Lgr5 Expression Research Articles

Intestinal epithelial cells differentiated from iPS cells (iPS-IECs) suppressed bacterial translocation in an organ model of alcohol-induced intestinal epithelial dysfunction

Abstract Microbiota translocated from the intestine causes infectious complications in alcoholics, frequently. In the present study, the effect of iPS-IEC transplantation on gut-associated bacterial translocation was investigated in an organoid model of intestinal epithelial burrier (IEB) dysfunction in patients with alcohol use disorder. iPS-IECs (a mixture of Paneth cells, goblet cells, enteroendocrine cells, and enterocytes) were differentiated from iPS cells, originated from human peripheral CD38+ hematopoietic stem cells. Human gut organoids (HGO) were generated from iPS cells in 3D cultures for 2 weeks. HGO were alcoholized with 0.2% ethanol for 2 weeks (A/HGO). iPSIECs (5 × 103 cells) were co-cultured with 100 A/HGO for 3 days. IEB functions of A/HGO were tested by severities of Enterococcus faecalis (E. faecalis) invasion into the organoids. The gene expressions of tight junction proteins and intestinal cell markers of A/HGO were determined by real-time PCR and Western blotting. In the results, E. faecalis added to culture media invaded the A/HGO, while the pathogen did not invade the HGO in the same cultures. However, the pathogen did not invade the A/HGO co-cultured with iPS-IECs. Reduced expression of tight junction protein mRNAs was shown in A/HGO, while these mRNAs were expressed in A/HGO co-cultured with iPS-IECs. LGR5 and NOTCH1 gene expressions in A/HGO were shown to be a minimum, while these genes were expressed sufficiently in A/HGO co-cultured with iPS-ICs. These results indicate that the alcohol-induced IEB dysfunction of A/HGO is significantly improved in cultures added with iPSIECs.

Silencing LGR6 Attenuates Stemness and Chemoresistance via Inhibiting Wnt/β-Catenin Signaling in Ovarian Cancer

Leucine-rich-repeat-containing G protein-coupled receptors (LGRs) have been widely found to be implicated with development and progression in multiple cancer types. However, the clinical significance and biological functions of LGR6 in ovarian cancer remains unclear. In this study, LGR6 expression was mainly examined by immunohistochemistry. Functional assays in vitro and animal experiments in vivo were carried out to explore the effect of LGR6 on cancer stem cell (CSC) characteristics and chemotherapeutic responses in ovarian cancer cells. Luciferase assays and GSEA were used to discern the underlying mechanisms contributing to the roles of LGR6 in ovarian cancer. Here, we reported that LGR6 was upregulated in ovarian cancer, which positively correlated with poor chemotherapeutic response and progression survival in ovarian cancer patients. Loss-of-function assays showed that downregulating LGR6 abrogated the CSC-like phenotype and chemoresistance in vitro. More importantly, silencing LGR6 improved the chemoresistance of ovarian cancer cells to cisplatin in vivo. Mechanistic investigation further revealed that silencing LGR6 inhibited stemness and chemoresistance by repressing Wnt/β-catenin signaling. Collectively, our results uncover a novel mechanism contributing to LGR6-induced chemotherapeutic resistance in ovarian cancer, providing the evidence for LGR6 as a potential therapeutic target in ovarian cancer.

Corrigendum] Increased expression of Lgr5 is associated with chemotherapy resistance in human gastric cancer.

An interested reader drew to the attention of the Journal that the western blot featured in Fig.3B of the above paper also appeared as Fig.3D in the following publication, featuring many of the same authors: Xi HQ, Cai AZ, Wu XS etal: Leucine‑rich repeat‑containing G‑protein‑coupled receptor5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer. BrJCancer110: 2011‑2020, 2014. After having consulted the authors about this matter, they conceded that there was a data sharing violation here, and that the image should not have been reproduced in the above article without having received the prior permission of the British Journal of Cancer. This permission has now been sought after and obtained, and Fig.3 is reproduced opposite, now including the appropriate credit for the original source of Fig.3B. The authors apologize to the Editors of the British Journal of Cancer and Oncology Reports, and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 32: 181‑188, 2014; DOI: 10.3892/or.2014.3204].

Lgr5-overexpressing mesenchymal stem cells augment fracture healing through regulation of Wnt/ERK signaling pathways and mitochondrial dynamics.

