The RNA-Binding Protein Rbfox2: an Essential Regulator of EMT-Driven Alternative Splicing and a Mediator of Cellular Invasion

ORIGINAL ARTICLE The RNA-binding protein Rbfox2: an essential regulator of EMT-driven alternative splicing and a mediator of cellular invasion

C Braeutigam1,4, L Rago1,4, A Rolke1, L Waldmeier2, G Christofori2 and J Winter1,3

The epithelial–mesenchymal transition (EMT), a prerequisite for cancer progression and metastasis formation, is regulated not only at the transcriptional but also at the post-transcriptional level, including at the level of alternative pre-mRNA splicing. Several recent studies have highlighted the involvement of splicing factors, including epithelial splicing regulatory proteins (Esrps) and RNA- binding Fox protein 2 (Rbfox2), in this process. Esrps regulate epithelial-specific splicing, and their expression is downregulated during EMT. By contrast, the role of Rbfox2 is controversial because Rbfox2 regulates epithelial as well as mesenchymal splicing events. Here, we have used several established cell culture models to investigate the functions of Rbfox2 during EMT. We demonstrate that induction of an EMT upregulates the expression of Rbfox2, which correlates with an increase in Rbfox2-regulated splicing events in the cortactin (Cttn), Pard3 and dynamin 2 (Dnm2) transcripts. At the same time, however, the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is either reduced or lost completely by Rbfox2, which might be due, in part, to downregulation of the expression of the Esrps cooperative factors. Depletion of Rbfox2 during EMT did not prevent the activation of transforming growth factor-b signaling, the upregulation of mesenchymal markers or changes in cell morphology toward a mesenchymal phenotype. In addition, this depletion did not influence cell migration. However, depletion of Rbfox2 in cells that have completed an EMT significantly reduced their invasive potential. Taken together, our results suggest that during an EMT, Rbfox2-regulated splicing shifts from epithelial-to mesenchymal-specific events, leading to a higher degree of tissue invasiveness.

Oncogene (2014) 33, 1082–1092; doi:10.1038/onc.2013.50; published online 25 February 2013 Keywords: Rbfox2; epithelial–mesenchymal transition; EMT; alternative splicing

INTRODUCTION EMT-associated splicing is most likely regulated by several During epithelial–mesenchymal transition (EMT), cancer cells splicing factors, including Rbfox2, Esrps, Celf, Mbnl, hnRNPs, 15–17 originating from a primary carcinoma of epithelial origin gain Sam68 and SF2/ASF. Epithelial splicing regulatory proteins 1 migratory and invasive capabilities and acquire a mesenchymal and 2 (Esrp1 and Esrp2) are important regulators of epithelial- morphology.1,2 It is thought that EMT promotes invasion and specific splicing choices and are downregulated during the EMT. metastases; therefore, understanding the molecular basis of an Of note, siRNA-mediated depletion of Esrps in epithelial cells 18 EMT is crucial for developing effective cancer therapies. Although causes EMT-like morphological changes. The alternative splicing it was initially thought that an EMT is regulated mainly at the of a subset of Esrp target mRNAs is co-regulated by Rbfox2. Thus, transcriptional level, it is now widely accepted that post- both Esrps and Rbfox2 are essential regulators for epithelial- transcriptional regulation, including alternative pre-mRNA specific splicing of fibroblast growth factor receptor 2 and splicing, has an important role as well.3 Enah.4,19,20 In contrast to the Esrps, the role of Rbfox2 during The RNA-binding Fox (Rbfox) proteins (Rbfox1, Rbfox2 and the EMT is less well understood. Despite its established Rbfox3) are regulators of alternative splicing. All three proteins importance for epithelial-specific splicing of the targets contain a central RNA-recognition motif that recognizes the mentioned above, Rbfox2 expression was found to increase consensus sequence (U)GCAUG in the introns that flank target moderately following EMT in an established in vitro model, exons. Rbfox proteins promote exon skipping when they bind suggesting an active function during an EMT. In agreement with upstream of the alternative exon but inclusion when they bind this hypothesis, depletion of Rbfox2 in mesenchymal cells leads to downstream of this exon.4–10 Although all Rbfox proteins bind to a partial reversion toward an epithelial phenotype in the same the (U)GCAUG sequence, because of their differing expression model. Moreover, numerous EMT-regulated skipped exons have patterns these proteins are only partially redundant in vivo. Rbfox1 been found to overlap with Rbfox2 CLIP-Seq clusters in human cell is expressed in the brain, heart and ovaries, and Rbfox3 is lines.15 Thus, although it seems clear that Rbfox2 promotes expressed exclusively in the brain. By contrast, Rbfox2 is more epithelial-specific splicing in some cases, it appears to promote widely expressed and is the only member of the Rbfox family that mesenchymal-specific splicing in others. is found in mammary cell lines as well as in normal and breast In our study, we have used two transforming growth factor-b cancer tissue.11–14 (TGFb)-inducible in vitro models to study, in detail, the role of

