Journal articles
Brooks H, Li L, Addeo A, Stevens M, Comins C, Oltean S (2023). Detection of genomic mutations in blood and urine free circulating tumour DNA in patients with inoperable and metastatic lung adenocarcinoma harbouring an EGFR mutation in tissue: a UK pilot study.
Frontiers in Oncology,
13Abstract:
Detection of genomic mutations in blood and urine free circulating tumour DNA in patients with inoperable and metastatic lung adenocarcinoma harbouring an EGFR mutation in tissue: a UK pilot study
The development of methodologies to analyse circulating tumour DNA (ctDNA) in the blood or urine of cancer patients provides an invaluable resource that can be used for diagnosis and prognosis and to evaluate response to treatments. Lung cancer has seen in the last years a revolution in treatment strategy with the use of several classes of EGFR inhibitors. However, almost invariably, resistance to such therapies appears. In this paper, we describe a pilot, longitudinal study with 20 patients with confirmed EGFR mutations in tissue biopsy for lung cancer. The objective of the study was to determine whether ctDNA from plasma and/or urine could be used to monitor the EGFR mutational status of patients with confirmed EGFR mutation-positive non-small cell lung cancer (NSCLC) during treatment with EGFR inhibitors. Blood and urine were collected monthly over periods ranging from 6 to 16 months. CtDNA was analysed in each patient for the presence of several known mutations that predispose to resistance to EGFR inhibitors. We have proven that serial monitoring of ctDNA from both plasma and urine is feasible and that patients are willing to participate in this process. We have also shown that longitudinal ctDNA monitoring may detect resistance mutations before the development of radiological and clinical disease progression.
Abstract.
Li L, Zheng J, Stevens M, Oltean S (2022). A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts.
Mol Ther Methods Clin Dev,
25, 147-157.
Abstract:
A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts.
Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.
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Author URL.
Ayine ML, Stevens M, Oltean S (2022). IDF21-0319 Novel compounds found to regulate VEGF-A alternative splicing in diabetic podocytes. Diabetes Research and Clinical Practice, 186
Star E, Stevens M, Gooding C, Smith CWJ, Li L, Ayine ML, Harper SJ, Bates DO, Oltean S (2021). A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.
Oncogenesis,
10(5).
Abstract:
A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties
AbstractAlternative splicing of the vascular endothelial growth factor a (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.
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Uzor S, Porazinski SR, Li L, Clark B, Ajiro M, Iida K, Hagiwara M, Alqasem AA, Perks CM, Wilson ID, et al (2021). CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer.
Scientific Reports,
11(1).
Abstract:
CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer
AbstractDysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.
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Legge D, Li L, Moriarty W, Lee D, Szemes M, Zahed A, Panousopoulos L, Chung WY, Aghabi Y, Barratt J, et al (2021). The epithelial splicing regulator <i>ESRP2</i> is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor.
Molecular Oncology,
16(3), 630-647.
Abstract:
The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor
Wilms tumour (WT), an embryonal kidney cancer, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of foetal kidney with two WT cell lines, filtering our results to remove common cancer‐associated epigenetic changes and to enrich for genes involved in early kidney development. This identified four hypermethylated genes, of which ESRP2 (epithelial splicing regulatory protein 2) was the most promising for further study. ESRP2 was commonly repressed by DNA methylation in WT, and this occurred early in WT development (in nephrogenic rests). ESRP2 expression was reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was overexpressed in WT cell lines, it inhibited cellular proliferation in vitro, and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA‐seq of the ESRP2‐expressing WT cell lines identified several novel splicing targets. We propose a model in which epigenetic inactivation of ESRP2 disrupts the mesenchymal to epithelial transition in early kidney development to generate WT.
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Kitchen P, Lee KY, Clark D, Lau N, Lertsuwan J, Sawasdichai A, Satayavivad J, Oltean S, Afford S, Gaston K, et al (2020). A Runaway PRH/HHEX-Notch3-Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition.
Cancer Res,
80(4), 757-770.
Abstract:
A Runaway PRH/HHEX-Notch3-Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition.
Aberrant Notch and Wnt signaling are known drivers of cholangiocarcinoma (CCA), but the underlying factors that initiate and maintain these pathways are not known. Here, we show that the proline-rich homeodomain protein/hematopoietically expressed homeobox (PRH/HHEX) transcription factor forms a positive transcriptional feedback loop with Notch3 that is critical in CCA. PRH/HHEX expression is elevated in CCA, and depletion of PRH reduces CCA tumor growth in a xenograft model. Overexpression of PRH in primary human biliary epithelial cells is sufficient to increase cell proliferation and produce an invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 reveals that unlike Notch3, PRH directly activates canonical Wnt signaling. These data indicate that hyperactivation of Notch and Wnt signaling is independent of the underlying mutational landscape and has a common origin in dysregulation of PRH. Moreover, they suggest new therapeutic options based on the dependence of specific Wnt, Notch, and CDK4/6 inhibitors on PRH activity. SIGNIFICANCE: the PRH/HHEX transcription factor is an oncogenic driver in cholangiocarcinoma that confers sensitivity to CDK4/6 inhibitors.
