WNT11, a new gene associated with early-onset osteoporosis, is required for osteoblastogenesis

Monogenic early-onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in WNT11 (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G > A p.Ala73Thr) and (NM_004626.2:c.865G > A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor LGR5, was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but WNT3A recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2–LGR5 complex via the non-canonical Wnt pathway.

Correlation between Lgr5 Expression and 5-FU based Chemotherapy Response in Stage IV Colorectal Cancer Patients

Background. Colorectal cancer is the third commonest malignancy and the second leading cause of cancer death in the world. 5-Fluorouracyl-based chemotherapy is the primary treatment modality for colorectal cancer. Cancer stem cells are known to be responsible for chemotherapy resistance. Lgr5 is a colorectal cancer stem cell marker that is the target gene for Wnt signaling. Lgr5 potentiates the Wnt signaling pathway through inhibition of a regulator that inhibits Wnt signaling. Lgr5 overexpression is associated with a worse prognosis and chemotherapy resistance. This study was aimed to investigate the correlation between Lgr5 expression and 5-FU-based chemotherapy response in stage IV colorectal cancer patients at Dr. Mohammad Hoesin Hospital Palembang.Methods. This study used a correlative analysis study with a retrospective design using secondary data from medical records and paraffin blocks of stage IV colorectal cancer patients who received 5-FU-based chemotherapy from September 2018 to September 2020. The number of samples was 30 subjects consisting of 22 cases of negative responses and eight positive responses. All samples were stained with Lgr5 immunohistochemistry. Data analysis used the contingency coefficient correlation test.Results. Of the 30 research subjects, 20 subjects (66.7%) had high Lgr5 expression and ten subjects (33.3%) with low Lgr5 expression. Correlation analysis using the contingency coefficient test showed a weak correlation between Lgr5 expression and 5-FU based chemotherapy response with a positive direction, which means the higher the Lgr5 expression, the less response to chemotherapy.Conclusion. There is a weak correlation between Lgr5 expression and 5-FU based chemotherapy response in stage IV colorectal cancer patients at dr.Mohammad Hoesin Hospital Palembang.

Correlation between Lgr5 Expression and 5-FU based Chemotherapy Response in Stage IV Colorectal Cancer Patients

Background. Colorectal cancer is the third commonest malignancy and the second leading cause of cancer death in the world. 5-Fluorouracyl-based chemotherapy is the primary treatment modality for colorectal cancer. Cancer stem cells are known to be responsible for chemotherapy resistance. Lgr5 is a colorectal cancer stem cell marker that is the target gene for Wnt signaling. Lgr5 potentiates the Wnt signaling pathway through inhibition of a regulator that inhibits Wnt signaling. Lgr5 overexpression is associated with a worse prognosis and chemotherapy resistance. This study was aimed to investigate the correlation between Lgr5 expression and 5-FU-based chemotherapy response in stage IV colorectal cancer patients at Dr. Mohammad Hoesin Hospital Palembang.Methods. This study used a correlative analysis study with a retrospective design using secondary data from medical records and paraffin blocks of stage IV colorectal cancer patients who received 5-FU-based chemotherapy from September 2018 to September 2020. The number of samples was 30 subjects consisting of 22 cases of negative responses and eight positive responses. All samples were stained with Lgr5 immunohistochemistry. Data analysis used the contingency coefficient correlation test.Results. Of the 30 research subjects, 20 subjects (66.7%) had high Lgr5 expression and ten subjects (33.3%) with low Lgr5 expression. Correlation analysis using the contingency coefficient test showed a weak correlation between Lgr5 expression and 5-FU based chemotherapy response with a positive direction, which means the higher the Lgr5 expression, the less response to chemotherapy.Conclusion. There is a weak correlation between Lgr5 expression and 5-FU based chemotherapy response in stage IV colorectal cancer patients at dr.Mohammad Hoesin Hospital Palembang.

Intestinal Stem Cell Marker, ASCL2 Is a Novel Prognostic Predictor in Esophageal Adenocarcinoma