1Department of Molecular Embryology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; 2Department of Biomedicine, University of Basel, Basel, Switzerland and 3Institute of Human Genetics, University Medical Center of the Johannes Gutenberg, University of Mainz, Mainz, Germany. Correspondence: Dr J Winter, Department of Molecular Embryology, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, Freiburg 79108, Germany. E-mail: [email protected] 4These authors contributed equally to this work. Received 26 June 2012; revised 18 January 2013; accepted 21 January 2013; published online 25 February 2013 Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1083 Rbfox2 during an EMT. The expression of Rbfox2 increased RESULTS moderately following induction of the EMT and Rbfox2 regulated Two different Rbfox2 isoforms are expressed during EMT EMT-associated splicing. Rbfox2-depleted cells underwent an EMT To study the functions of Rbfox2 in the TGFb-dependent EMT, we with no obvious morphological differences from the control cells. used two mouse cell lines, which both underwent a complete EMT Yet, cell invasion was signiﬁcantly reduced in Rbfox2-depleted upon treatment with TGFb21–23 (Figure 1a). We used the well- tumor cells, suggesting that Rbfox2 is essential for epithelial cells characterized non-transformed mammary epithelial cell line to gain invasive properties during an EMT. NMuMG and an epithelial murine breast cancer cell line, PY2T,

Figure 1. Rbfox2 expression in TGFb-dependent EMT-models: (a) morphological changes of NMuMG and PY2T cells cultured in the presence of 2 ng/mlTGFb1 for 3 days. (b) Real time RT–PCR assays of Rbfox2 expression in NMuMG and PY2T cell lines at different time points after induction of an EMT by treatment with 2 ng/mlTGFb1. Total RNA was isolated from NMuMG and PY2T cells as indicated and was reversed transcribed to cDNA using random hexamer oligos. The expression of Rbfox2 was measured by real time RT–PCR, and mRNA expression was normalized to Tbp expression. (c) Immunofluorescence analyses of Rbfox2 subcellular localization in NMuMG and PY2T cell lines before and after induction of the EMT by TGFb-treatment. NMuMG and PY2T cells were treated with or without 2 ng/mlTGFb1 for 3 days and subjected to immunofluorescence analyses using a Rbfox2-specific antibody. As a control Rbfox2-specific siRNAs were transfected. (d–g) Identification of Rbfox2 isoforms expressed in NMuMG and PY2T cells. (d) Odyssey Immunoblotting analysis of Rbfox2 in different cell lines before and after induction of an EMT. Cells were lysed at the indicated time points and used for immunoblotting analysis using an antibody that detects all known Rbfox2 isoforms. b-actin was used as the loading control. Upper panel, representative immunoblot; lower panel, quantification of three independent experiments. (e) Arrangement of alternative first exons in the Rbfox2 gene. Solid bars, exons. Lines between exons, introns. There are four alternative first exons in the Rbfox2 gene (1A, 1D, 1E, 1F). Arrows, the primer pairs used in the RT–PCR experiments. (f) RT–PCR assay of Rbfox2 isoforms expressed in NMuMG and PY2T cells. Total RNA was isolated from NMuMG and PY2T cells as indicated and was reversed transcribed to cDNA using random hexamer oligos. The cDNA was then amplified by PCR using primers, as indicated in panel (e), and the products were resolved in 1% agarose gels. Subsequently, the products were cloned and sequenced. Asterisks, unspecific products. (g) Forty- eight hours after transfection with the indicated siRNAs, cells were lysed and subjected to immunoblotting analysis using an antibody that detects all known Rbfox2 isoforms; *Po0.05; ***Po0.001.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1082 – 1092 Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1084 which was established from a mammary tumor of a Polyoma middle-T transgenic mouse.24 In both the cell lines, Rbfox2 expression increased moderately, by 1.5–2-fold, following induction of an EMT by TGFb treatment, suggesting an active function of this protein during EMT (Figure 1b). Of note, upon removal of TGFb Rbfox2 expression decreased back to the level of untreated cells, indicating that this expression change was reversible (Supplementary Figure S1A). Immunofluorescence experiments using a commercially available anti-Rbfox2 antibody revealed that Rbfox2 localization was mainly restricted to the nucleus in both NMuMG and PY2T cells and that this localization did not change after induction of an EMT, which is in accordance with the known function of Rbfox2 as an alternative splicing factor (Figure 1c). The transcription of the Rbfox2 gene is initiated from several alternative promoters, producing Rbfox2 isoforms that differ in their N-terminal sequences. Although the functional differences of the different isoforms are poorly understood, it has been suggested that different isoforms may regulate alternative splicing with varying efficiencies.10 Moreover, dominant-negative Rbfox2 isoforms lacking part of the RNA recognition motif have been described.25 Therefore, a hypothetical switch in the production of Rbfox2 isoforms during an EMT could lead to a shift in alternative splicing of target exons. Immunoblotting analysis using an Rbfox2- specific antibody detected two Rbfox2 isoforms in lysates of both NMuMG and PY2T cell lines before and after induction of an EMT (Figure 1d). Quantification of the two isoforms revealed that the increase in Rbfox2 expression after the induction of an EMT was mainly due to an increase in the smaller Rbfox2 isoform (Figure 1d). To identify these isoforms, we carried out isoform- specific reverse transcriptase–PCR (RT–PCR) by combining forward primers located in each of the four alternative first exons of the Rbfox2 gene with a reverse primer located near the 30 end (Figure 1e). Although strong products appeared when forward primers corresponding to first exons 1A and 1F were used, primers located in first exons 1D and 1E did not reveal any products in the RT–PCR experiments (Figure 1f). To test for the presence of dominant-negative Rbfox2 isoforms, we performed RT–PCR experiments before and after induction of an EMT by TGFb treatment of NMuMG and PY2T cells. Remarkably, the skipping of exon 6, the splicing event leading to the production of dominant- negative Rbfox2 isoforms, was not detected in NMuMG cells before or after induction of an EMT in 21 and 25 sequenced clones, respectively. It was also not detected in PY2T cells after induction of an EMT in 17 sequenced clones, and it was detected in only 1 out of 16 sequenced clones in PY2T cells before induction of an EMT (data not shown). These results indicate that the skipping of exon 6 is a rare event in our EMT models, which should not influence Rbfox2 activity. To further confirm that the Rbfox2 isoforms identified by immunoblotting analysis were produced from transcripts containing Rbfox2 exons 1A or 1F, siRNAs specifically targeting each of these exons were transfected into NMuMG cells. Immunoblotting analysis of the obtained lysates showed the disappearance of the expected isoforms when compared with a control knockdown or a knockdown of all Rbfox2 isoforms (Figure 1g). The increase of isoform 1F and the decrease of isoform 1A during EMT could lead to a switch in the splicing of specific Rbfox2 targets. Hypothetically, the isoform 1A could mediate epithelial splicing events and isoform 1F could promote mesenchymal Figure 2. Alternative splicing of epithelial-specific Rbfox2 targets in splicing events. To test whether the differential expression of the TGFb-dependent EMT. (a–c) Alternative splicing of Enah exon 11a, two isoforms was controlled by transcriptional start site selection, Slk exon 13 and Tsc2 exon 25 at different time points after induction of the EMT in NMuMG and PY2T cells. EMT was induced by adding we carried out quantitative RT–PCR experiments with primers 2 ng/mlofTGFb1 to the culture medium. Schematics of all splicing specifically detecting isoforms 1A or 1F. In NMuMG cells, isoform events are shown. Solid bars, exons. Thin lines between exons, 1A was indeed significantly downregulated and isoform 1F was introns. Isoform-specific primers were designed to correspond either significantly upregulated at the RNA level 3 days and 4–5 weeks entirely to the respective alternatively spliced exons or to exon– after induction of an EMT, suggesting that in this cell line a switch exon junctions. Upper and lower panels show expression and in isoform expression was mediated at the transcriptional level splicing changes, respectively; *Po0.05; **Po0.01; ***Po0.001.