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Li L, Hobson L, Perry L, Clark B, Heavey S, Haider A, Sridhar A, Shaw G, Kelly J, Freeman A, et al (2020). Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer.
British Journal of Cancer,
123(6), 1024-1032.
Abstract:
Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer
Abstract
. Background
. The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4.
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. Methods
. We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5′ and 3′ splice sites of ERG’s exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo.
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. Results
. In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member β-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3′ splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3′ splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo.
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. Conclusions
. We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.
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Bowler E, Oltean S (2019). Alternative Splicing in Angiogenesis.
Int J Mol Sci,
20(9).
Abstract:
Alternative Splicing in Angiogenesis.
Alternative splicing of pre-mRNA allows the generation of multiple splice isoforms from a given gene, which can have distinct functions. In fact, splice isoforms can have opposing functions and there are many instances whereby a splice isoform acts as an inhibitor of canonical isoform function, thereby adding an additional layer of regulation to important processes. Angiogenesis is an important process that is governed by alternative splicing mechanisms. This review focuses on the alternative spliced isoforms of key genes that are involved in the angiogenesis process; VEGF-A, VEGFR1, VEGFR2, NRP-1, FGFRs, Vasohibin-1, Vasohibin-2, HIF-1α, Angiopoietin-1 and Angiopoietin-2.
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Munkley J, Li L, Krishnan SRG, Hysenaj G, Scott E, Dalgliesh C, Oo HZ, Maia TM, Cheung K, Ehrmann I, et al (2019). Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.
eLife,
8Abstract:
Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer
Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.
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Kikuchi R, Stevens M, Harada K, Oltean S, Murohara T (2019). Anti-angiogenic isoform of vascular endothelial growth factor-A in cardiovascular and renal disease.
,
88, 1-33.
Abstract:
Anti-angiogenic isoform of vascular endothelial growth factor-A in cardiovascular and renal disease
Accumulating evidence suggests that pathologic interactions between the heart and the kidney can contribute to the progressive dysfunction of both organs. Recently, there has been an increase in the prevalence of cardiovascular disease (CVD) and chronic kidney disease (CKD) due to increasing obesity rates. It has been reported that obesity causes various heart and renal disorders and appears to accelerate their progression. Vascular endothelial growth factor-A (VEGF-A) is a major regulator of angiogenesis and vessel permeability, and is associated with CVD and CKD. It is now recognized that alternative VEGF-A gene splicing generates VEGF-A isoforms that differ in their biological actions. Proximal splicing that includes an exon 8a sequence results in pro-angiogenic VEGF-A 165 a, whereas distal splicing inclusive of exon 8b yields the anti-angiogenic isoform of VEGF-A (VEGF-A 165 b). This review highlights several recent preclinical and clinical studies on the role of VEGF-A 165 b in CVD and CKD as a novel function of VEGF-A. This review also discusses potential therapeutic approaches of the use of VEGF-A in clinical settings as a potential circulating biomarker for CVD and CKD.
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Stevens M, Oltean S (2019). Modulation of Receptor Tyrosine Kinase Activity through Alternative Splicing of Ligands and Receptors in the VEGF-A/VEGFR Axis.
Cells,
8(4).
Abstract:
Modulation of Receptor Tyrosine Kinase Activity through Alternative Splicing of Ligands and Receptors in the VEGF-A/VEGFR Axis.
Vascular endothelial growth factor a (VEGF-A) signaling is essential for physiological and pathological angiogenesis. Alternative splicing of the VEGF-A pre-mRNA gives rise to a pro-angiogenic family of isoforms with a differing number of amino acids (VEGF-Axxxa), as well as a family of isoforms with anti-angiogenic properties (VEGF-Axxxb). The biological functions of VEGF-A proteins are mediated by a family of cognate protein tyrosine kinase receptors, known as the VEGF receptors (VEGFRs). VEGF-A binds to both VEGFR-1, largely suggested to function as a decoy receptor, and VEGFR-2, the predominant signaling receptor. Both VEGFR-1 and VEGFR-2 can also be alternatively spliced to generate soluble isoforms (sVEGFR-1/sVEGFR-2). The disruption of the splicing of just one of these genes can result in changes to the entire VEGF-A/VEGFR signaling axis, such as the increase in VEGF-A165a relative to VEGF-A165b resulting in increased VEGFR-2 signaling and aberrant angiogenesis in cancer. Research into this signaling axis has recently focused on manipulating the splicing of these genes as a potential therapeutic avenue in disease. Therefore, further research into understanding the mechanisms by which the splicing of VEGF-A/VEGFR-1/VEGFR-2 is regulated will help in the development of drugs aimed at manipulating splicing or inhibiting specific splice isoforms in a therapeutic manner.
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Stevens M, Oltean S (2019). Modulation of the Apoptosis Gene Bcl-x Function Through Alternative Splicing.
FRONTIERS IN GENETICS,
10 Author URL.
Melegh Z, Oltean S (2019). Targeting Angiogenesis in Prostate Cancer.
Int J Mol Sci,
20(11).
