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MiR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis.

ABSTRACT: MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of beta-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a 'miRNA sponge' in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.

SUBMITTER: Ma L

PROVIDER: S-EPMC2845545 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis.

Ma Li L Young Jennifer J Prabhala Harsha H Pan Elizabeth E Mestdagh Pieter P Muth Daniel D Teruya-Feldstein Julie J Reinhardt Ferenc F Onder Tamer T TT Valastyan Scott S Westermann Frank F Speleman Frank F Vandesompele Jo J Weinberg Robert A RA

Nature cell biology 20100221 3

MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of beta-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in ...[more]

PMID: 20173740

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Project description:Targeting glutamine metabolism has emerged as a potential therapeutic strategy for Myc overexpressing cancer cells. Myc proteins contribute to an aggressive neuroblastoma phenotype. Radiotherapy is one of the treatment modalities for high-risk neuroblastoma patients. Herein, we investigated the effect of glutamine deprivation in combination with irradiation in neuroblastoma cells representative of high-risk disease and studied the role of Myc member interplay in regulating neuroblastoma cell radioresistance. Methods: Cell proliferation and viability assays were used to establish the effect of glutamine deprivation in neuroblastoma cells expressing c-Myc or MycN. Gene silencing and overexpression were used to modulate the expression of Myc genes to determine their role in neuroblastoma radioresistance. qPCR and western blot investigated interplay between expression of Myc members. The impact of glutamine deprivation on cell response following irradiation was explored using a radiobiological 3D colony assay. DNA repair gene pathways as well as CSC-related genes were studied by qPCR array. Reactive Oxygen Species (ROS) and glutathione (GSH) levels were detected by fluorescence and luminescence probes respectively. Cancer-stem cell (CSC) properties were investigated by sphere-forming assay and flow cytometry to quantify CSC markers. Expression of DNA repair genes and CSC-related genes was analysed by mining publicly available patient datasets. Results: Our results showed that glutamine deprivation decreased neuroblastoma cell proliferation and viability and modulated Myc member expression. We then demonstrated for the first time that combined glutamine deprivation with irradiation led to a selective radioresistance of MYCN-amplified neuroblastoma cells. By exploring the underlying mechanism of neuroblastoma radioresistance properties, our results highlight interplay between c-Myc and MycN expression suggesting compensatory mechanisms in Myc proteins leading to radioresistance in MYCN-amplified cells. This result was associated with the ability of MYCN-amplified cells to dysregulate the DNA repair gene pathway, maintain GSH and ROS levels and to increase the CSC-like population and properties. Conversely, glutamine deprivation led to radiosensitization in non-MYCN amplified cell lines through a disruption of the cell redox balance and a trend to decrease in the CSC-like populations. Mining publicly available gene expression dataset obtained from pediatric neuroblastoma patients, we identified a correlation pattern between Myc members and CSC-related genes as well as a specific group of DNA repair gene pathways. Conclusions: This study demonstrated that MycN and c-Myc tightly cooperate in regulation of the neuroblastoma CSC phenotypes and radioresistance upon glutamine deprivation. Pharmacologically, strategies targeting glutamine metabolism may prove beneficial in Myc-driven tumors. Consideration of MycN/c-Myc status in selecting neuroblastoma patients for glutamine metabolism treatment will be important to avoid potential radioresistance.

Project description:N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3? phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/?-catenin signaling resulting from inactivation of GSK3?.Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

Project description:BackgroundGrowing evidence shows that long non-coding RNAs (lncRNAs) play significant roles in cancer development. However, the functions of most lncRNAs in human gastric cancer are still not fully understood. Here, we explored the role of a novel c-Myc-activated lncRNA, LINC01050, in gastric cancer progression.MethodsThe expression of LINC01050 in the context of gastric cancer was assessed using The Cancer Genome Atlas datasets. Its functions in gastric cancer were investigated through gain- and loss-of-function experiments combined with the Cell Counting Kit-8 assays, colony-forming assays, Transwell assays, flow cytometry, Western blot analyses, and xenograft tumor and mouse metastasis models. Potential LINC01050 transcription activators were screened via bioinformatics and validated by chromatin immunoprecipitation and luciferase assays. The interaction between LINC01050 and miR-7161-3p and the targets of miR-7161-3p were predicted by bioinformatics analysis and confirmed by a luciferase assay, RNA immunoprecipitation, RNA pull-down, and rescue experiments.ResultsLINC01050 was significantly up-regulated in gastric cancer, and its high expression was positively correlated with a poor prognosis. The transcription factor c-Myc was found to directly bind to the LINC01050 promoter region and activate its transcription. Furthermore, overexpression of LINC01050 was confirmed to promote gastric cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition in vitro and tumor growth in vivo. At the same time, its knockdown inhibited gastric cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition in vitro along with tumor growth and metastasis in vivo. Moreover, mechanistic investigations revealed that LINC01050 functions as a molecular sponge to absorb cytosolic miR-7161-3p, which reduces the miR-7161-3p-mediated translational repression of SPZ1, thus contributing to gastric cancer progression.ConclusionsTaken together, our results identified a novel gastric cancer-associated lncRNA, LINC01050, which is activated by c-Myc. LINC01050 may be considered a potential therapeutic target for gastric cancer.

Dataset Information

MiR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis.

Publications

miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis.

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