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1.  A new function of the splicing factor SRSF2 in the control of E2F1-mediated cell cycle progression in neuroendocrine lung tumors 
Cell Cycle  2013;12(8):1267-1278.
The transcription factor E2F1 belongs to the E2F family and plays a crucial role during cell cycle progression and apoptosis. Ser/Arg-Rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing events. We previously identified the SR protein SRSF2 as a new transcriptional target of E2F1 and demonstrated that both proteins cooperate to induce apoptosis in non-small cell lung carcinoma. In this study, we postulated that SRSF2 is also involved in the proliferative functions of E2F1. Using IHC, we first demonstrate that SRSF2 and its phosphorylated form (P-SRSF2) are overexpressed in neuroendocrine lung tumors that are highly proliferative tumors expressing high levels of E2F1. Importantly, we show a direct correlation between cyclin E, an E2F1-target gene controlling S phase, and P-SRSF2 proteins levels (p = 0.0083), suggesting a role of SRSF2 in E2F1-mediated cellular proliferation. Accordingly, using neuroendocrine lung carcinoma cell lines, we demonstrate that SRSF2 is a cell cycle-regulated protein involved in entry and progression into S phase. We also provide evidence that SRSF2 interacts with E2F1 and stimulates its transcriptional control of cell cycle target genes such as cyclin E. Finally, we show that inhibition of AKT signaling pathway prevents SRSF2 phosphorylation and activity toward E2F1 transcriptional function. Taken together, these results identify a new role of SRSF2 in the control of cell cycle progression and reinforce the functional link between SRSF2 and E2F1 proteins.
doi:10.4161/cc.24363
PMCID: PMC3674091  PMID: 23518498
AKT; cellular proliferation; E2F1; neuroendocrine lung tumors; p45SKP2; SRSF2
2.  Abnormal Expression of the Pre-mRNA Splicing Regulators SRSF1, SRSF2, SRPK1 and SRPK2 in Non Small Cell Lung Carcinoma 
PLoS ONE  2012;7(10):e46539.
Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III–IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma.
doi:10.1371/journal.pone.0046539
PMCID: PMC3468597  PMID: 23071587
3.  The translational landscape of the splicing factor SRSF1 and its role in mitosis 
eLife  2014;3:e02028.
The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.
DOI: http://dx.doi.org/10.7554/eLife.02028.001
eLife digest
Genes contain the instructions to make proteins. These instructions are first transcribed to produce an intermediate molecule called a messenger RNA (mRNA), which is then translated to produce the protein. However, gene sequences are often interrupted by ‘introns’, sections of DNA that do not code for protein, and these introns must be removed from the mRNA molecules via a process called ‘splicing’ before the protein is produced.
Splicing can also be used to ‘mix and match’ sections of gene sequences to produce slightly different versions of the same protein in a process called ‘alternative splicing’. SRSF1 is one of a family of proteins that control both types of gene splicing but also promotes the translation of specific mRNAs. To date only a few of the genes whose translation is regulated by SRSF1 have been identified.
Here, Maslon, Heras et al. have used human cells that artificially produce more SRSF1 protein than normal to identify those genes whose translation is regulated by SRSF1. Over 1500 ‘target genes’ were found; many of which encoded proteins that are involved in cell division—and cells with less SRSF1 than normal failed to divide properly. Maslon, Heras et al. also found a link between alternative splicing and protein translation: many of the mRNAs that were spliced differently in cells that over-produced SRSF1 were also genes whose translation was affected by SRSF1.
Since uncontrolled cell division, or defects in mRNA splicing or protein synthesis are all often linked to cancer, these discoveries might provide new insights into the mechanisms underlying this disease.
DOI: http://dx.doi.org/10.7554/eLife.02028.002
doi:10.7554/eLife.02028
PMCID: PMC4027812  PMID: 24842991
translation; splicing; SR proteins; human
4.  Correlation of SRSF1 and PRMT1 expression with clinical status of pediatric acute lymphoblastic leukemia 
Background
Acute lymphoblastic leukemia (ALL) is the most frequently-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 children with ALL, SFRS1 was found to be up-regulated. Serine/arginine-rich splicing factor 1 (SRSF1, also termed SF2/ASF), encoded by the SFRS1 gene, had been shown to be a pro-oncoprotein. Our previous study indicated that SRSF1 can be methylated by protein arginine methyltransferase 1 (PRMT1) in vitro; however, the biological function of SRSF1 and PRMT1 in pediatric ALL are presently unknown.