Oncogene (2014) 1082 – 1092 & 2014 Macmillan Publishers Limited Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1085 (Supplementary Figure S2A). In PY2T cells, however, this was not (Slk) and tuberous sclerosis protein 2 (Tsc2) transcripts, respec- the case. Rather, both isoforms were upregulated after induction of tively, are included in epithelial cells and skipped in mesenchymal an EMT (Supplementary Figure S2A). To test whether the decrease cells.15 To test whether the presence of Rbfox2 could prevent in isoform 1A expression that we had observed in NMuMG cells was mesenchymal-specific splicing in our models following TGFb a cell-specific effect, we analyzed the expression of Rbfox2 isoforms treatment, we studied the splicing changes in these target exons. in another EMT model. MTdeltaEcad cells were generated by Thus, we developed an exon-specific quantitative RT–PCR assay establishing a cell line from the mammary tumors of MMTV-Neu using primers that either specifically anneal to the alternative transgenic mice, in which both E-cadherin alleles were flanked by exons or span the exon–exon junctions (Supplementary Table S2). LoxP recombination sites (MTflEcad). MTdeltaEcad cells were In contrast to NMuMG cells, PY2T cells upregulated Enah generated from this cell line by expressing Cre recombinase, expression dramatically 4–5 weeks following TGFb treatment. thereby leading to genetic ablation of E-cadherin, which was Interestingly, the presence of Rbfox2 could not prevent an followed by an upregulation of mesenchymal markers and a increased skipping of exon 11a of the Enah transcript in these change in cell morphology toward a fibroblastic appearance.23 cells, which was most evident 4–5 weeks following TGFb Expression of both Rbfox2 isoforms increased in the mesenchymal treatment (Figure 2a). Consistently, the presence of Rbfox2 could MTdelEcad cells at both RNA and protein levels when compared also not prevent an increased skipping of exon 13 of the Slk with the epithelial MTflEcad cells (Supplementary Figure S2A and B). transcript following TGFb treatment in NMuMG and PY2T cells and These results suggest that a switch in Rbfox2 isoform expression of exon 25 of the Tsc2 transcript following TGFb treatment in PY2T might not be a general hallmark of an EMT. Rather, it might cells (Figures 2b and c). All these splicing changes reversed back be a cell type-specific event or be important early after EMT to levels of untreated cells upon removal of TGFb, however, the induction. A further analysis of putative isoform-specific functions kinetics of this reversal varied between the different exons turned out to be technically challenging because transfection of (Supplementary Figure S1B). To test whether Rbfox2 had lost siRNAs targeting the isoform-specific regions caused various activity towards these targets in TGFb-treated cells, we transfected off-target effects. Rbfox2 siRNAs into NMuMG and PY2T cells before and after 3 days and after 4–5 weeks of TGFb treatment. Knock down of Rbfox2 in NMuMG cells led to a decrease of Enah exon 11a inclusion at all The presence of Rbfox2 does not prevent mesenchymal-specific time points tested, whereas in PY2T cells, knock down of Rbfox2 splicing in the Enah, Slk and Tsc2 transcripts only affected the splicing of Enah exon 11a before and after 3 days Rbfox2 promotes epithelial-specific splicing in a number of but not after 4–5 weeks of TGFb treatment (Figure 3a). The loss of transcripts. Exons 11a, 13 and 25 of the Enah, STE20-like kinase activity of Rbfox2 toward splicing of exon 11a of the Enah