Fracture remains one of the most common traumatic conditions in orthopedic surgery. The use of mesenchymal stem cells (MSCs) to augment fracture repair is promising. Leucine-rich repeat-containing GPCR 5 (Lgr5), a transmembrane protein, has been identified as a novel adult stem cell marker in various organs and tissues. However, the roles of Lgr5 in MSCs are not fully understood. In this study, we investigated cellular functions of Lgr5 in MSCs and its potential implications in treating fracture. Lgr5-overexpressing MSCs (MSCLgr5) were established in murine SV40 promoter-driven luciferase reporter MSC line through virus transfection. Results of real-time quantitative PCR and Western blot analysis confirmed the increased expression of Lgr5 in MSCLgr5. MSCLgr5 exhibited increased osteogenic capacity, which may result from elevated expression of β-catenin and phosphorylated ERK1/2 within the nuclear region of cells. In contrast, inhibition of Lgr5 expression decreased the osteogenic differentiation ability of MSCs, accompanied with increased mitochondrial fragmentation and reduced expression of β-catenin. Local transplantation of MSCLgr5 at fracture sites accelerated fracture healing via enhanced osteogenesis and angiogenesis. MSCLgr5 stimulated the tube formation capacity of HUVECs in a Matrigel coculture system in vitro significantly. Taken together, results suggest that Lgr5 is implicated in the cellular processes of osteogenic differentiation of MSCs through regulation of Wnt and ERK signaling pathways and mitochondrial dynamics in fusion and fission. Inhibition of Lgr5 expression induced increased mitochondrial fragmentation and suppression of osteogenesis. MSCLgr5 exhibited enhanced therapeutic efficacy for fracture healing, which may serve as a superior cell source for bone tissue repair.-Lin, W., Xu, L., Pan, Q., Lin, S., Feng, L., Wang, B., Chen, S., Li, Y., Wang, H., Li, Y., Wang, Y., Lee, W. Y. W., Sun, D., Li, G. Lgr5-overexpressing mesenchymal stem cells augment fracture healing through regulation of Wnt/ERK signaling pathways and mitochondrial dynamics.

Decrease of n6/n3 PUFA Ratio Augmented Growth and Improved Markers of Intestinal Barrier Integrity in Small Intestinal Organoids Derived from Naïve and Alcohol‐Fed Mice

IntroductionIntestinal stem cells (ISCs) play an important role in gut homeostasis and function, including the maintenance of gut barrier integrity. Disruption of the intestinal barrier, specifically tight junctions, results in “leakage” of microbe‐derived compounds/toxins such as lipopolysaccharide (LPS) into the blood stream leading to endotoxemia and liver injury. We have previously demonstrated that both alcohol and nutritional factors are key players in alcohol‐induced intestinal and liver pathology. In the present study, we aimed to investigate the effects of alcohol and modulation of the n6/n3 PUFA ratio on ISC homeostasis. To this end, we used transgenic fat‐1 mice, which endogenously decrease the n6/n3 ratio by converting n6 PUFAs to n3.Hypothesisintestinal organoids derived from fat‐1 mice will show improved growth and proliferation, as well as attenuation of alcohol‐associated alterations in markers of tight junctions and antimicrobial defense.Materials and MethodsIntestinal crypts were isolated from small intestines of naïve or chronic ethanol‐fed WT C57BL/6 and fat‐1 mice. Crypts were cultured for seven days in StemCell IntestiCult growth media supplemented with gentamicin. Growth of ex vivo 3‐dimensional organoids was monitored daily by measuring cross‐sectional area, which was calculated digitally in ImageJ software. RNA was isolated with the Qiagen RNeasy kit, converted to cDNA, and used to quantify ISC growth/proliferation, tight junction, and antimicrobial defense markers by RT‐qPCR.ResultsCrypts derived from naïve fat‐1 mice grew organoids that were significantly larger than those derived from naïve WT mice after four days of culture, whereas ethanol had no significant effect. ISC markers Lgr5 and Bmi1 were significantly upregulated in organoids derived from fat‐1 as compared to WT mice in both pair‐fed and ethanol‐fed animals. No difference between WT and fat‐1 mice was seen in the expression of Axin2, a downstream target of the pro‐growth/proliferation Wnt/β‐catenin pathway. Expression of tight junction proteins ZO1 and Occludin were upregulated in ethanol‐fed fat‐1 mice as compared to their ethanol‐fed WT littermates. Bpi1, an antimicrobial peptide, was significantly increased in both ethanol‐fed and pair‐fed fat‐1 mice compared to WT animals. A similar trend was seen with Muc2, a marker of intestinal goblet cells.ConclusionsDecrease in the n6/n3 PUFA ratio and a significant increase in endogenous n3 PUFAs resulted in augmented growth of small intestinal organoids derived from naïve fat‐1 compared to WT mice. Chronic alcohol exposure compromised growth and proliferation in WT but not fat‐1 mice, as shown by expression of Lgr5 and Bmi1. Similarly, expression of tight junction and antimicrobial defense markers was greater in mice with higher endogenous n3 PUFA levels.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of in vitro fermentation derived microbial metabolites on intestinal enteroid function