Abstract:
Targeting Angiogenesis in Prostate Cancer.
Prostate cancer is the most commonly diagnosed cancer among men in the Western world. Although localized disease can be effectively treated with established surgical and radiopharmaceutical treatments options, the prognosis of castration-resistant advanced prostate cancer is still disappointing. The objective of this study was to review the role of angiogenesis in prostate cancer and to investigate the effectiveness of anti-angiogenic therapies. A literature search of clinical trials testing the efficacy of anti-angiogenic therapy in prostate cancer was performed using Pubmed. Surrogate markers of angiogenic activity (microvessel density and vascular endothelial growth factor a (VEGF-A) expression) were found to be associated with tumor grade, metastasis, and prognosis. Six randomizedstudies were included in this review: two phase II trials on localized and hormone-sensitive disease (n = 60 and 99 patients) and four phase III trials on castration-resistant refractory disease (n = 873 to 1224 patients). Although the phase II trials showed improved relapse-free survival and stabilisation of the disease, the phase III trials found increased toxicity and no significant improvement in overall survival. Although angiogenesis appears to have an important role in prostate cancer, the results of anti-angiogenic therapy in castration-resistant refractory disease have hitherto been disappointing. There are various possible explanations for this lack of efficacy in castration-resistant refractory disease: redundancy of angiogenic pathways, molecular heterogeneity of the disease, loss of tumor suppressor protein phosphatase and tensin homolog (PTEN) expression as well as various VEGF-A splicing isoforms with pro- and anti-angiogenic activity. A better understanding of the molecular mechanisms of angiogenesis may help to develop effective anti-angiogenic therapy in prostate cancer.
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Jumbe SL, Porazinski SR, Oltean S, Mansell JP, Vahabi B, Wilson ID, Ladomery MR (2019). The Evolutionarily Conserved Cassette Exon 7b Drives ERG's Oncogenic Properties.
Transl Oncol,
12(1), 134-142.
Abstract:
The Evolutionarily Conserved Cassette Exon 7b Drives ERG's Oncogenic Properties.
The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.
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Stevens M, Star E, Lee M, Innes E, Li L, Bowler E, Harper S, Bates DO, Oltean S (2019). The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo.
RNA Biol,
16(12), 1672-1681.
Abstract:
The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo.
Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo.
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Stevens M, Neal CR, Craciun EC, Dronca M, Harper SJ, Oltean S (2019). The natural drug DIAVIT is protective in a type II mouse model of diabetic nephropathy.
PLoS One,
14(3).
Abstract:
The natural drug DIAVIT is protective in a type II mouse model of diabetic nephropathy.
There is evidence to suggest that abnormal angiogenesis, inflammation, and fibrosis drive diabetic nephropathy (DN). However, there is no specific treatment to counteract these processes. We aimed to determine whether DIAVIT, a natural Vaccinium myrtillus (blueberry) and Hippophae Rhamnoides (sea buckthorn) extract, is protective in a model of type II DN. Diabetic db/db mice were administered DIAVIT in their drinking water for 14 weeks. We assessed the functional, structural, and ultra-structural phenotype of three experimental groups (lean+vehicle, db/db+vehicle, db/db+DIAVIT). We also investigated the angiogenic and fibrotic pathways involved in the mechanism of action of DIAVIT. Diabetic db/db mice developed hyperglycaemia, albuminuria, and an increased glomerular water permeability; the latter two were prevented by DIAVIT. db/db mice developed fibrotic glomeruli, endothelial insult, and glomerular ultra-structural changes, which were not present in DIAVIT-treated mice. Vascular endothelial growth factor a (VEGF-A) splicing was altered in the db/db kidney cortex, increasing the pro-angiogenic VEGF-A165 relative to the anti-angiogenic VEGF-A165b. This was partially prevented with DIAVIT treatment. Delphinidin, an anthocyanin abundant in DIAVIT, increased the VEGF-A165b expression relative to total VEGF-A165 in cultured podocytes through phosphorylation of the splice factor SRSF6. DIAVIT, in particular delphinidin, alters VEGF-A splicing in type II DN, rescuing the DN phenotype. This study highlights the therapeutic potential of natural drugs in DN through the manipulation of gene splicing and expression.
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Stevens M, Payne M, Innes E, Torregrossa RO, Wood M, Stevens MKO, Whiteman MO, Oltean SO (2018). A novel mitochondria‐targeted hydrogen sulfide delivery molecule prevents uraemia and diabetes‐induced renal cell oxidative stress. The FASEB Journal, 32(S1), 619.10-619.10.
Stevens M, Oltean S (2018). Assessment of Kidney Function in Mouse Models of Glomerular Disease.
J Vis Exp(136).
Abstract:
Assessment of Kidney Function in Mouse Models of Glomerular Disease.
The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.
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Stevens M, Oltean S (2018). Modulation of VEGF-A Alternative Splicing as a Novel Treatment in Chronic Kidney Disease.
Genes (Basel),
9(2).
Abstract:
Modulation of VEGF-A Alternative Splicing as a Novel Treatment in Chronic Kidney Disease.
