Isoform Switching Research Articles

The Estrogen Receptor α Signaling Pathway Controls Alternative Splicing in the Absence of Ligands in Breast Cancer Cells.

Simple SummaryAberrant alternative splicing is now considered a hallmark of cancer, including breast cancer. This results in the production of novel tumor-specific splice RNA variants, and the activation of biological processes such as epithelial-to-mesenchymal transition, leading to more aggressive phenotypes. The purpose of this study was to explore the role of estrogen receptor α in regulating the expression of RNA-binding proteins in luminal breast cancer cells and to determine the effects of its downregulation at the isoform level by exploring changes in isoform usage and alternative splicing. The findings of this study unravel a novel layer of gene regulation mediated by estrogen receptor α, which is fundamental for breast cancer cell growth as well as epithelial-to-mesenchymal transition. Finally, we foresee that this novel feature should be considered when studying the functional roles of estrogen receptor α in the onset and progression of breast cancer.Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. Methods: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. Results: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3′end processing. ApoERα depletion results in 758 isoform switching events with effects on 3′end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. Conclusion: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level.

Abstract LBA020: Targeting FGFR2c isoform, a novel therapeutic target with FGFR inhibitor in endometrial cancer

Abstract Purpose: Endometrial cancer (EC) is the most frequently diagnosed gynaecological cancer. The majority of women with EC are treated surgically and have a good outcome, however 25-30% of patients presenting with metastases or recurrent disease do not have effective therapies and have <12 months survival. Recent investigations demonstrated radio-chemotherapy has little benefit for women with high-risk EC within the deficient mismatch repair (dMMR) and p53 wild type (p53wt) molecular subtypes. We reported isoform switching from the FGFR2b (epithelial) splice-isoform to the FGFR2c (mesenchymal) splice-isoform in 40% of dMMR and 30% of p53wt ECs. This isoform switching was associated with adverse clinicopathologic markers, shorter recurrence free survival and disease specific survival in the Canadian cohort used to identify and validate the ProMisE molecular risk stratification approach. The objectives of the current study were i) to identify patient derived xenograft (PDX) models with FGFR2c expression and develop PDX derived organoids (PDXOs) for preclinical drug testing, ii) assess the efficacy of the BGJ398 FGFR inhibitor (FGFRi) in FGFR2c expressing models and generate preclinical data that would support an early phase clinical trial in FGFR2c stratified EC patients. Method: BaseScope RNA ISH was used to detect FGFR2c expression in patient tumours, PDX models and PDXOs. PDXOs derived from three independent PDX tumours were established from each of five PDX models (3 showing high FGFR2c expression, 2 showing low/no FGFR2c expression). Each PDXO culture was treated with 300nM BGJ398 (pan-FGFRi) or DMSO for 72 h and assessed using a live-dead assay and confocal microscopy. PDXs from three models were engrafted subcutaneously into 8 weeks female NSG mice and when tumours reached 100-150mm3, mice were randomized (4 mice/arm) and treated with 30mg/Kg BGJ398 or vehicle daily for 21 days. Tumours were measured 3x/week and mice sacrificed when tumours reached 900mm3. Results and conclusion: FGFR2c expression was higher in PDXs representing the dMMR and p53wt subtype compared to p53mut subtype and similar expression levels were seen between patient tumours, PDXs and PDXOs. In vitro FGFRi with BGJ398 showed significant cell death occurred in PDXOs with high FGFR2c expression (p< 0.0001, 2-way ANOVA). These in vitro findings were validated in vivo using PDX68 carrying a FGFR2 C383R mutation and PDX52 and PDX59, both showing FGFR2 isoform switching. Significant tumour growth inhibition and a ~2-fold increase in survival was seen in all three models (PDX68, p<0.0001 and p<0.007; PDX52, p<0.02 and P<0.03; and PDX59 P<0.0001 and P<0.0006 respectively). In conclusion, our investigation revealed FGFRi (BGJ398) was effective in EC PDX models representing both mutational activation and isoform switching of FGFR2. As FGFR isoform switching occurs most commonly in the dMMR subtype where immune checkpoint inhibitors (ICIs) are approved, we propose the combination of ICIs and FGFRi may be more effective in women with FGFR2 activation compared to ICIs alone. Citation Format: Asmerom T Sengal, Vanessa Bonazzi, Olga Kondrashova, Lewis Perrin, Naven Chetty, Deborah Smith, Antonio Gil-Moreno, Eva Colas, Pamela M Pollock. Targeting FGFR2c isoform, a novel therapeutic target with FGFR inhibitor in endometrial cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA020.