Figure 3. Alternative splicing of epithelial-speciﬁc Rbfox2 targets in Rbfox2-depleted cells. (a) Rbfox2-dependent inclusion of exon 11a of the Enah mRNA at different time points after induction of the EMT. The cells were treated with 2 ng/mlofTGFb1 for 0 or 3 days or 4–5 weeks and then transfected with the indicated siRNAs. Forty-eight hours after transfection, total RNA was isolated, and cDNA synthesis and real time PCR were performed. (b) Rbfox2-dependent inclusion of the exon 13 of the Slk mRNA. The cells were treated and processed as described in panel (a). (c) Rbfox2-dependent inclusion of exon 25 of the Tsc2 mRNA. The cells were treated and processed as described in panel (a); *Po0.05; **Po0.01; ***Po0.001.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1082 – 1092 Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1086 transcript in PY2T cells 4–5 weeks after TGFb treatment suggests that Rbfox2, although necessary, is not sufficient to regulate this splicing event. In contrast to splicing of Enah exon 11a, knock down of Rbfox2 in PY2T cells led to a further decrease of alternative exon inclusion of the Slk and Tsc2 transcripts even after 4–5 weeks following TGFb treatment (Figures 3b and c). This indicates that, although Rbfox2 retains activity towards these targets, it cannot prevent an increased skipping of these exons following TGFb treatment. Other splicing factors that act in a cooperative or antagonistic fashion may co-regulate splicing of the epithelial-specific exons in the Enah, Slk and Tsc2 transcripts. In agreement with this suggestion, it has been shown that the splicing of exons 11a, 13 and 25 of the Enah, Slk and Tsc2 transcripts, respectively, is not only controlled by the binding of Rbfox2 to conserved UGCAUG motifs but also by the binding of Esrp1 and Esrp2 to UGG-rich motifs. Hence, a cooperative function of Rbfox2 and Esrps in the splicing of these exons has been proposed.18 We found that although NMuMG cells expressed exclusively Esrp2 (Supplementary Figure S4B26), PY2T cells expressed both Esrp1 and Esrp2 before induction of an EMT. Of note, 4–5 weeks after induction of an EMT, PY2T cells displayed dramatically downregulated expression of both Esrps, whereas NMuMG cells still expressed approximately 50% of the level of Esrp2 compared with untreated cells (Supplementary Figure S4A and B). These results suggest that Esrps may act cooperatively with Rbfox2 in TGFb-treated NMuMG cells. However, other, as yet to be identified, factors may, in addition, co-regulate the inclusion or skipping of the epithelial-specific exons.

Rbfox2 regulates EMT-associated alternative splicing An increase in Rbfox2 expression during the EMT should be reflected by an upregulation of Rbfox2-regulated splicing choices. We chose three different Rbfox2 target exons as model systems to study Rbfox2-regulated splicing during EMT. Several studies have shown that Rbfox2 regulates the skipping of exon 18 and a 12-nt exon in Pard3 and Dnm2 gene expression, respectively, as well as the inclusion of exon 11 in Cttn gene expression.4,7 Although the functional consequences of the alternative splicing events of the Pard3 and Cttn mRNAs are unclear, the 12-nt exon of the Dnm2 transcript that is skipped after the induction of EMT has been functionally linked to the localization and functioning of the GTPase Dnm2 at the GOLGI apparatus. How this affects the induction or progression of the EMT, however, remains unclear. The skipping of exon 18 and of the 12-nt exon of the Pard3 and Dnm2 transcripts, respectively, was enhanced in NMuMG and PY2T cells 3 days and 4–5 weeks following TGFb treatment (Supplementary Figure S4A and B). Cttn exon 11 inclusion was increased in NMuMG cells 3 days and 4–5 weeks after induction of an EMT and in PY2T cells 4–5 weeks after induction of an EMT (Figure 4c). All splicing changes reversed back to the levels of untreated cells upon removal of TGFb; however, the kinetics of this reversal varied between the different exons (Supplementary FigureS 1B). Conversely, the skipping of exon 18 and of the 12-nt exon of the Pard3 and Dnm2 transcripts, respectively, and the inclusion of Cttn exon 11 was reduced significantly in NMuMG and PY2T cells transfected with Rbfox2 siRNAs at all time points analyzed (Figures 5a–c), indicating that Rbfox2 is an important regulator of EMT-driven alternative Figure 4. Alternative splicing of mesenchymal-specific Rbfox2 targets splicing. To exclude the possibility of promiscuous effects on in TGFb-dependent EMT. (a–c) Alternative splicing of Pard3 exon 18, alternative splicing in Rbfox2 knockdown cells, we analyzed the Dnm2 12-nt exon and Cttn exon 11 at different time points after splicing of exons v8–v10 in the CD44 transcript, which is induction of the EMT in NMuMG and PY2T cells. EMT was induced by not a described Rbfox2 target. Although the skipping of exons adding 2 ng/mlofTGFb1 to the culture medium. Schematics of all v8–v10 was enhanced in both the NMuMG and PY2T cells splicing events are shown. Solid bars, exons. Thin lines between exons, introns. Isoform-specific primers were designed to correspond following TGFb treatment, the knock down of Rbfox2 did not alter either entirely to the respective alternatively spliced exons or to the splicing of these exons in any of the two cell lines exon–exon junctions. Upper and lower panels show expression and (Supplementary Figure S3A and B). splicing changes, respectively; *Po0.05; **Po0.01; ***Po0.001.

