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

Development and validation of cyclic peptide probe for gastric cancer based on phage display technique

AbstractCancer‐targeting diagnostics should have a sensitive and specific binding affinity. To achieve this, biomarker development is critical. This study aimed to develop and validate a 7‐mer cyclic peptide probe that can target gastric cancer. We developed this probe based on LGR5 (leucine‐rich repeat‐containing G‐protein coupled receptor 5)‐specific targeting, which is a marker for gastric cancer stem cells. An LGR5 targeting peptide sequence that was developed using phage display technology resulted in a cyclic peptide, C‐YLASRVH‐C (named YLA). We conjugated this peptide with fluorescent probes to validate its specific targeting ability for gastric cancer. The fluorescence‐labeled YLA peptide exhibited 3.0‐fold higher fluorescence intensity in a gastric cancer cell line (MKN45) than it did in a normal cell line (CCD841 cells). In contrast, pancreatic and colorectal cancer cells did not show significant fluorescence intensity with the YLA peptide. To verify its tumor‐targeting affinity, we developed a control peptide, C‐YLASAVH‐C (named YLASA) using an ALA scanning experiment. Whole‐body imaging of a gastric cancer xenograft model showed higher fluorescence intensity of tumors in the YLA peptide group than in the control peptide group. Moreover, ex vivo imaging of tumor tissues exhibited 6.8‐fold higher fluorescence intensity in the YLA peptide group compared to that in the YLASA control peptide group. In conclusion, we confirmed that the YLA peptide probe functions as a specific diagnostic probe for gastric cancer. We anticipate that it will play a theranostic role through further development.

Rspo2 exacerbates rheumatoid arthritis by targeting aggressive phenotype of fibroblast-like synoviocytes and disrupting chondrocyte homeostasis via Wnt/β-catenin pathway

BackgroundThe aggressive phenotype of fibroblast-like synoviocytes (FLS) has been identified as a contributing factor to the exacerbation of rheumatoid arthritis (RA) through the promotion of synovitis and cartilage damage. Regrettably, there is currently no effective therapeutic intervention available to address this issue. Recent research has shed light on the crucial regulatory role of R-spondin-2 (Rspo2) in cellular proliferation, cartilage degradation, and tumorigenesis. However, the specific impact of Rspo2 on RA remains poorly understood. We aim to investigate the function and mechanism of Rspo2 in regulating the aggressive phenotype of FLS and maintaining chondrocyte homeostasis in the context of RA.MethodsThe expression of Rspo2 in knee joint synovium and cartilage were detected in RA mice with antigen-induced arthritis (AIA) and RA patients. Recombinant mouse Rspo2 (rmRspo2), Rspo2 neutralizing antibody (Rspo2-NAb), and recombinant mouse DKK1 (rmDKK1, a potent inhibitor of Wnt signaling pathway) were used to explore the role and mechanism of Rspo2 in the progression of RA, specifically in relation to the aggressive phenotype of FLS and chondrocyte homeostasis, both in vivo and in vitro.ResultsWe indicated that Rspo2 expression was upregulated both in synovium and articular cartilage as RA progressed in RA mice and RA patients. Increased Rspo2 upregulated the expression of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), as the ligand for Rspo2, and β-catenin in FLS and chondrocytes. Subsequent investigations revealed that intra-articular administration of rmRspo2 caused striking progressive synovitis and articular cartilage destruction to exacerbate RA progress in mice. Conversely, neutralization of Rspo2 or inhibition of the Wnt/β-catenin pathway effectively alleviated experimental RA development. Moreover, Rspo2 facilitated FLS aggressive phenotype and disrupted chondrocyte homeostasis primarily through activating Wnt/β-catenin pathway, which were effectively alleviated by Rspo2-NAb or rmDKK1.ConclusionsOur data confirmed a critical role of Rspo2 in enhancing the aggressive phenotype of FLS and disrupting chondrocyte homeostasis through the Wnt/β-catenin pathway in the context of RA. Furthermore, the results indicated that intra-articular administration of Rspo2 neutralizing antibody or recombinant DKK1 might represent a promising therapeutic strategy for the treatment of RA.

LGR5 As a Potential Therapeutic Target for Breast Cancer: A Systematic Review and Meta-analysis.