Purpose: Stem cell markers play a significant role in esophageal adenocarcinoma carcinogenesis via Barrett’s esophagus; however, its utility as a prognostic biomarker has not been established. Methods: We analyzed the immunohistochemical expression of Ascl2 and Lgr5 utilizing whole slides (WS; 35 cases) and tissue microarray (TMA; 64 cases of esophageal adenocarcinoma with adjacent normal squamous epithelium, Barrett's esophagus, and dysplasia). Two pathologists semi-quantitatively scored stained slides independently/blindly, and the results were correlated with clinicopathologic factors and outcomes. Results: In WS, 51% and 57% expressed high Ascl2 and high Lgr5; in TMA, 69% and 88% expressed high Ascl2 and high Lgr5, respectively. In TMA, high Ascl2 and low Lgr5 expression significantly correlated to a higher number of involved lymph nodes (p=0.027 and p=0.0039), and Lgr5 expression significantly correlated to the pathological stage (p=0.0032). Kaplan-Meier analysis showed a negative impact of high Ascl2 expression on overall survival (OS; WS p=0.0168, TMA p=0.0276) as well as progression-free survival (PFS; WS p=0.000638, TMA p=0.0466), but not Lgr5. Multivariate Cox regression analysis revealed that Ascl2 expression is an independent prognostic factor for esophageal adenocarcinoma (OS; WS p=0.25, TMA p=0.011. PFS; WS p=0.012, TMA p=0.038). Analysis of the TCGA dataset showed that ASCL2 mRNA levels were correlated to nodal status but not overall survival.Conclusion: Intestinal stem cell marker, Ascl2 is an independent prognostic factor in esophageal adenocarcinoma. High Ascl2 levels were associated with nodal status in both immunohistochemical and mRNA expression. These findings may contribute to the development of prognosis-based subclassification of esophageal adenocarcinoma.

LGR5 expression is associated with prognosis in poorly differentiated gastric adenocarcinoma

Background Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is an important cancer stem cell marker in gastric cancer. However, no detailed studies are available on LGR5 expression in poorly differentiated gastric adenocarcinoma (PD-AC). Therefore, we investigated the relationship between LGR5 expression and clinicopathological data in PD-AC. Methods LGR5 mRNA expression levels were quantified in 41 PD-AC specimens using a highly sensitive RNAscope in situ hybridization technique. Epstein–Barr virus (EBV) infection was also detected by EBV in situ hybridization. Results LGR5 expression levels were measured in 38 of 41 PD-AC cases, and 17 cases were identified as LGR5 high. The frequency of EBV positivity tended to be higher in the LGR5-low group than in the LGR5-high group (P = 0.0764). Furthermore, the frequency of vascular invasion tended to be higher in the LGR5-high group than in the LGR5-low group (P = 0.0764). The overall survival of PD-AC patients in the LGR5-high group was significantly lower than in the LGR5-low group (log-rank test, P = 0.0108). The Cox proportional hazard regression model revealed that the LGR5-low group (HR = 0.29; 95% CI: 0.11–0.74; P = 0.01) showed independently better OS for PD-AC. Conclusions Quantifying the levels of LGR5 expression may facilitate defining prognosis in Japanese patients with PD-AC. Further study of LGR5 in this context is warranted.

MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

Most colitis associated carcinomas lack expression of LGR5: a preliminary study with implications for unique pathways of carcinogenesis compared to sporadic colorectal carcinoma

Background: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a component of the Wnt receptor complex, is thought to lineage label gastric and intestinal stem cells. LGR5 expression is increased in colorectal carcinoma (CRC) compared to normal tissue. Colitis associated colorectal adenocarcinoma (CAC) often shows distinct morphologic and molecular phenotypes compared to sporadic cases. However, the expression profile of LGR5, and by extension the potential role of an intestinal stem cell phenotype, has not been well described in a series of human CAC. Method: RNA in situ hybridization (ISH) for LGR5 expression on 30 CACs (12 cases with conventional morphology and 18 cases with non-conventional type morphology) from 29 inflammatory bowel disease (IBD) patients was performed and compared the expression profile to a control group of 10 sporadic CRCs. Immunohistochemistry for beta-catenin and SATB2 was performed on the 30 CACs. Result: LGR5 was positive in 30% (9/30) of CAC cases and 90% (9/10) of sporadic CRCs (p=0.002). A large majority (89%) of LGR5 positive CACs were of the conventional histologic type, and conventional type CAC showed a significantly higher LGR5 score (median 3.0; interquartile range 1.75-3.25) than non-conventional type CAC (median 1.5; interquartile range 1.00-2.00) (p = 0.034). CAC with conventional morphology did have a lower level of LGR5 expression than sporadic CRC. Sporadic CRCs showed a significantly higher LGR5 level score than non-conventional type CACs (p <0.001). Nuclear translocation of beta-catenin was strongly associated with LGR5 expression (p=0.003), however no significant association was identified between SATB2 expression and LGR5 expression status in CACs. Conclusion: These findings suggest that the wider spectrum of tumor morphology in CAC may be associated with absence of a LGR5 -expressing intestinal stem cell phenotype.