Oncogene (2014) 1082 – 1092 & 2014 Macmillan Publishers Limited Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1087

Figure 5. Alternative splicing of mesenchymal-speciﬁc Rbfox2 targets in Rbfox2-depleted cells. (a) Rbfox2-dependent skipping of exon 18 of the Pard3 mRNA at different time points after induction of the EMT. The cells were treated with 2 ng/mlofTGFb1 for 0 or 3 days or 4–5 weeks and then transfected with the indicated siRNAs. Forty-eight hours after transfection, total RNA was isolated, and cDNA synthesis and real time PCR were performed. (b) Rbfox2-dependent skipping of the 12-nt exon of the Dnm2 mRNA. The cells were treated and processed as described in panel (a). (c) Rbfox2-dependent inclusion of exon 11 of the Cttn mRNA. The cells were treated and processed as described in panel (a); *Po0.05; **Po0.01; ***Po0.001.

Depletion of Rbfox2 does not prevent morphological changes suggest that Rbfox2 is not essential for cells to acquire a during the EMT mesenchymal phenotype after induction of an EMT. Upon TGFb treatment, NMuMG and PY2T cells undergo drastic morphological changes and upregulate mesenchymal markers, Depletion of Rbfox2 leads to reduced invasion such as N-cadherin and vimentin. Important consequences of EMT include altered cell migration and To test whether Rbfox2 expression was essential for acquiring a invasion. To assess the effect of Rbfox2 depletion on cell migration mesenchymal phenotype, we depleted Rbfox2 from both the cell and invasion, we transfected control and Rbfox2 siRNAs into lines by transfection of specific siRNAs. Then, 48 h after NMuMG and PY2T cells that had been exposed to long-term TGFb transfection, an EMT was induced by TGFb treatment. Immuno- treatment (4–5 weeks) and thus had completed an EMT blotting analysis revealed efficient knock down of Rbfox2 (Figure 7a). Forty-eight hours after transfection, the cells were under these conditions (Figure 6a). Several observations indicated used for morphological analysis and for in vitro migration and that cells transfected with Rbfox2 siRNA underwent an EMT. Both invasion assays. Morphologically, Rbfox2-depleted cells were Rbfox2-depleted cells and cells treated with control siRNAs indistinguishable from control siRNA-transfected cells upregulated Phospho-Smad2 1 h after TGFb treatment, indicating (Supplementary Figure S5A and B; data not shown). Two that TGFb signaling could be activated in these cells (Figure 6b). approaches were used to study migration: wound-healing assays Moreover, the upregulation of several EMT markers was unaf- and transwell migration assays (Figures 7b and c; data not shown). fected by the depletion of Rbfox2, as evidenced by western In neither of the two approaches could we detect a difference in blot analyses using specific antibodies. No difference was the migration of NMuMG or PY2T cells between the control detected in the expression of N-cadherin and vimentin in Rbfox2 knockdown and Rbfox2 knockdown cells, suggesting that Rbfox2 knockdown cells when compared with control knockdown cells depletion does not impair the migratory behavior of cells that (Figures 6c and d). Morphologically, Rbfox2-depleted cells were have undergone an EMT. indistinguishable from control siRNA-transfected cells and To assess the effect of Rbfox2 depletion on cell invasion, we acquired a spindle-shaped fibroblastic morphology upon TGFb used an invasion assay in Matrigel-coated transwell chambers. We treatment. Both control knockdown and Rbfox2 knockdown cells found no differences in cell invasion by NMuMG cells between the formed stress fibers and focal adhesions, as evidenced by control siRNA and Rbfox2 siRNA-transfected cells (Figure 7d). phalloidin and vinculin staining of NMuMG and PY2T cells 48 h Interestingly, PY2T cells depleted of Rbfox2 showed significantly after TGFb treatment (Figures 6e and f). In summary, our results reduced invasion compared with the control knockdown cells

& 2014 Macmillan Publishers Limited Oncogene (2014) 1082 – 1092 Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1088 (Figure 7e), indicating that Rbfox2-dependent splicing is important for PY2T cells to maintain an invasive phenotype. To test whether the reduced invasion after Rbfox2 knock down was specific to PY2T cells or was a general phenomenon, we repeated the invasion assays with MTdeltaEcad cells. Immunoblotting analysis revealed efficient knock down of Rbfox2 in these cells (Figure 7a). As observed in PY2T cells, Rbfox2 depletion reduced the invasiveness of MTdeltaEcad cells significantly, indicating that Rbfox2 regulates invasiveness in different experimental systems (Figure 7f).

