Leucine-rich Repeat-containing G-protein-coupled Receptor 5 Expression Research Articles

Antitumor Activity of a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting LGR5 in Preclinical Models of Neuroblastoma.

Neuroblastoma (NB) is a cancer of the peripheral nervous system found in children under 15 years of age. It is the most frequently diagnosed cancer during infancy, accounting for ~12% of all cancer-related deaths in children. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a membrane receptor that is associated with the primary tumor formation and metastasis of cancers in the gastrointestinal system. Remarkably, high levels of LGR5 are found in NB tumor cells, and high LGR5 expression is strongly correlated with poor survival. Antibody-drug conjugates (ADCs) are monoclonal antibodies that are covalently linked to cell-killing cytotoxins to deliver the payloads into cancer cells. We generated an ADC with an anti-LGR5 antibody and pyrrolobenzodiazepine (PBD) dimer-based payload SG3199 using a chemoenzymatic conjugation method. The resulting anti-LGR5 ADC was able to inhibit the growth of NB cells expressing LGR5 with high potency and specificity. Importantly, the ADC was able to completely inhibit the growth of NB xenograft tumors in vivo at a clinically relevant dose for the PBD class of ADCs. The findings support the potential of targeting LGR5 using the PBD class of payload for the treatment of high-risk NBs.

TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells.

Mutations in components of the Wnt/β-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.

Correlation of LGR5 expression and clinicopathological features in intrahepatic cholangiocarcinoma

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a known cancer stem cell marker. However, there are no reported analyses of LGR5 mRNA expression in normal liver and liver cancer tissues. Here, we evaluated LGR5 expression by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 25 samples of intrahepatic cholangiocarcinoma (ICC) selected from the medical archives at our hospital. LGR5 expression levels were divided into high and low expression groups by the five-grade scoring system, and clinicopathological features were analyzed. Low LGR5 expression was identified in some normal hepatocytes and bile duct cells. In addition, LGR5 expression was identified in all bile duct cancer samples except one case. Well-differentiated to moderately-differentiated adenocarcinoma tended to show higher LGR5 expression than poorly-differentiated adenocarcinoma (P = 0.0561), and the large duct type showed significantly higher LGR5 expression levels than the small duct type (P = 0.0225). Patients in the high LGR5 expression group tended to have good overall survival (OS) (P = 0.0623). The Cox proportional hazard regression model revealed that the high LGR5 expression group showed independently better OS for ICC (P = 0.0285). High LGR5 expression is possibly a good prognosis factor in ICC. However, the detailed mechanism of LGR5 in this disease remains unclear, and further analysis is warranted.

The clinical and prognostic significance of LGR5 in GC: A meta-analysis of IHC assay and bioinformatics analysis

Recently, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a newly identified cancer stem cell marker and Wnt target gene. However, the role of LGR5 in gastric cancer (GC) remains uncertain. This study was performed to investigate the effect of LGR5 expression in GC. The eligible studies were searched via electronic databases. The odds ratios (ORs) with 95% confidence intervals (CIs) or hazard ratios (HRs) with 95% CIs were applied to estimate the effect of LGR5. Further bioinformatics validation data were used to confirm our results. Eleven studies consisting of 2,646 GC patients were identified. LGR5 expression was not associated with age, gender, tumor stage, T stage, tumor size, lymphatic invasion, lymph node metastasis, and distal metastasis. LGR5 expression was related to tumor type (intestinal vs. diffuse: OR = 2.25, P = 0.032). LGR5 expression was negatively correlated with tumor grade (grade 3-4 vs. grade 1-2: OR = 0.40, P = 0.033). Further TCGA validation data also showed similar findings, and LGR5 expression was also found to have a negative association with tumor grade. LGR5 expression was associated with worse overall survival (OS) using multivariate Cox analysis (HR = 2.54, P = 0.009). Further bioinformatics data showed that LGR5 expression was still correlated with shorter OS in 876 GCs. LGR5 expression was negatively correlated with tumor grade and its expression was higher in intestinal-type than in diffuse-type. Moreover, LGR5 may be a potential prognostic factor for survival prediction in GC.

