SRSF1 Expression Research Articles

Eucalyptal A inhibits glioma by rectifying oncogenic splicing of MYO1B mRNA via suppressing SRSF1 expression

Glioma is the most common primary intracranial tumor, in which glioblastoma (GBM) is the most malignant and lethal. However, the current chemotherapy drugs are still unsatisfactory for GBM therapy. As the natural products mainly extracted from Eucalyptus species, phloroglucinol-terpene adducts have the potential to be anti-cancer lead compounds that attracted increasing attention. In order to discover the new lead compounds with the anti-GBM ability, we isolated Eucalyptal A with a phloroglucinol-terpene skeleton from the fruit of E. globulus and investigated its anti-GBM activity in vitro and in vivo. Functionally, we verified that Eucalyptal A could inhibit the proliferation, growth and invasiveness of GBM cells in vitro. Moreover, Eucalyptal A had the same anti-GBM activity in tumor-bearing mice as in vitro and prolonged the overall survival time by maintaining mice body weight. Further mechanism research revealed that Eucalyptal A downregulated SRSF1 expression and rectified SRSF1-guided abnormal alternative splicing of MYO1B mRNA, which led to anti-GBM activity through the PDK1/AKT/c-Myc and PAK/Cofilin axes. Taken together, we identified Eucalyptal A as an important anti-GBM lead compound, which represents a novel direction for glioma therapy.

Copy Number Loss of 17q22 Is Associated with Enzalutamide Resistance and Poor Prognosis in Metastatic Castration-Resistant Prostate Cancer.

The purpose of this study was to measure genomic changes that emerge with enzalutamide treatment using analyses of whole-genome sequencing and RNA sequencing. One hundred and one tumors from men with metastatic castration-resistant prostate cancer (mCRPC) who had not been treated with enzalutamide (n = 64) or who had enzalutamide-resistant mCRPC (n = 37) underwent whole genome sequencing. Ninety-nine of these tumors also underwent RNA sequencing. We analyzed the genomes and transcriptomes of these mCRPC tumors. Copy number loss was more common than gain in enzalutamide-resistant tumors. Specially, we identified 124 protein-coding genes that were more commonly lost in enzalutamide-resistant samples. These 124 genes included eight putative tumor suppressors located at nine distinct genomic regions. We demonstrated that focal deletion of the 17q22 locus that includes RNF43 and SRSF1 was not present in any patient with enzalutamide-naïve mCRPC but was present in 16% (6/37) of patients with enzalutamide-resistant mCRPC. 17q22 loss was associated with lower RNF43 and SRSF1 expression and poor overall survival from time of biopsy [median overall survival of 19.3 months in 17q22 intact vs. 8.9 months in 17q22 loss, HR, 3.44 95% confidence interval (CI), 1.338-8.867, log-rank P = 0.006]. Finally, 17q22 loss was linked with activation of several targetable factors, including CDK1/2, Akt, and PLK1, demonstrating the potential therapeutic relevance of 17q22 loss in mCRPC. Copy number loss is common in enzalutamide-resistant tumors. Focal deletion of chromosome 17q22 defines a previously unappreciated molecular subset of enzalutamide-resistant mCRPC associated with poor clinical outcome.

SRSF1 mediates cytokine-induced impaired imatinib sensitivity in chronic myeloid leukemia.

Patients with chronic myeloid leukemia (CML) who are treated with tyrosine kinase inhibitors (TKIs) experience significant heterogeneity regarding depth and speed of responses. Factors intrinsic and extrinsic to CML cells contribute to response heterogeneity and TKI resistance. Among extrinsic factors, cytokine-mediated TKI resistance has been demonstrated in CML progenitors, but the underlying mechanisms remain obscure. Using RNA-sequencing, we identified differentially expressed splicing factors in primary CD34+ chronic phase (CP) CML progenitors and controls. We found SRSF1 expression to be increased as a result of both BCR-ABL1- and cytokine-mediated signaling. SRSF1 overexpression conferred cytokine independence to untransformed hematopoietic cells and impaired imatinib sensitivity in CML cells, while SRSF1 depletion in CD34+ CP CML cells prevented the ability of extrinsic cytokines to decrease imatinib sensitivity. Mechanistically, PRKCH and PLCH1 were upregulated by elevated SRSF1 levels, and contributed to impaired imatinib sensitivity. Importantly, very high SRSF1 levels in the bone marrow of CML patients at presentation correlated with poorer clinical TKI responses. In summary, we find SRSF1 levels to be maintained in CD34+ CP CML progenitors by cytokines despite effective BCR-ABL1 inhibition, and that elevated levels promote impaired imatinib responses. Together, our data support an SRSF1/PRKCH/PLCH1 axis in contributing to cytokine-induced impaired imatinib sensitivity in CML.

