RBBP6 maintains glioblastoma stem cells through CPSF3-dependent alternative polyadenylation
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ABSTRACT:
SUBMITTER: Lin P
PROVIDER: S-EPMC10951364 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundGlioblastomas are universally lethal brain tumors containing tumor-propagating glioblastoma stem cells (GSCs). EGFR gene amplification or mutation is frequently detected in GBMs and is associated with poor prognosis. However, EGFR variants in GSCs and their role in the maintenance of GSCs and progression of GBM are unclear.MethodsEGFR variants were detected through bioinformatic HISAT-StringTie-Ballgown pipeline and verified through 5' RACE, RT-PCR, ribonuclease protection, and northern blotting assays. EGFRx function was investigated through neurosphere, cell viability, intracranial xenograft and RNA-seq assays. EGFRx-STAT5 signaling was investigated through western blotting, coimmunoprecipitation, immunofluorescence, luciferase reporter, RT-PCR and CUT&Tag assays.ResultsWe identified a novel EGFR variant (EGFRx), that is specifically expressed in GSCs. Unlike the EGFRvIII variant, which lacks exons 2-7, EGFRx is characterized by the absence of exons 2-14, and encodes an EGFR protein that does not possess the entire extracellular ligand-binding domain. We observed that EGFRx exhibits significant glycosylation, is required for GSC self-renewal, proliferation, and tumorigenesis, and highly active in glioblastomas compared to normal brain tissue. Mechanistically, EGFRx constitutively and specifically activates STAT5 in GSCs through spontaneous asymmetric dimerization of the kinase domain.ConclusionsEGFRx plays essential roles in the maintenance of the GSC phenotype through constitutive activation of STAT5 and promotes GBM progression, suggesting that EGFRx-STAT5 signaling represents a promising therapeutic target for GBM.
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Project description:BackgroundCancer stem cells (CSCs) have unique metabolic characteristics and are hypothesized to contribute significantly to the recurrence and drug resistance of glioblastoma multiforme (GBM). However, the reliance on mitochondrial metabolism and the underlying mechanism of glioblastoma stem cells (GSCs) remains to be elucidated.MethodsTo quantify differential mitochondrial protein expression between GSCs and differentiated cells, a mass spectrum screen was applied by the Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) technique. Functional experiments including CCK8, neurosphere formation, flow cytometry, transwell, and wound healing assays were conducted to evaluate GBM cell malignant phenotype. The potential molecular mechanism of FDFT1 was screened by RNA-seq analyses. The candidate target genes were validated through RT-qPCR and western blot analyses.ResultsAs a top candidate, FDFT1 protein expression in GSCs was elevated relative to their differentiated counterparts. Functionally, the knockdown of FDFT1 suppressed the GBM cell proliferation and migration, while simultaneously enhancing sensitivity to temozolomide. Treatment with both the FDFT1 inhibitor (YM-53601) and simvastatin (an HMG-CoA reductase inhibitor) induced apoptosis in GSCs. Mechanistically, FDFT1 was transcriptionally regulated by SREBP2 but not SREBP1. Furthermore, FDFT1 activates the AKT pathway to regulate tumor metabolism and maintain the stemness of tumor cells.ConclusionsGSCs exhibit a dependency on FDFT1-mediated mevalonate metabolism. Inhibition of FDFT1 could represent a potent strategy to eliminate GSCs.
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Project description:BackgroundGlioblastoma multiforme (GBM) is characterized by its cellular complexity, with a microenvironment consisting of diverse cell types, including oligodendrocyte precursor cells (OPCs) and neoplastic CD133 + radial glia-like cells. This study focuses on exploring the distinct cellular transitions in GBM, emphasizing the role of alternative polyadenylation (APA) in modulating microRNA-binding and post-transcriptional regulation.ResultsOur research identified unique APA profiles that signify the transitional phases between neoplastic cells and OPCs, underscoring the importance of APA in cellular identity and transformation in GBM. A significant finding was the disconnection between differential APA events and gene expression alterations, indicating that APA operates as an independent regulatory mechanism. We also highlighted the specific genes in neoplastic cells and OPCs that lose microRNA-binding sites due to APA, which are crucial for maintaining stem cell characteristics and DNA repair, respectively. The constructed networks of microRNA-transcription factor-target genes provide insights into the cellular mechanisms influencing cancer cell survival and therapeutic resistance.ConclusionsThis study elucidates the APA-driven regulatory framework within GBM, spotlighting its influence on cell state transitions and microRNA network dynamics. Our comprehensive analysis using single-cell RNA sequencing data to investigate the microRNA-binding sites altered by APA profiles offers a robust foundation for future research, presenting a novel approach to understanding and potentially targeting the complex molecular interplay in GBM.