Clinicopathologic and prognostic significance of LGR5, a cancer stem cell marker in patients with colorectal cancer.

Aim:To analyze the clinicopathologic and prognostic significance of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a cancer stem cell marker expression in a cohort of colorectal cancer patients (CRC).Patients & methods:A total of 76 formalin-fixed paraffin-embedded tissue blocks of primary or metastatic tumors from 49 CRC patients were collected for duration 2009–2015. LGR5 expression was assessed through immunohistochemical staining of a tissue microarray.Results:LGR5 was significantly over expressed in CRC tissue samples and found to be a statistically significant independent prognostic marker for an improved overall survival.Conclusion:LGR5 expression was higher in colorectal cancer than in normal tissue. LGR5 was an independent prognostic marker for better clinical outcomes and might be used as a potential therapeutic target in CRCs.

Correlation of clinicopathological features and leucine-rich repeat-containing G-protein-coupled receptor 5 expression in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. Previous studies have established leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) as a cancer stem cell marker in gastrointestinal cancers. However, few reports have examined LGR5 in PDAC. Here we examined LGR5 expression and its clinicopathological significance in PDAC. We evaluated LGR5 expression in 78 PDAC patients who underwent surgical resection in our institution using RNAscope, a newly described RNA in situ hybridization technique. All 78 PDAC cases expressed LGR5 in cancer tissues, and LGR5 expression was prominent in the gland-forming part. LGR5 expression was significantly higher in patients with low histological grade (G1–G2) (p < 0.001) and early clinical stage (p = 0.004). Univariate analysis showed that low LGR5 expression (p = 0.034) was significantly associated with worse overall survival. However, LGR5 expression did not remain a predictor of prognosis in multivariate analysis (p = 0.639). All PDAC cases showed LGR5 expression to varying degrees, indicating LGR5 might be a cancer stem cell marker of PDAC, as in gastrointestinal cancer. Reduced LGR5 expression in tumor cells was associated with worse prognosis in PDAC. Further studies are required to elucidate the relationship between tumor progression and LGR5 expression in PDAC.

Overexpression of LGR-5 as a Predictor of Poor Outcome in Patients with Hepatocellular Carcinoma.

Hepatocarcinogenesis and distant metastasis pose major challenges for physicians. They are regulated by several genes, such as AKT, JUK, Wnt, and P53, and their expression activates several important processes such as cell proliferation, migration, motility, and interaction in the microenvironment. The leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR-5) is a novel biomarker, particularly in stem cells, and is involved in embryogenesis, tumor development, and tumor cell signal transduction. Here, we investigated LGR-5 expression using immunohistochemistry and analyzed the correlation between clinical features and prognosis in patients with hepatocellular carcinoma (HCC). We found that LGR-5 expression was higher in tumor tissues than in normal liver tissues, and that high LGR-5 expression possibly favored poor outcomes in HCC, especially in well/moderate differentiation grade, hepatitis C virus (HCV)-negative, and hepatitis B virus (HBV)-positive groups. Thus, the LGR-5 marker is suggested to be a routine biomarker for poor prognosis, thereby providing a platform for anti-LGR-5-targeted therapy in the future.

3089 Clinicopathologic and Prognostic Significance of LGR5, a Cancer Stem cell Marker in Peritoneal Metastasis of a Colorectal Origin