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment.

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Underexpression of SRSF3 and its target gene RBMX predicts good prognosis in patients with head and neck cancer.

Head and neck cancer collectively is one of the most common cancer types in the world. Oral squamous cell carcinoma (OSCC) is the most common subtype of head and neck cancer. SRSF3 is a proto-oncogene and is overexpressed in patients with OSCC. However, the relationship between SRSF3 expression and the clinical outcomes of patients with head and neck cancer remains unclear. By using the cBioPortal for Cancer Genomics, a public online tool originally developed at Memorial Sloan Kettering Cancer Center (New York, NY, USA), it was revealed that patients with head and neck cancer with an underexpression of SRSF3 showed better overall and disease-/progression-free survival rates. Moreover, 227 genes were found to be significantly coexpressed with SRSF3 in head and neck cancer. Then, in combination with the analysis of a previous splice-array dataset that included significantly changed genes after the silencing of SRSF3, four potential target genes of SRSF3 were identified. RBMX and HNRNPL were further confirmed to be target genes of SRSF3. Moreover, the underexpression of RBMX was determined to be significantly associated with a favorable overall survival rate among patients, while patients with an underexpression of both SRSF3 and RBMX is a subgroup of individuals with better prognoses than all other patients. These results suggest that the underexpression of SRSF3 and that of its target RBMX can be used as potential biomarkers to predict favorable overall survival among head and neck cancer patients.

Genome Wide Transcriptomic Analysis Identifies Dysregulated Splicing Factor Profile with Molecular and Functional Role in Multiple Myeloma

Alternative splicing (AS) is a critical post-transcriptional event, which affects the number of cellular processes. Aberrant splicing of some genes has been reported in multiple myeloma (MM). However, to date, whole-transcriptome-wide AS study has not been performed. We used deep RNA-sequencing data from 16 normal plasma cells (NPC) and 360 newly-diagnosed MM patients to describe the landscape of the alternative splicing events and the molecular mechanisms driving aberrant AS in MM. Global splicing analysis showed that mutually exclusive exon (MXE) (n=510) and Skipped Exon (SE) (n=417) are the most frequent splicing events in MM compared to NPC. Among these events, ~54% were observed in genes which are not differentially expressed between MM and NPC and 46% of the AS events (SE, MXE, retained intron, alternative 3'/5' sites) were observed in differentially expressed genes targeting 203 unique genes. AS affected RNA transcription regulation genes such as IKZF1, IKZF3, and key regulatory elements in MM including, IRF3, IRF4, or key transcription factors such as MEF2C, XPB1, STAT2, and ILF3. In general, AS targetted DNA replication, cell cycle, and apoptosis pathways. MM subgroups showed a heterogeneity for AS events. Monosomy 14, t(4;14), del1p and del17p had the highest number of unique (not observed in other subgroups) AS events compared to NPC.To understanding the molecular mechanisms driving aberrant alternate splicing we next investigated115 splicing factors (SF) in MM and associated them with AS events. We observed that ~40% of SF were dysregulated (dysregulated expression and/or copy number alteration) in MM compared to NPC, including SRSF, PCBP and RBM families. To understand the key binding regions, we have performed SF binding motif enrichment analysis around AS events and found that SRFS1, SRSF9, and PCB1 motifs to be enriched among the splicing events. Importantly, SRSF1 expression was linked with survival in two independent MM datasets.We therefore explored functional role of SRSF1 in MM with perturbation studies. While upregulation of SRSF1 expression significantly increased the cell growth and survival, conversely downregulation of SRSF1 inhibited the both. To dissect the mechanisms of SRSF1-mediated MM growth induction, we utilized SRSF1 mutants lacking either of the 2 RNA-recognition motifs or the serine/argine-rich C-terminal domain involved in protein-protein interactions, and recruitment of spliceosome components. We also utilized a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. These studies suggested that SRSF1-regulated AS effects MM cell proliferation. We surprisingly also found that even NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation.We next investigated alternative splicing pattern changes induced by SRSF1 knock down.When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affect's genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR, E2F and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells.Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of oncogenic E2F1 in MM cells. Consistent with these findings, we observed greater in vitro loss of viability in a large panel of MM cell lines compared with PBMCs from healthy volunteers, following exposure to the splicing modulator pladeniolide. In summary, this study for the first time reports a detailed splicing landscape in myeloma and highlights the biological and clinical importance of alternative splicing events. Moreover, these results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM, highlighting the need to further understand the splicing pattern in myeloma initiation and progression. Disclosures Anderson: Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Celgene: Consultancy.