Breast cancer is the world's most common malignancy. Despite significant advances in the diagnosis and treatment of the disease, the associated mortality rate is still high. Tumor initiating cells known as cancer stem cells with unique abilities are suspected responsible for therapy failure and poor prognosis. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker that promotes aggressive features in breast cancer cells. So, the aim of this study was to perform a systematic review and meta-analysis to evaluate LGR5 as a therapeutic target for breast cancer. This systematic review and meta-analysis were performed using databases of Web of Science, Scopus, and PubMed. We searched these databases with LGR5 and Breast Cancer and related keywords based on the mesh database until Oct12, 2021. All studies that reported the rate of LGR5 high expression with Immunohistochemistry in breast cancer patients were included in this review. We used the STATA and random effect models for data analysis. Finally, 7 studies including 2632 breast cancer samples were studied. The pooled prevalence of LGR5 high expression in breast cancer was 48.6 % (CI95%: 40.5-56.7%, I2=0.0) and in triple negative was 48.6% (CI95%: 38.4-58.7%, I2= 0.0). Our findings show that the rate of LGR5 high expression in breast cancer in general and especially in triple-negative was considerable and it seems that this is a potential therapeutic target for breast cancer.

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.

RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.

Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2 , impacting PAR2 -induced signaling and colon cancer growth. RNF43 co-associates with PAR2 , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased β-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts β-catenin signaling and colon cancer growth.

Characterization of human nasal organoids from chronic rhinosinusitis patients.

ABSTRACTPatient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.

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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.

OSR1 suppresses acute myeloid leukaemia cell proliferation by inhibiting LGR5-mediated JNK signalling

Odd-skipped related transcription factor 1 (OSR1) is implicated in various pathophysiologic processes, such as embryonic heart and urogenital formation, and functions as a tumour suppressor in diverse tumours. Regardless, the regulatory role and mechanism of OSR1 in acute myeloid leukaemia are scarce. Firstly, the CD34-positive blasts or the normal blasts were isolated from the plasma samples of acute myeloid leukaemia patients or healthy donors, respectively. Expression of OSR1 analysis by western blot and qRT-PCR showed that OSR1 was reduced in CD34-positive blasts and acute myeloid leukaemia cell lines. Secondly, acute myeloid leukaemia cell lines were transfected with pcDNA vector or shRNA for the over-expression or silence of OSR1, respectively. Functional assays demonstrated that ectopic expression of OSR1 decreased cell viability and repressed the proliferation of acute myeloid leukaemia cells, while promoted the cell apoptosis. Silence of OSR1 contributed to the proliferation of acute myeloid leukaemia cells and suppressed the cell apoptosis. Thirdly, over-expression of OSR1 decreased protein expression of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) and JNK phosphorylation in the acute myeloid leukaemia cells. Ectopic expression of LGR5 attenuated OSR1 over-expression-induced decrease of LGR5 and JNK phosphorylation. Lastly, ectopic expression of LGR5 attenuated OSR1 over-expression-induced decrease of cell viability and proliferation in acute myeloid leukaemia cells. In conclusion, OSR1 functioned as a tumour suppressor in acute myeloid leukaemia cells by inhibiting LGR5-mediated activation of JNK signalling.

Active elimination of intestinal cells drives oncogenic growth in organoids.

SummaryCompetitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner.

Vitamin D3 suppresses intestinal epithelial stemness via ER stress induction in intestinal organoids

BackgroundVitamin D3 is important for normal function of the intestinal epithelial cells (IECs). In this study, we aimed to investigate the effects of vitamin D3 on the differentiation, stemness, and viability of healthy IECs in intestinal organoids.MethodsIntestinal organoids derived from mouse small intestine were treated with vitamin D3, and the effects on intestinal stemness and differentiation were evaluated using real-time PCR and immunofluorescence staining of the distinct lineage markers. Cell viability was analyzed using viability and apoptosis assays.ResultsVitamin D3 enhanced IEC differentiation into the distinct lineages of specialized IECs, including Paneth, goblet, and enteroendocrine cells and absorptive enterocytes. Decreased expression levels of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) and the presence of several LGR5-green fluorescent protein (GFP)-positive cells were observed in vitamin D3-treated organoids derived from LGR5-GFP mice. The formation of the crypt-villus structure was also inhibited by vitamin D3, suggesting that vitamin D3 suppresses intestinal cell stemness. Furthermore, the expression levels of unfolded protein response genes, C/EBP homologous protein (CHOP), and activating transcription factor 6 (ATF6) were upregulated in vitamin D3-treated organoids. Moreover, vitamin D3 promoted apoptotic cell death in intestinal cells, which may be associated with the decrease in intestinal stemness. LGR5 gene expression, ISC number, and apoptotic cell death were partially recovered in the presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA), suggesting that intestinal stemness suppression and intestinal apoptosis occurred via ER stress activation.ConclusionsOur study provides important insights into the effects of vitamin D3 on the induction of IEC differentiation and apoptotic cell death, and inhibition of intestinal stemness accompanied by ER stress augmentation.

