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WNT/β-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair β-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of β-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as β-catenin activity and its target gene VEGFA. Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed β-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since β-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and β-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. The current TNM (Tumor, Node, and Metastasis) classification approach is suboptimal in determining the prognosis of CRC patients. The prognosis for CRC is affected by a variety of features that are present at the initial diagnosis. Herein, we performed a systematic exploration and established a novel five-panel gene signature as a prognostic and early diagnosis biomarker after performing differential gene expression analyses in five independent in silico CRCs cohort and independently validating it in one clinical cohort, using immunohistochemistry. Four genes (BDNF, PTGS2, GSK3B, and CTNNB1) were significantly upregulated and one gene (HPGD) was significantly downregulated in primary tumor tissues compared with adjacent normal tissues throughout all the five in silico datasets. The univariate CoxPH analysis yielded a five-gene signature that accurately predicted overall survival (OS) and recurrence-free survival (RFS) in the in silico training (AUC = 0.73 and 0.69, respectively) and one independent in silico validation cohort (AUC = 0.69 and 0.74, respectively). This five-gene signature demonstrated significant associations with poor OS in independent clinical validation cohorts of colon cancer (CC) patients (AUC = 0.82). Intriguingly, a risk stratification model comprising of the five-gene signature together with TNM stage and gender status achieved an even superior AUC of 0.89 in the clinical cohorts. On the other hand, the circulating mRNA expression of the upregulated four-gene signature achieved a robust AUC = 0.83 with high sensitivity and specificity as a diagnosis marker in plasma from CRC patients. We have identified a novel, five-gene signature as an independent predictor of OS, which in combination with TNM stage and gender offers an easy-to-translate and facile assay for the personalized risk-assessment in CRC patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is one of the most common type of cancer and the third leading cause of cancer-related death. CRC is associated with inflammatory bowel disease. We have earlier shown that high levels of the inflammatory receptor CysLT1 goes with poor prognosis for CRC patients. In this study, we found that high levels of neutrophils (CD66b) and brain-derived neurotropic factor (BDNF) goes with poor prognosis for colon cancer patient. We discovered a strong positive correlation between CysLT1, CD66b and BDNF. Our data support that these three proteins can be used as a combined biomarker for CC patients. The tumor microenvironment has been recognized as a complex network in which immune cells play an important role in cancer progression. We found significantly higher CD66b neutrophil expression in tumor tissue than in matched normal mucosa in the Malmö colon cancer (CC) cohort and poorer survival of stage I-III patients with high CD66b expression. Additionally, mice lacking CysLT1R expression (cysltr1−/−) produce less brain-derived neurotrophic factor (BDNF) compared to WT mice and Montelukast (a CysLT1R antagonist)-treated mice also reduced BDNF expression in a mouse xenograft model with human SW480 CC cells. CD66b and BDNF expression was significantly higher in patient tumor tissues than in the matched normal mucosa. The univariate Cox PH analysis yielded CD66b and BDNF as an independent predictor of overall survival, which was also found in the public TCGA-COAD dataset. We also discovered a strong positive correlation between CD66b, BDNF and CysLT1R expression in the Malmö CC cohort and in the TCGA-COAD dataset. Our data suggest that CD66b/BDNF/CysLT1R expression as a prognostic combined biomarker signature for CC patients. [ABSTRACT FROM AUTHOR]