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Supplementary Figure S1

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.

The mechanisms that orchestrate the development of a multicellular organism from a single common ancestor cell have fascinated people for centuries. We are still a long way from truly understanding the robust programs that allow building of our specialized organs that regulate digestion, respiration, and hemodynamics and that guard us from invasion by other unsolicited organisms. However, in the past three decades, many of the genes that play a role in patterning our bodies have been identified. We have learned much about the mechanisms involved in development by characterizing the nature of these patterning genes and the way they work together. In this chapter, we will discuss patterning mechanisms, introduce the Hedgehog pathway and then summarize our current understanding of the role of Hedgehog signaling in development and maintenance of the gastrointestinal (GI) tract.

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.

Background & Aims Recently, we have shown that micro-metastases, in the hypoxic transition zone surrounding lesions generated by radiofrequency ablation (RFA), display strongly accelerated outgrowth. CD95 is best known for its ability to induce apoptosis but can also promote tumorigenesis in apoptosis-resistant tumor cells. Therefore, we tested whether CD95 signaling plays a role in accelerated outgrowth of colorectal liver metastases following RFA. Methods Hypoxia-induced invasion was assessed in three-dimensional EGFP-expressing C26 tumor cell cultures by confocal microscopy. CD95 localization was tested by immunofluorescence. Invasion and outgrowth of liver metastases following RFA were analyzed by post-mortem confocal microscopy and by morphometric assessment of tumor load. Neutralization of CD95L was performed by using antibody MFL4. CD95 was suppressed by lentiviral RNA interference. The role of host CD95L was assessed using gld mice. Results Micro-metastases in the hypoxic transition zone following RFA displayed a highly invasive phenotype and increased expression of CD95 and CD95L. Hypoxia-induced tumor cell invasion in vitro increased the expression of CD95 and CD95L and induced translocation of CD95 to the invasive front. In vitro invasion, metastasis invasion, and accelerated tumor growth in the transition zone were strongly suppressed by neutralizing CD95L or by suppressing tumor cell CD95. In contrast, metastasis invasion and outgrowth were unaffected in gld mice. Conclusions Hypoxia causes autocrine activation of CD95 on colorectal tumor cells, thereby promoting local invasion and accelerated metastasis outgrowth in the hypoxic transition zone following RFA. Further pre-clinical work is needed to assess the role of CD95L neutralization, either alone or in combination with chemotherapy, in limiting aggressive recurrence of liver metastases following RFA.

CD95 and its ligand (CD95L) are widely expressed in colorectal tumors, but their role in shaping tumor behavior is unclear. CD95 activation on tumor cells can lead to apoptosis, while CD95L attracts neutrophils, suggesting a function in tumor suppression. However, CD95 can also promote tumorigenesis, at least in part by activating non-apoptotic signaling pathways that stimulate tumor cell proliferation, invasion and survival. In addition, CD95 signaling in stromal cells and tumor-infiltrating inflammatory cells has to be taken into account when addressing the function of CD95 and its ligand in colorectal tumor biology. We present a model in which the tumor-suppressing and tumor-promoting activities of CD95/CD95L together determine colorectal tumor behavior. We also discuss how these multiple activities are changing our view of CD95 and CD95L as potential therapeutic targets in the treatment of colorectal cancer. We conclude that locking CD95 in apoptosis-mode may be a more promising anti-cancer strategy than simply inhibiting or stimulating CD95.

Background & Aims Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. Methods Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor–related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. Results CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras–dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras–driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras–proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras–deficient cells against apoptosis. Conclusions Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1–driven metastasis formation.

Background & Aims Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. Methods Glioma-associated oncogene ( Gli1 ) -CreERT2 and Patched ( Ptch )- lacZ reporter mice were crossed with Apc Min mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc Min and Ptch-lacZ-Apc Min mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc ( Apc HET ) mice, which develop adenomas after administration of β-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc Min mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). Results Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc HET mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc HET mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. Conclusions Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.