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.

Clinical Significance of Tumour CD44v and MIST1 Expression in Patients With Non-small-cell Lung Cancer.

Cancer stem cells (CSCs) are involved in cancer metastasis and relapse. Therefore, identification of CSC biomarkers might help determine the success of a treatment. In this study, we examined the expression of four CSC markers: Cluster of differentiation 44 variant (CD44v), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), frizzled 7 (FZD7), and muscle, intestine and stomach expression 1 (MIST1), in cancer tissues of patients with non-small-cell lung cancer at >5 years after resection, and its clinical significance. We examined the expression of each CSC marker in 360 patients with NSCLC (n=360) who underwent curative resection by immunohistochemical analysis of tissue microarrays, and determined its relationship with survival. High expression of MIST1 was related to better overall survival (p<0.05); high CD44v expression was associated with poor overall and recurrence-free survival (p<0.001 for both) and thus, CD44v was defined as an independent prognostic factor (p<0.05), according to a multivariate analysis. Tumoral CD44v expression might be a useful prognostic marker for patients after curative resection of NSCLC.

Preclinical Evaluation of the Novel Small-Molecule MSI-N1014 for Treating Drug-Resistant Colon Cancer via the LGR5/β-catenin/miR-142-3p Network and Reducing Cancer-Associated Fibroblast Transformation.

Colorectal cancer represents one of the most prevalent malignancies globally, with an estimated 140,000 new cases in the United States alone in 2019. Despite advancements in interventions, drug resistance occurs in virtually all patients diagnosed with late stages of colon cancer. Amplified epidermal growth factor receptor (EGFR) signaling is one of the most prevalent oncogenic drivers in patients and induces increased Janus kinase (JAK)/signal transduction and activator of transcription (STAT) and β-catenin functions, all of which facilitate disease progression. Equally important, cancer-associated fibroblasts (CAFs) transformed by cancer cells within the tumor microenvironment (TME) further facilitate malignancy by secreting interleukin (IL)-6 and augmenting STAT3 signaling in colon cancer cells and promoting the generation of cancer stem-like cells (CSCs). Based on these premises, single-targeted therapeutics have proven ineffective for treating malignant colon cancer, and alternative multiple-targeting agents should be explored. Herein, we synthesized a tetracyclic heterocyclic azathioxanthone, MSI-N1014, and demonstrated its therapeutic potential both in vitro and in vivo. First, we used a co-culture system to demonstrate that colon cancer cells co-cultured with CAFs resulted in heightened 5-fluorouracil (5-FU) resistance and tumor sphere-forming ability and increased side populations, accompanied by elevated expression of cluster of differentiation 44 (CD44), β-catenin, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and ATP-binding cassette super-family G member 2 (ABCG2). MSI-N1014 suppressed cell viability, colony formation, and migration in both DLD1 and HCT116 cells. MSI-N1014 treatment led to decreased expressions of oncogenic markers, including mammalian target of rapamycin (mTOR), EGFR, and IL-6 and stemness markers such as CD44, β-catenin, and LGR5. More importantly, MSI-N1014 treatment suppressed the transformation of CAFs, and was associated with decreased secretion of IL-6 and vascular endothelial growth factor (VEGF) by CAFs. Furthermore, MSI-N1014 treatment resulted in significantly reduced oncogenic properties, namely the migratory ability, tumor-sphere generation, and resistance against 5-FU. Notably, an increased level of the tumor suppressor, miR-142-3p, whose targets include LGR5, IL-6, and ABCG2, was detected in association with MSI-N1014 treatment. Finally, we demonstrated the therapeutic potential of MSI-N1014 in vivo, where combined treatment with MSI-N1014 and 5-FU led to the lowest tumor growth, followed by MSI-N1014 only, 5-FU, and the vehicle control. Tumor samples from the MSI-N1014 group showed markedly reduced expressions of LGR5, β-catenin, IL-6, and mTOR, but increased expression of the tumor suppressor, miR-142-3p, according to qRT-PCR analysis. Collectively, we present preclinical support for the application of MSI-N1014 in treating 5-FU-resistant colon cancer cells. Further investigation is warranted to translate these findings into clinical settings.

The kinase PERK represses translation of the G-protein–coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells

ObjectiveLiver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of...

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.