OBJECTIVES/SPECIFIC AIMS: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is expressed on Wnt/β-catenin-dependent adult stem cell populations of the colon. Cancer stem cells are hypothesized to be the driving force behind tumor progression and metastasis, making them attractive therapeutic targets. Our aim was to analyze the clinicopathologic and prognostic significance of LGR5 expression in a cohort of colorectal cancer patients with peritoneal metastasis. METHODS/STUDY POPULATION: A total of 49 Formalin-fixed paraffin-embedded (FFPE) tissue blocks of primary or metastatic tumors and their respective normal tissues were collected from the tissue bank for time period 2009-2015. LGR5 expression was assessed at the protein level through immunohistochemical (IHC) staining of tissue microarray (TMA) constructs consisting of pairs of tumor and normal colon tissue. The correlation between LGR5 expression and clinicopathologic parameters and prognosis was assessed by statistical analysis. RESULTS/ANTICIPATED RESULTS: Of the 49 patient sample, 30(61.22%) were female vs. 19 (38.78%) males. Age range at initial diagnosis ranged from 31.7 years to 84.4 years, with a median age of 61.29 years. Duration of follow-up ranged from 1 – 9 years with a median of 5 years.LGR5 expression was higher in colorectal cancer than in normal mucosa. In univariate survival analysis overexpression of LGR5 was significantly associated with improved survival (p=0.002).Of significance, LGR5 positivity was an independent prognostic marker for better prognosis in a multivariate survival analysis adjusting for prognostic variables age, stage, gender, tumor histology and grade (HR 2.67. 95% CI 1.01-7.00, P = 0.046). DISCUSSION/SIGNIFICANCE OF IMPACT: LGR5 was significantly over expressed in colorectal cancer compared to normal tissues. LGR5 was noted to be an independent prognostic variable for an improved survival outcome in colorectal cancer patients with peritoneal metastasis, making LGR5 a potential therapeutic target in colorectal cancer patients with peritoneal metastasis.

The biological functions of LGR5 in promoting non-small cell lung cancer progression

Background: The Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), which is used as a marker of adult stem cells and colorectal cancer stem cells (CSCs), is closely associated with the progression of non-small cell lung cancer (NSCLC). This study aimed to identify the clinical significance and biological function of LGR5 in NSCLC. Methods: Quantitative reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the expression of LGR5 and stemness-related genes in 22 NSCLC patients, and the clinical significance of LGR5 in NSCLC progression was estimated by statistical analysis. LGR5 overexpressing A549- and H1299-transfected cells were established, and CCK-8 and clone formation assays were used to test the proliferation ability. A wound-healing assay was utilized to clarify the migration ability. The invasion ability was confirmed via the Transwell assay kit. Results: LGR5 expression was markedly higher in NSCLC tissues than in the matched adjacent normal tissues, and had a trend to associate with tumor size, lymph node metastasis, and TNM stage. The proliferation rates, clone formation rates, wound healing rates, number of invasive cells, and the NOTCH1 expression of the LGR5 overexpressing groups, were significantly higher than those of the control groups. Conclusions: LGR5 plays an essential role in NSCLC tumorigenesis and is closely associated with the proliferation, metastasis, and invasion of NSCLC cells. LGR5 may promote NSCLC progression via NOTCH1 and could be a new target for gene-targeted therapies for NSCLC.

G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A.

BackgroundTargeting cancer stem cells (CSCs) in breast cancer (BrCa) may improve treatment outcome and patient prognosis. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a well-recognized adult stem cell and CRC marker, and previous reports have suggested the cancer-promoting role of LGR5 in breast cancer, but the mechanism remains unclear.Material/MethodsPotential LGR5-associating genes were explored using STRING database, and LGR5 overexpression and knockdown was constructed in MCF-7 and MDA-MB-453 human BrCa cells, respectively. PKA catalytic subunit activation and PKA kinase activity in human BrCa cells was examined by Western blot and PKA kinase activity assay, respectively. Protein expression level or activation of β-catenin and GSK-3β in human BrCa cells was investigated by Western blot. Cell proliferation, colony formation, Transwell migration, cisplatin sensitivity, and in vivo tumor formation of human BrCa cells were examined.ResultsLGR5 overexpression increased PKA activation and its kinase activity in human BrCa cells, which was decreased by LGR5 knockdown. LGR5 expression level or PKA kinase activity were correlated with β-catenin Ser 552 phosphorylation but inversely correlated with GSK-3β Ser9 phosphorylation in human BrCa cells in vitro. LGR5/PKA increased cell proliferation, colony formation, Transwell migration, and cisplatin resistance in vitro, as well as tumor formation in vivo, of human BrCa cells.ConclusionsLGR5 activates the Wnt/β-catenin signaling pathway in human BrCa cells in vitro via PKA.

