Deregulation Of Alternative Splicing Research Articles

Alternative splicing (AS) regulation as a novel therapeutic target in soft-tissue sarcoma (STS).

11546 Background: There is an urgent clinical need to identify novel treatments for advanced STS, since all the clinical trials testing chemotherapy combinations have failed to improve survival compared to doxorubicin (dox) alone. AS deregulation has been associated with cancer initiation, progression and resistance to therapy. This deregulation can occur due to mutations or imbalanced expression or activity of splicing factors (SF). The alteration SF can alter thousands of pre-mRNAs, increasing cellular complexity and facilitating tumour progression. CDC-like kinases (CLK) that phosphorylate serine-arginine-rich SF (SRSFs), are key regulators of AS. SM09419 is an oral pan CLK/DYRK inhibitor that has been shown to inhibit SRSF phosphorylation, selectively modify spliceosome activity and decrease tumour growth by multiple mechanisms including the downregulation of Wnt/β-catenin signalling, a pathway that we reported to be important in sarcoma. Methods: A panel of 9 STS cell lines, including LMS (CP0024, IEC005, AA, SK-UT-1), LPS (93T449), synovial sarcoma (MCP037) and UPS (MCP016, MCP021, MCP025) were used to analyse the effect of SM09419 (provided by Biosplice) or PRI-724 (Wnt/β-catenin inhibitor) on cell viability (MTS assays). TOP flash β-catenin/TCF-responsive reporter assay was performed to evaluate the inhibition of the Wnt/β-catenin pathway in response to SM09419. In vivo experiments tested the combination of SM09419 (25mg/kg 5 days on and 2 off, for 21 days cycle) plus IP dox (5mg/kg once a week for 21 days cycle) in LMS (IEC005) and synovial sarcoma (FJD-SS-001) PDX models. Results: Our results showed that SM09419 had superior activity in STS cells compared to specific Wnt/β-catenin inhibitors with IC50 values starting at 29.8 nM in SK-UT-1 cell line, whereas PRI-724 IC50 values were clearly higher, starting from 5.18 µM in 93T449 cells. SM09419 treatment significantly inhibited the transcriptional activity of β-catenin by 40% in the MCP021 cells, demonstrating the regulatory activity of AS inhibitors on this pathway. I n vivo studies confirmed the higher efficacy of SM09419 in monotherapy compared to dox, in inhibiting tumour growth (p < 0.05), which was potentiated when combined with dox. The combination was increasingly active in synovial sarcoma with tumour regressions in 3 out of 4 mice, including one complete response. Treatment schedule was adapted to a single IP administration of dox (5mg/kg) and 5 administrations of SM09419 (25mg/kg), to improve the tolerability of the combination. Conclusions: The effects of pan CLK/DYRK inhibition on growth and survival of STS cancer cell lines and PDX models, indicate that can STS may be therapeutically addressed with these AS-modulators. The combination of SM09419 with dox was active in STS preclinical models known for their resilience toward treatment. Based on these results, the exploration of such combinations in clinical trials is warranted.

Integrative analysis of synovial sarcoma transcriptome reveals different types of transcriptomic changes.

Background: Synovial sarcoma (SS) is a rare and aggressive cancer that can come from distinct soft tissue types including muscle and ligaments. However, the transcriptomic landscape of SS is still poorly understood. This study aimed to systematically dissect the changes in SS transcriptome from different perspectives. Methods: We performed deep total RNA sequencing on ten paired Synovial sarcoma and tumor-adjacent tissues to systematically dissect the transcriptomic profile of SS in terms of gene expression, alternative splicing, gene fusion, and circular RNAs. Results: A total of 2,309 upregulated and 1,977 downregulated genes were identified between SS and tumor-adjacent tissues. Those upregulated genes could lead to the upregulation of the cell cycle, ribosome, and DNA replication pathways, while the downregulated genes may result in the downregulation of a set of metabolic biological processes and signaling pathways. Moreover, 2,511 genes (including 21 splicing factors) were differentially alternative spliced, indicating that the deregulation of alternative splicing could be one important factor that contributes to tumorigenesis. Additionally, we identified the known gene fusions of SS18-SSX1/SSX2 as well as 11 potentially novel gene fusions. Interestingly, 49 circular RNAs were differentially expressed and their parental genes could function in muscle contraction and muscle system processes. Conclusions: Collectively, our comprehensive dissection of the transcriptomic changes of SS from both transcriptional and post-transcriptional levels provides novel insights into the biology and underlying molecular mechanism of SS.