Rbfox2 targets promote the invasive phenotype Aberrant splicing of mesenchymal-specific Rbfox2 targets might be causative for the reduced invasiveness of Rbfox2 knockdown cells. To identify Rbfox2 targets with known functions during invasion, we screened the available data sets of EMT-associated splicing.15 This screen identified 28 transcripts whose splicing patterns changed during EMT and whose protein products have a described function during invasion. Thirteen of the 28 transcripts contained conserved Rbfox2-binding sites in their introns or exons (Table 1). Of note, this list of 13 transcripts included splicing changes in the mesenchymal-specific Rbfox2 target Dnm2 and the epithelial-specific Rbfox2 targets Enah, Slk and Tsc2, which we had already verified in our models in the quantitative RT–PCR assay (Figures 2b, 3b, 4a–c and 5a–c). In addition, the mesenchymal- specific Rbfox2 targets Pard3 and Cttn, which were not included in the data set but identified in our quantitative RT–PCR assay, are well-known regulators of invasion (Table 127,28). In summary, 6 of the overall 15 transcripts with conserved Rbfox2-binding sites were mesenchymal-specific Rbfox2 targets (Table 1).

DISCUSSION We have used several cellular systems to study the functions of the alternative splicing factor Rbfox2 during EMT. We show that induction of an EMT is associated with the increased expression of Rbfox2 in all the cell lines tested, which correlates with the upregulation of Rbfox2-regulated splicing events in the Pard3, Dnm2 and Cttn transcripts, whereas the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is reduced or lost completely by Rbfox2. Notably, although depletion of Rbfox2 before the onset of an EMT does not inhibit this process, the depletion of Rbfox2 after cells have completed an EMT significantly reduces the invasiveness of these cells. The moderate upregulation of Rbfox2 expression that we observed after induction of an EMT is in agreement with a recently 15 Figure 6. NMuMG and PY2T cells undergo an EMT in the absence of published study by Shapiro et al., which showed that the Rbfox2. (a) Efficient Rbfox2 depletion in NMuMG and PY2T cells using expression of Rbfox2 increases by approximately 15% following Rbfox2-specific siRNAs. Forty-eight hours after the transfection of the EMT in a mammary epithelial cell line that undergoes an EMT specific siRNAs, cells were lysed and subjected to immunoblotting after the induction of Twist expression by tamoxifen treatment. analysis using a Rbfox2-specific antibody. Antibodies detecting However, as we can further show, the sole increase in Rbfox2 Gapdh (glyceraldehyde 3-phosphate dehydrogenase) or Actin were expression is not always predictive of an increase in Rbfox2- used as loading controls. (b) Activation of the TGFb-pathway in the absence of Rbfox2. Forty-eight hours after the transfection of specific regulated splicing events. Thus, Rbfox2-dependent epithelial- siRNAs, NMuMG and PY2T cells were treated with 2 ng/mlTGFb1for specific splicing of the Enah and Slk transcripts did not increase 1 h. Then, the cells were lysed, and western blot analyses were in NMuMG cells, and Rbfox2-dependent splicing of the Enah, Slk performed with antibodies specifically detecting Smad2/3 or and Tsc2 transcripts was completely abolished or reduced in PY2T phospho-Smad2. (c, d) Upregulation of N-cadherin (Ncad) and cells. Skipping exons 11a, 13 and 25 of the Enah, Slk and Tsc2 vimentin. Forty-eight hours after transfection with specific siRNAs, transcripts, respectively, during EMT has been observed in other NMuMG or PY2T cells were treated with 2 ng/mlTGFb1 for another models as well.15,19 Although these splicing events are certainly 48 h. Then, the cells were lysed, and immunoblotting analysis were regulated by Rbfox2, other splicing factors that act cooperatively performed using antibodies specifically detecting Ncad (c)or may be equally important. The splicing of exon 11a of the Enah vimentin (d). (e, f) Rbfox2-depleted cells form stress fibers and focal adhesions. NMuMG and PY2T cells plated on coverslips were treated transcript is controlled by a conserved sequence located as described in panel (c) and processed for immunofluorescence approximately 1.8 kb downstream of this exon. Similar to the analysis using an Rbfox2-specific antibody to control for knockdown Rbfox2-binding site UGCAUG, this sequence contains UGG-rich efficiency, a vinculin-specific antibody to analyze the formation of motifs, which are bound by the epithelial-specific splicing factors focal adhesions and Phalloidin488 to visualize actin stress fibers. Esrps to promote the inclusion of exon 11a. Also, inclusion of Untreated cells are shown for comparison. exons 13 and 25 of the Slk and Tsc2 transcripts, respectively, is

Oncogene (2014) 1082 – 1092 & 2014 Macmillan Publishers Limited Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1089

