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Previous Article | Next Article 
Molecular and Cellular Biology, January 2006, p. 362-370, Vol. 26, No. 1
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.1.362-370.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Center for Cancer Research,1 Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 021392
Received 28 June 2005/ Returned for modification 11 August 2005/ Accepted 8 October 2005
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ABSTRACT |
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INTRODUCTION |
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Interestingly, the peptides encoded by these variable exons are located in the extracellular domain of the protein. Some of these alternative spliced variants interact with growth factors and render cells responsive to extracellular stimuli (2, 17, 33, 37-39, 43). The CD44 splice variant containing variable exon 6 (v6) can form a complex with the extracellular hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met (33). Formation of this CD44 v6-HGF-Met complex stimulates the activation of Met through autophosphorylation and further activates Met-dependent Ras signaling, probably through the association of ERM (ezrin-radixin-moesin) proteins to the cytoplasmic tail of CD44 (33). The net result is that the CD44 v6-HGF-Met complex activates Ras signaling and promotes cell proliferation. Interestingly, Ras activation also stimulates transcription of CD44 and promotes inclusion of its variable exons (14, 19). This constitutes a CD44-mediated positive feedback loop in the activation of Ras signaling if a factor such as HGF is present. Alternatively, the tumor suppressor protein merlin can also bind to the cytoplasmic tail of CD44. Binding of merlin to CD44 disrupts the interaction between ERM and CD44 and therefore inhibits Ras activation (29). As a result, Ras-dependent CD44 alternative splicing is inhibited, the positive feedback loop is disrupted, and cell growth diminishes. Consistent with the above observations, the majority of CD44 expressed in resting lymphocytes is in the constitutively spliced form (standard form), whereas the variants are commonly observed only during activation.
When overexpressed in human colon cancer cells, the standard form of CD44 acts as a tumor suppressor and inhibits the potential for metastasis (5, 34). Furthermore, a significant body of evidence indicates a correlation between metastasis and dysregulation of CD44 variants. For example, ectopically expressed CD44 v4-7 variant (containing variable exons 4, 5, 6, and 7) confers metastatic potential to nonmetastatic cells (11). Conversely, high levels of expression of CD44 variants correlate with tumors with a poor clinical prognosis (12, 13, 20, 30, 31, 46). Despite the significance of the association of CD44 variants with a variety of malignancies, insight into the regulation of CD44 alternative splicing remains incomplete.
Regulation of CD44 alternative splicing has been studied following mitogen stimulation. Using a splicing construct containing the CD44 v5 exon flanked by its introns and two constitutive exons, Konig and colleagues found that inclusion of v5 is activated by the Ras-Raf-MEK-Erk (extracellular signal-regulated kinase) signaling cascade (23, 45). Addition of the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (PMA), a Ras signaling activator, or ectopic expression of the above signaling molecules elevates the inclusion of v5. Analysis of cis-acting elements in the 117-nucleotide (nt) v5 exon revealed the presence of both exonic splicing enhancers (ESEs) and exonic splicing silencers. These cis-acting elements were identified by consecutively substituting 10-nt mutations through the v5 region (19). Interestingly, in contrast to the broad distribution of experimentally identified exonic splicing silencers, the v5 ESEs are located in two 10-nt segments. The most defective mutant was created by the substitution of the v5 ATGAAGAGGA sequence with random sequence. Mutational substitution of this region reduced basal level inclusion of the v5 exon and decreased the response to PMA stimulation. The proteins that specifically interact with this ESE in this context have yet to be identified. Recently, a nuclear RNA binding protein, Sam68, was shown to bind to a region of v5 containing the site AAAAUU (21, 23). The activation of Ras signaling by PMA, which phosphorylates and in turn activates Sam68, stimulates v5 exon inclusion (23). Deletion of the Sam68 binding site does not inactivate the PMA response, consistent with the notion that other factors in addition to Sam68 are also important.