Mechanisms of action of intranuclear microRNAs. Part 2. MicroRNA-mediated regulation of ncRNA transcriptome and alternative splicing

The scientific review presents the mechanisms of action of intranuclear miRNAs, namely microRNA-mediated regulation of the non-coding RNA (ncRNA) transcriptome and alternative splicing. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka databases. It is emphasized that a significant effect on the composition of the transcriptome is provided by microRNAs in the RNA-induced silencing complex that induce posttranscriptional degradation of long ncRNAs localized in the cell nucleus. Scientists believe that long ncRNAs are involved in the epigenetic regulation of gene silencing through chromatin remodeling. It is shown that long ncRNAs are actively involved in the development of some liver diseases. It is reported that the most important mechanism for expanding the spectrum of the transcriptome in the cell is the alternative splicing of pre-mRNA. The authors present the results of scientific studies that show that pre-mRNA of more than 90 % of human genes are subjected to alternative splicing. It is presented that splicing is performed by a specialized macromolecular formation — suprasplicesome, which is a megacomplex (21 MDA) of nuclear ribonucleroprotein. It is shown that scientists have proposed two models of epigenetic regulation of splicing: kinetic and recruitment one. The authors reveal the main provisions of these models. MicroRNAs are actively involved in splicing. Liver disease may be based on a deficiency of splicing factors and deregulation of alternative splicing caused by the action of miRNAs. Disorders of alternative splicing, which stimulate proliferation, prevent apoptosis and support cell transformation, are a pathognomonic phenomenon in malignant tumors. Thus, the constituent mechanisms of action of intranuclear miRNAs are alteration of the ncRNAs transcriptome and participation in the regulation of alternative splicing. MicroRNA-mediated regulation of the stability of long ncRNAs causes a change in the spectrum of activity of expression of epigenetically regulated genes. Long ncRNAs are actively involved in the development of some liver diseases. Alternative splicing is an integral part of cell differentiation and contributes to the formation of tissue specificity. Alternative splicing and generation of various isoforms of proteins determine molecular consequences that cause the development of various pathological conditions.

The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma

Deregulation of alternative splicing is implicated as a relevant source of molecular heterogeneity in cancer. However, the targets and intrinsic mechanisms of splicing in hepatocarcinogenesis are largely unknown. Here, we report a functional impact of a Splicing Regulatory Glutamine/Lysine-Rich Protein 1 (SREK1) variant and its regulator, Serine/arginine-rich splicing factor 10 (SRSF10). HCC patients with poor prognosis express higher levels of exon 10-inclusive SREK1 (SREK1L). SREK1L can sustain BLOC1S5-TXNDC5 (B-T) expression, a targeted gene of nonsense-mediated mRNA decay through inhibiting exon-exon junction complex binding with B-T to exert its oncogenic role. B-T plays its competing endogenous RNA role by inhibiting miR-30c-5p and miR-30e-5p, and further promoting the expression of downstream oncogenic targets SRSF10 and TXNDC5. Interestingly, SRSF10 can act as a splicing regulator for SREK1L to promote hepatocarcinogenesis via the formation of a SRSF10-associated complex. In summary, we demonstrate a SRSF10/SREK1L/B-T signalling loop to accelerate the hepatocarcinogenesis.