Vascular endothelial growth factor a (VEGF-A) is a prominent pro-angiogenic and pro-permeability factor in the kidney. Alternative splicing of the terminal exon of VEGF-A through the use of an alternative 3' splice site gives rise to a functionally different family of isoforms, termed VEGF-Axxxb, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-Axxx/VEGF-Axxxb isoform balance has recently been reported in several kidney pathologies, including diabetic nephropathy (DN) and Denys-Drash syndrome. Using mouse models of kidney disease where the VEGF-A isoform balance is disrupted, several reports have shown that VEGF-A165b treatment/over-expression in the kidney is therapeutically beneficial. Furthermore, inhibition of certain splice factor kinases involved in the regulation of VEGF-A terminal exon splicing has provided some mechanistic insight into how VEGF-A splicing could be regulated in the kidney. This review highlights the importance of further investigation into the novel area of VEGF-A splicing in chronic kidney disease pathogenesis and how future studies may allow for the development of splicing-modifying therapeutic drugs.
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Bates DO, Beazley-Long N, Benest AV, Ye X, Ved N, Hulse RP, Barratt S, Machado MJ, Donaldson LF, Harper SJ, et al (2018). Physiological Role of Vascular Endothelial Growth Factors as Homeostatic Regulators.
Compr Physiol,
8(3), 955-979.
Abstract:
Physiological Role of Vascular Endothelial Growth Factors as Homeostatic Regulators.
The vascular endothelial growth factor (VEGF) family of proteins are key regulators of physiological systems. Originally linked with endothelial function, they have since become understood to be principal regulators of multiple tissues, both through their actions on vascular cells, but also through direct actions on other tissue types, including epithelial cells, neurons, and the immune system. The complexity of the five members of the gene family in terms of their different splice isoforms, differential translation, and specific localizations have enabled tissues to use these potent signaling molecules to control how they function to maintain their environment. This homeostatic function of VEGFs has been less intensely studied than their involvement in disease processes, development, and reproduction, but they still play a substantial and significant role in healthy control of blood volume and pressure, interstitial volume and drainage, renal and lung function, immunity, and signal processing in the peripheral and central nervous system. The widespread expression of VEGFs in healthy adult tissues, and the disturbances seen when VEGF signaling is inhibited support this view of the proteins as endogenous regulators of normal physiological function. This review summarizes the evidence and recent breakthroughs in understanding of the physiology that is regulated by VEGF, with emphasis on the role they play in maintaining homeostasis. © 2017 American Physiological Society. Compr Physiol 8:955-979, 2018.
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Stevens M, Neal CR, Salmon AHJ, Bates DO, Harper SJ, Oltean S (2018). Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury.
Nephron,
139(1), 51-62.
Abstract:
Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury.
BACKGROUND/AIMS: Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b). METHODS: the Pod-DTR mouse was crossed with a mouse that over-expressed VEGF-A165b specifically in the podocytes (Neph-VEGF-A165b). Wild type (WT), Pod-DTR, Neph-VEGF-A165b and Pod-DTR X Neph-VEGF-A165b mice were treated with several doses of DT (1, 5, 100, and 1,000 ng/g bodyweight). Urine was collected and the glomerular water permeability (LpA/Vi) was measured ex vivo after 14 days. Structural analysis and podocyte marker expression were also assessed. RESULTS: Pod-DTR mice developed an increased glomerular LpA/Vi 14 days after administration of DT (all doses), which was prevented when the mice over-expressed VEGF-A165b. No major structural abnormalities, podocyte ablation or albuminuria was observed in Pod-DTR mice, indicating this to be a mild model of podocyte disease. However, a change in expression and localisation of nephrin within the podocytes was observed, indicating disruption of the slit diaphragm in the Pod-DTR mice. This was prevented in the Pod-DTR X Neph-VEGF-A165b mice. CONCLUSION: Although only a mild model of podocyte injury, over-expression of the anti-permeability VEGF-A165b isoform in the podocytes of Pod-DTR mice had a protective effect. Therefore, this study further highlights the therapeutic potential of VEGF-A165b in glomerular disease.
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Oltean S, Coward R, Collino M, Baelde H (2017). Diabetic Nephropathy: Novel Molecular Mechanisms and Therapeutic Avenues.
BIOMED RESEARCH INTERNATIONAL,
2017 Author URL.
Barratt SL, Blythe T, Jarrett C, Ourradi K, Shelley-Fraser G, Day MJ, Qiu Y, Harper S, Maher TM, Oltean S, et al (2017). Differential Expression of VEGF-Axxx Isoforms is Critical for Development of Pulmonary Fibrosis.
Am J Respir Crit Care Med,
196(4), 479-493.
Abstract:
Differential Expression of VEGF-Axxx Isoforms is Critical for Development of Pulmonary Fibrosis.
RATIONALE: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES: to establish VEGF-A isoform expression and functional effects in IPF. METHODS: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS: IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. CONCLUSIONS: These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.
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Author URL.
Midgley AC, Oltean S, Hascall V, Woods EL, Steadman R, Phillips AO, Meran S (2017). Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype.