Methods
Matched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 57 patients were collected in order to evaluate the expression patterns of SRSF1 and PRMT1. The potential oncogenic mechanism of SRSF1 and PRMT1 in leukemogenesis was also investigated.
Results
We identified significant up-regulation of SRSF1 and PRMT1 in the ND samples. Importantly, the expression of SRSF1 and PRMT1 returned to normal levels after CR, but rebounded in the RE samples. Our observation that SRSF1 could predict disease relapse was of particular interest, although the expression patterns of SRSF1 and PRMT1 were independent of the cytogenetic subtypes. In pre-B-cell lines, both SRSF1 and PRMT1 expression could be efficiently attenuated by the clinical chemotherapy agents arabinoside cytosine (Ara-c) or vincristine (VCR). Moreover, SRSF1 and PRMT1 were associated with each other in leukemia cells in vivo. Knock-down of SRSF1 resulted in an increase in early apoptosis, which could be further induced by chemotherapeutics.
Conclusions
Our results indicate that SRSF1 serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients by cooperating with PRMT1.
doi:10.1186/1756-8722-5-42
PMCID: PMC3459738  PMID: 22839530
Acute lymphoblastic leukemia; Splicing factor SRSF1; Protein arginine methyltransferase 1 (PRMT1); Alternative splicing; Arginine methylation
5.  ONCOGENIC SPLICING FACTOR SRSF1 IS A CRITICAL TRANSCRIPTIONAL TARGET OF MYC 
Cell reports  2012;1(2):110-117.
The SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here we show that SRSF1 is a direct target of the transcription-factor oncoprotein MYC. These two oncogenes are significantly co-expressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two non-canonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC’s oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC, and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.
doi:10.1016/j.celrep.2011.12.001
PMCID: PMC3334311  PMID: 22545246
6.  Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation 
PLoS ONE  2013;8(3):e59137.
SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.
doi:10.1371/journal.pone.0059137
PMCID: PMC3594168  PMID: 23536862
7.  Expression of Tra2β in Cancer Cells as a Potential Contributory Factor to Neoplasia and Metastasis 
The splicing regulator proteins SRSF1 (also known as ASF/SF2) and SRSF3 (also known as SRP20) belong to the SR family of proteins and can be upregulated in cancer. The SRSF1 gene itself is amplified in some cancer cells, and cancer-associated changes in the expression of MYC also increase SRSF1 gene expression. Increased concentrations of SRSF1 protein promote prooncogenic splicing patterns of a number of key regulators of cell growth. Here, we review the evidence that upregulation of the SR-related Tra2β protein might have a similar role in cancer cells. The TRA2B gene encoding Tra2β is amplified in particular tumours including those of the lung, ovary, cervix, stomach, head, and neck. Both TRA2B RNA and Tra2β protein levels are upregulated in breast, cervical, ovarian, and colon cancer, and Tra2β expression is associated with cancer cell survival. The TRA2B gene is a transcriptional target of the protooncogene ETS-1 which might cause higher levels of expression in some cancer cells which express this transcription factor. Known Tra2β splicing targets have important roles in cancer cells, where they affect metastasis, proliferation, and cell survival. Tra2β protein is also known to interact directly with the RBMY protein which is implicated in liver cancer.
doi:10.1155/2013/843781
PMCID: PMC3723085  PMID: 23935626
8.  The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival 
Nucleic Acids Research  2013;42(5):3218-3227.
NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.
doi:10.1093/nar/gkt1307
PMCID: PMC3950702  PMID: 24369428
9.  Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation 
Nucleic Acids Research  2014;42(6):4019-4030.
Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.
doi:10.1093/nar/gkt1387
PMCID: PMC3973337  PMID: 24442672
10.  Regulation of Mcl-1 by SRSF1 and SRSF5 in Cancer Cells 
PLoS ONE  2012;7(12):e51497.
