High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer.

In normal colon tissue, oestrogen receptor alpha (ERα) is expressed at low levels, while oestrogen receptor beta (ERß) is considered the dominant subtype. However, in colon carcinomas, the ERα/ß ratio is often increased, an observation that prompted us to further investigate ERα's role in colorectal cancer (CRC). Here, we assessed ERα nuclear expression in 351 CRC patients. Among them, 119 exhibited positive ERα nuclear expression, which was significantly higher in cancer tissues than in matched normal tissues. Importantly, patients with positive nuclear ERα expression had a poor prognosis. Furthermore, positive ERα expression correlated with increased levels of the G-protein coupled cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear ß-catenin, both known tumour promoters. In mouse models, ERα expression was decreased in Cysltr1-/- CAC (colitis-associated colon cancer) mice but increased in ApcMin/+ mice with wild-type Cysltr1. In cell experiments, an ERα-specific agonist (PPT) increased cell survival via WNT/ß-catenin signalling. ERα activation also promoted metastasis in a zebrafish xenograft model by affecting the tight junction proteins ZO-1 and Occludin. Pharmacological blockade or siRNA silencing of ERα limited cell survival and metastasis while restoring tight junction protein expression. In conclusion, these findings highlight the potential of ERα as a prognostic marker for CRC and its role in metastasis.

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.

Tumour-suppressive effect of oestrogen receptor ß in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor ß (ERß) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERß expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERß and its selective agonist. CRC patients with high ERß expression had significantly higher levels of membrane-associated ß-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear ß-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERß expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERß expression and ß-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERß expression in three different colon cancer mouse models; ERß expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERß-agonist ERB-041 reduced CysLT1 R, active ß-catenin, and COX-2 levels but increased phospho-ß-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERß expression had significantly more distant metastasis at the time of diagnosis than patients with high ERß expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERß's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases.

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4 ). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4 ) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, ß-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of ß-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4 . The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT.

High tumor mast cell density is associated with longer survival of colon cancer patients.

BACKGROUND: Inflammatory cells and inflammatory mediators play an important role in colorectal cancer (CRC). Previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have a poorer prognosis, and Cysltr1-/- mice display fewer intestinal polyps. However, the role of mast cells (MCs) in colon cancer progression remains unclear. The aim of the present study was to explore the relevance of MCs in CRC. MATERIAL AND METHODS: A tissue microarray from 72 CRC patients was stained with MC anti-tryptase and -chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCs in patients and mice. RESULTS: Patient colon cancer tissue had in comparison with normal colon tissue a reduced number of MCs, predominantly of chymase-positive cells. Further analysis revealed that patients with a relative high MCD in their cancer tissues showed significantly longer overall survival compared to those with a low MCD [hazard ratio (HR) 0.539; 95% confidence interval (CI), 0.302-0.961]. Similar results were observed in subgroups of patients with either no distant metastasis (p = 0.004), or <75 years (p = 0.015) at time of diagnosis. Multivariate Cox analysis showed that MCD independently correlated with reduced risk of death in colon cancer patients (HR 0.380; 95% CI 0.202-0.713). Additionally, a negative correlation was found between cytoplasmic CysLTR1 expression and number of MCs. In agreement, in the CAC mouse model, Cysltr1-/- mice showed significantly higher MCs in their polyp/tumor areas compared with wild-type mice. CONCLUSION: A high MCD in cancer tissue correlated with longer patient survival independently from other risk factors for CRC. The concept that MCs have an anti-tumor effect in CRC is further supported by the findings of a negative correlation with CysLTR1 expression in patients and a high MCD in colon polyps/tumors from CysLTR1-/- mice.

Combined Estrogen Alpha and Beta Receptor Expression Has a Prognostic Significance for Colorectal Cancer Patients.