Sci Signal,
10(506).
Abstract:
Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype.
The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine-rich (SR) protein-mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis.
Abstract.
Author URL.
Bates DO, Morris JC, Oltean S, Donaldson LF (2017). Pharmacology of Modulators of Alternative Splicing.
Pharmacol Rev,
69(1), 63-79.
Abstract:
Pharmacology of Modulators of Alternative Splicing.
More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed.
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Author URL.
Bullock N, Oltean S (2017). The many faces of SRPK1.
J Pathol,
241(4), 437-440.
Abstract:
The many faces of SRPK1.
Serine-arginine protein kinase 1 (SRPK1) phosphorylates proteins involved in the regulation of several mRNA-processing pathways, including alternative splicing. SRPK1 has been recently reported to be overexpressed in multiple cancers, including prostate cancer, breast cancer, lung cancer, and glioma. Several studies have shown that inhibition of SRPK1 has anti-tumoural effects, and SRPK1 has therefore become a new candidate for targeted therapies. Interestingly, in terms of molecular mechanism, SRPK1 seems to act heterogeneously, and has been reported to affect several processes in different cancers, e.g. angiogenesis in prostate and colon cancer, apoptosis in breast and colon cancer, and migration in breast cancer. A recent report adds to this puzzle, showing that the main effect of SRPK1 overexpression in non-small-cell lung carcinoma is to stimulate a stem cell-like phenotype. This pleiotropy might be related to preferential activation of different downstream signalling pathways by SRPK1 in various cancers. © 2016 the Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Author URL.
Kollareddy M, Sherrard A, Park JH, Szemes M, Gallacher K, Melegh Z, Oltean S, Michaelis M, Cinatl J, Kaidi A, et al (2017). The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.
Cancer Lett,
403, 74-85.
Abstract:
The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.
Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase a inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers.
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Author URL.
Stevens M, Neal CR, Salmon AHJ, Bates DO, Harper SJ, Oltean S (2017). VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.
J Physiol,
595(19), 6281-6298.
Abstract:
VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.
KEY POINTS: Progressive depletion of all vascular endothelial growth factor a (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor a (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165. The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.
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Author URL.
Stevens M, Oltean S (2016). Alternative Splicing in CKD.
J Am Soc Nephrol,
27(6), 1596-1603.
Abstract:
Alternative Splicing in CKD.
Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit.
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Author URL.
Mavrou A, Oltean S (2016). SRPK1 inhibition in prostate cancer: a novel anti-angiogenic treatment through modulation of VEGF alternative splicing.
Pharmacol Res,
107, 276-281.
Abstract:
SRPK1 inhibition in prostate cancer: a novel anti-angiogenic treatment through modulation of VEGF alternative splicing.
Prostate cancer remains one of the leading causes of cancer death in men around the world, regardless of intense research and development of novel therapies in the last 10 years. One of the new avenues that has been tested - inhibition of angiogenesis - has been disappointing so far in clinical studies in spite of strong evidence that determinants of angiogenesis (e.g. vascular endothelial growth factor) are strongly associated with disease progression. One of the reasons for these outcomes may be our poor understanding of the biology of angiogenesis in prostate cancer (and probably other cancers as well) resulting in inhibition of both detrimental and favourable molecules. We discuss here novel targeted and more specific approaches to inhibit angiogenesis in prostate cancer as well as a completely new therapeutic modality to do this - modulation of alternative splicing - that may be applicable to other molecules/biological processes as well.
Abstract.
Bullock N, Potts J, Simpkin AJ, Koupparis A, Harper SJ, Oxley J, Oltean S (2016). Serine-arginine protein kinase 1 (SRPK1), a determinant of angiogenesis, is upregulated in prostate cancer and correlates with disease stage and invasion.
J Clin Pathol,
69(2), 171-175.
Abstract:
Serine-arginine protein kinase 1 (SRPK1), a determinant of angiogenesis, is upregulated in prostate cancer and correlates with disease stage and invasion.
Vascular endothelial growth factor (VEGF) undergoes alternative splicing to produce both proangiogenic and antiangiogenic isoforms. Preferential splicing of proangiogenic VEGF is determined by serine-arginine protein kinase 1 (SRPK1), which is upregulated in a number of cancers. In the present study, we aimed to investigate SRPK1 expression in prostate cancer (PCa) and its association with cancer progression. SRPK1 expression was assessed using immunohistochemistry of PCa tissue extracted from radical prostatectomy specimens of 110 patients. SRPK1 expression was significantly higher in tumour compared with benign tissue (p
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Hamdollah Zadeh MA, Amin EM, Hoareau-Aveilla C, Domingo E, Symonds KE, Ye X, Heesom KJ, Salmon A, D'Silva O, Betteridge KB, et al (2015). Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.
Mol Oncol,
9(1), 167-178.
Abstract:
Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.
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-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, 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.
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Bunni J, Shelley-Fraser G, Stevenson K, Oltean S, Salmon A, Harper SJ, Carter JG, Bates DO (2015). Circulating levels of anti-angiogenic VEGF-A isoform (VEGF-Axxxb) in colorectal cancer patients predicts tumour VEGF-A ratios.