In this study, we show that SRm160, a splicing activator of pre-mRNAs containing GAA repeats (4, 8), specifically stimulates inclusion of CD44 v5 in a Ras-dependent manner. Mutation of the above ATGAAGAGGA sequence abolishes SRm160's stimulation. SRm160 and Sam68 are detected in a complex that probably regulates CD44 alternative splicing. Moreover, we show that small interfering RNA (siRNA) knockdown of CD44 v5 decreases tumor cell invasion. Reduction of SRm160 inhibits alternative splicing of endogenous CD44 variants and results in a decrease in tumor cell invasiveness. These observations support a link between regulation of alternative splicing and tumor cell invasiveness.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA interference (RNAi) experiments were performed in HeLa cells using 100 pmol of siRNAs (control, SRm160, Sam68, or CD44 v5) and 5 µl of oligofectamine for each well of a six-well plate. For cotransfection with the CD44 v5 reporter gene, siRNA-treated HeLa cells were trypsinized and plated in 12-well plates 24 h after the first siRNA transfection. Twenty-four hours after the cells were plated, the CD44 reporter construct was cotransfected with an H-Ras V12 plasmid and the same siRNAs transfected in six-well plates using Lipofectamine 2000. Cells were harvested 30 to 36 h posttransfection. siRNA sequences of the sense strands are as follows: SRm160-1, CGACCCAAGAGAUCCCAUGdTdG; SRm160-2, GAGACGUUCACCUUCAUUAdTdT; Sam68-1, CGGAUAUGAUGGAUGAUAUdTdT; Sam68-2, CGCAGAACAAAGUUACGAAdTdT; CD44 v5-1, CCACUGCUUAUGAAGGAAAdTdT; and CD44v5-2, AUGAGCAUCAUGAGGAAdTdT.
RT-PCR. RNA was prepared using a QIAGEN RNeasy kit. A total of 0.25 µg of RNA was used in a reverse transcription (RT) reaction in a 20-µl volume for 1 h. Two microliters of the resulting cDNA was subjected to PCRs using specific primers. Each cycle of PCRs included 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C. Thirty cycles were performed to detect v5 inclusion using the v5 luciferase minigene construct. For endogenous CD44, different cycles of PCR were performed, as follows: 30 cycles for v8-10; 31 cycles for v3r; 32 cycles for v3, v7, v6r, and v7r; 34 cycles for v2r; and 35 cycles for v2, v4, v5, and v6. Twenty-nine cycles were used for amplifying standard CD44 and loading control dihydrofolate reductase (DHFR). PCR products were analyzed on a 1.5% agarose gel. SRm160 was amplified using the forward primer 5'-GTCGTGTTTCTGTGTCTCCAG and the reverse primer 5'-TATGCTTGGATGATAATGAAGGTG. DHFR was amplified using the forward primer 5'-GTTCTGCTGTAACGAGCGG and the reverse primer 5'-AGGCATCATCTAGACTTCTGG. The primers for amplifying the CD44 luciferase minigene and endogenous CD44 variants using forward individual variable exon primers were described previously (18, 45). A forward primer pairing to the 5' constitutive region is 5'-CATCCCAGACGAAGACAGTC. Reverse primers base pairing to specific variable exons are as follows: v2r, 5'-TGTGAAGATGATTCTTTGACTC; v3r, 5'-CATCATCAATGCCTGATCCAGA; v6r, 5'-CAGCTGTCCCTGTTGTCGAA; and v7r, 5'-TCCTGCTTGATGACCTCGTC.
Immunoprecipitation. 293T cells transfected with hemagglutinin (HA)-tagged Sam68 or Flag-tagged SRm160 were lysed in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% NP-40, 50 mM NaF, supplemented with protease inhibitors [complete tablet from Roche]). Cell lysates were precleared with protein A beads and incubated with HA or Flag antibody-conjugated beads (Roche and Sigma, respectively). The immunoprecipitated complexes were washed five times with NP-40 buffer and eluted in 2x sodium dodecyl sulfate (SDS) loading buffer by incubation at 95°C for 5 min. The eluates were loaded onto an SDS-polyacrylamide gel for Western blot analysis.