Localization of leucine-rich repeat-containing G-protein-coupled receptor 5- and Ki67-positive periodontal cells expressing runt-related transcription factor 2 during tooth movement

PurposeMechanical stimuli induce the proliferation and osteogenic differentiation of periodontal ligament (PDL) progenitor cells. However, the localization of formative and proliferative cells in response to orthodontic force in the PDL is unknown. We aimed to elucidate the expression patterns and localization of the cell proliferation- and osteogenesis-associated markers in the PDL during tooth movement. Materials and methodsFive-week-old male Sprague–Dawley rats had their right maxillary first molars (rM1s) moved mesially using a 10-gf titanium-nickel alloy closed-coil spring. Left maxillary first molars (lM1s) served as controls. Immunohistochemical analyses of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), Ki67, and runt-related transcription factor 2 (RUNX2) were performed in the periapical region of M1. mRNA levels of Ki67, Runx2, and β-catenin were quantified by real-time reverse transcription polymerase chain reaction (qRT-PCR). In vitro human PDL stem cells (hPDLSCs) were cyclically stretched for 24h, and MTT assay and western blot analysis of LGR5 were performed. ResultsImmunohistochemistry showed that LGR5 and Ki67 were partially co-expressed with RUNX2 in the periapical PDL. Immunoreactivities of LGR5, Ki67, and RUNX2 were significantly greater in the PDL of the rM1 than in that of the lM1 on day 3. qRT-PCR showed that Ki67 and Runx2 mRNA levels, but not β-catenin levels, increased on the mesial side of the periapical PDL on day 3. Western blot analysis revealed that 24h of cyclic strain stimulated LGR5 expression in hPDLSCs. ConclusionOur findings suggested that orthodontic force induced LGR-5- and Ki67-positive cells expressing RUNX2 in the PDL during tooth movement.

Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

STUDY QUESTIONIs human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated?SUMMARY ANSWER LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated.WHAT IS KNOWN ALREADYThe human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia.STUDY DESIGN, SIZE, DURATIONThe study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women’s hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture.PARTICIPANTS/MATERIALS, SETTING, METHODSIn this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC).MAIN RESULTS AND THE ROLE OF CHANCE LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays.LARGE SCALE DATAWe did not generate our own large scale data but interrogated publically available large scale data sets.LIMITATIONS, REASONS FOR CAUTIONIn the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent.WIDER IMPLICATIONS OF THE FINDINGSThese data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

LGR5 promotes cancer stem cell traits and chemoresistance in cervical cancer

Cancer stem cells (CSCs), also known as tumor-initiating cells, contribute to tumorigenesis, resistance to chemoradiotherapy and recurrence in human cancers, suggesting targeting CSCs may represent a potential therapeutic strategy. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) has recently been found to be a bona fide marker of colorectal CSCs. Our previous study showed that LGR5 functions as a tumor promoter in cervical cancer by activating the Wnt/β-catenin pathway. However, very little is known about the function or contribution of LGR5 to cervical CSCs. Here, we have modulated the expression of LGR5 using an overexpression vector or short hairpin RNA in cervical cancer cell lines. We demonstrated that elevated LGR5 expression in cervical cancer cells increased tumorsphere-forming efficiency; conferred chemoresistance to cisplatin treatment; augmented cell migration, invasion and clonogenicity; and elevated the levels of stem cell-related transcription factors in vitro. Furthermore, modulated LGR5+ cells, unlike LGR5− cells, were highly tumorigenic in vivo. In addition, the modulated LGR5+ cells could give rise to both LGR5+ and LGR5− cells in vitro and in vivo, thereby establishing a cellular hierarchy. Finally, we found that the increased tumorsphere-forming efficiency induced by LGR5 could be regulated through the inhibition or activation of the Wnt/β-catenin pathway in cervical cancer cells. Taken together, these results indicate that LGR5 has a vital oncogenic role by promoting cervical CSC traits and may represent a potential clinical target.