Downregulation of SRSF3 by antisense oligonucleotides sensitizes oral squamous cell carcinoma and breast cancer cells to paclitaxel treatment.

Paclitaxel (PTX) is widely used in the chemotherapy of many cancers, including breast cancer and oral squamous cell carcinoma (OSCC). However, many patients respond poorly to PTX treatment. The SRSF3 oncogene and several splicing factors play important roles in OSCC tumorigenesis. This study aimed to understand the function of splicing factors in PTX treatment and improve the therapeutic effects of PTX treatment. Splicing factors regulated by PTX treatment were screened in CAL 27 cell by reverse transcription polymerase chain reaction. The function of SRSF3 in PTX treatment was analyzed by gain-of-function or loss-of-function assay in OSCC cell lines CAL 27 and SCC-9 and breast cancer cell line MCF-7. Alternative splicing of SRSF3 exon 4 in cancer tissues or cells was analyzed by RT-PCR and online program TSVdb. SRSF3-specific antisense oligonucleotide (ASO) SR-3 was used to downregulate SRSF3 expression and enhance the effect of PTX treatment. PTX treatment decreased SRSF3 expression, and SRSF3 overexpression rescued the growth inhibition caused by PTX in both OSCC and breast cancer cells. Moreover, we found that PTX treatment could repress SRSF3 exon 4 (containing an in-frame stop codon) exclusion and then decrease the SRSF3 protein expression. Increased exclusion of SRSF3 exon 4 is correlated with poor survival in OSCC and breast cancer patients. SR-3 downregulated SRSF3 protein expression and significantly increased the sensitivity of cancer cells to PTX treatment. SRSF3 downregulation by ASO sensitizes cancer cells to PTX treatment.

Serine/Arginine-Rich Splicing Factor 3 Modulates the Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2.

Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA trans-factor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was "rescued" by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. IMPLICATIONS: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/9/1920/F1.large.jpg.

Cortisol-induced SRSF3 expression promotes GR splicing, RACK1 expression and breast cancer cells migration

Recent data have demonstrated that triple negative breast cancer (TNBC) with high glucocorticoid receptor (GR) expression are associated to therapy resistance and increased mortality. Given that GR alternative splicing generates mainly GRα, responsible of glucocorticoids action, we investigated its role in the regulation of RACK1 (Receptor for Activated C Kinase 1), a scaffolding protein with a GRE (Glucocorticoid Response Element) site on its promoter and involved in breast cancer cells migration and invasion. We provide the first evidence that GRα transcriptionally regulates RACK1 by a mechanism connected to SRSF3 splicing factor, which promotes GRα, essential for RACK1 transcriptional regulation and consequently for cells migration. We also establish that this mechanism can be positively regulated by cortisol. Hence, our data elucidate RACK1 transcriptional regulation and demonstrate that SRSF3 involvement in cells migration implies its role in controlling different pathways thus highlighting that new players have to be considered in GR-positive TNBC.