The HNRNPA2B1–MST1R–Akt axis contributes to epithelial-to-mesenchymal transition in head and neck cancer

AbstractThe deregulation of splicing factors and alternative splicing are increasingly viewed as major contributory factors in tumorigenesis. In this study, we report overexpression of a key splicing factor, heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1), and thereby misregulation of alternative splicing, which is associated with the poor prognosis of head and neck cancer (HNC). The role of HNRNPA2B1 in HNC tumorigenesis via deregulation of alternative splicing is not well understood. Here, we found that the CRISPR/Cas9-mediated knockout of HNRNPA2B1 results in inhibition of HNC cells growth via the misregulation of alternative splicing of MST1R, WWOX, and CFLAR. We investigated the mechanism of HNRNPA2B1-mediated HNC cells growth and found that HNRNPA2B1 plays an important role in the alternative splicing of a proto-oncogene, macrophage stimulating 1 receptor (MST1R), which encodes for the recepteur d'origine nantais (RON), a receptor tyrosine kinase. Our results indicate that HNRNPA2B1 mediates the exclusion of cassette exon 11 from MST1R, resulting in the generation of RON∆165 isoform, which was found to be associated with the activation of Akt/PKB signaling in HNC cells. Using the MST1R-minigene model, we validated the role of HNRNPA2B1 in the generation of RON∆165 isoform. The depletion of HNRNPA2B1 results in the inclusion of exon 11, thereby reduction of RON∆165 isoform. The decrease of RON∆165 isoform causes inhibition of Akt/PKB signaling, which results in the upregulation of E-cadherin and downregulation of vimentin leading to the reduced epithelial-to-mesenchymal transition. The overexpression of HNRNPA2B1 in HNRNPA2B1 knockout cells rescues the expression of the RON∆165 isoform and leads to activation of Akt/PKB signaling and induces epithelial-to-mesenchymal transition in HNC cells. In summary, our study identifies HNRNPA2B1 as a putative oncogene in HNC that promotes Akt/PKB signaling via upregulation of RON∆165 isoform and promotes epithelial to mesenchymal transition in head and neck cancer cells.HNRNPA2B1 regulates the splicing of MST1R and promotes the expression of a cancer-specific isoform, RON∆165, in head and neck cancer cells. RON∆165 activates the Akt/PKB pathway and leads to increased expression of epithelial to mesenchymal transition regulators such as TWIST2, E-cadherin, vimentin, and ZEB1 and promotes the invasive behavior of head and neck cancer.

Hypoxia-induced TGF-β-RBFOX2-ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer.

Hypoxic microenvironment heralds epithelial–mesenchymal transition (EMT), invasion and metastasis in solid tumors. Deregulation of alternative splicing (AS) of several cancer-associated genes has been instrumental in hypoxia-induced EMT. Our study in breast cancer unveils a previously unreported mechanism underlying hypoxia-mediated AS of hMENA, a crucial cytoskeleton remodeler during EMT. We report that the hypoxia-driven depletion of splicing regulator ESRP1 leads to skipping of hMENA exon 11a producing a pro-metastatic isoform, hMENAΔ11a. The transcriptional repression of ESRP1 is mediated by SLUG, which gets stimulated via hypoxia-driven TGF-β signaling. Interestingly, RBFOX2, an otherwise RNA-binding protein, is also found to transcriptionally repress ESRP1 while interacting with SLUG. Similar to SLUG, RBFOX2 gets upregulated under hypoxia via TGF-β signaling. Notably, we found that the exosomal delivery of TGF-β contributes to the elevation of TGF-β signaling under hypoxia. Moreover, our results show that in addition to hMENA, hypoxia-induced TGF-β signaling contributes to global changes in AS of genes associated with EMT. Overall, our findings reveal a new paradigm of hypoxia-driven AS regulation of hMENA and insinuate important implications in therapeutics targeting EMT.

TRA2A Promoted Paclitaxel Resistance and Tumor Progression in Triple-Negative Breast Cancers via Regulating Alternative Splicing.