Identification of an mRNA isoform switch for HNRNPA1 in breast cancers

Roles of HNRNPA1 are beginning to emerge in cancers; however, mechanisms causing deregulation of HNRNPA1 function remain elusive. Here, we describe an isoform switch between the 3′-UTR isoforms of HNRNPA1 in breast cancers. We show that the dominantly expressed isoform in mammary tissue has a short half-life. In breast cancers, this isoform is downregulated in favor of a stable isoform. The stable isoform is expressed more in breast cancers, and more HNRNPA1 protein is synthesized from this isoform. High HNRNPA1 protein levels correlate with poor survival in patients. In support of this, silencing of HNRNPA1 causes a reversal in neoplastic phenotypes, including proliferation, clonogenic potential, migration, and invasion. In addition, silencing of HNRNPA1 results in the downregulation of microRNAs that map to intragenic regions. Among these miRNAs, miR-21 is known for its transcriptional upregulation in breast and numerous other cancers. Altogether, the cancer-specific isoform switch we describe here for HNRNPA1 emphasizes the need to study gene expression at the isoform level in cancers to identify novel cases of oncogene activation.

Abstract P170: SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.

Accurate expression quantification from nanopore direct RNA sequencing with NanoCount.

Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. However, there are a lack of tools specifically designed for DRS and its ability to identify differential expression in complex organisms is poorly characterised. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. Differential expression of 231 genes, 333 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. NanoCount quantification of thousands of novel isoforms discovered with DRS likewise enabled identification of their differential expression. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms.

Probing Isoform Switching Events in Various Cancer Types: Lessons From Pan-Cancer Studies.

Alternative splicing is an essential regulatory mechanism for gene expression in mammalian cells contributing to protein, cellular, and species diversity. In cancer, alternative splicing is frequently disturbed, leading to changes in the expression of alternatively spliced protein isoforms. Advances in sequencing technologies and analysis methods led to new insights into the extent and functional impact of disturbed alternative splicing events. In this review, we give a brief overview of the molecular mechanisms driving alternative splicing, highlight the function of alternative splicing in healthy tissues and describe how alternative splicing is disrupted in cancer. We summarize current available computational tools for analyzing differential transcript usage, isoform switching events, and the pathogenic impact of cancer-specific splicing events. Finally, the strategies of three recent pan-cancer studies on isoform switching events are compared. Their methodological similarities and discrepancies are highlighted and lessons learned from the comparison are listed. We hope that our assessment will lead to new and more robust methods for cancer-specific transcript detection and help to produce more accurate functional impact predictions of isoform switching events.

RBPMS, an RNA-Binding Protein That Mediates Cardiomyocyte Binucleation and Cardiovascular Development

SummaryNoncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that genetic deletion of R NA b inding p rotein with m ultiple s plicing ( Rbpms ), an uncharacterized RNA binding and splicing factor, causes perinatal lethality in mice due to congenital cardiovascular defects. Loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates isoform switching of the heart-specific LIM domain protein Pdlim5 . Loss of Rbpms leads to abnormal accumulation of Pdlim5 -short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of Rbpms in this process.