Am J Cancer Res,
5(6), 2083-2089.
Abstract:
Circulating levels of anti-angiogenic VEGF-A isoform (VEGF-Axxxb) in colorectal cancer patients predicts tumour VEGF-A ratios.
PURPOSE: Bevacizumab as an adjunct to chemotherapy improves survival for some patients with metastatic colorectal cancer. Immunohistochemical staining of samples from the registration ECOG E3200 trial of bevacizumab with FOLFOX demonstrated that only patients with carcinomas expressing low levels of VEGF-A165b, an anti-angiogenic splice variant of the Vascular Endothelial Growth Factor family of proteins, benefited from bevacizumab treatment. To identify a more useful biomarker of response we tested the hypothesis that circulating VEGF-A165b levels correlate with immunohistochemical staining. EXPERIMENTAL DESIGN: 17 patients with biopsy proven colorectal adenocarcinoma had pre-operative blood samples drawn. They underwent resection and had post-resection blood drawn. The plasma was analysed for levels of VEGF-Axxxb using enzyme-linked immunosorbent assay (ELISA) and the tumour blocks stained for VEGF-Axxxb and pan-VEGF-A. The normalised ratio of VEGF-Axxxb expression to that of panVEGF-A expression scored by IHC was calculated and correlated with plasma VEGF-A165b levels. RESULTS: Plasma levels of VEGF-Axxxb significantly correlated with the VEGF-Axxxb:panVEGF-A ratio (r=0.594, P
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Author URL.
Oltean S (2015). Modulators of alternative splicing as novel therapeutics in cancer.
World J Clin Oncol,
6(5), 92-95.
Abstract:
Modulators of alternative splicing as novel therapeutics in cancer.
Alternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g. pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics.
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Mavrou A, Brakspear K, Hamdollah-Zadeh M, Damodaran G, Babaei-Jadidi R, Oxley J, Gillatt DA, Ladomery MR, Harper SJ, Bates DO, et al (2015). Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.
Oncogene,
34(33), 4311-4319.
Abstract:
Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.
Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor a (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa.
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Munkley J, Oltean S, Vodák D, Wilson BT, Livermore KE, Zhou Y, Star E, Floros VI, Johannessen B, Knight B, et al (2015). The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.
Oncotarget,
6(33), 34358-34374.
Abstract:
The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.
Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.
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Oltean S, Qiu Y, Ferguson JK, Stevens M, Neal C, Russell A, Kaura A, Arkill KP, Harris K, Symonds C, et al (2015). Vascular Endothelial Growth Factor-A165b is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.
J Am Soc Nephrol,
26(8), 1889-1904.
Abstract:
Vascular Endothelial Growth Factor-A165b is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.
Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.
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Author URL.
Oltean S, Bates DO (2014). Hallmarks of alternative splicing in cancer.
Oncogene,
33(46), 5311-5318.
Abstract:
Hallmarks of alternative splicing in cancer.
The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.
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Gammons MV, Lucas R, Dean R, Coupland SE, Oltean S, Bates DO (2014). Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.
Br J Cancer,
111(3), 477-485.
Abstract:
Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.
BACKGROUND: Current therapies for metastatic melanoma are targeted either at cancer mutations driving growth (e.g. vemurafenib) or immune-based therapies (e.g. ipilimumab). Tumour progression also requires angiogenesis, which is regulated by VEGF-A, itself alternatively spliced to form two families of isoforms, pro- and anti-angiogenic. Metastatic melanoma is associated with a splicing switch to pro-angiogenic VEGF-A, previously shown to be regulated by SRSF1 phosphorylation by SRPK1. Here, we show a novel approach to preventing angiogenesis-targeting splicing factor kinases that are highly expressed in melanomas. METHODS: We used RT-PCR, western blotting and immunohistochemistry to investigate SRPK1, SRSF1 and VEGF expression in tumour cells, and in vivo xenograft assays to investigate SRPK1 knockdown and inhibition in vivo. RESULTS: in both uveal and cutaneous melanoma cell lines, SRPK1 was highly expressed, and inhibition of SRPK1 by knockdown or with pharmacological inhibitors reduced pro-angiogenic VEGF expression maintaining the production of anti-angiogenic VEGF isoforms. Both pharmacological SRPK1 inhibitors and SRPK1 knockdown reduced growth of human melanomas in vivo, but neither affected cell proliferation in vitro. CONCLUSIONS: These results suggest that selective blocking of pro-angiogenic isoforms by inhibiting splice-site selection with SRPK1 inhibitors reduces melanoma growth. SRPK1 inhibitors may be used as therapeutic agents.
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Bates DO, Mavrou A, Qiu Y, Carter JG, Hamdollah-Zadeh M, Barratt S, Gammons MV, Millar AB, Salmon AHJ, Oltean S, et al (2013). Detection of VEGF-A(xxx)b isoforms in human tissues.
PLoS One,
8(7).
Abstract:
Detection of VEGF-A(xxx)b isoforms in human tissues.
Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.