Up-regulation of the apoptosis-regulatory gene Mcl-1 (myeloid cell leukemia-1) occurs in different cancer types and is linked with drug resistance to cancer therapies. It is well known that Mcl-1 pre-mRNA undergoes alternative splicing events to produce two functionally distinct proteins, Mcl-1S (pro-apoptotic) and Mcl-lL (anti-apoptotic); the latter isoform is predominant in different cancers including breast and ovarian cancer cells. In the present study we report that the RNA-binding protein (RBP) and proto-oncogene SRSF1 (serine and arginine-rich splicing factor 1) influences splicing of Mcl-1 in both MCF-7 and MDA-MB-231 breast cancer cells and JAR choriocarcinoma cells; we also show for the first time that another RBP SRSF5 affects splicing of Mcl-1 in the MCF-7 cells. Moreover, we report that SRSF1 is involved in other aspects of Mcl-1 regulation with knockdown of SRSF1, by RNAi, resulting in a significant decrease in Mcl-1 protein levels in MCF-7 cells but an increase in JAR cells, respectively, by potentially affecting protein stability and translation of Mcl-l. The key findings from this study highlight the importance of the cellular context of different cancer cells for the function of multifunctional RBPs like SRSF1 and have implications for therapeutic approaches employed to target Mcl-1.
doi:10.1371/journal.pone.0051497
PMCID: PMC3524227  PMID: 23284704
11.  HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition 
Nucleic Acids Research  2013;41(18):8665-8679.
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.
doi:10.1093/nar/gkt579
PMCID: PMC3794575  PMID: 23863836
12.  Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces cellular senescence 
Molecular cell  2013;50(1):56-66.
SUMMARY
Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor-suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal-protein complex—separate from the ribosome—that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS, and demonstrate a link between spliceosomal and ribosomal components—functioning independently of their canonical roles—to monitor cellular physiology and cell-cycle progression.
doi:10.1016/j.molcel.2013.02.001
PMCID: PMC3628402  PMID: 23478443
13.  A dedicated microarray for in-depth analysis of pre-mRNA splicing events: application to the study of genes involved in the response to targeted anticancer therapies 
Molecular Cancer  2014;13:9.
Alternative pre-mRNA splicing (AS) widely expands proteome diversity through the combinatorial assembly of exons. The analysis of AS on a large scale, by using splice-sensitive microarrays, is a highly efficient method to detect the majority of known and predicted alternative transcripts for a given gene. The response to targeted anticancer therapies cannot easily be anticipated without prior knowledge of the expression, by the tumor, of target proteins or genes. To analyze, in depth, transcript structure and levels for genes involved in these responses, including AKT1-3, HER1-4, HIF1A, PIK3CA, PIK3R1-2, VEGFA-D and PIR, we engineered a dedicated gene chip with coverage of an average 185 probes per gene and, especially, exon-exon junction probes. As a proof of concept, we demonstrated the ability of such a chip to detect the effects of over-expressed SRSF2 RNA binding protein on the structure and abundance of mRNA products in H358 lung cancer cells conditionally over-expressing SRSF2. Major splicing changes were observed, including in HER1/EGFR pre-mRNA, which were also seen in human lung cancer samples over-expressing the SRSF2 protein. In addition, we showed that variations in HER1/EGFR pre-mRNA splicing triggered by SRSF2 overexpression in H358 cells resulted in a drop in HER1/EGFR protein level, which correlated with increased sensitivity to gefitinib, an EGFR tyrosine kinase inhibitor. We propose, therefore, that this novel tool could be especially relevant for clinical applications, with the aim to predict the response before treatment.
doi:10.1186/1476-4598-13-9
PMCID: PMC3899606  PMID: 24428911
DNA chip; Targeted anticancer therapies; Pre-mRNA splicing; SRSF2
14.  Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion 
Molecular Biology of the Cell  2013;24(20):3164-3176.
Matching sets of human primary fibroblasts cocultured with placenta explants are used to compare tissue capacities to support trophoblast invasion. Substituting endometrium with dermis dramatically reduces EVCT interstitial invasion, a phenomenon related to the ECM fibronectin content, FN alternative splicing, and expression of the SR protein SRSF1.
Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA–) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.
doi:10.1091/mbc.E13-03-0142
PMCID: PMC3806663  PMID: 23966470
15.  Protein Kinase A–Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein 
Genes & Cancer  2011;2(8):841-851.
Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein’s capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.
doi:10.1177/1947601911430226
PMCID: PMC3278900  PMID: 22393468
pre-mRNA splicing regulation; SRSF1; PKA; phosphorylation
16.  SRSF1 and SRSF9 RNA binding proteins promote Wnt signalling-mediated tumorigenesis by enhancing β-catenin biosynthesis 
EMBO Molecular Medicine  2013;5(5):737-750.