Alternative polyadenylation dependent function of splicing factor SRSF3 contributes to cellular senescence.

Down-regulated splicing factor SRSF3 is known to promote cellular senescence, an important biological process in preventing cancer and contributing to individual aging, via its alternative splicing dependent function in human cells. Here we discovered alternative polyadenylation (APA) dependent function of SRSF3 as a novel mechanism explaining SRSF3 downregulation induced cellular senescence. Knockdown of SRSF3 resulted in preference usage of proximal poly(A) sites and thus global shortening of 3′ untranslated regions (3′ UTRs) of mRNAs. SRSF3-depletion also induced senescence-related phenotypes in both human and mouse cells. These 3′ UTR shortened genes were enriched in senescence-associated pathways. Shortened 3′ UTRs tended to produce more proteins than the longer ones. Simulating the effects of 3′ UTR shortening by overexpression of three candidate genes (PTEN, PIAS1 and DNMT3A) all led to senescence-associated phenotypes. Mechanistically, SRSF3 has higher binding density near proximal poly(A) site than distal one in 3′ UTR shortened genes. Further, upregulation of PTEN by either ectopic overexpression or SRSF3-knockdown induction both led to reduced phosphorylation of AKT and ultimately senescence-associated phenotypes. We revealed for the first time that reduced SRSF3 expression could promote cellular senescence through its APA-dependent function, largely extending our mechanistic understanding in splicing factor regulated cellular senescence.

Oncogenic splicing factor SRSF3 regulates ILF3 alternative splicing to promote cancer cell proliferation and transformation.

Alternative RNA splicing is an important focus in molecular and clinical oncology. We report here that SRSF3 regulates alternative RNA splicing of interleukin enhancer binding factor 3 (ILF3) and production of this double-strand RNA-binding protein. An increased coexpression of ILF3 isoforms and SRSF3 was found in various types of cancers. ILF3 isoform-1 and isoform-2 promote cell proliferation and transformation. Tumor cells with reduced SRSF3 expression produce aberrant isoform-5 and -7 of ILF3. By binding to RNA sequence motifs, SRSF3 regulates the production of various ILF3 isoforms by exclusion/inclusion of ILF3 exon 18 or by selection of an alternative 3′ splice site within exon 18. ILF3 isoform-5 and isoform-7 suppress tumor cell proliferation and the isoform-7 induces cell apoptosis. Our data indicate that ILF3 isoform-1 and isoform-2 are two critical factors for cell proliferation and transformation. The increased SRSF3 expression in cancer cells plays an important role in maintaining the steady status of ILF3 isoform-1 and isoform-2.

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.

Proteomic Characterization of Transcription and Splicing Factors Associated with a Metastatic Phenotype in Colorectal Cancer.

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.

Theophylline exhibits anti-cancer activity via suppressing SRSF3 in cervical and breast cancer cell lines.

Caffeine, theophylline, and theobromine are the most well-known members of methylxanthines. Caffeine-induced serine/arginine-rich splicing factor 2, SRSF2, and SRSF3 are required for the alternative splicing of a subset of cancer-associated genes. However, it remains to be investigated whether and how theophylline and theobromine as well as caffeine exert their antitumor effects through mediating the alternative splicing process. Here, we reveal that theophylline down-regulated SRSF3 expression and switched p53 from alpha into a beta isoform as caffeine did in HeLa and MCF-7 cells via the reverse-transcriptase polymerase chain reaction and Western blot analysis. Further functional studies show that theophylline induced cellular apoptosis, senescence, and decreased colony formation. Interestingly, theophylline had a suppressive effect on cellular proliferation, whereas caffeine enhanced cellular proliferation rates via the 5-bromo-2-deoxyuridine analysis. Theophylline and caffeine had no effect on MCF-10A cells, which is a normal breast cell line. Our results provide an insight that theophylline as well as caffeine could be repurposed as antitumor leading compounds via the downregulation of splicing factor SRSF3 and its target genes.

Long Noncoding RNA MALAT1 Promotes Hepatocellular Carcinoma Development by SRSF1 Upregulation and mTOR Activation.

