15 research outputs found
Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance
© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy
An investigation into the alternative splicing of vascular endothelial groAvth factor A [VEGFA)gene
EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors
Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in proangiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFα treatment favoured PSS (increasing VEGFxxx) whereas TGFβ1 favoured DSS, increasing VEGFxxxb levels. TGFβ1 induced DSS selection was prevented by inhibition of p38 MAPK and the CIk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3′UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro- versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFβ1-induced DSS selection, and identify SRp55 as a key regulatory splice factor
Loss of BRMS1 Promotes a Mesenchymal Phenotype through NF-κB-Dependent Regulation of Twist1
Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms: A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS*
Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF165 and less VEGF165b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies
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MSI2 Is Required for Maintaining the Activated Myelodysplastic Syndrome Stem Cell
Abstract + The first four authors contributed equally to this project. Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change <= 0). Furthermore, Gene Set Enrichment Analysis (GSEA) demonstrated a more progenitor like gene expression signature, enrichment in an NRAS activated signature, and a reduced quiescent phenotype. Unsupervised hierarchical clustering of the NHD13/MSI2 LSK gene signature in MDS patients resulted in four distinct clusters. Clusters segregated MSI2 high expressing MDS patients and this "MSI2 high cluster" predicted poor survival. In summary, our findings suggest that MSI2 plays a critical functional role in the maintenance of the hematopoietic stem and progenitor compartment in MDS and highlights it as a novel therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare
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MSI2 is required for maintaining activated myelodysplastic syndrome stem cells
Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease
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RNA Binding Protein Syncrip Regulates the Leukemia Stem Cell Program
Abstract RNA binding proteins (RBPs) tightly control mRNA abundance, stability and translation while mutations or altered expression of specific factors can drive malignancy. Yet, the identity of the RBPs that govern myeloid stem cells remains poorly characterized. We and others have recently demonstrated that MUSASHI-2 (MSI2) is a central regulator of the cancer stem cell program in myeloid leukemia. Therefore, we curated a list of 127 MSI2 direct protein interactors and associated genes to perform an in vivo shRNA screen using MLL-AF9 leukemia cells. We identified shRNAs corresponding to 24 genes that were significantly depleted in vivo after sequencing and comparing their representation from day 16 to day 0. We confirmed knockdown and demonstrated marked reduction in myeloid colony formation in vitro after depleting 7 hits identified in our screen. Additionally, we tested these genes in normal bone marrow c-Kit positive cells and found that the most differentially required gene in leukemia cells compared to normal cells was SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein). SYNCRIP is an RNA binding protein that has been implicated in various RNA regulatory processes but its role in the hematopoietic system is virtually unknown. Depletion of SYNCRIP with shRNAs in murine MLL-AF9 leukemia cells resulted in an increase in myeloid differentiation, apoptosis and delayed leukemogenesis in vivo (median survival of 35 days; control versus 61 days shRNA#1 knockdown was selected against, and "not reached" shRNA#2). To further assess SYNCRIP function in vivo, we developed a germline Syncrip knockout (KO) by injecting Cas9-DNA and Syncrip - guides RNAs into embryos and harvested E13 fetal liver cells. After Syncrip deletion was verified by immunoblotting, we observed normal numbers of HSCs and equivalent engraftment in lethally irradiated animals in both primary and secondary transplants. In contrast, we observed a delay in leukemeogenesis (median survival of 87.5 days; WT versus 118 days KO) in recipient mice after transplantation of MLL-AF9 transformed LSKs. Notably, non-deleted leukemia cells outcompeted the SYNCRIP deleted cells based on a reemergence of SYNCRIP expression. These data suggest that SYNCRIP is differentially required in myeloid leukemia cells compared to normal cells. Furthermore, we found that SYNCRIP was highly expressed in wide variety of human AML cell lines and in primary AML patients (n=4/5). SYNCRIP depletion with shRNAs resulted in reduced cell proliferation and the induction of apoptosis in human AML cell lines (MOLM13, NOMO-1, KASUMI-1 and NB4) and a marked decrease in engraftment of primary AML patient cells. To gain insights into SYNCRIP function, we performed RNA-sequencing of leukemia cells depleted for SYNCRIP. Gene set enrichment analysis (GSEA) negatively enriched for the MLL-AF9, HOXA9 and stem cell programs in SYNCRIP-KD cells and positively enriched for MSI2's direct mRNA binding targets and a MSI2 deficient LSC signature. Reciprocal immunoblotting in the presence or absence of RNAse demonstrated that SYNCRIP and MSI2 interaction is RNA dependent. We validated their shared targets by performing SYNCRIP RNA-immunoprecipitation (RIP) for previously identified MSI2's direct mRNAs targets (HOXA9 and c-MYC). SYNCRIP depletion resulted in reduced protein abundance of HOXA9 and c-MYC. Forced MSI2 expression partially rescued the colony formation and HOXA9 expression in SYNCRIP-KD cells. To assess the functional downstream targets of SYNCRIP in leukemia, we overexpressed HOXA9 and c-MYC in SYNCRIP-KD cells and observed that HOXA9 expression but not c-MYC partially rescues the effect of SYNCRIP depletion on myeloid colony formation. Mechanistically, we showed that SYNCRIP regulates translation of HOXA9 without affecting HoxA9 mRNA stability. Overall, we provide a strategy for interrogating the functional RNA binding network in leukemia using shRNA screening. Additionally, we validated SYNCRIP as a novel RBP that controls the leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in myeloid leukemia. Disclosures Melnick: Janssen: Research Funding. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Järås:Cantargia AB: Equity Ownership