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Project description:The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3' untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3'-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3' UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.
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Project description:Purpose: To identify all of the APA targets of CFIm25 on a global scale and develop an algorithm that can idenitify APA events from standard RNA-seq data Methods: RNA from HeLa cells treated with control siRNA and CFIm25 siRNA were subject to RNA-Seq. Using a custom-designed algorithm to mine RNA-seq data for novel APA events regulated by CFIm25. Results: We identified over 1,400 genes with shortened 3’UTRs after CFIm25 knockdown. Importantly, we show that as a consequence of APA, many of these mRNAs have greatly enhanced protein expression due to the loss of destabilizing features within the 3’UTR. Conclusions: Our study underscored the critical function of the CFIm complex members in governing APA and establish a previously unknown link between APA and metabolic pathways important for tumor progression. Hela cell line mRNA profiles of control treated and CFIm25 Knockdown were generated by RNA-Seq using Illumina GAIIx.
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Project description:Glioblastoma stem cells (GSCs) are pivotal in the recurrence and drug resistance of glioblastoma multiforme (GBM). However, precision therapeutic and diagnostic markers for GSCs have not been fully established. Here, using bioinformatics and experimental analysis, we identified P4HB, a protein disulfide isomerase, as a serum marker that maintains stemness in GSCs through the Wnt/β-catenin signaling pathway. Transcriptional silencing of P4HB induces apoptosis and diminishes stem cell-like characteristics in GSCs. Treatments with the chemical CCF624 or the China National Medical Products Administration (NMPA)-approved securinine significantly prolonged survival in patient-derived xenograft mouse models, underscoring P4HB's potential as a therapeutic target and presenting an expedited path to clinical application through drug repurposing. Additionally, elevated P4HB levels in patient serum were found to correlate with disease progression, underscoring its utility as a biomarker and its promise for precision medicine.
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Project description:Noncoding RNAs regulate transcription of gene expression and play an important role in the pathogenesis of glioblastomas. These tumors are heterogeneous with some glioma stem cells (GSCs) that are highly tumorigenic subpopulations of cells contributing to recurrence and treatment resistance. In this study, GSCs were established by neurosphere cultures of primary glioblastoma cells and validated by the expression of GSC marker CD133. The expression of the long noncoding RNA HOTAIRM1 was detected using real-time quantitative reverse transcription PCR (qRT-PCR). The role of HOTAIRM1 in the proliferation, apoptosis, stemness, and tumorigenicity of GSCs was investigated by soft agar colony formation, flow cytometry, TUNEL analysis, sphere formation, and in vivo xenograft models through silencing of HOTAIRM1. The expression of HOTAIRM1 and the neighboring HOX genes were analyzed by qRT-PCR in different grades of gliomas and nontumor tissues. We found that HOTAIRM1 is significantly elevated in GSCs. The silencing of HOTAIRM1 significantly impairs the proliferation, apoptosis, self-renewal, tumorigenesis of GSCs. In addition, HOTAIRM1 is significantly upregulated in gliomas and associated with tumor grade and patient survival. HOTAIRM1 neighboring genes, HOXA1, HOXA2, and HOXA3, are also significantly upregulated in gliomas and correlate with the expression of HOTAIRM1. Among them, HOXA2 and HOXA3 were identified as being upregulated in GSCs and contributed to the self-renewal of these stem cells. Taken together, our results demonstrate that HOTAIRM1 plays a critical role in the self-renewal of GSCs. These data also suggest that overexpression of HOTAIRM1 can be a negative prognostic factor for patient survival in malignant glioma and may be a promising potential therapeutic target.
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