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. 2018 Nov 1;78(21):6098-6106.
doi: 10.1158/0008-5472.CAN-17-3600. Epub 2018 Sep 19.

Heterozygosity of Chaperone Grp78 Reduces Intestinal Stem Cell Regeneration Potential and Protects against Adenoma Formation

Jooske F van Lidth de Jeude  1 , Claudia N Spaan  1 , Bartolomeus J Meijer  1 , Wouter L Smit  1 , Tanya T D Soeratram  1 , Mattheus C B Wielenga  1 , B Florien Westendorp  1 , Amy S Lee  2 , Sander Meisner  1 , Jacqueline L M Vermeulen  1 , Manon E Wildenberg  1 , Gijs R van den Brink  1 , Vanesa Muncan  1 , Jarom Heijmans  3   4
Affiliations

Heterozygosity of Chaperone Grp78 Reduces Intestinal Stem Cell Regeneration Potential and Protects against Adenoma Formation

Jooske F van Lidth de Jeude et al. Cancer Res. .

Abstract

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098-106. ©2018 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1.
Figure 1.
Grp78 heterozygous organoids exhibit loss of stem cell markers, increase in UPR markers, and impaired growth. A, Brightfield and fluorescent images of wild-type and Grp78+/− organoids at day 4 after passaging. B, qRT-PCR analysis of floxed Grp78Δ5–7 mRNA in organoids depicted in A. C, Western blot analysis for Grp78 protein in nonrecombined and recombined organoids. Actin was used as equal loading control. D, Quantification of Western blot analysis in C, relative to control. E, Organoid growth assessed by the percentage of organoids with two or more buds at indicated times after passaging. F, qRT-PCR analysis for UPR components and CBC markers. wt, wild type; veh, vehicle. ns, not significant; **, P < 0.01; ***, P < 0.001. Original magnification, ×400.
Figure 2.
Figure 2.
Grp78 heterozygous mice show unaltered proliferation and stem cell numbers in the intestine. A, LacZ staining on intestines of mice showing recombination one month after induction of Cre-mediated recombination with tamoxifen. B, qRT-PCR analysis offloxed Grp78Δ5−7 mRNA. C, Western blot analysis for Grp78 protein in nonrecombined and recombined small intestine. Actin was used as equal loading control. D, Quantification of Western blot analysis in C, relative to control. E, Quantification of BrdU-positive cells. F, ISH for stem cell marker Olfm4. G, Quantification of Olfm4+ve cells. H, qRT-PCR analysis of a panel of UPR components and CBC-stem cell markers Lgr5 and Olfm4. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Original magnifications, ×200 and ×400.
Figure 3.
Figure 3.
Regeneration of Grp78 heterozygous intestinal epithelium after γ-irradiation is favored from nonrecombined stem cells, showing loss of self-renewal capacity in heterozygous intestines. A, LacZ staining on small intestines of mice showing recombination 48 hours and 96 hours after irradiation. B, Percentage of crypts that are nonrecombined in wild-type control animals and Grp78het(IEC) animals, 48 hours and 96 hours after irradiation. C, qRT-PCR for expression of LacZ mRNA. D, IHC for cleaved-caspase-3 at 48 hours postirradiation. Red arrows, positive cells. E, Quantification of cleaved-caspase-3–positive cells. ns, not significant; *, P < 0.05; **, P < 0.01. Original magnification, ×400.
Figure 4.
Figure 4.
Reduced adenoma formation in Grp78 heterozygous mice. A, Quantification of IHC for BrdU on mice with indicated genotypes. B, Number of adenomas in small and large intestines of mice with indicated genotypes. All animals were sacrificed at the age of 20 weeks, 16 weeks after recombination with tamoxifen. C, Average adenoma size in millimeters (mm) of all adenomas. D, Representative images (hematoxylin and eosin staining) showing adenomas in the entire small intestine of indicated genotypes. ns, not significant; **, P < 0.01.
Figure 5.
Figure 5.
Reduced protein synthesis in Grp78 heterozygous organoids and increased ER stress in presence of Wnt pathway activating compound CHIR-99021. A, 35S-methionine labeling assay of organoids of indicated genotypes, grown in normal culture medium or with the addition of 5 μmol/L CHIR-99021. B, Western blot analysis for c-Myc and Grp78 protein in organoids, with the addition of 5 μmol/L CHIR-99021. C, Quantification of Western blot analysis in B, relative to control. veh, vehicle. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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References

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