A single‐stage batch in vitro fermentation system has been designed to simulate distal colon microbial activity. This system allows for examination of bacterial function. Outputs include filtered fecal supernatant (FS) that can be used for metabolite identification and examining host response. The goal of this study was to treat human duodenal and colonic enteroids with FS to determine how microbial metabolites alter cell proliferation, differentiation, and expression of other intestinal factors. Three‐dimensional (3D) human duodenal and colonic enteroids were treated with 0, 0.1, 1, 10, 25, and 50% FS or 1μg/mL lipopolysaccharide for 24 h. Cell lysates were used for RNA extraction and mRNA analysis. Visually, increasing FS concentration caused increased browning of enteroids as signs of cell differentiation. At 25 and 50% FS, structural disruption occurred, with fargreater effect at 50% potentially indicating cell death. Expression of LGR5, a stem cell marker, decreased with FS concentration and was lowest at 10% (P < 0.01). EdU staining confirmed this as cell proliferation was significantly decreased when treated with concentrations >1% FS. Expression of enterocyte and Paneth cells markers were increased by greater than 14‐fold at the 10 and 25% concentrations for alkaline phosphatase (ALPI), and six‐fold at 25% for lysozyme (LYZ), respectively (P < 0.01). Mucin 2 (MUC2) expression was increased by eight and 50‐fold at the 10 and 25% concentrations (P< 0.01) respectively, and two and three‐fold increases for mucin 3a (MUC3A; P < 0.01) suggesting an overall increase in goblet cell number. Expression of E‐Cadherin (ECAD), occludin(OCLN), claudin‐3 (CLDN3), and tight junction protein 1 (TJP1) were increased two to four‐fold and six to 23‐fold in the 10 and 25% FS groups respectively (P < 0.01). In a two‐dimensional duodenal enteroid monolayer grown on transwell inserts, there was a 13 and 25%increase in transepithelial electrical resistance (TER) at 10 and 25% FS concentrations respectively over a 24 h period (P< 0.01), and a three‐fold increase after 72 h with 25% FS (P < 0.01). In 3D colonic enteroids treated with FS there was a reduction in LGR5 expression while ALPI and LYZ were upregulated ten and three‐fold, respectively, at the 25% FS concentration. Expression of MUC2, MUC3A, Regenerating islet‐derived protein 3 alpha (REG3A), and trefoil factor 3 (TFF3) were substantially increased (>8‐fold) in the 25% FS group (P < 0.01), although in the10% FS group increases were two to three‐fold (P< 0.01) and TFF3 was not different from other treatments. The 10% FS group also saw an increase in ECAD and TJP1 expression while CLDN3 was decreased (P < 0.01). Interestingly, the 25% FS group saw a two to four‐fold increase in ECAD, OCLN, CLDN3 and TJP1 (P < 0.01). Overall, treatment with FS alters enteroid cell differentiation, and expression of mucin and tight junction protein mRNA, likely similar to a typical intestinal phenotype.Support or Funding InformationDepartment of Defense Combat Feeding Research and Engineering Program and The Applied Research for the Advancement of Science and Technology Priorities (ARAP) Program on SyntheticBiology for Military Environments (SBME), through the Office of the Secretary of Defense.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differentiation antagonizing non-protein coding RNA modulates the proliferation, migration, and angiogenesis of glioma cells by targeting the miR-216a/LGR5 axis and the PI3K/AKT signaling pathway.