Wnt/β-catenin signalling is widely implicated in embryogenesis, tissue homeostasis and tumorigenesis. The key event in Wnt signalling activation is β-catenin accumulation, which is controlled by both its production and degradation. However, much more emphasis has been placed on the understanding of its degradation. Here, we show that the synthesis of β-catenin protein, which requires a group of serine/arginine-rich splicing factors (SRSF), also contributes to its tumorigenic activity. Overexpression of SRSF1 and SRSF9 promote β-catenin accumulation via the recruitment of β-catenin mRNA and by enhancing its translation in an mTOR-dependent manner. We further demonstrate that, like SRSF1, SRSF9 is also an oncogene, and is frequently overexpressed in multiple types of human tumours. Finally, our results suggest that promoting degradation and blocking production of β-catenin synergistically reduce β-catenin levels under pathological conditions and that a combinational therapy could be a promising approach for the treatment of cancer patients.
doi:10.1002/emmm.201202218
PMCID: PMC3662316  PMID: 23592547
β-catenin synthesis; oncogene; SRSF1; SRSF9; Wnt signalling
17.  AR-A 014418 Used against GSK3beta Downregulates Expression of hnRNPA1 and SF2/ASF Splicing Factors 
Journal of Oncology  2014;2014:695325.
Glioblastoma is one of the most aggressive forms of primary brain tumors of glial cells, including aberrant regulation of glycogen synthase kinase 3β (GSK3β) and splicing factors deregulation. Here, we investigate the role of small molecule AR-A014418 and Manzamine A against GSK3 kinase with factual control on splicing regulators. AR-A 014418, 48 hrs posttreatment, caused dose (25–100 μM) dependent inhibition in U373 and U87 cell viability with also inhibition in activating tyrosine phosphorylation of GSK3alpha (Tyr 279) and beta (Tyr 216). Furthermore, inhibition of GSK3 kinase resulted in significant downregulation of splicing factors (SRSF1, SRSF5, PTPB1, and hnRNP) in U87 cells with downregulation of antiapoptotic genes such as BCL2, BCL-xL, Survivin, MCL1, and BMI1. Similarly, downregulation of splicing factors was also observed in U373 glioma cell after using SiRNA against AKT and GSK3beta kinase. In addition, potential roles of AR-A014418 in downregulation of splicing factors were reflected with decrease in Anxa7 (VA) variant and increase in Anxa7 WT tumor suppressor transcript and protein. The above results suggest that inhibition of GSK3beta kinase activation could be the beneficial strategy to inhibit the occurrence of alternative cancer escape pathway via downregulating the expression of splicing regulators as well as apoptosis.
doi:10.1155/2014/695325
PMCID: PMC3914408  PMID: 24550987
18.  The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes 
Genome Biology  2012;13(3):R17.
Background
The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.
Results
SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay.
Conclusions
SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members.
doi:10.1186/gb-2012-13-3-r17
PMCID: PMC3439968  PMID: 22436691
19.  SRSF1 (SRp30a) regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells 
Molecular cancer research : MCR  2011;9(7):889-900.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the four exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
doi:10.1158/1541-7786.MCR-11-0061
PMCID: PMC3140550  PMID: 21622622
ceramide; non-small cell lung cancer; RNA trans-factor; tumor repressor; oncogene; ASF/SF2; SRp30a; SRSF1; chemotherapy; erlotinib; daunorubicin; cisplatinum; paclitaxel
20.  The Splicing Factor SRSF1 as a Marker for Endothelial Senescence 
Aging is the major risk factor per se for the development of cardiovascular diseases. The senescence of the endothelial cells (ECs) that line the lumen of blood vessels is the cellular basis for these age-dependent vascular pathologies, including atherosclerosis and hypertension. During their lifespan, ECs may reach a stage of senescence by two different pathways; a replicative one derived from their preprogrammed finite number of cell divisions; and one induced by stress stimuli. Also, certain physiological stimuli, such as transforming growth factor-β, are able to modulate cellular senescence. Currently, the cellular aging process is being widely studied to identify novel molecular markers whose changes correlate with senescence. This review focuses on the regulation of alternative splicing mediated by the serine–arginine splicing factor 1 (SRSF1, or ASF/SF2) during endothelial senescence, a process that is associated with a differential subcellular localization of SRSF1, which typically exhibits a scattered distribution throughout the cytoplasm. Based on its senescence-dependent involvement in alternative splicing, we postulate that SRSF1 is a key marker of EC senescence, regulating the expression of alternative isoforms of target genes such as endoglin (ENG), vascular endothelial growth factor A (VEGFA), tissue factor (T3), or lamin A (LMNA) that integrate in a common molecular senescence program.
