Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

Neuromedin U induces an invasive phenotype in CRC cells expressing the NMUR2 receptor.

BACKGROUND: Successful colorectal cancer (CRC) therapy often depends on the accurate identification of primary tumours with invasive potential. There is still a lack of identified pathological factors associated with disease recurrence that could help in making treatment decisions. Neuromedin U (NMU) is a secretory neuropeptide that was first isolated from the porcine spinal cord, and it has emerged as a novel factor involved in the tumorigenesis and/or metastasis of many types of cancers. Previously associated with processes leading to CRC cell invasiveness, NMU has the potential to be a marker of poor outcome, but it has not been extensively studied in CRC. METHODS: Data from The Cancer Genome Atlas (TCGA) were used to analyse NMU and NMU receptor (NMUR1 and NMUR2) expression in CRC tissues vs. normal tissues, and real-time PCR was used for NMU and NMU receptor expression analysis. NMU protein detection was performed by immunoblotting. Secreted NMU was immunoprecipitated from cell culture-conditioned media and analysed by immunoblotting and protein sequencing. DNA demethylation by 5-aza-CdR was used to analyse the regulation of NMUR1 and NMUR2 expression. NMU receptor activity was monitored by detecting calcium mobilisation in cells loaded with fluo-4, and ERK1/2 kinase activation was detected after treatment with NMU or receptor agonist. Cell migration and invasion were investigated using membrane filters. Integrin expression was evaluated by flow cytometry. RESULTS: The obtained data revealed elevated expression of NMU and NMUR2 in CRC tissue samples and variable expression in the analysed CRC cell lines. We have shown, for the first time, that NMUR2 activation induces signalling in CRC cells and that NMU increases the motility and invasiveness of NMUR2-positive CRC cells and increases prometastatic integrin receptor subunit expression. CONCLUSIONS: Our results show the ability of CRC cells to respond to NMU via activation of the NMUR2 receptor, which ultimately leads to a shift in the CRC phenotype towards a more invasive phenotype.

Implications of ABCC4-Mediated cAMP Eflux for CRC Migration.

Colorectal cancer (CRC) presents significant molecular heterogeneity. The cellular plasticity of epithelial to mesenchymal transition (EMT) is one of the key factors responsible for the heterogeneous nature of metastatic CRC. EMT is an important regulator of ATP binding cassette (ABC) protein expression; these proteins are the active transporters of a broad range of endogenous compounds and anticancer drugs. In our previous studies, we performed a transcriptomic and functional analysis of CRC in the early stages of metastasis induced by the overexpression of Snail, the transcription factor involved in EMT initiation. Interestingly, we found a correlation between the Snail expression and ABCC4 (MRP4) protein upregulation. The relationship between epithelial transition and ABCC4 expression and function in CRC has not been previously defined. In the current study, we propose that the ABCC4 expression changes during EMT and may be differentially regulated in various subpopulations of CRC. We confirmed that ABCC4 upregulation is correlated with the phenotype conversion process in CRC. The analysis of Gene Expression Omnibus (GEO) sets showed that the ABCC4 expression was elevated in CRC patients. The results of a functional study demonstrated that, in CRC, ABCC4 can regulate cell migration in a cyclic nucleotide-dependent manner.

Erratum: Neuromedin U: A Small Peptide in the Big World of Cancer. Cancers 2019, 11, 1312.

The authors wish to make the following corrections to this paper [...].

Neuromedin U: A Small Peptide in the Big World of Cancer.

Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells.

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.