Treatment of triple-negative breast cancer (TNBC) has been challenging, and paclitaxel resistance is one of the major obstacles to the better prognosis. Deregulation of alternative splicing (AS) may contribute to tumor progression and chemotherapy resistance. Human AS factor TRA2 has two separate gene paralogs encoding TRA2A and TRA2B proteins. TRA2B is associated with cancer cell survival and therapeutic sensitivity. However, the individual role of TRA2A in cancer progression has not been reported. Here we report that TRA2A facilitates proliferation and survival and migration and invasion of TNBC cells. In addition, TRA2A promotes paclitaxel resistance of TNBC by specifically controlling cancer-related splicing, which is independent of other splicing factors. TRA2A overexpression could promote AS of CALU, RSRC2, and PALM during paclitaxel treatment of TNBC cells. The isoform shift of RSRC2 from RSRC2s to RSRC2l leads to a decreased RSRC2 protein expression, which could contribute to TNBC paclitaxel resistance. TRA2A can regulate RSRC2 AS by specifically binding upstream intronic sequence of exon4. Strikingly, TRA2A expression is increased dramatically in patients with TNBC, and has a close relationship with decreased RSRC2 expression; both are associated with poor survival of TNBC. Collectively, our findings suggest that paclitaxel targets the TRA2A-RSRC2 splicing pathway, and deregulated TRA2A and RSRC2 expression may confer paclitaxel resistance. In addition to providing a novel molecular mechanism of cancer-related splicing dysregulation, our study demonstrates that expression of TRA2A in conjunction with RSRC2 may provide valuable molecular biomarker evidence for TNBC clinical treatment decisions and patient outcome. Mol Cancer Ther; 16(7); 1377-88. ©2017 AACR.

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.

Abstract 2124: Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer

Abstract Lung cancer is the most common cause of death by cancer worldwide. Deregulation of alternative splicing (AS) is becoming increasingly relevant for the understanding of the molecular mechanisms of lung tumor progression. Genome-wide microarrays containing probes in exons and junctions can be used to identify differential AS events. We developed an algorithm, called ExonPointer, which was optimized to detect AS events using this kind of platforms. Using this technique, we successfully detected splicing events regulated by the oncogene serine/arginine-rich splicing factor 1 (SRSF1) in lung cancer cell lines. Moreover, four of these genes, ATP11C, IQCB1, TUBD1 and PRRC2C, showed a significantly different pattern of AS in primary non-small cell lung cancer (NSCLC) compared with normal lung tissue. In the case of PRRC2C, we also demonstrated that the deregulation of the splicing event was important for tumor progression. We next applied the same technique to target differential AS events in NSCLC primary tissues (13 lung squamous cell carcinomas and 9 lung adenocarcinomas). Their paired normal lung tissues were used as reference. The validation rate for the top 20 events identified by ExonPointer was 70%. Gene cluster analyses showed an enrichment of clusters implicated in cancer development such as Cellular Growth and Proliferation, or Cell Death and Survival. Among others, we observed accumulation of a tumor-specific splice variant in Mitogen-Activated Protein Kinase 9 (MAPK9 or JNK2). The tumor-specific variant for this gene contains an alternative exon 2 which causes a frame shift introducing a premature stop codon. Functional analyses using isoform-specific siRNAs showed that this variant did not affect tumor growth or progression. However, its expression significantly correlated with the expression of Regulator of Nonsense Transcripts 3A and B (UPF3A and UPF3B) in 42 lung cancer cell lines. These proteins belong to the Nonsense Mediated Decay (NMD) pathway, which eliminates aberrant mRNA transcripts, suggesting that the presence of some aberrant AS variants in lung cancer are caused by a malfunction of NMD. In conclusion, we have developed a reliable tool for the high throughput analysis of AS, and have identified new splicing events in lung cancer that can be studied as potential diagnostic markers and therapeutic targets. Citation Format: Fernando J. de Miguel, Ravi D. Sharma, Pablo Reclusa, María J. Pajares, Angel Rubio, Ruben Pio, Luis M. Montuenga. Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2124. doi:10.1158/1538-7445.AM2015-2124