Matrigel invasion assay. Biocoat Matrigel invasion chambers (Becton Dickinson) were used to assess the invasiveness of HeLa cells. Two days after siRNA treatment, 5 x 104 cells were resuspended in 250 µl of serum-free Dulbecco's modified Eagle's medium and added to the cell culture inserts of the invasion chambers. Fetal bovine serum (10%) was used as a chemoattractant and placed in the lower wells. After 22 to 24 h, cells on the upper surface of the membrane were removed using cotton swabs, and the filters were fixed by immersion in 4% formaldehyde for 10 min. After two washes with water, the invaded cells were then stained with 0.2% crystal violet. Excess dye was rinsed off two times with water. The stained cells were photographed using a digital camera and counted.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether SRm160 has an important role in regulating CD44 alternative splicing, we used a CD44 v5 reporter minigene construct (23, 45). This minigene contains a luciferase-based splicing reporter, in which the CD44 v5 exon and its flanking introns were inserted upstream of an intact Photinus luciferase gene (Fig. 1A). Inclusion of the v5 exon produces a fusion protein containing the coding sequence of the Photinus luciferase, whereas exclusion of the v5 exon positions a stop codon upstream of the luciferase coding region. Thus, the luciferase activity directly indicates the levels of CD44 v5 inclusion. Previous work has demonstrated that the ratios of the v5 inclusion and exclusion are consistent whether they are measured by RT-PCR of spliced mRNA or luciferase assays (45).
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3.7-fold in cells that overexpressed H-Ras V12. These results are consistent with the idea that the stimulation of v5 inclusion by SRm160 is dependent on Ras signaling. The stimulation of luciferase expression from the CD44 v5 minigene is an indirect measure of alternative splicing. To directly assay the v5 inclusion in mRNA, we analyzed the ratios of variants by RT-PCR with primers flanking the v5 exon (Fig. 1A; the location of primers relative to spliced mRNAs is indicated by arrows). Both the included and excluded variants can be detected in one PCR, with the former yielding a longer product. In the absence of Ras activation, the levels of v5 inclusion were similar in cells ectopically overexpressing SRm160 or control vector (0.20 and 0.13, respectively). However, in the presence of cotransfected activated Ras, overexpression of SRm160 stimulated the level of v5 inclusion to 0.75 (Fig. 1D). These results show that SRm160 activity can regulate CD44 alternative splicing through processes influenced by Ras signaling.
A potential mechanism explaining the activity of SRm160 in stimulation of the v5 exon is the presence of GAA-containing ESEs. This was addressed by testing the activity of a mutant form of v5 exon where the 10-nt GAA-containing ESE segment (ATGAAGAGGA) was replaced with a random sequence (19). In agreement with previously described results (19), we observed, in the presence of activated Ras, an 50% reduction in inclusion of the v5 exon when this GAA mutant was compared to the wild-type v5 construct. More importantly, the SRm160 stimulatory effect on v5 inclusion was almost entirely abolished by this mutation (0.10 versus 0.15, without and with SRm160, respectively) (Fig. 1D). A control mutation with a 10-nt substitution immediately downstream of this GAA segment displayed stimulatory effects very similar to the wild-type v5 construct upon SRm160 overexpression (ratios of inclusion to exclusion: 0.23 versus 1.09, without and with SRm160, respectively) (data not shown). To test whether the 10-nt GAA-containing ESE segment is sufficient to allow Ras-dependent alternative splicing, we used a reporter construct, pSXN (6, 9), that contains an alternatively spliced 33-nt exon 2. Random-sequence insertion in this exon 2 predominantly results in exon skipping. Interestingly, insertion of the 10-nt GAA segment into the pSXN exon 2 promoted inclusion of this exon. This inclusion was moderately stimulated (1.6-fold ± 0.1-fold) in response to SRm160 in a Ras signaling-dependent manner (data not shown). These observations indicate that the v5 GAA-containing ESEs and possibly other elements are important for SRm160's stimulation of v5 inclusion.