LGR5 is associated with tumor aggressiveness in papillary thyroid cancer.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker and a down-stream target in Wnt/β-catenin signaling. In human papillary thyroid cancer (PTC), over activation of Wnt/β-catenin has been associated with tumor aggressiveness. Using established human cell lines (TPC-1, KTC-1, Nthy-ori-3-1), we report LGR5 and R-spondin (RSPO1-3) overexpression in PTC and manipulate LGR5 and Wnt/β-catenin signaling via both pharmacologic and genetic interventions. We test the association of LGR5 tumor expression with markers of PTC aggressiveness using a Discovery Cohort (n = 26 patients) and a Validation Cohort (n = 157 patients). Lastly, we explore the association between LGR5 and the BRAFV600E mutation (n = 33 patients). Our results reveal that LGR5 and its ligand, RSPO, are overexpressed in human PTC, whereby Wnt/β-catenin signaling regulates LGR5 expression and promotes cellular migration. In two separate cohorts of patients, LGR5 and RSPO2 were associated with markers of tumor aggressiveness including: lymph node metastases, vascular invasion, increased tumor size, aggressive histology, advanced AJCC TNM stage, microscopic extra thyroidal extension, capsular invasion, and macroscopic invasion. As a biomarker, LGR5 positivity predicts lymph node metastasis with 95.5% sensitivity (95% CI 88.8%-98.7%) and 61% specificity (95% CI: 48.4%-72.4%) and has a negative predictive value (NPV) of 91.3% (95% CI 79.2%-97.5%) for lymph node metastatic disease. In human PTC, LGR5 is also strongly associated with the BRAFV600E mutation (p = 0.005). We conclude that overexpression of LGR5 is associated with markers of tumor aggressiveness in human PTC. LGR5 may serve as a future potential biomarker for patient risk stratification and loco regional metastases in PTC.

The prognostic role of Leucine-rich repeat-containing G-protein-coupled receptor 5 in gastric cancer: A systematic review with meta-analysis

The prognostic value of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) in gastric cancer remains controversial. To further investigate this relationship, we performed meta-analyses to systematically review the association between LGR5 expression and various clinical parameters in gastric cancer patients. Eligible studies from PubMed, Embase, Web of Science, CNKI (Chinese National Knowledge Infrastructure), Wangfang (Database of Chinese Ministry of Science & Technology) and CBM (China Biological Medicine) databases were evaluated to investigate the association of LGR5 expression with overall survival (OS) and clinicopathological features of gastric cancer. LGR5 overexpression was significantly associated with poor OS in patients with gastric cancer (HR 1.66, 95% CI 1.02-2.69). LGR5 overexpression was also significantly associated with TNM stage (TIII/TIV vs TI/TII: OR 5.42, 95% CI 1.02-28.72) and lymph node metastasis (positive vs negative: OR 2.30, 95% CI 1.06-5.0). Our meta-analysis indicates that LGR5 may be a predictive factor for invasion and metastasis, and poor prognosis in patients with gastric cancer.

Recent advances in pancreatology.

Recent advances in pancreatology.

Cytoplasmic expression of LGR5 in pancreatic adenocarcinoma

Background: CD133 has been identified as a cancer stem cell marker for pancreatic ductal adenocarcinoma. Although leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5), a marker of intestinal stem cells, has been shown to be on a higher level of the stem cell hierarchy than CD133, the expression and function of LGR5 in pancreatic cancer tissue remains unclear. This study investigated tissue expression of LGR5 and CD133 in resected pancreatic cancer tissue.Methods: LGR5 and CD133 expression was immunohistochemically examined in 9 patients with pancreatic ductal adenocarcinoma who underwent resection.Results: LGR5 was expressed in the cytoplasm of pancreatic cancer cells in 4 of 9 cases. CD133 was not detected in cancerous tissue. In non-neoplastic tissue, LGR5 was expressed in the basolateral membrane of a subset of endocrine cells. Conversely, CD133 was expressed in the apical membrane of small duct cells. Co-localization of LGR5 and CD133 was not found in either neoplastic or non-neoplastic tissue. LGR5 expression in pancreatic cancer cells showed no statistically significant correlation with survival after surgery.Conclusion: We have demonstrated that LGR5 is expressed in the cytoplasm of pancreatic adenocarcinoma cells, and the basolateral membrane of a subset of endocrine cells of the human pancreas. Further investigation is required to clarify any prognostic significance of LGR5 expression.