Purpose: DANCR plays an important role in various types of cancer. However, its role in gliomas remains unclear. In the present study, we aimed to investigate the mechanism underlying the role of DANCR in gliomas.Methods: DANCR expression was measured by qRT-PCR, and expression of LGR5, PI3K, AKT, and phosphorylated AKT (p-AKT) was detected by western blotting. The combination of miR-216a and DANCR was quantified by Luciferase reporter assays. Proliferation, apoptosis and cell cycle, migration and invasion, and angiogenesis of glioma cells were measured by MTT, flow cytometry, Transwell, and Tube formation assays, respectively.Results: DANCR expression was significantly higher in glioma cells than in normal human astrocytes. Silencing of DANCR inhibited proliferation, migration, invasion, and angiogenesis of glioma cells, promoted apoptosis, blocked the cell cycle at the G1/S transition, and reduced LGR5, PI3K, and p-AKT expression. We identified miR-216a as a direct target of DANCR. Silencing of DANCR in glioma cells increased miR-216a expression. Further, miR-216a suppression increased proliferation, migration, invasion, and angiogenesis and inhibited apoptosis of glioma cells transfected with DANCR-targeting siRNA. In addition, miR-216a suppression compromised inhibition of the G1/S transition caused by DANCR silencing. Furthermore, suppression of miR-216a increased accumulation of LGR5, PI3K, AKT, and p-AKT in glioma cells transfected with DANCR-targeting siRNA.Conclusion: DANCR modulates growth and metastasis by targeting the miR-216a/LGR5 axis and PI3K/AKT signaling pathway.

A human enteroid model to study the effect of short‐chain fatty acids on the intestinal epithelium

Short‐chainfatty acids (SCFA) are dietary fiber derived bacterial metabolites thought to be beneficial to thehost. Of high interest is butyrate (BUT) which is the main fuel source for absorptivecells in the colon; as well as acetate (ACET) and propionate (PROP). Effects of BUT on intestinal physiology have been studied in several in vitro models, however, data in primary cells, includingintestinal stem cells (ISCs) and their distinct progenies is still not fullycharacterized. Recent advances inenteroid models have allowed these mechanisms to be studied in a more physiologically relevant human model. Therefore, our objective was to examine effects of BUT, ACET, and PROP on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using human 2‐dimensional (2D) and 3‐dimensional (3D) enteroid models. Duodenal 2D and 3D enteroids were treated with 10 mM BUT, ACET, or PROP for 24 h. Gene expression of stem cell marker Lgr5 was increased more than2‐fold in 3D enteroids treated with ACET. Conversely, enteroids treated with BUT and PROP had a greater than 10‐fold decrease in Lgr5 compared to controls (CON). Enterocyte biomarker alkaline phosphatase (Alpi) was increased due to all SCFA treatments (P<0.05). Gene expression of goblet cell marker mucin‐2 (Muc2) was increased 2 and 5‐fold due to ACET and BUT treatments respectively, but not due to PROP treatment. Paneth cell marker lysozyme (Lyz) increased due to BUT treatment and enteroendocrine cell marker chromogranin‐A (Chga) was increased 170‐fold in BUT treated enteroidsalone. Tuft cell marker Dclk1 was elevated due to ACET (7‐fold) and BUT (14‐fold) treatment compared to CON (P<0.05). Anti‐microbial molecule gene expression of Reg3b and Reg3g were not increased due to ACET treatment, however, were increased due to BUT (2.5‐fold and3‐fold, respectively) treatment. Defb1 gene expression increased 66‐fold and 75‐fold respectively in BUT and PROP treated enteroids (P<0.05). Tight junction marker expression of Ocln was only elevated in ACET treated organoids (4‐fold; P<0.05). However, Cldn3 and Tjp1 were increased due to both ACET and BUT treatments (P<0.05). 2Denteroid monolayers treated with BUT had a 2.4‐fold increase in relative transepithelial electrical resistance (TEER) compared to CON (P<0.05). PROP treatment also increased TEER (P < 0.05), while ACET had no effect. Protein expression of Interleukin‐8 in the media was induced by exposure to BUT (1.8‐fold increase) and PROP (1.5‐fold increase), but not ACET(P<0.05). Similarly, Interleukin‐18expression was increased by BUT (1.7‐fold increase) and PROP(1.3‐fold increase), while ACET was not different from CON. Intestinal Trefoil factor 3 expression in the media was increased1.3‐fold due to BUT treatment only (P<0.05). In conclusion, we determined that human enteroids are valuable and physiologically relevant models to study the effects of bacterial metabolites based upon the responses we examined. This work will be useful for future microbiomeand nutritional work using enteroid models.Support or Funding InformationFunding was provided by the Department of Defense Combat Feeding Research and Engineering Program through project “Gut Microbiomeand Performance Nutrition (TB 15–19).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ZO‐1‐Deficient Enterocytes Undergo Abortive Proliferative Expansion in Response to Mucosal Injury