Knockdown of SRm160 inhibits inclusion of the CD44 v5 exon.
To further investigate the role of SRm160, we determined the effects of SRm160 reduction on v5 inclusion. SRm160 siRNA transfection was used to reduce the level of this protein. Transient transfection of the SRm160 siRNA produced an 10-fold knockdown of SRm160 mRNA as measured by RT-PCR analysis relative to cells transfected with a control siRNA (Fig. 2A). Protein analysis using immunoblotting with an SRm160-specific antibody showed that the levels of a series of bands of the SRm160 protein were undetectable in cells treated with SRm160 siRNA (Fig. 2B). These clustered polypeptide bands for SRm160 have been described previously and probably represent different degrees of phosphorylation of this highly repetitive SR protein. In total, these results indicate that RNAi knockdown was successful.
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SRm160 interacts with Sam68. The Src substrate and RNA binding protein Sam68 have been reported to be important for stimulation of v5 inclusion by Ras activation (23). Since both SRm160 and Sam68 stimulate this inclusion, it is possible that these two proteins might act in the same pathway and could interact with each other. To test this possibility, immunoprecipitation experiments were performed. A plasmid containing Flag-tagged SRm160 was transfected into 293T cells. Proteins that interact with Flag-tagged SRm160 protein were precipitated using anti-Flag antibody-coupled beads. The presence of Sam68 in the immunoprecipitated fraction was detected by immunoblotting using a Sam68 antibody. Indeed, the result shows that SRm160 and Sam68 coimmunoprecipitate (Fig. 3A). The above observation was further confirmed by immunoprecipitation using lysates from cells that overexpressed HA-tagged Sam68 protein and anti-HA antibody-coupled beads (Fig. 3B). Again, we observed that SRm160 immunoprecipitated with Sam68. Moreover, this coimmunoprecipitation was resistant to extensive RNase treatment (data not shown), indicating that SRm160 and Sam68 association was not dependent on RNA bridging. We conclude that these two proteins can associate through protein-protein interactions. In addition, we observed that overexpression of Sam68 did not rescue the reduction of v5-luciferase inclusion caused by SRm160 siRNA treatment, suggesting that these two proteins may be functionally cooperative (data not shown).
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Because of the involvement of Sam68 in CD44 alternative splicing and the interaction between Sam68 and SRm160 described above, it was important to determine whether Sam68 is important for inclusion of the same subset of CD44 variable exons as SRm160. To examine this, we knocked down Sam68 expression by siRNAs and examined the effect on CD44 inclusion. RT-PCR analysis showed about a fourfold reduction of Sam68 RNA expression in Sam68 siRNA-treated cells, and Western blot analysis showed a similar reduction of Sam68 protein in these cells (Fig. 4C). Notably, we observed very similar to CD44 inclusion patterns in cells treated with Sam68 siRNA those treated with SRm160 siRNA, with slight differences (Fig. 4B). For example, inclusion of v2 was more affected in Sam68 siRNA- than in SRm160 siRNA-treated cells, with a sevenfold versus a threefold reduction, respectively. Inclusion of v5 was also less affected in the same comparison, with a threefold versus a sixfold reduction, respectively. These results indicate that knockdown of Sam68 impaired the inclusion of endogenous CD44 variable exons to degrees similar to those found with the knockdown of SRm160. Together with the coimmunoprecipitation studies, these results indicate that SRm160 and Sam68 are important for regulation of alternative splicing of the endogenous CD44 gene in HeLa cells.