Abstract 14268: PKM2 to PKM1 Isoform Switching in Fibroblasts Attenuates Cardiac Dysfunction in Infarcted Mice

Introduction: The muscle isoform of pyruvate kinase, PKM, has two splice variants, with PKM2 having important roles in cardiomyocytes during development. The goal of this study was to understand the role of PKM2 in the adult failing heart. Methods: To determine how PKM2 expression changes after myocardial infarction (MI), wild-type (WT) mice were subjected to permanent coronary ligation followed by measurement of PKM2 abundance and localization by Western blotting and immunofluorescence imaging. To delineate the role of PKM2 in fibroblasts after MI, PKM2 fl/fl mice were bred with a fibroblast-specific, tamoxifen-inducible Cre driver (Col1a2-CreER; Cre). The Cre + and Cre - PKM2 fl/fl mice of both sexes were treated with tamoxifen (i.p. 20 mg/kg/d; 5 d), followed by tamoxifen washout and MI surgery. Four weeks after MI, cardiac function was examined by echocardiography. Results: Hearts from infarcted WT mice had 10-fold higher levels of PKM2 compared with sham WT hearts (n=8/gp, p<0.05). Immunofluorescence staining revealed PKM2 localized to areas of replacement fibrosis in infarcted hearts, and immunoblotting of isolated, adult cell populations showed PKM2 expressed in fibroblasts, smooth muscle cells, and endothelial cells, but not in cardiomyocytes. Furthermore, PKM2 levels were higher in cardiac fibroblasts isolated from infarcted mice compared with sham mice (n=3/group; p=0.005). Cardiac fibroblasts isolated from tamoxifen-treated Cre + /PKM2 fl/fl mice had decreased PKM2 expression and increased PKM1 expression. In naïve mice, PKM2 to PKM1 isoform switching did not affect cardiac function for up to 5 wk after tamoxifen administration. Although survival was not different between Cre - and Cre + PKM2 fl/fl mice after MI (n=18-20/gp; p=0.6383), PKM2 to PKM1 switching in Cre + PKM2 fl/fl male mice improved cardiac function compared with Cre - PKM2 fl/fl controls (n=7-12/gp), with a 15% improvement in ejection fraction (p=0.03), a decrease in LVIDd (p=0.04), EDV (p=0.04), and ESV (p=0.03), and an increase in LVPWd (p=0.01) and LVPWs (p=0.01); however, in female mice, PKM isoform switching did not improve cardiac function. Conclusions: Fibroblast-specific switching of PKM2 to PKM1 prior to MI prevents cardiac functional decline in male mice.

ADAR1 Splicing Modulation As a Mechanism to Eradicate Immunologically Silent Leukemia Stem Cells

Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. DisclosuresCrews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Cigarette smoking-associated isoform switching and 3′ UTR lengthening via alternative polyadenylation

Cigarette smoking induces a profound transcriptomic and systemic inflammatory response. Previous studies have focused on gene level differential expression of smoking, but the genome-wide effects of smoking on alternative isoform regulation have not yet been described. We conducted RNA sequencing in whole-blood samples of 454 current and 767 former smokers in the COPDGene Study, and we analyzed the effects of smoking on differential usage of isoforms and exons. At 10% FDR, we detected 3167 differentially expressed genes, 945 differentially used isoforms and 160 differentially used exons. Isoform switch analysis revealed widespread 3′ UTR lengthening associated with cigarette smoking. The lengthening of these 3′ UTRs was consistent with alternative usage of distal polyadenylation sites, and these extended 3′ UTR regions were significantly enriched with functional sequence elements including microRNA and RNA-protein binding sites. These findings warrant further studies on alternative polyadenylation events as potential biomarkers and novel therapeutic targets for smoking-related diseases.

Genomic and Transcriptomic Differences at the Pyruvate Kinase M (PKM) Locus in Patient-Derived Glioblastoma Multiforme (GBM) Cell Lines

