LGR5 positivity defines stem-like cells in colorectal cancer

Abstract

Like normal colorectal epithelium, colorectal carcinomas (CRCs) are organized hierarchically and include populations of cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is associated with these stem cells in normal colorectal epithelium; however, the precise function of LGR5 in CRC remains largely unknown. Here, we analyzed the functional and molecular consequences of short hairpin RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed Lgr5-EGFP-IRES-CreERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS), which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization. Silencing of LGR5 in SW480 CRC cells resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells, including LGR5. Silencing of LGR5 reduced proliferation, migration and colony formation in vitro and tumorigenicity in vivo. In accordance with these results, NOTCH signaling was downregulated upon LGR5 silencing. In AOM/DSS-induced colon tumors, Lgr5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal colon cells. Array comparative genomic hybridization revealed no genomic imbalances in either tumor cell fraction. Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.

From the Cover: PhIP/DSS-Induced Colon Carcinogenesis in CYP1A-Humanized Mice and the Possible Role of Lgr5+ Stem Cells

“…In addition, it appears that a few of these scattered Lgr5-EGFP-expressing stem cells are promoted to form cell clusters, which progress to high-grade dysplastic lesions and adenocarcinomas. These findings are in contrast to recent studies that showed Lgr5-EGFP expression is restricted to a small population of scattered cells in colon tumors induced by AOM/DSS (Barker et al, 2009;Hirsch et al, 2014). Although exact reason for the discrepancy is currently unclear and need to be further studied, we speculate that the greatly variegated expression of Lgr5-EGFP in the colon crypts may play a fundamental role.…”

Targeting colorectal cancer (stem-like) cells using LGR5 directed antibody drug conjugates

“…Of note, Lgr5 mRNA expression was significantly elevated in intestinal tumors relative to matched normal mucosa, with a similar magnitude as previously reported in an inflammation driven mouse model of colonic tumorigenesis (AOM/DSS model) (21,24). Also, the fraction of Lgr5 positive tumor cells was comparable in the AKV model, in the AOM/DSS model (21,24) Taken together, Junttila and colleagues could show that targeting of LGR5 positive colorectal cancer cells with ADCs is feasible without causing significant side effects to normal tissue in mice and rats, though linker and drug selection was critical to achieve acceptable toxicity profiles. Moreover, anti-LGR5 specific ADCs significantly prolonged survival in a genetically engineered mouse model of intestinal tumorigenesis, decreasing tumor size and reducing tumor growth rate.…”

LGR5 promotes the proliferation of colorectal cancer cells via the Wnt/β-catenin signaling pathway

“…When activated and accumulated in the cytoplasm, β-catenin is transferred to the nucleus, where it activates numerous nuclear transcription factors, such as transcription factor (TCF)/lymphoid enhancer-binding factor, which results in the activation of downstream target molecules. Abnormal expression of these molecules may result in abnormal proliferation and tumorigenesis (33). APC is an important tumor suppressor that downregulates the transcriptional activity of β-catenin by the following three mechanisms: i) Reducing the levels of cytoplasmic β-catenin by binding to Axin; ii) promoting the export of nuclear β-catenin; and iii) sequestering β-catenin, preventing it from binding to TCF (34).…”