SOX2 modulates alternative splicing in transitional cell carcinoma

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor.     

Splicing-active nuclear extracts from rat brain

In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies. These tissue-specific extracts promote the neuron-specific pathway of splicing, and display characteristic changes in hnRNP protein function and expression. These extracts can be used in combination with affinity selection and depletion/complementation assays to identify regulatory factors and to characterize their interactions and effects on spliceosome assembly. The ability to prepare extracts from brain regions at a range of postnatal ages provides opportunities to address related questions as a function of cell differentiation. These neuronal extracts may also be valuable for the development of in vitro assays to elucidate other neuron-specific RNA processing pathways, such as 3' end formation, RNA editing, or miRNA maturation.     

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3′ splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis-acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3′ splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.     

Splicing factors are differentially expressed in tumors

Although alternative splicing of many genes has been found associated with different stages of tumorigenesis and splicing variants have been characterized as tumor markers, it is still not known whether these examples are sporadic or whether there is a broader association between the two phenomena. In this report we evaluated, through a bioinformatics approach, the expression of splicing factors in both normal and tumor tissues. This was possible by integrating data produced by proteomics, serial analysis of gene expression (SAGE) and microarray experiments. We observed a significant shift in the expression of splicing factors in tumors in both SAGE and microarray data, resulting from a large amount of experiments. We discuss that this supports the notion of a broader association between alternative splicing and cell transformation, and that splicing factors may be involved in oncogenic pathways.     

Detection and measurement of alternative splicing using splicing-sensitive microarrays

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.     

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.     

Analysis of integrin mRNA in human and rodent tumor cells.

Several human and rodent tumor cell lines were examined for the presence of integrin mRNAs by dot- and Northern-blot analysis. All tumor cells tested expressed mRNAs for alpha 5, alpha IIb, beta 1 and beta 3. The mRNA of beta 2 integrin was not detectable and that of alpha V integrin was found only in certain cells. Northern blotting was carried out in three selected tumor cell lines: Clone A, HEL and B16a. An apparent difference in the mRNA species coding for the alpha IIb beta 3 integrin, but not for alpha 5 and beta 1, was found. Our result suggests that alternative splicing of integrin genes may be one of the important mechanisms in regulating alpha IIb beta 3 expression and function in different tumor cells.     

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.     

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) represents an anomalous subset of breast cancer with a greatly reduced (30%) 5-year survival rate. The enhanced mortality and morbidity of TNBC arises from the high metastatic rate, which requires the acquisition of AnR, a process whereby anchorage-dependent cells become resistant to cell death induced by detachment. In this study TNBC cell lines were selected for AnR, and these cell lines demonstrated dramatic enhancement in the formation of lung metastases as compared with parental cells. Genetic analysis of the AnR subclones versus parental cells via next generation sequencing and analysis of global alternative RNA splicing identified that the mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was altered in AnR TNBC cells. Specifically, increased inclusion of exon 4 into the mature mRNA to produce the CPEB2B isoform was observed in AnR cell lines. Molecular manipulations of CPEB2 splice variants demonstrated a key role for this RNA splicing event in the resistance of cells to anoikis. Specifically, down-regulation of the CPEB2B isoform using siRNA re-sensitized the AnR cell lines to detachment-induced cell death. The ectopic expression of CPEB2B in parental TNBC cell lines induced AnR and dramatically increased metastatic potential. Importantly, alterations in the alternative splicing of CPEB2 were also observed in human TNBC and additional subtypes of human breast cancer tumors linked to a high metastatic rate. Our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key mechanism in AnR and a driving force in TNBC metastasis.     

Abnormal alternative splicing promotes tumor resistance in targeted therapy and immunotherapy

Abnormally alternative splicing events are common hallmark of diverse types of cancers. Splicing variants with aberrant functions play an important role in cancer development. Most importantly, a growing body of evidence has supported that alternative splicing might play a significant role in the therapeutic resistance of tumors. Targeted therapy and immunotherapy are the future directions of tumor therapy; however, the loss of antigen targets on the tumor cells surface and alterations in drug efficacy have resulted in the failure of targeted therapy and immunotherapy. Interestingly, abnormal alternative splicing, as a strategy to regulate gene expression, is reportedly involved in the reprogramming of cell signaling pathways and epitopes on the tumor cell surface by changing splicing patterns of genes, thus rendering tumors resisted to targeted therapy and immunotherapy. Accordingly, increased knowledge regarding abnormal alternative splicing in tumors may help predict therapeutic resistance during targeted therapy and immunotherapy and lead to novel therapeutic approaches in cancer. Herein, we provide a brief synopsis of abnormal alternative splicing events in cancer progression and therapeutic resistance.     

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform

Background Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system.Results In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian.ConclusionsDrosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.     

SRSF5 functions as a novel oncogenic splicing factor and is upregulated by oncogene SRSF3 in oral squamous cell carcinoma

Alternative splicing of precursor messenger RNA has been increasingly associated with tumorigenesis. The serine/arginine-rich protein (SR) family plays key roles in the regulation of pre-mRNA alternative splicing. Increasing evidence has demonstrated that the SR protein family is involved in tumorigenesis. However, the functions and mechanisms of SR proteins in tumourigenesis remain largely unknown. In the present study, we discovered that serine/arginine-rich splicing factor 5 (SRSF5) is a novel oncogenic splicing factor that is overexpressed in oral squamous cell carcinoma (OSCC) tissues and cells, being crucial for OSCC cell proliferation and tumor formation. Overexpression of SRSF5 transformed immortal rodent fibroblasts to form tumors in nude mice, while downregulation of SRSF5 in oral squamous cell lines retarded cell growth, cell cycle progression, and tumor growth. The expression of SRSF5 is controlled by an autoregulation mechanism. Serine/arginine-rich splicing factor 3 (SRSF3) has been identified as an oncogene. We found that SRSF5 is a novel target of SRSF3. SRSF3 impairs the autoregulation of SRSF5 and promotes SRSF5 overexpression in cancer cells. Altogether, the present study demonstrated that SRSF5 is a novel oncogene that is upregulated by SRSF3 in OSCC cells.