Glioblastoma multiforme (GBM), a particularly devastating primary CNS tumor, alters the expression and activity of multiple metabolic genes to enhance its ability to grow and divide, buffer against oxidative stress, and resist apoptosis. The shift from oxidative metabolism in the mitochondria to aerobic glycolysis is known as the Warburg effect, a phenomenon mediated by gene expression changes like the isoform switch at the pyruvate kinase muscle-type (PKM) locus from PKM1 to PKM2. Novel pharmaceuticals targeting this tumor-specific isoform have entered clinical trials, but the effect of genetic or transcriptomic background on the effectiveness of these drugs is unknown. The purpose of this work is to characterize the PKM locus at the level of DNA and mRNA.Our lab has previously collected and curated patient-derived GBM cell lines and characterized them with high-throughput transcriptome sequencing (RNA-seq). Herein, RNA-seq reads were mapped to the genome. Limiting analysis to the PKM locus, variant calling including single nucleotide polymorphism (SNP) calling is performed, and absolute expression and relative isoform abundance is measured and compared across lines.In this analysis of 16 GBM cell lines, multiple SNPs are identified that are predicted to be deleterious to the function of the protein. Additionally, concordant with previous findings in astrocytomas, the PKM2 isoform is the dominant pyruvate kinase transcript in all cell lines profiled. One cell line, in particular, had substantially higher PKM expression compared to the other cell lines.Previous studies have queried the mutational burden of PKM in other cancer types, but no mutational analysis has been performed on GBM. This work suggests genetic variation at this important locus. Targeting tumor-specific metabolism can increase the effectiveness of current standard-of-care therapies while leaving normal tissue intact. This characterization of the PKM locus at the level of DNA variants and gene expression will inform planned future work using a novel pharmaceutical targeting PKM2.K.J. Lee: None. C. Willey: None.

S-glutathionylation, a redox switch disrupting angiogenic balance to promote the preeclampsia phenotype

Abstract Background Elevation of circulating anti-angiogenic factors is pivotal in the development of the preeclampsia (PE) phenotype of incomplete vascular remodelling, hypertension and kidney dysfunction during pregnancy. Oxidative stress is explicitly linked to PE with high levels measurable in the placenta. Yet antioxidant therapy has failed, in some cases worsening pregnancy outcomes. The modulation of protein activity by reversible oxidative post-translational modifications (oxPTM) under low levels of reactive oxygen species is emerging as an important “redox-switch” mechanism in cardiovascular diseases, although oxPTM have not been investigated in the context of PE. Of significance, S-glutathionylation is a common oxPTM which reversal by glutaredoxin (Grx) is predominant in preeclamptic placenta and was associated with attenuated revascularisation and sFlt-1 elevation in mice. Purpose We aimed to identify the molecular basis for how S-glutathionylation reversal by Grx may contribute to pregnancy-induced vascular complications by modulating angiogenic signalling at the maternofoetal interface. Methods We combined physiological in vivo assessment with bioinformatics proteomic analysis and exon-level microarray to investigate the role of S-glutathionylation in the development of PE phenotype. In vitro studies using primary endothelial cells (EC) and iPS-derived trophoblasts investigated the effects of oxPTM reversal on angiogenic signalling in individual placental cell types and the functional consequences were assessed in 3D models replicating early-pregnancy events. Results Overexpressing Grx transgenic mice (TG) developed gestational hypertension, kidney dysfunction and elevated plasma levels of the anti-angiogenic factor sFlt-1 compared to their littermate controls (WT) during timed pregnancy. Grx-mediated oxPTM reversal in EC disrupted angiogenic sprouting and promoted anti-angiogenic signals by increasing sFlt-1:PlGF ratio and decreasing endoglin levels. The rise in sFlt-1 was associated with an isoform switch promoting sFlt-e15a over sFlt-i13. In trophoblasts, Grx overexpression inhibited migration and syncytialisation and modulated angiogenic balance in a cell type-specific manner. The sFlt1-e15a:PlGF ratio was increased in syncytiotrophoblasts and decreased in extra-villous trophoblasts, while endoglin expression was decreased in both cell types. A genome-wide exon-level profiling of TG vs WT mice placenta revealed a global alteration of alternative splicing events. Conclusion Grx-mediated removal of oxPTM directly disrupts placental angiogenic balance via dysregulation of sFlt-1 isoforms, which may promote the PE phenotype of impaired vascular remodelling, hypertension and kidney dysfunction during pregnancy. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020 - Marie Skłodowska-Curie grant agreement (iPLACENTA)