Intestinal damage can be caused by physical, infectious, and immune‐mediated injury. Rapid repair, which is essential for return to mucosal homeostasis, depends on integration of epithelial migration, as in restitution, proliferation and differentiation. These are, in turn, regulated by growth factors and other stimuli that promote expansion of stem and transit‐amplifying cells. Barrier restoration is essential to recovery, but it is not known if tight junction proteins also contribute to critical events prior to junctional repair.AIMTo determine the contributions of the intestinal epithelial tight junction protein zonula occludens‐1 (ZO‐1) to mucosal wound repair.RESULTSIntestinal epithelial ZO‐1 expression is reduced in biopsies from inflammatory bowel disease (IBD) patients and mice with experimental IBD. To determine if this ZO‐1 loss contributes to disease pathogenesis, we created intestinal epithelial ZO‐1‐deficient (Tjp1f/fx villin‐Cre) mice (ZO1IEC.KO). These mice are fertile, reproduce with Mendelian genetics, and, in the absence of stress, mice grow and gain weight normally and are indistinguishable from ZO‐1‐expressing (Tjp1f/f) mice (ZO1IEC.WT). However, 2% DSS induced greater mucosal damage and weight loss in ZO1IEC.KO, relative to ZO1IEC.WT, mice, likely reflecting competition between injury and repair, as 3% DSS induced similar weight loss in ZO1IEC.KO and ZO1IEC.WT mice. Consistent with inadequate repair, ZO1IEC.KO mice failed to recover, even 4 weeks after DSS discontinuation. Labeling with EdU (2h before sacrifice) demonstrated blunted proliferative responses in ZO1IEC.KO, relative toZO1IEC.WT, mice. More striking however, was the loss and nuclear fragmentation, suggestive of apoptosis, of BrdU‐labeled epithelial cells (24h before sacrifice) in ZO1IEC.KO mice. Partial rescue by the caspase‐3 inhibitor z‐DEVD‐FMK indicates that this abortive proliferation contributes to the enhanced severity of DSS colitis in ZO1IEC.KO mice. These defects were not limited to the colon, as ZO1IEC.KO mice also displayed delayed and abortive proliferative responses following T cell activation‐induced (anti‐CD3) small intestinal mucosal damage that were partially rescued by z‐DEVD‐FMK. ZO1IEC.KO mice demonstrated greater Olfm4+ stem cells loss than ZO1IEC.WT mice, and analyses of gene expression showed reduced expression of Lgr5, Ascl2, Oflm4, Bmi1, Lrig1, Wnt3, Dll1, Jag1, Dll4, Notch1, and Hes1, reflecting stem cell loss and defective wnt and notch signaling. In contrast, Tgfbr2 and Smad4 expression were relatively increased in ZO1IEC.KO mice.CONCLUSIONThese data indicate that ZO‐1 plays critical roles in stem cell activation and epithelial survival that are distinct from contributions to tight junction structure. We therefore conclude that non‐canonical ZO‐1 functions are central to stem cell survival, epithelial proliferation, and repair that are necessary for recovery after intestinal mucosal damage. These data further suggest that ZO‐1 loss may contribute to the defective wound healing observed in human and experimental IBD.Support or Funding InformationSupported by NIH R01 DK061931.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunohistochemical Analysis of LGR5 and TROY Expression in Gastric Carcinogenesis Demonstrates an Inverse Trend

Immunohistochemical Analysis of LGR5 and TROY Expression in Gastric Carcinogenesis Demonstrates an Inverse Trend

Polymethoxylated Flavones Target Cancer Stemness and Improve the Antiproliferative Effect of 5-Fluorouracil in a 3D Cell Model of Colorectal Cancer.

