The UPR Transducer IRE1 Promotes Breast Cancer Malignancy by Degrading Tumor Suppressor microRNAs
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ABSTRACT: To understand the mechanistic basis by which inositol-requiring enzyme 1 (IRE1) is involved in luminal breast cancer malignancy, we analyzed the transcriptomic signature that IRE1 regulates in breast cancer. We suppressed the activity of IRE1 RNase in luminal breast cancer SUM52 line by adenoviral-based over-expression of the IRE1 dominant-negative K599A or K907A, and then performed RNA-sequencing (RNA-seq) analysis with IRE1 dominant-negative and control SUM52 cells. Through the RNA-seq analysis, we identified 98 genes that were commonly upregulated (65 genes) or downregulated (33 genes) in K599A-expressing SUM52 (SUM52-K599A) or K907A-expressing SUM52 (SUM52-K907A) cells.
Project description:To explore the regulatory mechanism by which inositol-requiring enzyme 1 (IRE1) regulates oncogenic factors, particularly RAB3B, in luminal breast cancer cells, we blocked IRE1 activity in breast cancer cell lines by using the IRE1 inhibitor 4μ8C or expressing IRE1 dominant-negative for miRNA microarray analysis. SUM52 and SUM225 lines with high-level IRE1 expression were treated with 4μ8C for 2 days. The IRE1 kinase dominant-negative mutant K599A or K907A was also used to suppress IRE1 kinase or RNase activity in SUM52 cells. The miRNA microarray analysis revealed a landscape change in miRNA expression profiling in IRE1-inhibited luminal breast cancer cells. Using a criterion of p < 0.05 in miRNA analysis, we identified 41 miRNAs in both SUM52 and SUM225 cells that were altered after inhibiting IRE1 activity. Additionally, we exogenously overexpressed wild-type IRE1 in human nontumorigenic mammary epithelial MCF10A cells and then performed miRNA array assays. When we combined miRNA data from both IRE1 inhibition models in breast cancer cells and exogenous overexpression of IRE1 in MCF10A cells (p<0.05), we identified 5 miRNAs (3607-3p, 374a-5p, 4764-3p, 516a-3p, and 6073) that were upregulated in IRE1-inhibited breast cancer cells and downregulated in MCF10A-IRE1 cells.
Project description:Dysregulation of inositol-requiring enzyme 1 (IRE1), the primary transducer of Unfolded Protein Response (UPR), has been observed in tumor initiation and progression, but the underlying mechanism remains to be further elucidated. In this study, we identified that the IRE1 gene is frequently amplified and over-expressed in aggressive luminal B breast cancer cells and that IRE1 upregulation is significantly associated with worse overall survival of patients with breast cancer. IRE1 processes and mediates degradation of a subset of tumor suppressor microRNAs (miRNAs), including miR-3607, miR-374a, and miR-96, via a mechanism called Regulated IRE1-Dependent Decay (RIDD). IRE1-dependent degradation of tumor suppressor miR-3607 leads to elevation of RAS oncogene GTPase RAB3B in breast cancer cells. Inhibition of IRE1 endoribonuclease activity with the pharmacological compound 4μ8C or genetic approaches effectively suppresses luminal breast cancer cell proliferation and aggressive cancer phenotypes. Our work revealed the IRE1-RIDD-miRNAs pathway that promotes malignancy of luminal breast cancer.
Project description:We investigate the contribution of IRE1 signaling to the modulation of U87 glioma cells transcriptome upon various stresses. To this end, IRE1 control and IRE1 dominant negative expressing U87 glioma cells were subjected to environmental or chemical challenges and their transcriptome monitored using Affymetrix microarrays. Stress-induced transcriptome modulation in function of IRE1 proficiency/deficiency
Project description:We investigate the contribution of IRE1 signaling to the modulation of U87 glioma cells transcriptome upon various stresses. To this end, IRE1 control and IRE1 dominant negative expressing U87 glioma cells were subjected to environmental or chemical challenges and their transcriptome monitored using Affymetrix microarrays.
Project description:Transcriptome analysis was performed from human U87 glioblastoma cell clones: U87 IRE1.NCK DN (U87dn, IRE1 dominant negative) and U87 control (U87ctrl, empty plasmid). Cells were grown in DMEM supplemented with 10% FBS and glutamine for 16 hours in culture prior mRNA isolation and analyses U87dn cells expressing a dominant negative transgene of IRE1alpha were compared to U87ctrl cells transfected with the corresponding empty plamid to identify genes associated to tumor invasion and angiogenesis.
Project description:Transcriptome analysis was performed from human U87 glioblastoma cell clones: U87 IRE1.NCK DN (U87dn, IRE1 dominant negative) and U87 control (U87ctrl, empty plasmid). Cells were grown in DMEM supplemented with 10% FBS and glutamine for 16 hours in culture prior mRNA isolation and analyses
Project description:The IRE1 Rnase domain has been implicated in the pathology of triple negative breast cancer (TNBC), a disease with limited treatment options. The IRE1 Rnase mediates it's effects on the transcriptome via activation of the trancription factor XBP1s and via direct cleavage of mRNA through a process called RIDD. The processes through which the RNase domain contributes to TNBC is not fully understood. We used a novel small molecule inhibitor of the IRE1 Rnase to find novel targets of IRE1 in TNBC, in an effort to elucidate how it contributes to the disease.
Project description:We measured steady-state mRNA levels by microarray hybridization, comparing WT, (delta)ire1, (delta)gcn4, and (delta)gcn2 cells treated with 2 mM DTT for 30 min (by which time the UPR is qualitatively complete) to untreated samples of the same genotype. WT cells were taken as a positive control for UPR induction, and (delta)ire1 cells as a negative control. Fold change in expression of a given gene was computed as the ratio of mRNA level in the treated sample to the level in an untreated sample of the same genotype. Values reported here are the log2 fold change. Keywords = unfolded protein response Keywords = UPR Keywords = ire1 Keywords = gcn4 Keywords = gcn2