ire1 pathway in the HCC model. a Real-time PCR of Grp78, Grp94 and P58IPK during hepatocarcinogenesis. Orange line compares DEN-treated groups at different time points. From W25, different tissue compartments were isolated: green, non-HCC; blue, surrounding; red, tumors. Dashed lines compare these compartments at W30. Relative fold change was calculated using the ΔΔCT method. b Immunoblotting for UPR-mediated proteins. Results are representative of two independent experiments. Densitometric analysis of the p-Ire1:Ire1 ratio is indicated. c Immunostaining for Grp78 in livers treated for 30 W. Arrows indicate tumor. Positive control received a single injection with tunicamycin. Scale bar 100 µm. d Real-time PCR of Ire1-mediated splicing activity, Erdj4, Canx and Edem1. The Ire1-mediated splicing of Xbp1 mRNA is calculated as the relative ratio of spliced Xbp1 mRNA over total Xbp1 mRNA. Horizontal axis in (a) and (d) indicates the number of weeks of DEN treatment. *p < 0.05, **p < 0.01. (Color figure online)
Mentions: Every 5 weeks (W) after DEN administration, mice were killed for the analysis of tumor progression and the expression of the (co-)chaperones Grp78, Grp94 and P58IPK (Fig. 1a). At W25, tumor nodules were observed in a background of fibrosis (Fig. S1). The expression of Grp78 was upregulated at W10 (mRNA: p < 0.05; protein: Fig. 1b) but reduced again at W15 (mRNA: p < 0.05), and once tumors were established, Grp78 mRNA was elevated in the nodules compared to the surrounding (p < 0.05) and non-HCC tissue (p < 0.01). By immunohistochemistry, we demonstrated an inhomogeneous pattern of Grp78-positive HCC cells within the nodules, but only a few Grp78-positive cells in the surrounding tissue (Fig. 1c). Grp94 mRNA followed a similar temporal pattern, i.e., increased from W25 only in the surrounding and tumor tissue (p < 0.05, Fig. 1a). Accordingly, in addition to a tendency to increase at W10, co-chaperone P58IPK exhibited upregulation in the surrounding tissue and nodules from W25 (Fig. 1a).Fig. 1

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