Polymethoxylated flavones (PMFs) from citrus fruits are reported to present anticancer potential. However, there is a lack of information regarding their effect on cancer stem cell (CSC) populations, which has been recognized as responsible for tumor initiation, relapse, and chemoresistance. In this study, we evaluated the effect of an orange peel extract (OPE) and its main PMFs, namely, nobiletin, sinensetin, tangeretin, and scutellarein tetramethylether in targeting cell proliferation and stemness using a 3D cell model of colorectal cancer composed of HT29 cell spheroids cultured for 7 days in stirred conditions. Soft agar assay, ALDH1 activity, and relative quantitative gene expression analysis of specific biomarkers were carried out to characterize the stemness, self-renewal, and mesenchymal features of HT29 cell spheroids. Then, the impact of OPE and PMFs in reducing cell proliferation and modulating cancer stemness and self-renewal was assessed. Results showed that, when compared with monolayer cultures, HT29 cell spheroids presented higher ALDH1 activity (81.97% ± 5.27% compared to 63.55% ± 17.49% for 2D), upregulation of CD44, PROM1, SOX9, and SNAI1 genes (1.83 ± 0.34, 2.54 ± 0.51, 2.03 ± 0.15, and 6.12 ± 1.59 times) and high self-renewal capability (352 ± 55 colonies compared to 253 ± 42 for 2D). Incubation with OPE (1 mg/mL) significantly inhibited cell proliferation and modulated cancer stemness and self-renewal ability: colony formation, ALDH1 activity, and the expression of cancer stemness biomarkers PROM1 and LGR5 were significantly reduced (0.66 ± 0.15 and 0.51 ± 0.14 times, respectively). Among all PMFs, tangeretin was the most efficient in targeting the CSC population by decreasing colony formation and the expression of PROM1 and LGR5. Scutellarein tetramethylether was shown to modulate markers of mesenchymal/metastatic transition (increasing CDH1 and reducing ZEB1 and SNAI1) and nobiletin was capable of downregulating PROM1 and SNAI1 expression. Importantly, all PMFs and OPE were shown to synergistically interact with 5-fluorouracil, improving the antiproliferative response of this drug.

Immunohistochemical Expression of CD133 and LGR5 in Ulcerative Colitis-associated Colorectal Cancer and Dysplasia.

Cluster of differentiation 133 (CD133) and leu cine-rich orphan G-protein-coupled receptor 5 (LGR5) are the most putative stem cell markers for colorectal cancer (CRC), and are associated with poor prognosis of patients with CRC. However, the role of CD133 and LGR5 in the inflammation-dysplasia-carcinoma sequence has not been fully elucidated. We examined the expression of CD133 and LGR5 in ulcerative colitis-associated CRC (UC-CRC; n=20) and UC-associated colorectal dysplasia (n=16) by immunohistochemistry. The rate of CD133-positive cases in UC-CRC was significantly higher than that in dysplasia (p=0.026), but that of LGR5 expression was not. Moreover, LGR5 expression was significantly positively associated with p53 expression (p=0.03), whereas CD133 expression positively correlated with p53 expression, but not significantly (p=0.10). CD133 may play an important role in tumor development in the context of the inflammation-dysplasia-carcinoma sequence. LGR5-positive cancer stem cells may play a critical role in the development of UC-CRC, particularly upon loss of p53 function.

Overexpression of cancer stem cell marker Lgr5 in colorectal cancer patients and association with clinicopathological findings.

Background:To determine the expression of cancer stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) in colorectal carcinoma samples compared to normal adjacent tissue and any possible association with clinicopathological findings.Methods:This study was performed on forty samples of cancerous colorectal tissues (case group) and their adjacent normal mucosa (control group) in Imam Khomeini Hospital (Sari, Mazandaran, Iran). Expression of Lgr5 in tissue sections was done by immunohistochemistry. Statistical analysis was carried out using SPSS software.Results:Forty colorectal cancer patients including 21 males (57.8±11.6 years) and 19 females (58.4±12.77 years) were enrolled. Lgr5 was overexpressed in tumoral samples than normal adjacent tissues (77.5% vs 27.5%, p<0.001). Also, no association was found between primary tumor, regional lymph nodes, invasion, histological type, grade, distant metastasis and IHC results. Patients with low Lgr5 expression had a better survival rate than patients with high expression but this was not statistically significant (p=0.121).Conclusion:The higher immunoreactivity of Lgr5 in colorectal cancer tissues may indicate its role as a cancer stem cell marker in tumor carcinogenesis and patient’s survival however; Lgr5 is not associated with pathological prognostic variables.