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

PD37-11 TROPOMYOSIN ISOFORM SWITCHING IN UROTHELIAL BLADDER CANCER AS A DIAGNOSTIC BIOMARKER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-11 TROPOMYOSIN ISOFORM SWITCHING IN UROTHELIAL BLADDER CANCER AS A DIAGNOSTIC BIOMARKER Nada Humayun-Zakaria, Douglas Ward, Ben Abbotts, Maurice Zeegers, KK Cheng, Nick James, Richard Bryan, and Roland Arnold Nada Humayun-ZakariaNada Humayun-Zakaria More articles by this author , Douglas WardDouglas Ward More articles by this author , Ben AbbottsBen Abbotts More articles by this author , Maurice ZeegersMaurice Zeegers More articles by this author , KK ChengKK Cheng More articles by this author , Nick JamesNick James More articles by this author , Richard BryanRichard Bryan More articles by this author , and Roland ArnoldRoland Arnold More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The urgent unmet need for biomarker discovery for the early detection and surveillance of bladder cancer in parallel with advancements in high throughput sequencing has fuelled understanding of molecular biology and pathology, paving the way for ‘precision medicine’. Transcriptomic analyses of Tropomyosin (TPM) have revealed differential expression of isoforms with ‘isoform switching’ in the presence of urothelial bladder cancer (UBC) making them promising candidates as diagnostic biomarkers. METHODS: RNA-sequence quantification was performed on 3 cancer datasets (Cancer Cell Line Encyclopaedia (CCLE), The Cancer Genome Atlas (TCGA) and West Midland’s Bladder Cancer Prognosis Program (BCPP)) and one non-cancer dataset (GEO Accession number GSE133624) using a bespoke bioinformatics pipeline – ‘IsoTPM’. Wilcoxon rank sum determined p-values for pairwise-comparison of isoform expression in cancer vs non-cancer. Subsequent qPCR analysis with isoform-specific primers was performed on in-house CCLE cell lines followed by Sanger sequencing. Evaluation of TPM isoform expression at protein level was achieved using mass spectrometry (MS) on CCLE and BCPP samples to delineate peptides of interest. RESULTS: The differential expression (DE) of two TPM1 isoforms, TPM1-201 and TPM1-206, appeared as dominant transcripts in non-cancer and cancer datasets, respectively. TPM4 analyses revealed high abundance of TPM4-218 in cancer datasets in comparison to TPM4-221 found exclusively in non-cancer. Additionally, TPM4-205 was expressed in non-muscle-invasive tumours (BCPP). qPCR analysis showed the relative expressions of TPM1-206 and TPM4-218 in a range of cell lines. MS revealed ∼20 peptides, including splice-junction exons from TPM4-218 and TPM1-206, from both cancer datasets (BCPP and CCLE). CONCLUSIONS: Comparative analysis of non-cancer and cancer datasets demonstrates that TPM1-201 isoform switching to TPM1-206 accompanies early bladder tumorigenesis and/or epithelial-mesenchymal-transition (EMT). TPM4-218 is upregulated in all cancer datasets accompanied by downregulation of TPM4-221 also suggesting an association with bladder tumorigenesis. Source of Funding: QEHB charities funded NH-Z's research © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e659-e660 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nada Humayun-Zakaria More articles by this author Douglas Ward More articles by this author Ben Abbotts More articles by this author Maurice Zeegers More articles by this author KK Cheng More articles by this author Nick James More articles by this author Richard Bryan More articles by this author Roland Arnold More articles by this author Expand All Advertisement Loading ...