doi:10.3389/fphys.2012.00054
PMCID: PMC3314196  PMID: 22470345
alternative splicing; endothelial senescence; SRSF1; endoglin; progerin; VEGF; tissue factor
21.  The major reverse-transcriptase-incompetent splice variant of the human telomerase protein inhibits telomerase activity but protects from apoptosis 
Cancer research  2013;73(9):2817-2828.
hTERT (TERT), the catalytic protein subunit of telomerase, is subjected to numerous alternative splicing events, but the regulation and function of these splice variants is obscure. Full-length hTERT includes conserved domains that encode reverse transcriptase activity, RNA binding and other functions. The major splice variant termed α+β− or β-deletion is highly expressed in stem and cancer cells, where it codes for a truncated protein lacking most of the reverse transcriptase domain but retaining the known RNA binding motifs. In a breast cancer cell panel, we found that β-deletion was the hTERT transcript that was most highly expressed. Splicing of this transcript was controlled by the splice regulators SRSF11, HNRNPH2 and HNRNPL and the β-deletion transcript variant was associated with polyribosomes in cells. When ectopically overexpressed, β-deletion protein competed for binding to hTR (TERC) RNA, thereby inhibiting endogenous telomerase activity. Overexpressed β-deletion protein localized to the nucleus and mitochondria and it protected breast cancer cells from cisplatin-induced apoptosis. Our results reveal that a major hTERT splice variant can confer a growth advantage to cancer cells independent of telomere maintenance, suggesting hTERT makes multiple contributions to cancer pathophysiology.
doi:10.1158/0008-5472.CAN-12-3082
PMCID: PMC3643995  PMID: 23610451
Telomerase; hTERT; alternative splicing; apoptosis; breast cancer cells
22.  Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation 
PLoS ONE  2007;2(7):e615.
Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.
doi:10.1371/journal.pone.0000615
PMCID: PMC1910610  PMID: 17637831
23.  Heat Shock-Induced SRSF10 Dephosphorylation Displays Thermotolerance Mediated by Hsp27▿  
Molecular and Cellular Biology  2010;31(3):458-465.
Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this “thermotolerance” of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.
doi:10.1128/MCB.01123-10
PMCID: PMC3028621  PMID: 21135127
24.  Inhibition of eIF2α dephosphorylation inhibits ErbB2-induced deregulation of mammary acinar morphogenesis 
BMC Cell Biology  2009;10:64.
Background
The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in ~30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2α inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2α can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2α phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.
Results
We tested whether ErbB2 signaling influenced eIF2α signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt) Neu/ErbB2 or a constitutively active (CA) variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2α signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2α or induction of its downstream target ATF4. However, inhibition of eIF2α dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells.
Conclusion
Depending on the strength of ErbB2 signaling there is a differential regulation of CHOP and eIF2α phosphorylation. ErbB2 uncouples in basal conditions eIF2α phosphorylation from CHOP induction. However, this signal was restored by salubrinal treatment in Wt-ErbB2 expressing MCF10A cells as these DCIS-like structures underwent luminal clearing. In CA-ErbB2 structures apoptosis is not induced by salubrinal and instead a state of quiescence with reduced proliferation was achieved. Treatments that stabilize P-eIF2α levels may be effective in treating ErbB2 positive cancers without severely disrupting normal tissue function and structure.
doi:10.1186/1471-2121-10-64
PMCID: PMC2754445  PMID: 19754954
25.  DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer 
PLoS Medicine  2009;6(5):e1000068.
Ann Killary and colleagues describe a new gene that is genetically altered in breast tumors, and that may provide a new breast cancer prognostic marker.
Background
Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.
Methods and Findings
Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER−, PR−, HER-2−) of breast cancers with poor prognosis.
Conclusions
Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.