Lgr5-mediated p53 Repression through PDCD5 leads to doxorubicin resistance in Hepatocellular Carcinoma

The devastating prognosis of hepatocellular carcinoma (HCC) is partially attributed to chemotherapy resistance. Accumulating evidence suggests that the epithelial-mesenchymal transition (EMT) is a key driving force of carcinoma metastasis and chemoresistance in solid tumors. Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), as an EMT inducer, is involved in the potentiation of Wnt signaling in HCC. This study proposes uncovering the roles of Lgr5 in Doxorubicin (Dox) resistance of HCC to improve treatment efficacy for HCC.Methods: We investigated the expression and significance of Lgr5 in HCC tissue and different cell lines. The effect of Lgr5 in EMT and Dox resistance was analyzed in HCC cells and implanted HCC tumor models. A two-hybrid analysis, using the Lgr5 gene as the bait and a HCC cDNA library, was used to screen targeted proteins that interact with Lgr5. The positive clones were identified by coimmunoprecipitation (Co-IP) and Glutathione-S-transferase (GST) pull-down. The impact of the interaction on Dox resistance was investigated by a series of assays in vitro and in vivo .Result: We found that Lgr5 was upregulated and positively correlated with poor prognosis in HCC. Additionally, it functioned as a tumor promoter to increase cell migration and induce EMT in HCC cells and increase the resistance to Dox. We identified programmed cell death protein 5 (PDCD5) as a target gene of Lgr5 and we found that PDCD5 was responsible for Lgr5-mediated Dox resistance. Further analysis with Co-IP and GST pull-down assays showed that the N-terminal extracellular domain of Lgr5 could directly bind to PDCD5. Lgr5 induced p53 degradation by blocking the nuclear translocation of PDCD5 and leading to the loss of p53 stabilization. Lgr5 showed a protection against the inhibition of Dox on the growth of tumor subcutaneously injected. Moreover, Lgr5 suppressed Dox-induced apoptosis via the p53 pathway and attenuated the cytotoxicity of Dox to HCC.Conclusion: Lgr5 induces the EMT and inhibits apoptosis, thus promoting chemoresistance by regulating the PDCD5/p53 signaling axis. Furthermore, Lgr5 may be a potential target gene for overcoming Dox resistance.

BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells.

ABSTRACTTo decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with β-catenin, it is perhaps surprising, given the importance of deregulated Wnt/β-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of β-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced β-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes LGR5 and ASCL2. In contrast, the expression of canonical Wnt targets Myc and cyclin D1 remained unchanged. Furthermore, we show that BCL-3 increases the functional stem cell phenotype, as shown by colorectal spheroid and tumoursphere formation in 3D culture conditions. We propose that BCL-3 acts as a driver of the stem cell phenotype in CRC cells, potentially promoting tumour cell plasticity and therapeutic resistance. As recent reports highlight the limitations of directly targeting cancer stem cells (CSCs), we believe that identifying and targeting drivers of stem cell plasticity have significant potential as new therapeutic targets.This article has an associated First Person interview with the first author of the paper.

Surface expression marker profile in colon cancer cell lines and sphere-derived cells suggests complexity in CD26+ cancer stem cells subsets.

ABSTRACTTaking advantage of eight established cell lines from colorectal cancer patients at different stages of the disease and the fact that all of them could form spheres, cell surface biomarkers of cancer stem cells and epithelial-mesenchymal transition were tested. The aim was to investigate cancer stem cells and metastatic stem cells in order to provide functional characterization of circulating tumor cells and promote the development of new anti-metastatic therapies. Our model showed an important heterogeneity in EpCAM, CD133, CD44, LGR5, CD26 and E-cadherin expression. We showed the presence of a subset of E-cadherin+ (some cells being E-cadherinhigh) expressing CD26+ (or CD26high) together with the well-known CSC markers LGR5 and EpCAMhigh, sometimes in the absence of CD44 or CD133. The already described CD26+/E-cadherinlow or negative and CD26+/EpCAM−/CD133− subsets were also present. Cell division drastically affected the expression of all markers, in particular E-cadherin, so new-born cells resembled mesenchymal cells in surface staining. CD26 and/or dipeptidyl peptidase 4 inhibitors have already shown anti-metastatic effects in pre-clinical models, and the existence of these CD26+ subsets may help further research against cancer metastasis.

Use of l-pNIPAM hydrogel as a 3D-scaffold for intestinal crypts and stem cell tissue engineering.