Several long noncoding RNAs (lncRNA) are abrogated in cancer but their precise contributions to oncogenesis are still emerging. Here we report that the lncRNA MALAT1 is upregulated in hepatocellular carcinoma and acts as a proto-oncogene through Wnt pathway activation and induction of the oncogenic splicing factor SRSF1. Induction of SRSF1 by MALAT1 modulates SRSF1 splicing targets, enhancing the production of antiapoptotic splicing isoforms and activating the mTOR pathway by modulating the alternative splicing of S6K1. Inhibition of SRSF1 expression or mTOR activity abolishes the oncogenic properties of MALAT1, suggesting that SRSF1 induction and mTOR activation are essential for MALAT1-induced transformation. Our results reveal a mechanism by which lncRNA MALAT1 acts as a proto-oncogene in hepatocellular carcinoma, modulating oncogenic alternative splicing through SRSF1 upregulation. Cancer Res; 77(5); 1155-67. ©2016 AACR.

Overexpression of Antiangiogenic Vascular Endothelial Growth Factor Isoform and Splicing Regulatory Factors in Oral, Laryngeal and Pharyngeal Squamous Cell Carcinomas

Background:Overexpression of proangiogenic vascular endothelial growth factor A family VEGFAxxx is associated with tumor growth and metastasis. The role of the alternatively spliced antiangiogenic family VEGFAxxxb is poorly investigated in head and neck squamous cell carcinomas (HNSCCs). The antiangiogenic isoform binds to bevacizumab and its expression level could influence the treatment response and progression-free survival. In this study, the relative expression of VEGFAxxx and VEGFA165b isoforms and splicing regulatory factors genes was investigated in a series of HNSCCs.Methods:VEGFAxxx, VEGFA165b, SRSF6, SRSF5, SRSF1 and SRPK1 gene expression was quantified by quantitative real time PCR in 53 tissue samples obtained by surgery from HNSCC patients. Protein expression was evaluated by immunohistochemistry.Results:VEGFAxxx and VEGFA165b were overexpressed in HNSCCs. Elevated protein expression was also confirmed. However, VEGFA isoforms demonstrated differential expression according to anatomical sites. VEGFAxxx was overexpressed in pharyngeal tumors while the VEGFA165b isoform was up-regulated in oral tumors. The VEGFA165b isoform was also positively correlated with expression of the splicing regulatory genes SRSF1, SRSF6 and SRSF5.Conclusions:We concluded that VEGFAxxx and VEGFA165b isoforms are overexpressed in HNSCCs and the splicing regulatory factors SRSF1, SRSF6, SRSF5 and SRPK1 may contribute to alternative splicing of the VEGFA gene. The findings for the differential expression of the antiangiogenic isoform in HNSCCs could facilitate effective therapeutic strategies for the management of these tumors.

