LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer.

WNT/ß-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair ß-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of ß-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as ß-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 ß-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since ß-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 ß-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients.

Identification of a Novel Five-Gene Signature as a Prognostic and Diagnostic Biomarker in Colorectal Cancers.

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.

High PGD2 receptor 2 levels are associated with poor prognosis in colorectal cancer patients and induce VEGF expression in colon cancer cells and migration in a zebrafish xenograft model.

BACKGROUND: Despite intense research, the prognosis for patients with advanced colorectal cancer (CRC) remains poor. The prostaglandin D2 receptors DP1 and DP2 are explored here as potential therapeutic targets for advanced CRC. METHODS: A CRC cohort was analysed to determine whether DP1 and DP2 receptor expression correlates with patient survival. Four colon cancer cell lines and a zebrafish metastasis model were used to explore how DP1/DP2 receptor expression correlates with CRC progression. RESULTS: Analysis of the clinical CRC cohort revealed high DP2 expression in tumour tissue, whereas DP1 expression was low. High DP2 expression negatively correlated with overall survival. Other pathological indicators, such as TNM stage and metastasis, positively correlated with DP2 but not DP1 expression. In accordance, the in vitro results showed high DP2 expression in four CC-cell lines, but only one expressed DP1. DP2 stimulation resulted in increased proliferation, p-ERK1/2 and VEGF expression/secretion. DP2-stimulated cells exhibited increased migration in the zebrafish metastasis model. CONCLUSION: Our results support DP2 receptor expression and signalling as a therapeutic target in CRC progression based on its expression in CRC tissue correlating with poor patient survival and that it triggers proliferation, p-ERK1/2 and VEGF expression and release and increased metastatic activity in CC-cells.

Brain-Derived Neurotrophic Factor, Neutrophils and Cysteinyl Leukotriene Receptor 1 as Potential Prognostic Biomarkers for Patients with Colon Cancer.

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.

Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition.

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

A potential anti-tumor effect of leukotriene C4 through the induction of 15-hydroxyprostaglandin dehydrogenase expression in colon cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2, which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2, and is often down-regulated in cancer. Interestingly, CRC patients expressing high levels of the cysteinyl leukotriene 2 (CysLT2) receptor have a good prognosis; therefore, we investigated a potential link between CysLT2 signaling and the tumor suppressor 15-PGDH in colon cancer cells.We observed a significant up-regulation of 15-PGDH after treatment with LTC4, a CysLT2 ligand, in colon cancer cells at both the mRNA and protein levels, which could be reduced by a CysLT2 antagonist or a JNK inhibitor. LTC4 induced 15-PGDH promoter activity via JNK/AP-1 phosphorylation. Furthermore, we also observed that LTC4, via the CysLT2/JNK signaling pathway, increased the expression of the differentiation markers sucrase-isomaltase and mucin-2 in colon cancer cells and that down-regulation of 15-PGDH totally abolished the observed increase in these markers.In conclusion, the restoration of 15-PGDH expression through CysLT2 signaling promotes the differentiation of colon cancer cells, indicating an anti-tumor effect of CysLT2 signaling.

Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells.

The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in prostaglandin E2 catabolism and is down-regulated in colorectal cancer (CRC) tissue. Canonical Wnt signaling is frequently elevated in colon cancers and has been shown to down-regulate 15-PGDH expression. Therefore, we have in the current study investigated if the non-canonical ligand WNT5A relates to increased expression of 15-PGDH in colon cancer cells. In the same cohort of patients, we demonstrated a parallel and significant loss of 15-PGDH and WNT5A protein expression in CRC tissues compared with matched normal colon tissues. Furthermore, patients with low 15-PGDH/WNT5A expression in their tumors showed reduced survival compared with patients with high 15-PGDH/WNT5A expression. To investigate if WNT5A signaling directly affects 15-PGDH expression, we performed in vitro analyses of colon cancer cells (HT-29 and Caco-2). Both cell lines, when treated with recombinant WNT5A (rWNT5A) or Foxy-5, a WNT5A-mimicking peptide, responded by increasing their expression of 15-PGDH mRNA and protein. Our investigations showed that rWNT5A and Foxy-5 induced this increased expression of 15-PGDH through reduced ß-catenin signaling as well as increased JNK/AP-1 signaling in colon cancer cells. WNT5A signaling also induced increased 15-PGDH expression in a breast cancer cell line both in vitro and in vivo. In agreement, WNT5A signaling also increased the expression of the differentiation markers sucrose-isomaltase and mucin-2 in colon cancer cells. Our results show that WNT5A signaling regulates 15-PGDH expression, thus uncovering a novel mechanism by which WNT5A acts as a tumor suppressor and suggests that increased 15-PGDH expression could be used as an indicator of a positive response to Foxy-5 in patients treated with this WNT5A agonist.