Intestinal stem cells hold great potential in tissue regeneration of the intestine, however, there are key limitations in their culture in vitro. We previously reported a novel synthetic non-biodegradable hydrogel as a 3D culture model for intestinal epithelium using Caco2 and HT29-MTX cells. Here, we investigated the potential of this system as a 3D scaffold for crypts and single intestinal stem cells to support long-term culture and differentiation. Intestinal crypts were extracted from murine small intestines and Lgr5+ stem cells isolated by magnetic activated cell sorting. Crypts and stem cells were suspended within Matrigel or l-pNIPAM for 14 days or suspended within Matrigel for 7 days then released, dissociated, and suspended within, or on l-pNIPAM hydrogel for 28 days. Cellular behaviour and phenotype were determined by histology and immunohistochemistry for stem cell and differentiation markers: Lgr5, E-cadherin MUC2 chromograninA and lysozymes. Isolated crypts and Lgr5+ intestinal stem cells formed enteroids with a central lumen surrounded by multiple crypt-like buds when cultured in Matrigel. In contrast, when crypts and stem cells were directly suspended within, or layered on l-pNIPAM hydrogel under dynamic culture conditions they formed spherical balls of cells, with no central lumen. When enteroids were initially formed in Matrigel from crypts or single Lgr5+ intestinal stem cells and dissociated into small fragments or single cells and transferred to l-pNIPAM hydrogel they formed new larger enteroids with numerous crypt-like buds. These crypt-like buds showed the presence of mucin-producing cells, which resembled goblet cells, scattered throughout their structures. Immunohistochemistry staining also showed the expression of Lgr5 and differentiation markers of all the main intestinal cell types including: enterocytes, goblet cells, enteroendocrine and Paneth cells. This demonstrated that l-pNIPAM hydrogel supported long-term culture of crypts and Lgr5+ stem cells and promoted intestinal cell differentiation.

Clinical relevance of Lgr5 expression in colorectal cancer patients

Clinical relevance of Lgr5 expression in colorectal cancer patients

Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium.

Endometriosis is characterized by the abnormal presence of endometrium outside of the uterus, resulting in pelvic pain and infertility. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has been postulated to be a marker of stem cells in the endometrium. However, LGR5+ cells have a macrophage-like phenotype in this tissue, so it is unclear what role LGR5+ cells actually play in the endometrium. Macrophages serve an important function in the endometrium to maintain fertility, while LGR5+ cells generally have a role in tumor progression and are involved in invasion in some cancers. We sought to determine whether LGR5+ cells vary across the menstrual cycle in women with endometriosis and whether there are implications for LGR5 in the aggressiveness of endometriosis and reproductive outcomes. We performed immunofluorescence, flow cytometry, and primary culture in vitro experiments on eutopic and ectopic endometrium from healthy and endometriosis patients and observed that neither LGR5+ cells nor LGR5 expression varied throughout the cycle. Interestingly, we observed that LGR5+ cell percentage overexpressing CD163 (anti-inflammatory marker) was higher in healthy endometrium, suggesting that in endometriosis, endometrium presents a more pro-inflammatory phenotype that likely leads to poor obstetric outcomes. We also observed higher levels of LGR5+ cells in ectopic lesions compared to eutopic endometrium and specifically in deep infiltrating endometriosis, indicating that LGR5 could be involved in progression and aggressiveness of the disease.

Immunohistochemical Analysis of LGR5 and TROY Expression in Gastric Carcinogenesis Demonstrates an Inverse Trend

Background:Two of the Wnt signaling pathway target genes, tumor necrosis factor receptor family member (TROY) and leucine-rich G-protein coupled receptor (LGR5), are involved in the generation and maintenance of gastrointestinal epithelium. A negative modulatory role has recently been assigned to TROY, in this pathway. Here, we have examined their simultaneous expression in gastric carcinogenesis. Methods:Tumor and paired adjacent tissues of intestinal-type gastric cancer (GC) patients (n = 30) were evaluated for LGR5 and TROY expression by immunohistochemistry. The combination of the percentage of positively stained cells and the intensity of staining was defined as the composite score and compared between groups. The obtained findings were re-evaluated in a mouse model. Results:TROY expression in the tumor tissue was significantly lower than that of the adjacent tissue (2.5 ± 0.9 vs. 3.3 ± 0.9, p = 0.004), which was coincident with higher LGR5 expression (3.6 ± 1.1 vs. 2.7 ± 0.9, p = 0.001). This observation was prominent at stages II/III of GC, leading to a statistically significant mean difference of expression between these two molecules (p = 0.005). In the H. pylori infected-mouse model, this inverse expression was observed in transition from early (8-16 w) to late (26-50 w) time points, post treatment (p = 0.002). Conclusion:Our data demonstrates an inverse trend between TROY down-regulation and LGR5 up-regulation in GC tumors, as well as in response to H. pylori infection in mice. These findings support a potential negative modulatory role for TROY on LGR5 expression.