Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2013 May;34(5):1150-7.
doi: 10.1093/carcin/bgt020. Epub 2013 Jan 24.

"V体育安卓版" LGR5 promotes survival in human colorectal adenoma cells and is upregulated by PGE2: implications for targeting adenoma stem cells with NSAIDs

Manal R A Al-Kharusi  1 , Helena J M Smartt, Alexander Greenhough, Tracey J Collard, Elizabeth D Emery, Ann C Williams, Chris Paraskeva
Affiliations

LGR5 promotes survival in human colorectal adenoma cells and is upregulated by PGE2: implications for targeting adenoma stem cells with NSAIDs

Manal R A Al-Kharusi et al. Carcinogenesis. 2013 May.

Abstract

Cyclooxygenase-2 is overexpressed in the majority of colorectal tumours leading to elevated levels of prostaglandin E2 (PGE2), promoting many hallmarks of cancer. Importantly, PGE2 is reported to enhance Wnt/β-catenin signalling in colorectal carcinoma cells and in normal haematopoietic stem cells where it promotes stem cell function. Although Wnt signalling plays a crucial role in intestinal stem cells, the relationship between PGE2 and intestinal stem cells is unclear. Given that the key intestinal cancer stem cell marker LGR5 (leucine-rich G-protein coupled receptor 5) is a Wnt target and PGE2 enhances Wnt signalling, the focus of this study was to investigate whether PGE2 regulated LGR5 expression in colorectal adenoma cells and whether LGR5 was important for tumour cell survival. PGE2 upregulated LGR5 protein in adenoma (RG/C2) and carcinoma (DLD-1) cell lines. LGR5 knockdown induced cell death in RG/C2 and AA/C1 adenoma cells, suggesting that LGR5 has an important survival-promoting role in adenoma cells. Indeed, we detected LGR5 protein expression in 4 of 4 human adenoma cell lines. Furthermore, LGR5 small interfering RNA inhibited the survival-promoting effects of PGE2 in RG/C2, suggesting that PGE2 promotes adenoma cell survival, at least in part, by increasing LGR5 expression. These studies, therefore, show the first link between PGE2 and LGR5 in human colorectal adenoma and carcinoma cells and demonstrate a survival-promoting role of LGR5 VSports手机版. As non-steroidal anti-inflammatory drugs (NSAIDs) cause adenomas to regress in FAP patients, these studies could have important implications for the mechanism by which NSAIDs are chemopreventive, as lowering PGE2 levels could reduce LGR5 expression and survival of LGR5(+) adenoma stem cells. .