LGR5 Is a Negative Regulator of Tumourigenicity, Antagonizes Wnt Signalling and Regulates Cell Adhesion in Colorectal Cancer Cell Lines

BackgroundLGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is the most established marker for intestinal stem cells. Mouse models show that LGR5+ cells are the cells of origin of intestinal cancer, and LGR5 expression is elevated in human colorectal cancers, however very little is known about LGR5 function or its contribution to the stem cell phenotype and to colorectal cancer.Principal FindingsWe have modulated the expression of LGR5 by RNAi (inhibitory RNAs) or overexpression in colorectal cancer cell lines. Paradoxically, ablation of LGR5 induces increased invasion and anchorage-independent growth, and enhances tumourigenicity in xenografts experiments. Conversely, overexpression of LGR5 augments cell adhesion, reduces clonogenicity and attenuates tumourigenicity. Expression profiling revealed enhanced wnt signalling and upregulation of EMT genes upon knockdown of LGR5, with opposite changes in LGR5 overexpressing cells. These findings suggest that LGR5 is important in restricting stem cells to their niche, and that loss of LGR5 concomitant with activated wnt signalling may contribute to the invasive phenotype of colorectal carcinomas.

Abstract 4640: High expression of the stem cell marker LGR5 is associated with a poor clinical outcome in patients with Ewing's sarcoma

Abstract Ewing's sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that are most commonly diagnosed in childhood and adolescence. Unfortunately, drug-resistant relapse is not uncommon and is associated with very poor outcomes. No clinical or pathologic features currently exist that reliably predict chemosensitivity or relapse in newly diagnosed patients. In this study we used whole genome expression microarray profiling to identify potential markers of drug resistance and relapse in ESFT. Affymetrix human exon (HuEx) arrays were used to profile a highly drug resistant case of primary, localized ESFT before and after induction therapy. Comparison of gene summarized data from pre- and post-therapy samples with 10 chemosensitive tumors identified 309 genes to be differentially expressed in the resistant case at diagnosis (fold change&amp;gt;2). Of the 309 differentially expressed genes at diagnosis, 130 demonstrated an even greater change in expression post-induction therapy suggesting that these genes might be markers of the most highly drug-resistant cells. Intriguingly, two stem cell associated genes, PROM1 (prominin 1) and LGR5 (leucine-rich-repeat containing G-protein-coupled receptor 5), were included in the list of putative chemoresistance markers. Expression of PROM1 and LGR5 were 18-fold and 17-fold higher, respectively, at diagnosis and 31-fold and 21-fold higher post-induction therapy in the drug resistant case. To determine if either gene is associated with chemoresistant disease or relapse in ESFT we next evaluated whether their expression correlated with outcome in a large cohort of institutional and Children's Oncology Group patients. Clinical correlates analysis of HuEx gene expression data obtained from 53 tumors revealed no correlation between PROM1 expression and outcome. In contrast, increased expression of LGR5 was associated with worse event-free (p=0.008) and overall survival (p=0.046). Variable but detectable expression of LGR5 in ESFT tumors and cell lines was validated using quantitative RT-PCR. Finally, LGR5 expression in ESFT cells was compared to human embryonic and somatic stem cells and fibroblasts. Significantly, LGR5 was found to be highly expressed by neural crest stem cells but not by bone-marrow derived mesenchymal stem cells, differentiated neural crest cells, or embryonic fibroblasts. These data suggest that LGR5 might be useful as a predictor of outcome in ESFT and implicate a primitive neural crest stem cell phenotype in the developmental origins of drug-resistant and relapsed disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4640.