Editors' Summary
Background
Each year, more than one million women discover that they have breast cancer. This type of cancer begins when cells in the breast that line the milk-producing glands or the tubes that take the milk to the nipples (glandular and ductal epithelial cells, respectively) acquire genetic changes that allow them to grow uncontrollably and to move around the body (metastasize). The uncontrolled division leads to the formation of a lump that can be detected by mammography (a breast X-ray) or by manual breast examination. Breast cancer is treated by surgical removal of the lump or, if the cancer has started to spread, by removal of the whole breast (mastectomy). Surgery is usually followed by radiotherapy or chemotherapy. These “adjuvant” therapies are designed to kill any remaining cancer cells but can make patients very ill. Generally speaking, the outlook for women with breast cancer is good. In the US, for example, nearly 90% of affected women are still alive five years after their diagnosis.
Why Was This Study Done?
Although breast cancer is usually diagnosed in women in their 50s or 60s, some women develop breast cancer much earlier. In these women, the disease is often very aggressive. Compared to older women, young women with breast cancer have a lower overall survival rate and their cancer is more likely to recur locally or to metastasize. It would be useful to be able to recognize those younger women at the greatest risk of cancer recurrence so that they could be offered intensive surveillance and adjuvant therapy; those women at a lower risk could have gentler treatments. To achieve this type of “stratification,” the genetic changes that underlie breast cancer in young women need to be identified. In this study, the researchers discover a gene that is genetically altered (by mutations or deletion) in early-onset breast cancer and then investigate whether its expression can predict outcomes in women with this disease.
What Did the Researchers Do and Find?
The researchers used “suppression subtractive hybridization” to identify a new gene in a region of human Chromosome 1 where loss of heterozygosity (LOH; a genetic alteration associated with cancer development) frequently occurs. They called the gene DEAR1 (ductal epithelium-associated RING Chromosome 1) to indicate that it is expressed in ductal and glandular epithelial cells and encodes a “RING finger” protein (specifically, a subtype called a TRIM protein; RING finger proteins such as BRCA1 and BRCA2 have been implicated in early cancer development and in a large fraction of inherited breast cancers). DEAR1 expression was reduced or lost in several ductal carcinomas in situ (a local abnormality that can develop into breast cancer) and advanced breast cancers, the researchers report. Furthermore, many breast tumors carried DEAR1 missense mutations (genetic changes that interfere with the normal function of the DEAR1 protein) or had lost both copies of DEAR1 (the human genome contains two copies of most genes). To determine the function of DEAR1, the researchers replaced a normal copy of DEAR1 into a breast cancer cell that had a mutation in DEAR1. They then examined the growth of these genetically manipulated cells in special three-dimensional cultures. The breast cancer cells without DEAR1 grew rapidly without an organized structure while the breast cancer cells containing the introduced copy of DEAR1 formed structures that resembled normal breast acini (sac-like structures that secrete milk). In normal human mammary epithelial cells, the researchers silenced DEAR1 expression and also showed that without DEAR1, the normal mammary cells lost their ability to form proper acini. Finally, the researchers report that DEAR1 expression (detected “immunohistochemically”) was frequently lost in women who had had early-onset breast cancer and that the loss of DEAR1 expression correlated with reduced local recurrence-free survival, a strong family history of breast cancer and with a breast cancer subtype that has a poor outcome.
What Do These Findings Mean?
These findings indicate that genetic alteration and loss of expression of DEAR1 are common in breast cancer. Although laboratory experiments may not necessarily reflect what happens in people, the results from the three-dimensional culture of breast epithelial cells suggest that DEAR1 may regulate the normal acinar structure of the breast. Consequently, loss of DEAR1 expression could be an early event in breast cancer development. Most importantly, the correlation between DEAR1 expression and both local recurrence in early-onset breast cancer and a breast cancer subtype with a poor outcome suggests that it might be possible to use DEAR1 expression to identify women with early-onset breast cancer who have an increased risk of local recurrence so that they get the most appropriate treatment for their cancer.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000068.
This study is further discussed in a PLoS Medicine Perspective by Senthil Muthuswamy
The US National Cancer Institute provides detailed information for patients and health professionals on all aspects of breast cancer, including information on genetic alterations in breast cancer (in English and Spanish)
The MedlinePlus Encyclopedia provides information for patients about breast cancer; MedlinePlus also provides links to many other breast cancer resources (in English and Spanish)
The UK charities Cancerbackup (now merged with MacMillan Cancer Support) and Cancer Research UK also provide detailed information about breast cancer
doi:10.1371/journal.pmed.1000068
PMCID: PMC2673042  PMID: 19536326

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