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Validation of the LGR5 antibody for western blotting analysis (A–E). Validation of the LGR5 antibody used in this study. (A) Western blotting demonstrates that both LGR5 and HA antibodies recognize transiently expressed exogenous FLAG-HA-tagged LGR5 in LS174T. Note FLAG-HA-tagged LGR5 runs above the endogenous LGR5 doublet apparent in control LS174T cells in the LGR5 increased exposure blot. (B) Quantitative PCR confirms effective siRNA-mediated knockdown of LGR5 mRNA in RG/C2 cells. The LGR5 transcript level is relative to the Control siRNA, which is given an arbitrary value of 1. Sequencing the amplification product confirmed 100% identity to human LGR5 gene (data not shown). (C) The ~100kDa signal detected with a candidate LGR5 western blotting antibody is reduced by 20nM LGR5 siRNA using both smartpool (SP) siRNA and the four individual siRNA sequences (1–4) of the deconvoluted pool at 48h post-transfection. (D) Quantitative PCR demonstrates a reduction by 20nM β-catenin siRNA of the mRNA levels of LGR5, a Wnt/β-catenin target gene. The LGR5 transcript level is relative to the Control siRNA, which is given an arbitrary value of 1. (E) Western blotting confirms effective β-catenin knockdown and a reduction in the candidate LGR5 band. α-Tubulin was used as a loading control. **P < 0.01.
Fig. 2.
Fig. 2.
LGR5 expression in colorectal adenoma- and carcinoma-derived cell lines LGR5 expression screens using adenoma-, transformed adenoma (TA)-, carcinoma- and metastasis (M)-derived colorectal cell lines. (A) A quantitative PCR mRNA expression screen shows a range of expression levels, with some carcinomas showing very low to undetectable LGR5 expression. The LGR5 transcript level of each cell line is compared with the lowest LGR5-expressing cell line, HCT-15, which is given an arbitrary value of 1. (B) A more extensive LGR5 protein expression screen by western blotting is shown and demonstrates consistently high LGR5 expression in adenoma-derived cell lines. α-Tubulin was used as a loading control.
Fig. 3.
Fig. 3.
PGE2 upregulates LGR5 protein in PGE2-sensitive cell lines. (A) An increase in cell yield and a decrease in cell death are observed after 72h of PGE2 treatment in RG/C2 cells with different PGE2 concentrations (0.75, 1 and 5 μM). (B) PGE2 increases LGR5 protein levels at 24, 48 and 72h at all three doses in RG/C2 cells. (C) PGE2 also increases cell yield and increases LGR5 protein levels in the carcinoma cell line DLD-1 after 72h. (D) PGE2 does not significantly increase cell yield in the PGE2 insensitive adenoma cell line AA/C1 after 72h. PGE2 does not regulate LGR5 protein levels at 72h in AA/C1 cells. α-Tubulin was used as a loading control. Data in (D) represent the average of two repeat experiments carried out in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 and NS= not significant.
Fig. 4.
Fig. 4.
PGE2 does not increase LGR5 mRNA expression. (A and B) PGE2 upregulates LGR5 protein but not mRNA. (A) Quantitative PCR shows that LGR5 mRNA levels are not increased in RG/C2 following 24, 48 or 72h PGE2 treatment, despite the increase in LGR5 protein apparent by western blotting in (B). The LGR5 transcript level is relative to its vehicle control at each time point, which are given arbitrary values of 1. (C) Twenty nanomolar of β-catenin siRNA does not prevent PGE2 from increasing LGR5 protein levels in RG/C2, despite decreasing basal levels. Densitometric analysis of LGR5 western blotting demonstrates a similar 2-fold increase in LGR5 protein both in the absence or in the presence of β-catenin siRNA (C). (D) Quantitative PCR confirms that PGE2 does not increase LGR5 mRNA levels either in the absence or in the presence of β-catenin siRNA. The LGR5 transcript level is relative to the Control siRNA, which is given an arbitrary value of 1. Data in (D) represent the average of two repeat experiments carried out in triplicate. *P < 0.05, **P < 0.01 and NS = not significant.
Fig. 5.
Fig. 5.
LGR5 siRNA reduces survival in colorectal adenoma-derived cell lines. (A and B) LGR5 siRNA-mediated knockdown by 20nM LGR5 smartpool (SP) siRNA decreases cell yield and increases cell death at 72h in (A) RG/C2 and (B) AA/C1 adenoma-derived cell lines. LGR5 knockdown efficiency was confirmed by western blotting. (C) LGR5 siRNA-mediated knockdown reduces the sensitivity of RG/C2 to PGE2 treatment as it prevents PGE2 from significantly reducing cell death in RG/C2 cells. (D) LGR5 western blotting confirms upregulation of LGR5 protein by PGE2 and effective LGR5 knockdown. α-Tubulin was used as a loading control. *P < 0.05; **P < 0.01; ***P < 0.001 and NS= not significant.
See this image and copyright information in PMC

V体育官网入口 - References

    1. Fearon E.R., et al. (1990). A genetic model for colorectal tumorigenesis. Cell, 61, 759–767 - "V体育2025版" PubMed
    1. Brown J.R., et al. (2005). COX-2: a molecular target for colorectal cancer prevention. J. Clin. Oncol., 23, 2840–2855 - PubMed (V体育安卓版)
    1. Kitamura T., et al. (2002). Inhibitory effects of mofezolac, a cyclooxygenase-1 selective inhibitor, on intestinal carcinogenesis. Carcinogenesis, 23, 1463–1466 - PubMed
    1. Eberhart C.E., et al. (1994). Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology, 107, 1183–1188 - PubMed
    1. Elder D.J., et al. (2002). Human colorectal adenomas demonstrate a size-dependent increase in epithelial cyclooxygenase-2 expression. J. Pathol., 198, 428–434 - PubMed

Publication types

V体育平台登录 - MeSH terms

LinkOut - more resources