Targeting splicing in prostate cancer

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform specific siRNAs, and splice switching oligonucleotides

Alternative splicing in the hippo pathway-implications for disease and potential therapeutic targets

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Alternative splicing is a well-studied gene regulatory mechanism that produces biological diversity by allowing the production of multiple protein isoforms from a single gene. An involvement of alternative splicing in the key biological signalling Hippo pathway is emerging and offers new therapeutic avenues. This review discusses examples of alternative splicing in the Hippo pathway, how deregulation of these processes may contribute to disease and whether these processes offer new potential therapeutic targets

The Scd6/Lsm14 protein xRAPB has properties different from RAP55 in selecting mRNA for early translation or intracellular distribution in Xenopus oocytes

© 2015 Elsevier B.V. Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. Scd6/Lsm14 proteins, typified by RAP55, function in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules. In Xenopus laevis, the RAP55 orthologue xRAPA fulfils these functions. Here we describe the properties of a variant of xRAPA, xRAPB, which is a member of the Lsm14B group. xRAPB differs from xRAPA in various respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the DDX6 helicase Xp54; it is detected in polysomes and stalled translation initiation complexes; its over-expression leads to selective binding to translatable mRNA species without evidence of translation repression or mRNA degradation. Since both Xp54 and xRAPA are repressors of translation, activation appears to be effected through targeting of xRAPB/Xp54

Oligonucleotide-based systems:DNA, microRNAs, DNA/RNA aptamers

There is an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of DNA, RNA or a synthetic analogue to naturally occurring nucleic acids. This chapter will draw light upon various types of nucleic acids such as DNA, RNA (particularly microRNAs), their role and their application in biosensing. Also, it will cover DNA/RNA aptamers, which can be used as bioreceptors to a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides like PNA or LNA has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the lab-based status and the reality of biomedical applications

Going circular: History, present, and future of circRNAs in cancer

To date, thousands of highly abundant and conserved single-stranded RNA molecules shaped into ring structures (circRNAs) have been identified. CircRNAs are multifunctional molecules that have been shown to regulate gene expression transcriptionally and post-transcriptionally and exhibit distinct tissue- and development-specific expression patterns associated with a variety of normal and disease conditions, including cancer pathogenesis. Over the past years, due to their intrinsic stability and resistance to ribonucleases, particular attention has been drawn to their use as reliable diagnostic and prognostic biomarkers in cancer diagnosis, treatment, and prevention. However, there are some critical caveats to their utility in the clinic. Their circular shape limits their annotation and a complete functional elucidation is lacking. This makes their detection and biomedical application still challenging. Herein, we review the current knowledge of circRNA biogenesis and function, and of their involvement in tumorigenesis and potential utility in cancer-targeted therapy

Analysis of proteins bound to stored messenger RNA in 'Xenopus' oocytes

Regulation at the post-transcriptional level is gaining significance at a rapid pace. One example is the storage of messenger mRNA molecules in a translationally quiescent state, the so-called "masked messengers". Their existence has been known since the 1960s, but many details of their composition and structure have not yet been resolved. Masked messenger RNAs are particularly abundant in the oocytes of the African clawed toad Xenopus laevis. The aim of this study has been to examine the proteins bound to stored mRNAs in the oocytes, by focussing on the Y-box proteins which had already been identified as major components in mRNA masking, and by analyzing some of the other unidentified mRNP proteins. The YB proteins were studied in greater detail, gaining fresh information about their RNA-binding properties, defining distinct binding domains. The presence of an mRNP-associated protein kinase was confirmed, and binding assays suggested that phosphorylation influences the ability of the YB proteins to bind to mRNA. cDNA expression libraries were screened both with an RNA-binding assay and with an immunoscreening method, isolating a variety of known and novel cDNAs. Peptide sequencing of mRNP proteins revealed the presence of an RNA helicase distinct from the translation initiation factor eIF4A. It is postulated that the RNA helicase, in addition to the YB proteins, will be seen to have an important role in the formation and activity of the masked messenger RNA particles

Epigenenetic regulation of alternative splicing: How lncRNAs tailor the message

Alternative splicing is a highly fine-tuned regulated process and one of the main drivers of proteomic diversity across eukaryotes. The vast majority of human multi-exon genes is alternatively spliced in a cell type-and tissue-specific manner, and defects in alternative splicing can dramatically alter RNA and protein functions and lead to disease. The eukaryotic genome is also intensively transcribed into long and short non-coding RNAs which account for up to 90% of the entire tran-scriptome. Over the years, lncRNAs have received considerable attention as important players in the regulation of cellular processes including alternative splicing. In this review, we focus on recent discoveries that show how lncRNAs contribute significantly to the regulation of alternative splicing and explore how they are able to shape the expression of a diverse set of splice isoforms through several mechanisms. With the increasing number of lncRNAs being discovered and characterized, the contribution of lncRNAs to the regulation of alternative splicing is likely to grow significantly

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

The oncogenic transcription factor ERG represses the transcription of the tumour suppressor gene PTEN in prostate cancer cells

© 2017, Spandidos Publications. All rights reserved. The oncogene ETS-related gene (ERG) encodes a transcription factor with roles in the regulation of haematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion. The ERG oncogene is activated in >50% of prostate cancer cases, generally through a gene fusion with the androgen-responsive promoter of transmembrane protease serine 2. Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is often inactivated in cancer. ERG overexpression combined with PTEN inactivation or loss is often associated with aggressive prostate cancer. The present study aimed to determine whether or not ERG regulates PTEN transcription directly. ERG was demonstrated to bind to the PTEN promoter and repress its transcription. ERG overexpression reduced endogenous PTEN expression, whereas ERG knockdown increased PTEN expression. The ability of ERG to repress PTEN may contribute to a more cancer-permissive environment

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