Androgen Receptor Alternative Splicing and Human Infertility

About 95% of multi-exonic genes express more than one mRNA and downstream proteins by alternative splicing (AS) through the inclusion or exclusion of specific exons. Although AS provides a significant advantage in human evolution by increasing proteomic diversity, deregulation of AS can result in various pathologic conditions. The androgen receptor (AR), encoded by AR gene, is a steroid receptor transcription factor which mediates the cellular functions of androgen. The AR-mediated androgen actions play important and dual roles in the human reproduction development and function. Dysregulation of AR will result in human infertility. Multiple AR alternative splicing variants have been identified in different pathologies conditions, including androgen insensitivity syndrome, which will cause male infertility. More recently, our group has identified two AR splice variants expressed in granulosa cells from patients with polycystic ovary syndrome, which is one of the most common causes of female infertility. All of the aforementioned indicate that androgen receptor alternative splicing may be an important pathogenic mechanism in human infertility. The purpose of this review is to summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologies of the human infertility diseases, including polycystic ovary syndrome and androgen insensitivity syndrome.

Abstract 1623: The spliceosome as a new therapeutic target for anticancer treatment

Abstract Background: Bioinformatics analyses of pathways that are differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. In the present study, we identified spliceosome assembly components as candidate target for cancer treatment. The spliceosome, an intracellular ribonucleoprotein complex, is involved in eukaryotic pre-mRNA processing by splicing out intronic nucleic acids. Experimental procedures: Pathway analyses were done on exon and gene expression arrays datasets. Differential gene expression between malignant and benign lesions was applied to four public datasets. Small nuclear ribonucleoprotein E (SNRPE) downregulation was achieved by short interfering RNA technology in SKBr-3 and MDA-MB-231 human breast cancer cells. Functional effects of siRNA-induced knockdown of SNRPE expression on cell viability, cell cycle distribution and apoptosis were respectively determined using WST-1 assay, FACS analysis and annexin-V/7-AAD double-staining assay. Autophagy was investigated by electron microscopy (EM). We also evaluated alternative RNA splicing at the exon level using SpliceArray microarray. Results: Pathway analyses with BioCarta database identified spliceosome assembly component (SAC) as the most differentially expressed pathway between breast malignant and benign lesions (permutation p=0.002). The differential expression of SAC was confirmed in four different public dataset on breast, ovarian, lung and skin cancers. Based on these data we investigated whether SNRPE, a component of spliceosome, could be a candidate target. Knockdown of SNRPE protein expression resulted in a high inhibition of SKBr-3 (70%) and MDA-MB-231 (48%) cell proliferation as compared to non-targeting siRNA-transfected cells. Splice array analyses suggested a deregulation of alternative splicing, together with a dramatic reduction of mTOR transcripts. mTOR downregulation, together with a decrease in phosphorylation of downstream protein in the mTOR pathway (i.e 4E-BP1) was confirmed by western blot analyses. We then investigated the mechanisms of cell death. siRNA-mediatd SNRPE knockdown was not found to induce typical apoptosis as annexin-V-positive cells were not detected. However, the presence of numerous vesicular organelle of autophagy revealed by EM is strongly amplified in SNRPE-transfected cells. This observed deregulation of the mTOR pathway in SNRPE-depleted cells might in part explain the induction of autophagy following SNRPE silencing. Conclusion : Our data report that specific inactivation of SNRPE protein expression induces mTOR downregulation and autophagy in cancer cell lines, suggesting that the spliceosome could represent a new potential target that may open new perspectives in cancer therapy. Molecular mechanisms that lead to mTOR dowregulation by SNRPE extinction are being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1623. doi:10.1158/1538-7445.AM2011-1623

Alternative splicing deregulation leads to over expression of exon 13 P73 isoforms

Alternative splicing deregulation leads to over expression of exon 13 P73 isoforms