Figure 7. Rbfox2 depletion inhibits invasion but not migration in PY2T and MTdeltaEcad cells. (a) Efficient Rbfox2 depletion in NMuMG, PY2T and MTdeltaEcad cells using Rbfox2-specific siRNAs. NMuMG or PY2T cells were treated with 2 ng/mlTGFb1 for 4–5 weeks and then transfected with control or Rbfox2-specific siRNAs. Forty-eight hours after the transfection of specific siRNAs, cells were lysed and subjected to immunoblotting analysis using a Rbfox2-specific antibody. An antibody detecting Actin was used as loading control. (b,c) Scratch wound closure assay. NMuMG (b) or PY2T cells (c) were treated with 2 ng/mlTGFb1 for 4–5 weeks and then transfected with control or Rbfox2-specific siRNAs. Forty-eight hours after transfection, the cells were seeded into the wells of culture inserts (Ibidi) at a density of 6 Â 105 cells/ml. Twenty-four hours after seeding, the culture inserts were removed. Microphotographs were taken at 0, 6 and 24 h. (d–f) Invasion assays. NMuMG (d) or PY2T cells (e) were treated with 2 ng/mlTGFb1 for 4–5 weeks. Then, NMuMG, PY2T or MTdeltaEcad cells (f) were transfected with control or Rbfox2-specific siRNAs. Forty-eight hours after transfection, transwell invasion assays were carried out over 24 h; *Po0.05; **Po0.01. promoted by Esrps.18 In contrast to Rbfox2, the expression of the of exon 13 of the Slk transcript had already decreased at this time Esrps, that is, Esrp1 and Esrp2, has been reported to be point. In NMuMG cells, inclusion of Enah exon 11a did not downregulated upon induction of an EMT.18 Although the decrease at any time point following TGFb treatment and expression of both Esrps strongly decreased in PY2T cells that inclusion of Tsc2 exon 25 even increased 3 days following TGFb had completed an EMT, the expression of Esrp2, the only treatment. By contrast, inclusion of Slk exon 13 decreased strongly expressed Esrp in NMuMG cells, decreased by approximately after the induction of an EMT in NMuMG cells. Changes in 50%. The decrease in expression of Esrps correlated with the expression of other, as yet to be identified, splicing factors that decrease in alternative exon inclusion in the Enah, Slk and Tsc2 specifically regulate inclusion or skipping of the respective exons transcripts 4–5 weeks following TGFb treatment in PY2T cells, may account for these differences. suggesting that both Rbfox2 and Esrps are necessary, but not In contrast to the epithelial-specific splicing choices, Rbfox2- sufficient, to regulate these splicing events. In addition, other dependent mesenchymal-specific splicing of the Pard3, Dnm2 and splicing factors might be important as well. Three days after Cttn transcripts increased following the induction of an EMT and induction of an EMT in PY2T cells, Esrps were still expressed at the decreased in Rbfox2 knockdown cells. These results indicate that same levels as in untreated cells. However, the inclusion of exons Rbfox2 is an important regulator of mesenchymal-specific splicing 11a and 25 of the Enah and Tsc2 transcripts, respectively, but not choices during EMT.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1082 – 1092 Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1090 In summary, our results indicate that Rbfox2 is important in Table 1. Rbfox2 targets involved in invasion establishing the invasive phenotype through its regulation of Gene Conserved Rbfox2-regulated Ref. mesenchymal-speciﬁc splicing events during an EMT. Further Rbfox2-binding sites splicing studies are needed to clarify the role of Rbfox2 during tumor progression in humans and to evaluate Rbfox2 as a potential Dnm2 2 up Mesenchymal 35–37 target for cancer therapy. Etv1 1 up Mesenchymal 38 Fat 1 down Mesenchymal 39,40 Numb 1 up Mesenchymal 41 Cttn 2 down Mesenchymal 27 MATERIALS AND METHODS Pard3 3 up Mesenchymal 28 Cell culture 33,42 Enah 3 down Epithelial NMuMG, PY2T and MTdeltaEcad cells were cultured in Dulbecco’s modiﬁed Mbnl1 1up; 2 down Epithelial 43 44,45 Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, Scrib 1 up Epithelial penicillin, streptomycin, glutamine and non-essential amino acids and, in Slk 3 up Epithelial 46 47,48 the case of NMuMG cells, insulin. TGFb treatment was performed using Tsc2 2 down Epithelial 2 ng/ml of TGFb1 (PeproTech, Rocky Hill, NJ, USA; R&D Systems, Fgfr1 1 exon; 1 downstream Epithelial 49,50 51,52 Minneapolis, MN, USA) and TGFb was replenished every 3 days. Rbfox2 Fgfr2 1 up; 1 exon; 1 down Epithelial siRNAs were purchased from Dharmacon (Lafayette, CO, USA) and Qiagen Stx2 1 exon Unknown 53 54 (Hilden, Germany) (Supplementary Table S1). Transfections using Trans- Dock9 1 exon Unknown fectin (Bio-Rad, Mu¨nchen, Germany) were carried out according to the Abbreviation: Rbfox2, RNA-binding Fox protein 2. manufacturer’s instructions.

