Probes for LGR5

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

Leucine-rich repeat-containing G-protein coupled receptor-5 (Lgr5)+/olfactomedin-4 (Olfm4)+ intestinal stem cells (ISCs) in the crypt-base are crucial for homeostatic maintenance of the epithelium. The gut hormone, glucagon-like peptide-21-33 (GLP-2), stimulates intestinal proliferation and growth; however, the actions of GLP-2 on the Lgr5+ ISCs remain unclear. The aim of this study was to determine whether and how GLP-2 regulates Lgr5+ ISC cell cycle dynamics and number.Lgr5-eGFP-IRES-creERT2 mice were acutely administered human Gly2-GLP-2, or the GLP-2 receptor antagonist, GLP-23-33. Intestinal epithelial-insulin-like growth factor-1 receptor knockout and control mice were treated chronically with hGly2-GLP-2. Cell cycle parameters were determined by EdU, BrdU, Ki67 and phosphohistone-3 labeling and cell cycle gene expression.Acute hGly2-GLP-2 treatment increased the proportion of eGFP+EdU+/OLFM4+EdU+ cells by 11-22% (p<0.05), without affecting other cell cycle markers. hGly2-GLP-2 treatment also increased the ratio of eGFP+ cells in early-to-late S-phase by 97% (p<0.001), and increased the proportion of eGFP+ cells entering S-phase by 218% (p<0.001). hGly2-GLP-2 treatment induced jejunal expression of genes involved in cell cycle regulation (p<0.05), and increased expression of Mcm3 in the Lgr5-expressing cells by 122% (p<0.05). Conversely. GLP-23-33 reduced the proportion of eGFP+EdU+ cells by 27% (p<0.05), as well as the expression of jejunal cell cycle genes (p<0.05). Finally, chronic hGly2-GLP-2 treatment increased the number of OLFM4+ cells/crypt (p<0.05), in an intestinal epithelial insulin-like growth factor-1 receptor-dependent manner.These findings expand the actions of GLP-2 to encompass acute stimulation of Lgr5+ ISC S-phase entry through the GLP-2R, and chronic induction of Lgr5+ ISC expansion through downstream intestinal insulin-like growth factor-1 signaling.

Metaplasia and dysplasia in the corpus are reportedly derived from dedifferentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C-transcript expressing cells (PGC-transcript expressing cells) represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model.We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments, histological and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC-transcript expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC-transcript expressing cells with activated Kras, inactivated Apc and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice.Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC-transcript expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma, while Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma.The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.