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Oltean S, Gammons M, Hulse R, Hamdollah-Zadeh M, Mavrou A, Donaldson L, Salmon AH, Harper SJ, Ladomery MR, Bates DO, et al (2012). SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases.
Biochem Soc Trans,
40(4), 831-835.
Abstract:
SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases.
SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.
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Oltean S, Neal CR, Mavrou A, Patel P, Ahad T, Alsop C, Lee T, Sison K, Qiu Y, Harper SJ, et al (2012). VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice.
Am J Physiol Renal Physiol,
303(7), F1026-F1036.
Abstract:
VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice.
Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF(164) isoform, and the antiangiogenic VEGF(165)b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF(164) overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF(164) double transgenic mice. The consequences of simultaneous overexpression of VEGF(164) and VEGF(165)b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF(164)/nephrin-VEGF(165)b mice. Persistent VEGF(164) overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF(164) levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF(164)-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF(164) were not present in triple transgenic VEGF(164) and VEGF(165)b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF(164) overexpression. VEGF(164) overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF(165)b is capable of preventing VEGF(164)-induced changes in glomerular permeability and ultrastructure in vivo.
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Armstrong AJ, Marengo MS, Oltean S, Kemeny G, Bitting RL, Turnbull JD, Herold CI, Marcom PK, George DJ, Garcia-Blanco MA, et al (2011). Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers.
Mol Cancer Res,
9(8), 997-1007.
Abstract:
Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers.
During cancer progression, malignant cells undergo epithelial-mesenchymal transitions (EMT) and mesenchymal-epithelial transitions (MET) as part of a broad invasion and metastasis program. We previously observed MET events among lung metastases in a preclinical model of prostate adenocarcinoma that suggested a relationship between epithelial plasticity and metastatic spread. We thus sought to translate these findings into clinical evidence by examining the existence of EMT in circulating tumor cells (CTC) from patients with progressive metastatic solid tumors, with a focus on men with castration-resistant prostate cancer (CRPC) and women with metastatic breast cancer. We showed that the majority (> 80%) of these CTCs in patients with metastatic CRPC coexpress epithelial proteins such as epithelial cell adhesion molecule (EpCAM), cytokeratins (CK), and E-cadherin, with mesenchymal proteins including vimentin, N-cadherin and O-cadherin, and the stem cell marker CD133. Equally, we found that more than 75% of CTCs from women with metastatic breast cancer coexpress CK, vimentin, and N-cadherin. The existence and high frequency of these CTCs coexpressing epithelial, mesenchymal, and stem cell markers in patients with progressive metastases has important implications for the application and interpretation of approved methods to detect CTCs.
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Amin EM, Oltean S, Hua J, Gammons MVR, Hamdollah-Zadeh M, Welsh GI, Cheung M-K, Ni L, Kase S, Rennel ES, et al (2011). WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.
Cancer Cell,
20(6), 768-780.
Abstract:
WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.
Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.
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Qiu Y, Ferguson J, Oltean S, Neal CR, Kaura A, Bevan H, Wood E, Sage LM, Lanati S, Nowak DG, et al (2010). Overexpression of VEGF165b in podocytes reduces glomerular permeability.
J Am Soc Nephrol,
21(9), 1498-1509.
Abstract:
Overexpression of VEGF165b in podocytes reduces glomerular permeability.
The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF(165)b specifically in podocytes. We confirmed excess production of glomerular VEGF(165)b by reverse transcriptase-PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF(164)-overexpressing mice. Animals overexpressing VEGF(165)b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype.
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Qiu Y, Hoareau-Aveilla C, Oltean S, Harper SJ, Bates DO (2009). The anti-angiogenic isoforms of VEGF in health and disease.
Biochem Soc Trans,
37(Pt 6), 1207-1213.
Abstract:
The anti-angiogenic isoforms of VEGF in health and disease.
Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF(165)b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGF(xxx)b isoforms to the pro-angiogenic VEGF(xxx) isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGF(xxx)b isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.
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Oltean S, Febbo PG, Garcia-Blanco MA (2008). Dunning rat prostate adenocarcinomas and alternative splicing reporters: powerful tools to study epithelial plasticity in prostate tumors in vivo.
Clin Exp Metastasis,
25(6), 611-619.
Abstract:
Dunning rat prostate adenocarcinomas and alternative splicing reporters: powerful tools to study epithelial plasticity in prostate tumors in vivo.
Using alternative splicing reporters we have previously observed mesenchymal epithelial transitions in Dunning AT3 rat prostate tumors. We demonstrate here that the Dunning DT and AT3 cells, which express epithelial and mesenchymal markers, respectively, represent an excellent model to study epithelial transitions since these cells recapitulate gene expression profiles observed during human prostate cancer progression. In this manuscript we also present the development of two new tools to study the epithelial transitions by imaging alternative splicing decisions: a bichromatic fluorescence reporter to evaluate epithelial transitions in culture and in vivo, and a luciferase reporter to visualize the distribution of mesenchymal epithelial transitions in vivo.
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Bonano VI, Oltean S, Garcia-Blanco MA (2007). A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice.