Antibodies, immunoblotting and immunofluorescence Although Shapiro et al.15 found a partial reversion toward an Cells were lysed by the addition of Magic-Mix (48% urea, 15 mM Tris-HCl epithelial phenotype when they stably transfected Rbfox2-specific (pH 7.5), 8.7% glycerin, 1% sodium dodecyl sulfate, 0.004% bromophenol shRNAs, we could not detect such a phenotypic change in any of blue, 143 mM mercaptoethanol), centrifuged through Qia Shredder Columns (Qiagen) and boiled for 5 min at 95 1C. Thirty micrograms of the cell lines that had been depleted of Rbfox2 after an EMT was total protein from each sample were resolved on 10% SDS–PAGE (sodium completed. Different experimental approaches used in the two dodecyl sulfate-polyacrylamide gel electrophoresis) gels and blotted onto studies may be one reason for this. In addition, cell type-specific PVDF membranes. For immunoblotting using the Odyssey system, the gels changes in splicing patterns after depletion of Rbfox2 may were blotted and scanned according to the manufacturer’s instructions. account for the observed phenotypic differences. Despite no Immunofluorescence analyses were performed as described previously.34 obvious differences in cell morphology, cell invasion was The antibodies used in this study were anti-Rbm9 (Bethyl, Montgomery, TX, significantly reduced when Rbfox2 was depleted from PY2T and USA), anti-Vinculin (Sigma-Aldrich, Mu¨nchen, Germany), anti-Ncadherin MTdeltaEcad cells but was not reduced when Rbfox2 was (BD Transduction, Lexington, KY), anti-Vimentin (Sigma-Aldrich, Mu¨nchen, depleted from NMuMG cells. However, even in the presence of Germany) anti-Gapdh (Chemicon, Temecula, CA, USA), anti-Actin (Sigma- Aldrich), anti-Smad2/3 (Cell Signaling, Danvers, MA, USA) or anti-Phosho- Rbfox2, the non-transformed NMuMG cells were much less Smad2 (Cell Signaling). invasive than any of the other cell lines tested, which could explain why invasion could not be further decreased in this cell line upon depletion of Rbfox2. Alternative splicing has been Quantitative RT–PCR shown to contribute to every aspect of tumorigenesis, including Total RNA from NMuMG and PY2T cells was extracted using the High Pure resistance to apoptosis, and promotion of metastasis, RNA Isolation kit (Roche, Basel, Switzerland) and reverse transcribed using 29 angiogenesis and invasion. There are several examples of the RevertAid First Strand cDNA Synthesis kit (Fermentas, St Leon Rot, alternative splicing in genes that are involved in invasion, Germany). The resulting cDNA was used for qPCR analysis using Rox or the including CD44, Enah, TP53INP2 and others.29–33 Rbfox2 Sybr Green Mix (Applied Biosystems, Carlsbad, CA, USA). promotes splicing of the epithelial isoform of Enah and, therefore, this splicing event seems unlikely to mediate the invasive function of Rbfox2. To our knowledge, none of the other Migration assays splicing events has been shown to be regulated by Rbfox2. Thus, Forty-eight hours after transfection with control or Rbfox2 siRNAs, the cells the modulation of cell invasion by Rbfox2 may be mediated by were trypsinized, and 42 000 cells were seeded into each well of culture alternative splicing events in other genes that have yet to be inserts (Ibidi, Mu¨nchen, Germany). After 24 h, the inserts were removed, identified. In this study, we identified six mesenchymal-specific which created a cell-free gap of approximately 500 mm. The cells were observed by phase-contrast microscopy at 16 or 24 h. Rbfox2 targets, which are involved in invasion. Future studies will For the transwell migration assays, the cells were treated as described show how splicing changes in these targets may modulate the for invasion assays and added to control inserts (BD Biocoat Matrigel invasive phenotype. Invasion Chamber, Becton Dickinson, Heidelberg, Germany) instead of It has recently been demonstrated that many cancer-associated Matrigel-coated inserts. alternative splicing events are linked to Rbfox2-binding sites, that Rbfox2 is downregulated in ovarian cancer and that its splicing is shifted toward an inactive form in breast cancer.12 Notably, this Invasion assays study compared the expression of Rbfox2 between solid tumors of Forty-eight hours after transfection with the control or Rbfox2 siRNAs, the epithelial origin and normal tissues, which are composed of cells were trypsinized and 50 000 (NMuMG, MTdeltaEcad) or 25 000 (PY2T) mixtures of different cell types. Although these cell type cells were added to Matrigel-coated inserts (BD Biocoat Matrigel Invasion differences might affect differences in the expression of Rbfox2 Chamber; Becton Dickinson) in DMEM. These cells were placed in wells detected between normal and tumor tissue, our results clearly containing DMEM, 2% fetal bovine serum (MTdeltaEcad) or DMEM, 2% fetal indicate an upregulation of Rbfox2 during EMT of non- bovine serum, 5 ng/ml TGFb1 (PeproTech; R&D Systems; NMuMG, PY2T). After 24 h, the cells were removed from the upper surface of the transformed as well as tumorous mammary epithelial cells membrane using a cotton-tipped swab. The membranes were fixed in 4% (Figures 1b and d). Our data suggest that this upregulation will paraformaldehyde for 10 min and mounted using Fluoromount reagent most likely be a prerequisite for tumor cells to invade the with DAPI (4,6-diamidino-2-phenylindole; Southernbiotech, Birmingham, surrounding tissue. AL, USA).

Oncogene (2014) 1082 – 1092 & 2014 Macmillan Publishers Limited Rbfox2 and EMT-driven alternative splicing C Braeutigam et al 1091 CONFLICT OF INTEREST 24 Waldmeier L, Meyer-Schaller N, Diepenbruck M, Christofori G. Py2T murine breast The authors declare no conflict of interest. cancer cells, a versatile model of TGFbeta-induced EMT in vitro and in vivo. PLoS One 2012; 7: e48651. 25 Damianov A, Black DL. Autoregulation of Fox protein expression to produce dominant negative splicing factors. RNA 2010; 16: 405–416. ACKNOWLEDGEMENTS 26 Horiguchi K, Sakamoto K, Koinuma D, Semba K, Inoue A, Inoue S et al. TGF-beta This work was supported by the Max-Planck Society. We are grateful to Rolf Kemler drives epithelial-mesenchymal transition through deltaEF1-mediated down- for helpful discussions. regulation of ESRP. Oncogene 2012; 31: 3190–3201. 27 Kirkbride KC, Sung BH, Sinha S, Weaver AM. Cortactin: a multifunctional regulator of cellular invasiveness. Cell Adhes Migration 2011; 5: 187–198. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 1082 – 1092 & 2014 Macmillan Publishers Limited