An investigation into the regulation of transcription of the splice factor kinase gene SRPK1 in prostate and leukaemia cancer cell lines

The SRPK1 gene encodes a protein kinase that phosphorylates a class of splice factors called serine/arginine-rich proteins (SR proteins) modifying their ability to regulate alternative pre-mRNA splicing. Studies have linked high SRPK1 levels to tumour proliferation, growth and metastatic ability but little is known about how its expression is regulated. Different transcription factors including WT1 and ERG are thought to have some level of involvement in the regulation of SRPK1 transcription. The aim of this project was to study the transcriptional regulation of the splice factor kinase gene SRPK1.In this study, the regulation of SRPK1 expression was investigated in cancer cells and the results suggest that both the transcription factor WT1 and the co-repressor BASP1 are present on the SRPK1 promoter region. SRPK1 transcription is activated by WT1, but not when the WT1 binding site is mutated. Consistent with these findings, the knock-down of WT1 with siRNAs also resulted in decreased expression of endogenous SRPK1. The activity of the SRPK1 promoter in BASP1-expressing K562 cells or in PC3 cells transfected with a BASP1-expressing vector was repressed, consistent with BASP1 turning WT1 into a transcriptional repressor rather an activator. The effect of SRPK1 chemical inhibition via SPHINX and the overexpression of BASP1 on the alternative splicing of the vascular endothelial growth factor (VEGF) was also examined in this study; both SRPK1 inhibition and BASP1 expression promoted expression of anti-angiogenic VEGF. The transcription factor ERG, on the other hand, was also shown to bind to the SRPK1 promoter but it repressed its transcription. The overexpression of ERG isoforms reduced endogenous SRPK1 expression, whereas ERG knockdown increased SRPK1 expression. Finally, following manipulation of the expression of SRPK1, WT1, BASP1 and ERG in K562, PC3 and MG63 cell lines, changes in apoptosis, cell proliferation and migration were observed, suggesting that this regulatory axis could present a valid cancer therapy targe

SPHINX-based combination therapy as a potential novel treatment strategy for acute myeloid leukaemia

Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo. As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines; Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5 μg/ml, Kasumi-1 cells) and imatinib (up to 20 μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs

Altered VEGF splicing isoform balance in tumor endothelium involves activation of splicing factors Srpk1 and Srsf1 by the Wilms’ tumor suppressor Wt1

Angiogenesis is one hallmark of cancer. Vascular endothelial growth factor (VEGF) is a known inducer of angiogenesis. Many patients benefit from antiangiogenic therapies, which however have limitations. Although VEGF is overexpressed in most tumors, different VEGF isoforms with distinct angiogenic properties are produced through alternative splicing. In podocytes, the Wilms’ tumor suppressor 1 (WT1) suppresses the Serine/arginine-rich protein-specific splicing factor kinase (SRPK1), and indirectly Serine/arginine-rich splicing factor 1 (Srsf1) activity, and alters VEGF splicing. We analyzed VEGF isoforms, Wt1, Srpk1, and Srsf1 in normal and tumor endothelium. Wt1, Srpk1, Srsf1, and the angiogenic VEGF164a isoform were highly expressed in tumor endothelium compared to normal lung endothelium. Nuclear expression of Srsf1 was detectable in the endothelium of various tumor types, but not in healthy tissues. Inducible conditional vessel-specific knockout of Wt1 reduced Wt1, Srpk1, and Srsf1 expression in endothelial cells and induced a shift towards the antiangiogenic VEGF120 isoform. Wt1(−KTS) directly binds and activates both the promoters of Srpk1 and Srsf1 in endothelial cells. In conclusion, Wt1 activates Srpk1 and Srsf1 and induces expression of angiogenic VEGF isoforms in tumor endothelium

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth

Multi-Solvent Extraction Procedure for the Pioneer Fecal Metabolomic Analysis—Identification of Potential Biomarkers in Stable Kidney Transplant Patients

Metabolic alteration plays a functional role in kidney allograft complications. Metabolomics is a promising high-throughput approach in nephrology but is still limited by the lack of overlap in metabolite coverage. We performed an untargeted fecal metabolomic analysis of forty stable kidney allograft recipients and twenty non-transplant controls. First, we applied the ultra-high performance liquid chromatography (UHPLC) analysis coupled with the Diod Array detector. The potential biomarkers were then collected and identified by gas chromatography-mass spectrometry (GCMS). In order to allow for complete coverage of the fecal polar and non-polar metabolites, the performance of five organic solvents with increasing polarity was investigated successively. UHPLC analysis revealed that the fecal metabolite profiles following the five extractions were significantly different between controls and kidney allografts. GC-MS analysis showed that the best predictors’ metabolites belonged mainly to long-chain fatty acids, phenolic compounds, and amino acids. Collectively, our results showed the efficiency of our pioneer method to successfully discriminate stable kidney-transplant recipients from controls. These findings suggest that distinct metabolic profiles mainly affect fatty acid biosynthesis and amino acid metabolism. In such a context, the novel insights into metabolomic investigation may be a valuable tool that could provide useful new relevant biomarkers for preventing kidney transplant complications