Nat Protoc,
2(9), 2166-2181.
Abstract:
A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice.
Imaging technologies are influencing the way we study regulatory processes in vivo. Several recent reports use fluorescence minigenes to image alternative splicing events in living cells and animals. This type of reporter is being used to generate transgenic mice to visualize splicing regulation in diverse tissues and cell types. In this protocol, we describe how to develop animals that report on alternative splicing and how to assess reporter expression in excised organs and tissue sections. The entire procedure, from making the reporters to imaging organs and tissues in adult transgenic mice, should take approximately 1.5 years. Fluorescence reporters can be used to image many splicing decisions in normal tissues and organs and can be extended to the study of disease states.
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Col B, Oltean S, Banerjee R (2007). Translational regulation of human methionine synthase by upstream open reading frames.
Biochim Biophys Acta,
1769(9-10), 532-540.
Abstract:
Translational regulation of human methionine synthase by upstream open reading frames.
Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5' leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence.
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Oltean S, Sorg BS, Albrecht T, Bonano VI, Brazas RM, Dewhirst MW, Garcia-Blanco MA (2006). Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity.
Proc Natl Acad Sci U S A,
103(38), 14116-14121.
Abstract:
Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity.
In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.
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Bonano VI, Oltean S, Brazas RM, Garcia-Blanco MA (2006). Imaging the alternative silencing of FGFR2 exon IIIb in vivo.
RNA,
12(12), 2073-2079.
Abstract:
Imaging the alternative silencing of FGFR2 exon IIIb in vivo.
Alternative splicing multiplies genomic coding capacity and regulates proteomic composition. A well-studied example of this plasticity leads to the synthesis of functionally distinct isoforms of the Fibroblast Growth Factor Receptor-2 (FGFR2). The regulation of this isoform diversity necessitates the silencing of FGFR2 exon IIIb, which is mediated by flanking intronic splicing silencers and the polypyrimidine tract binding protein (PTB). To visualize this splicing decision in vivo, we developed mice harboring a green fluorescent protein construct that reports on the silencing of exon IIIb. The animals also harbor a red fluorescent protein reporter of constitutive splicing as an allelic control. This dual reporter system revealed that in various organs and cell types the silencing of exon IIIb required the intronic silencers. In neurons, which do not express PTB, we observed robust silencer-dependent repression of exon IIIb, suggesting that the neural paralog, brain PTB, can take over this function. In the epidermis, however, the intronic silencers were not required for efficient silencing. This work provides a first glimpse at splicing regulation among different cell types in vivo and promises the drafting of an anatomic map of splicing decisions.
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Oltean S, Banerjee R (2005). A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase.
J Biol Chem,
280(38), 32662-32668.
Abstract:
A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase.
Regulation of homocysteine, a sulfur-containing amino acid that is a risk factor for cardiovascular diseases, is poorly understood. Methionine synthase (MS) is a key enzyme that clears intracellular homocysteine, and its activity is induced by its cofactor, vitamin B12, at a translational level. In this study, we demonstrate that translation of MS, which has a long and highly structured 5'-untranslated region, is initiated from an internal ribosome entry site (IRES), which is modulated by B12. The minimal IRES element spans 71 bases immediately upstream of the initiation codon. Electrophoretic mobility shift analysis reveals the presence of a B12 -dependent protein-RNA complex and suggests the possibility that B12-dependent increase of IRES efficiency is mediated via a protein. Modulation of the IRES-dependent translation of an essential gene by the cofactor of the encoded enzyme represents a novel example of a gene-nutrient interaction.
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Spanu C, Oltean S (2003). Familial phosphoglycerate kinase deficiency associated with rhabdomyolysis and acute renal failure: abnormality in mRNA splicing?.
Nephrol Dial Transplant,
18(2), 445-446.
Author URL.
Oltean S, Banerjee R (2003). Nutritional modulation of gene expression and homocysteine utilization by vitamin B12.
J Biol Chem,
278(23), 20778-20784.
Abstract:
Nutritional modulation of gene expression and homocysteine utilization by vitamin B12.
Vitamins B12, B6, and folic acid converge at the homocysteine metabolic junction where they support the activities of two key enzymes involved in intracellular homocysteine management, methionine synthase (MS) and cystathionine beta-synthase. The molecular mechanism for the regulation of homocysteine metabolism by B12 supplementation has been investigated in this study. B12 supplementation does not alter mRNA or protein turnover rates but induces translational up-regulation of MS by shifting the mRNA from the ribonucleoprotein to the polysome pool. The B12-responsive element has been localized by deletion analysis using a reporter gene assay to a 70-bp region located at the 3' end of the 5'-untranslated region of the MS mRNA. The cellular consequence of the B12 response is a 2- and 3.5-fold increase in the flux of homocysteine through the MS-dependent transmethylation pathway in HepG2 and 293 cells, respectively. It is speculated that B12-induced up-regulation of MS may have evolved as an adaptive strategy for rapidly sequestering an essential and rare nutrient whose availability may have been limited in the evolutionary history of mammals, a problem that is exacerbated by the absence of this vitamin from the plant kingdom.
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