MP33-17 A GLUTAMINASE ISOFORM SWITCH DRIVES THERAPEUTIC RESISTANCE AND DISEASE PROGRESSION OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-17 A GLUTAMINASE ISOFORM SWITCH DRIVES THERAPEUTIC RESISTANCE AND DISEASE PROGRESSION OF PROSTATE CANCER Lingfan Xu Lingfan XuLingfan Xu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer is one of the most common cancer type in men. Although hormonal therapy, which targets the androgen receptor (AR) signaling, is efficacious initially, tumor recurrence will eventually recur. Better understanding the mechanism of therapy resistance and disease progression and therefore exploiting new therapeutic target is a pressing unmet issue. METHODS: We employed mass spectrometry and isotopomer tracing technology to determine metabolic flux and reprogramming in different stage of prostate cancer (primary, castration-resistant and neuroendocrine- transdifferentiated). Immunohistochemical staining on human tissue microarrays was used to determine the expression of the two isoforms of glutaminase (GLS1) across a panel of tissues diagnosed with different histology results. Genetic (e.g short hairpin RNA, crispr/cas9) and pharmacological (GLS1 inhibitor, CB-839) inhibition were employed to assess the biological function of GLS1 as well as its two splicing variants in prostate cancer. Animal models were established to longitudinally simulate the nature history of disease progression and drug therapeutic outcomes were also evaluated in vivo. RESULTS: We observe that hormonal therapy-resistant prostate cancer (including castration-resistant prostate cancer, CRPC and neuroendocrine prostate cancer, NEPC) is extremely addicted to glutamine instead of androgen, which results in the failure of AR-directed therapies. Furthermore, GLS1, the key enzyme for the glutamine metabolism, has two functionally different splicing variants (KGA and GAC), which are differentially expressed primary and recurrent stage of prostate cancer. KGA is the dominant isoform in hormone-sensitive prostate cancer whereas GAC, the much more enzymatically potent isoform, predominates in CRPC and NEPC. Furthermore, this isoform switch of GLS1 is evidently observed longitudinally in animal models along with tumor recurrence after being castrated. GLS1 selective inhibitor, CB-839, shows great inhibitory effect preferentially on GAC-dominant prostate cancer variants. CONCLUSIONS: An isoform switch of GLS1, from KGA to GAC, drives prostate cancer to be resistant to hormonal therapy, but dependent on glutamine due to the hyper capability of GAC for glutamine utilization. Therefore, GAC would be an ideal target independently of AR for those androgen/AR-indifferent prostate cancer patients. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e613-e613 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lingfan Xu More articles by this author Expand All Advertisement Loading ...

The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes.

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.

Neural Precursor Cells Expanded Inside the 3D Micro-Scaffold Nichoid Present Different Non-Coding RNAs Profiles and Transcript Isoforms Expression: Possible Epigenetic Modulation by 3D Growth.

Non-coding RNAs show relevant implications in various biological and pathological processes. Thus, understanding the biological implications of these molecules in stem cell biology still represents a major challenge. The aim of this work is to study the transcriptional dysregulation of 357 non-coding genes, found through RNA-Seq approach, in murine neural precursor cells expanded inside the 3D micro-scaffold Nichoid versus standard culture conditions. Through weighted co-expression network analysis and functional enrichment, we highlight the role of non-coding RNAs in altering the expression of coding genes involved in mechanotransduction, stemness, and neural differentiation. Moreover, as non-coding RNAs are poorly conserved between species, we focus on those with human homologue sequences, performing further computational characterization. Lastly, we looked for isoform switching as possible mechanism in altering coding and non-coding gene expression. Our results provide a comprehensive dissection of the 3D scaffold Nichoid’s influence on the biological and genetic response of neural precursor cells. These findings shed light on the possible role of non-coding RNAs in 3D cell growth, indicating that also non-coding RNAs are implicated in cellular response to mechanical stimuli.

ERR\u03b3 enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

IKK\u03b5 isoform switching governs the immune response against EV71 infection

The reciprocal interactions between pathogens and hosts are complicated and profound. A comprehensive understanding of these interactions is essential for developing effective therapies against infectious diseases. Interferon responses induced upon virus infection are critical for establishing host antiviral innate immunity. Here, we provide a molecular mechanism wherein isoform switching of the host IKKε gene, an interferon-associated molecule, leads to alterations in IFN production during EV71 infection. We found that IKKε isoform 2 (IKKε v2) is upregulated while IKKε v1 is downregulated in EV71 infection. IKKε v2 interacts with IRF7 and promotes IRF7 activation through phosphorylation and translocation of IRF7 in the presence of ubiquitin, by which the expression of IFNβ and ISGs is elicited and virus propagation is attenuated. We also identified that IKKε v2 is activated via K63-linked ubiquitination. Our results suggest that host cells induce IKKε isoform switching and result in IFN production against EV71 infection. This finding highlights a gene regulatory mechanism in pathogen-host interactions and provides a potential strategy for establishing host first-line defense against pathogens.