Homeostatic responses of colonic LGR5⁺stem cells following acutein vivoexposure to a genotoxic carcinogen

Abstract

Perturbations in DNA damage, DNA repair, apoptosis and cell proliferation in the base of the crypt where stem cells reside are associated with colorectal cancer (CRC) initiation and progression. Although the transformation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)(+) cells is an extremely efficient route towards initiating small intestinal adenomas, the role of Lgr5(+) cells in CRC pathogenesis has not been well investigated. Therefore, we further characterized the properties of colonic Lgr5(+) cells compared to differentiated cells in Lgr5-EGFP-IRES-creER(T2) knock-in mice at the initiation stage of carcinogen azoxymethane (AOM)-induced tumorigenesis using a quantitative immunofluorescence microscopy approach. At 12 and 24h post-AOM treatment, colonic Lgr5(+) stem cells (GFP(high)) were preferentially damaged by carcinogen, exhibiting a 4.7-fold induction of apoptosis compared to differentiated (GFP(neg)) cells. Furthermore, with respect to DNA repair, O(6)-methylguanine DNA methyltransferase (MGMT) expression was preferentially induced (by 18.5-fold) in GFP(high) cells at 24h post-AOM treatment compared to GFP(neg) differentiated cells. This corresponded with a 4.3-fold increase in cell proliferation in GFP(high) cells. These data suggest that Lgr5(+) stem cells uniquely respond to alkylation-induced DNA damage by upregulating DNA damage repair, apoptosis and cell proliferation compared to differentiated cells in order to maintain genomic integrity. These findings highlight the mechanisms by which colonic Lgr5(+) stem cells respond to cancer-causing environmental factors.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk

“…24 Mice were fed with n-3 PUFA and curcumin alone or in combination for 3 weeks, injected with AOM and analyzed to measure DNA damage (gamma H2AX ( γ H2AX)), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)), damage repair (methylguanine-methyl transferase (MGMT)) and proliferation (5-ethynyl-2′-deoxyuridine (EdU)) in crypts 12 and 24 h later as described previously. 4 Because the integration cassette in this mouse model is epigenetically silent in about 60–70% of crypts, 25 we analyzed only GFP-positive crypts using fluorescence microscopy. No differences between GFP high and GFP neg crypts in response to carcinogen and bioactive compounds were observed (data not shown).…”

“…It has been demonstrated that Lgr5 + stem cells are preferentially damaged by AOM in the distal colon compared with differentiated cells 4 and n-3 PUFA reduce AOM-induced DNA adducts, 26 which cause double-strand breaks (DSBs). 4 Therefore, to determine the chemoprotective effects of combinatorial bioactives on AOM-induced DNA damage in Lgr5 + stem cells as compared with differentiated cells in vivo , we measured γ H2AX, a marker of the double-strand breaks.…”

“…1 High fat diet (HFD)-induced obesity augments stemness and tumorigenicity of intestinal progenitors. 2 In addition, epithelial colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5) + stem cells respond to HFD 3 and genotoxic carcinogen, for example, AOM, 4 during the cancer initiation stage. It has also been demonstrated that colonic Lgr5 + stem cells are preferentially damaged by AOM compared with differentiated cells.…”

“…It has also been demonstrated that colonic Lgr5 + stem cells are preferentially damaged by AOM compared with differentiated cells. 4 A functional consequence of this status could enhance tumorigenesis. Thus, induction of apoptosis in DNA-damaged Lgr5 + stem cells is likely to be a useful marker for successful cancer prevention, and may hold promise for identifying novel and improved cancer-chemopreventive agents.…”

Association between red meat consumption and colon cancer: A systematic review of experimental results

“…This response has been demonstrated with other carcinogens that stimulate apoptotic removal of DNA damaged cells, which occurs throughout the crypt, including at the base of the crypt where the adult colon stem cells reside. [41][42][43] Wang et al 40 followed up this experiment using the same design in which they discovered that expression of select genes (e.g., NADPH oxidase, Nox) is elevated. They also noted that downregulation of Nox expression reduced cell cycle activity and allowed apoptosis to occur.…”