SRSF1 Is a Mediator of Radiation-Induced Alternative Splicing in B-Lymphocytes

Ionizing radiation is used in the treatment of Hodgkin and non-Hodgkin lymphomas (Spetch et al. 2014; Illidge et al. 2014). Despite its effectiveness, radiation is a "blunt" therapy that damages indiscriminately both cancer and normal cells, and can result in secondary malignancies (Dores et al. 2002). To better understand cellular response to radiation, we examined alternative splicing, and its regulation, in irradiated human cells.To accomplish this, we exposed cultured B-lymphocytes from 10 individuals to 10 Gy of ionizing radiation, and performed RNA sequencing before, and two and six hours after radiation treatment. With these data, we first identified alternative splicing events. From about 60 million reads per sample, we detected over 20,000 alternatively spliced events. In total, 1,600 events were radiation responsive (ANOVA, FDR<5%). The splicing events in irradiated cells belong mainly to three categories, each of which occurred in genes with distinct biological functions. Cassette exons, which were the most numerous splicing event, were found primarily in DNA damage response and apoptotic genes, while alternative first or last exons were found in chromatin assembly genes; retained introns occurred in genes involved in RNA processing and translation. Using these radiation-responsive events, we then examined the kinetics of this response. We found that changes in alternative splicing were acute, occurring within two hours of radiation treatment. Overall, the splicing events produced shorter transcripts in irradiated cells.Next, we looked for how alternative splicing is regulated in response to radiation. To identify putative mediators of this response, we determined the expression levels of over 60 trans-acting splicing factors. We found that the expression level of 26 splicing factors changed significantly in irradiated cells (ANOVA, FDR<5%). Of these putative mediators, we further examined serine/arginine-rich splicing factor (SRSF1)’s role in radiation response. First, we assessed SRSF1 expression in irradiated cells. Following radiation exposure, the transcript and protein expressions of SRSF1 decreased. Next, we performed motif enrichment analysis to identify target genes. SRSF1 RNA-binding sites were enriched in skipped cassette exons: specifically, of the 362 skipped cassette exons in irradiated cells, 93 (26%) contained SRSF1 binding motifs.Finally, we examined the link between SRSF1 and exon skipping in irradiated cells more closely. For example, in the transcripts for RNA-binding protein 3 (RBM3), SRSF1 binds an RBM3 exon that contains a premature termination codon (Sanford et al. 2009), and mediates exclusion of this exon. We confirmed experimentally that this results in an increase in RBM3 protein expression. Together, our data demonstrate that alternative splicing is a key part of cellular response to radiation, and SRSF1 plays a role in mediating exon skipping. In this presentation, we will describe radiation-induced alternative splicing by discussing the genes that are alternatively spliced, and its regulation by the splicing factor SRSF1. DisclosuresNo relevant conflicts of interest to declare.

SR proteins regulate V6 exon splicing of CD44 pre-mRNA.

CD44 pre-mRNA includes 20 exons, of which exons 1–5 (C1–C5) and exons 16–20 (C6–C10) are constant exons, whereas exons 6–15 (V1–V10) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V6 splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V6 minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V6 splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V6 splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V6 specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V6–10 and V6,8–10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V6 splicing.

Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder that causes systemic accelerated aging in children. This syndrome is due to a mutation in the LMNA gene that leads to the production of a truncated and toxic form of lamin A called progerin. Because the balance between the A-type lamins is controlled by the RNA-binding protein SRSF1, we have hypothesized that its inhibition may have therapeutic effects for HGPS. For this purpose, we evaluated the antidiabetic drug metformin and demonstrated that 48 h treatment with 5 mmol/l metformin decreases SRSF1 and progerin expression in mesenchymal stem cells derived from HGPS induced pluripotent stem cells (HGPS MSCs). The effect of metformin on progerin was then confirmed in several in vitro models of HGPS, i.e., human primary HGPS fibroblasts, LmnaG609G/G609G mouse fibroblasts and healthy MSCs previously treated with a PMO (phosphorodiamidate morpholino oligonucleotide) that induces progerin. This was accompanied by an improvement in two in vitro phenotypes associated with the disease: nuclear shape abnormalities and premature osteoblastic differentiation of HGPS MSCs. Overall, these results suggest a novel approach towards therapeutics for HGPS that can be added to the currently assayed treatments that target other molecular defects associated with the disease.

HnRNP L is important for the expression of oncogene SRSF3 and oncogenic potential of oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is the leading cause of death related to oral diseases. The mechanisms of OSCC development remain largely unknown. Heterogeneous nuclear ribonucleoprotein L (HnRNP L) is a multi-functional splicing factor. It has been reported to be an important regulator of apoptosis. However, the functions of hnRNP L in cancer need to be further explored. In the present study, we found that OSCC tissues expressed significantly higher levels of hnRNP L than normal tissues. Depletion of hnRNP L retarded cell growth, cell migration, and tumorigenesis of OSCC cells. HnRNP L regulates both the expression of oncogenic splicing factor SRSF3 and the alternative splicing of SRSF3 exon 4. Expression of hnRNP L is correlated with SRSF3 expression in OSCC tissues. These findings suggest that hnRNP L is important for the pathogenesis of OSCC and may be a novel potential therapeutic target of OSCC.