SRm160 and tumor cell invasion. CD44 variants have been implicated in tumor invasion and metastasis (16, 22). Therefore, it is tempting to speculate that the activity of SRm160 could influence tumor cell invasion by regulating the inclusion of CD44 variable exons. Thus, we tested whether reducing SRm160 levels in cells would affect their invasiveness. In order to validate an invasion assay while performing an interesting novel experiment, we tested whether inhibition of CD44 variants by RNAi would affect the invasive properties of cells. We chose to target variants containing v5 exon because v5 inclusion is elevated in some metastatic cells and tissues (12, 13, 20, 46, 47). We used human cervical carcinoma cells (HeLa) as these cells are active in the invasion assay and are highly transfectable (42). An siRNA designed to specifically target the v5 exon was transfected into HeLa cells. As shown in Fig. 5A, we observed a 3.5-fold knockdown of v5-containing variants in cells treated with this siRNA. A control siRNA, whose sequence does not match any known human genes, did not cause a reduction of v5 expression.
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Since reduction of SRm160 expression by siRNA knockdown inhibited v5 inclusion, we tested whether treatment with SRm160 siRNA would alter the invasiveness of cells. HeLa cells were treated with siRNAs specific to either SRm160, v5, or nonspecific control siRNA and tested as described above for invasiveness. Interestingly, cells treated with SRm160 siRNA showed a dramatic fivefold reduction in invasion activity (Fig. 5B and C), a reduction in invasiveness similar to that seen with v5 siRNA-treated cells. These results suggested that SRm160 activity could have an important role in tumor cell invasion by altering the level of CD44 variants in cells.
We have previously observed that SRm160 siRNA-treated cells proliferate more slowly than control siRNA-treated cells (unpublished observations). To confirm that the impaired invasiveness by SRm160 was not due to slower cell proliferation, the proliferation rate of these cells was monitored in parallel to the invasion assay. In the invasion assay period, 22 to 24 h, the cell numbers from these two populations differ by less than 15% (data not shown). Therefore, the reduction in cell number in the lower chamber following SRm160 siRNA treatment is not due to the decrease in rate of proliferation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability to silence genes by transfection of mammalian cells with siRNA permits a new test of the roles of particular proteins in control of CD44 alternative splicing. In the case of SRm160, we were able to show that knocking down the level of this protein by more than fivefold after treatment with siRNA resulted in a dramatic reduction in the level of inclusion of several of the alternatively spliced exons expressed from the endogenous CD44 gene. Specifically, inclusion of the v5 exon of CD44 was reduced by sixfold. The standard form of CD44 mRNA, however, was not affected. The activity of SRm160 as a splicing factor for each of the variant exons is unclear. Computational analysis indicates that most of the variable exons could contain ESEs of the GAA-type elements that might recruit SRm160. They also contain AT-rich elements that are potential Sam68 binding sites. It is also possible that the observed reduction in multiple variants after silencing of SRm160 could result from disruption of a positive feedback loop involved in CD44 alternative splicing (our unpublished observations). Treatment of cells with siRNA specific to Sam68 also reduced the levels of inclusion of CD44 variable exons, consistent with the observation that these two splicing factors interact. The similarity of the effects on inclusion of CD44 variable exons when either of these factors was silenced indicates that CD44 alternative splicing is probably regulated by SRm160 and Sam68 through a common pathway.
SRm160 is a coactivator of RNA splicing, which contains many serine and arginine repeats. Some fraction of SRm160 is associated with another highly repetitive protein, SRm300 (4). The GAA-containing ESEs have been shown to be important for recruiting SRm160 to pre-mRNA through interactions with the RNA binding protein Tra2-beta (8, 40). Interestingly, we observed that the function of SRm160 in stimulation of v5 inclusion is dependent on GAA-containing ESEs in this exon. Mutation of this GAA segment abolishes the stimulatory effect of SRm160. In addition, we have observed that SRm160 interacts with another RNA binding protein, Sam68, which has been shown to recognize an AAAUU-type sequence in v5. Since the simultaneous activities of these proteins are necessary for maximal stimulation of v5 inclusion, it is possible that recruitment of SRm160 and Sam68 to v5 pre-mRNA is cooperative. Another indication that Sam68 and SRm160 might have common activity are the recent reports that both factors can stimulate the 3'-end processing of some pre-mRNAs (24, 25, 27).
