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Supplementary Figure S1

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Supplementary Figure S3

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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Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3β inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic Kras(G12D). Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.

Modulation of global mRNA translation is essential for intestinal stem cell function. In colorectal cancer, proliferating stem-like cells are defined by high WNT signaling and display dysregulated translational control. It is currently unclear how factors that coordinate global translation affect stem cell behavior. Here we identified nucleotide exchange factor eIF2Be as a translation initiation factor involved in intestinal epithelial stemness. In eIF2Be Arg191His mice, which have a homozygous point mutation that intrinsically impairs eIF2Be’s enzymatic function, we demonstrate that dysfunctional eIF2Be affects small intestinal crypt formation, proliferation and stemness marker expression. By hyperactivating stem cell activity in ex vivo organoids, using a GSK3β inhibitor to mimic loss of tumor suppressor Apc , we demonstrate that eIF2Be is essential for clonogenic potential and associated with an increase in global translation. Finally, we observe high eIF2Be expression in human colonic adenoma tissues, exposing eIF2Be as a potential target in modulation of stemness.

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

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.

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.