We reported that high estrogen receptor beta (ERß) expression is independently associated with better prognosis in female colorectal cancer (CRC) patients. However, estrogen receptor alpha (ERα) is expressed at very low levels in normal colon mucosa, and its prognostic role in CRC has not been explored. Herein, we investigated the combined role of ERα and ERß expression in the prognosis of female patients with CRC, which, to the best of our knowledge, is the first study to investigate this topic. A total number of 306 primary CRCs were immunostained for ERα and ERß expression. A Cox regression model was used to evaluate overall survival (OS) and disease-free survival (DFS). The combined expression of high ERß + negative ERα correlates with longer OS (HR = 0.23; 95% CI: 0.11-0.45, P <0.0001) and DFS (HR = 0.10; 95% CI: 0.03-0.26, P < 0.0001) and a more favorable tumor outcome, as well as significantly higher expression of antitumorigenic proteins than combined expression of low ERß + positive ERα. Importantly, we found that low ERß expression was associated with local recurrence of CRC, whereas ERα expression was correlated with liver metastasis. Overall, our results show that the combined high ERß + negative ERα expression correlated with a better prognosis for CRC patients. Our results suggest that the combined expression of ERα and ERß could be used as a predictive combination marker for CRC patients, especially for predicting DFS.

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.

Association of the oestrogen receptor beta with hormone status and prognosis in a cohort of female patients with colorectal cancer.

BACKGROUND: The oestrogen receptor beta (ERß) is the predominant oestrogen receptor in the normal colon mucosa and has been reported to exert anti-proliferative and pro-apoptotic effects. However, the role of ERß in colorectal cancer (CRC) progression remains unclear. AIM: To investigate the role of ERß and its association with hormone status and lifestyle indicators in a female cohort of patients with CRC. METHODS: Tissue microarrays of primary CRC tumour samples from 320 female patients were conducted with a monoclonal anti-ERß antibody. The staining intensity was evaluated using immunohistochemistry. The association of ERß expression with overall survival, disease-free survival, hormone status and lifestyle was evaluated, and effect estimators with 95% confidence intervals (CIs) were reported. RESULTS: Among the 314 samples with successfully detected ERß, 182 (58%) had low expression and 132 (42%) had high expression. The Cox multivariate analysis indicated that patients with high ERß expression had a decreased risk of overall mortality by 50% (hazard ratio [HR], 0.50; CI, 0.30-0.83) and of cancer recurrence by 76% (HR, 0.24; CI, 0.11-0.52) after adjusting for age, tumour-node-metastasis stage and tumour intravascular invasion. Furthermore, high ERß expression was significantly correlated with shorter breastfeeding time and longer use of hormone replacement therapy. No association was found between ERß expression and lifestyle indicators. CONCLUSION: Elevated ERß expression is independently associated with a better prognosis and hormone status but not lifestyle indicators in female CRC patients.

Macronutrient composition and gestational weight gain: a systematic review.

BACKGROUND: Abnormal gestational weight gain is associated with unfavorable pregnancy outcomes. Several risk factors have been identified, but the effect of macronutrient intake during pregnancy on gestational weight gain has not been systematically evaluated in both high-income countries and low- and middle-income countries. OBJECTIVE: We conducted a systematic review of the literature in 8 different databases (until 12 August 2015) to assess whether energy intake and macronutrient intake (i.e., protein, fat, and carbohydrate) during pregnancy were associated with gestational weight gain (following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines). RESULTS: Of 7623 identified references, we included 56 articles (46 observational studies and 10 trials, 28 of which were in high-income countries and 28 of which were in low- and middle-income countries). Eleven of the included articles were of high quality (20%). Results of 5 intervention and 7 high-quality observational studies suggested that higher energy intake during pregnancy is associated with higher gestational weight gain (n = 52). Results from observational studies were inconsistent for protein intake (n = 29) and carbohydrate intake (n = 18). Maternal fat intake (n = 25) might be associated with gestational weight gain as suggested by observational studies, although the direction of this association might depend on specific types of fat (e.g., saturated fat). Macronutrient intake was not consistently associated with the prevalence of inadequate or excessive gestational weight gain. Associations were comparable for high-income countries and low- and middle-income countries. CONCLUSIONS: The current literature provides evidence that energy intake is associated with gestational weight gain, but the roles of individual macronutrients are inconsistent. However, there is a need for higher-quality research because the majority of these studies were of low quality.