The mechanism by which Ras activation stimulates CD44 alternative splicing has not been fully determined. Sam68 has been shown to be a target of Erk phosphorylation (23). In addition, the splicing factors, such as SRm160 and Tra2, that contain many phosphorylation sites may also be regulated by Ras signaling. As noted above, SRm160 can associate with SRm300, which is frequently highly phosphorylated in tumor verses normal cells (F. White, personal communication).
v5 inclusion, SRm160, and cell invasion. The expression of the CD44 v5 variant has been correlated with tumor malignancy in patients with poor clinical prognosis. The prognostic significance of v5 expression has been observed in human thymic epithelial neoplasms and gastric and renal carcinomas (12, 20, 46). Consistent with these clinical observations, our RNAi experiments show that v5 knockdown drastically impairs invasion by tumor cells. It is interesting that siRNA knockdown of v5 in HeLa cells in culture resulted in reduction of other variable exon inclusions (data not shown). This reduction is probably due to inhibition of the positive feedback loop that regulates CD44 alternative splicing (reviewed in the introduction). Thus, it is possible that the decrease in invasiveness of the cells after transfection of the v5 siRNA could also reflect decreases in other variable exons of CD44.
Silencing of SRm160 by siRNA treatment affects the inclusion of CD44 variable exons v2 to v9 without significantly decreasing the level of the constitutive mRNA isoform. The v4 and v5 exon inclusion was the most dramatically affected, with a reduction of five- to sixfold. As might be expected, the decrease in SRm160 causes a parallel decrease in cell invasiveness. Cells treated with SRm160 siRNA had a much lower invasive capacity than cells treated with control siRNA. The phenotype of SRm160 siRNA-treated cells was very similar to that produced by v5 siRNA treatment. This similarity suggests that the primary effect of SRm160 reduction on cell invasion is a consequence of the reduction in v5 levels. However, it is also possible that the reduction of SRm160 by siRNA could also affect invasion through other cellular pathways.
Previous work by others has described interactions between SRm160 and other tumor oncogenes, such as TLS/FUS and DEK (26, 28). TLS/FUS is a nucleic acid-binding protein, and its N-terminal half functions as a transcriptional activator domain in fusion oncoproteins found in human leukemias and liposarcomas (7, 15). Intriguingly, TLS/FUS interacts with SRm160, PTBP, and a subset of the classical SR proteins. DEK is a phosphoprotein that is often fused to the nucleoporin CAN in a subset of acute myeloid leukemias (44). Moreover, as mentioned previously, SRm300, the binding partner of SRm160, was identified as the protein that undergoes the most phosphorylation in cancer cells (F. White, personal communication). Furthermore, Sam68, another protein to interact with SRm160, associates with the oncogenic kinase Src during M phase (10, 41). The acetylated form of Sam68 has been implicated in tumor cell proliferation (1). In view of these results, it is tempting to speculate that SRm160 may play a role in influencing the malignancy of tumor cells.
It has been known that oncogenes and tumor suppressors control tumor growth and metastasis by regulating their downstream targets at the levels of transcriptional activation or repression. This study suggests that regulation of alternative splicing is yet another important mechanism in tumorigenesis. More than half of all human genes are estimated to undergo alternative splicing, some of which is regulated both temporally and spatially. The roles of CD44 isoforms in invasion clearly indicate that some of this alternative splicing is important in tumorigenesis.
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ACKNOWLEDGMENTS |
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This work was supported by U.S. Public Health Service grants R37-GM34277 (NIH) and P01-CA42063 (NCI) to P.A.S. and partially by Cancer Center Support (core) grant P30-CA14051 (NCI). C.C. is partially supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation.
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FOOTNOTES |
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