The WNT5A gene encodes two protein isoforms

To determine whether WNT5A encodes several protein isoforms, we analyzed sequences deposited in databases and searched for possible alternative transcripts. We found the WNT5A gene produces at least 3 possible WNT5A transcripts from alternative transcriptional start sites. One transcript initiates at exon 1 [19,20,28] and is predicted to encode a 380 amino-acid WNT5A protein precursor, from hereon referred to as WNT5A-L (Long) (Figures 1A, B; Figures S1A and S1B). The other two transcripts initiate 718 and 578 nucleotides upstream of exon 2, in an alternative exon 1β (Figure 1A; Figures S1A and S1B). Both exon 1β-initiated transcripts are predicted to encode a 365 or 360 amino acid (depending on use of start codon at position 16 or 21 relative to start codon of WNT5A-L) precursor protein, referred to as WNT5A-S (Short), and lack the first 15 or 20 N-terminal amino acids as compared with the WNT5A-L precursor ([29]; Figures 1A, B; Figures S1A and S1B).

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Figure 1. Description and biochemical chracterization of WNT5A isoforms.

A. Structure of the human WNT5A gene and alternative transcripts. Numbered boxes indicate the exons, with 5’ and 3’ untranslated (UTR) regions in grey and coding regions in black. nt: nucleotides, kb: kilo base. Blue and red arrows and dotted lines respectively indicate the positions of the different transcription initiation sites in exon 1 and exon 1β and splicing of exon 1- and exon 1β-initiated transcripts. Blue and red stars indicate the translation initiation codons for the WNT5A-L and WNT5A-S isoforms located in exon 1 and exon 2, respectively. Blue and red lightning bolt arrows indicate the position of the target sequences of WNT5A-L and WNT5A-S specific short interfering RNA (siRNA). B. Multiple amino acid sequence alignment of the N-terminus of the WNT5A precursor proteins. The blue M denotes the most likely start codon of WNT5A-L while the red Ms denote the most likely start codons of WNT5A-S. The grey arrows indicate the positions of signal peptide cleavage sites for both isoforms as predicted by SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). Blue and red arrows indicate the position of the observed first amino-acids of the mature WNT5A-L and WNT5A-S proteins, respectively, and thus the position of the observed signal peptide cleavage site for each isoform. C. Detection of purified WNT5A isoforms. Left panel: Immuno-blot for WNT5A demonstrates that both isoforms are of similar molecular weight (^≈43kDa). Right panel: Coomassie stained gel shows that the purified WNT5A preparations are pure with largely undetectable contaminant proteins. D. An anti-mouse Wnt5a antibody detects both isoforms (left panel) while an anti-rabbit WNT5A-L specific antibody (right panel) detects the WNT5A-L but not the WNT5A-S isoform. E. Triton X-114 phase separation demonstrates that both WNT5A isoforms partition to the hydrophobic/organic phase. WNT5A proteins were detected by immuno-blot analysis with an anti-Wnt5a antibody. F. Incubation of purified WNT5A isoforms at 37°C for the indicated times indicates that their in vitro stability is similar. WNT5A proteins were detected as in E and band intensities were quantified using ImageJ software.

https://doi.org/10.1371/journal.pone.0080526.g001

The overall exon-intron structure of the human WNT5A gene is highly conserved in other vertebrates, including mouse, chicken and zebrafish (Figure S2A). The WNT5A gene is split into 5 conserved exons. The length, sequence, and splice sites in codons across exons 2 - 5 are highly conserved. The untranslated region of exon 1 is more diverged, but the splice site is conserved. This exon-intron arrangement is also conserved in the closely related WNT5B gene, as annotated in mammalian genomes. Interestingly, the alternative exon 1β, which is embedded in the first intron, is also present in these vertebrates. In mice, exon 1β is detected in alternative Wnt5a transcripts and shares significant homology with the human sequence. In a pairwise alignment, the element encompassing the upstream regulatory region and exon 1 β are 95% conserved between human and mouse (Figure S2B). In birds (chicken and zebra finch), this alternative exon has not been annotated, but the presence of a highly conserved sequence with a splice donor at the 3’ end indicates that this exon 1β is present. The pairwise alignment between chicken and zebra finch of this element shows 96% conservation. Furthermore, two short sequences within these elements are highly conserved between mammals (mouse and human) and birds (chicken and zebra finch) (Figure 2B). Fish (zebrafish and stickleback) also contain a highly conserved element within this region. Although this element has not been annotated as an exon in any database, the elevated conservation (74%) suggests the presence of an additional exon in fish (Figure 2B). Together, the observed high degree of genomic sequence conservation suggests that alternative Wnt5a transcripts, similar to the ones described here, are present in multiple vertebrate species. Finally, alignment of the amino terminal amino acids of human, mouse and chicken Wnt5a shows that start codon M21 (position relative to start codon of WNT5A-L) is completely conserved (Figure 1B), suggesting that WNT5A-S initiates at this position.

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Figure 2. Effects of WNT5A isoforms on the β-catenin/TCF pathway and Disheveled proteins phosphorylation.

Both WNT5A proteins block Wnt3a-activation of β-catenin/TCF-driven transcriptional activity in HEK 293 (TOP-Luciferase, A.) and MDA-MB-231 (TOP-GFP reporter, B.). Cells were treated with purified Wnt3a and an increasing concentration of WNT5A for 24 hours (A) or 48 hours (B). C. Upon transfection, both WNT5A isoforms interfere with endogenous (MDA-MB-231, left panel) and WNT1-induced (HeLa, right panel) β-catenin/TCF-driven transcription in MDA-MB-231 (left panel) and HeLa cells (right panel). Cells were transfected with control, WNT5A-L or WNT5A-S expression vectors alone, or together with a WNT1 expression vector and assayed for β-catenin/TCF-driven TOP-Luciferase reporter activity. An expression vector encoding a dominant-negative form of TCF4 (ΔNTCF4) was used to interfere with the endogenous reporter activity in MDA-MB-231 cells. D. Both WNT5A isoforms promote DVL phosphorylation. L cells (left panel) and C2C12 cells (right panel) were treated with WNT5A isoforms (10 nM, 2 hours) and whole cell lysates were immuno-blotted with the indicated antibodies. Both WNT5A isoforms, as well as Wnt3a (10 nM, 2 hours), led to a mobility shift of Dvl1 and Dvl2 proteins, suggesting that Dvl proteins are post-translationally modified by phosphorylation in response to both canonical (Wnt3a) and non-canonical Wnt (WNT5A) signaling. Only Wnt3a induced accumulation of the β-catenin protein. Note: β-catenin accumulation in response to Wnt3a in C2C12 cells is not detectable in these whole cell lysates because these cells contain large amounts of membrane/adherens junction associated β-catenin.

https://doi.org/10.1371/journal.pone.0080526.g002

As is the case for most secreted proteins, Wnt precursors are cleaved at the amino terminus to remove the signal sequence thereby producing the mature polypeptide. A signal peptide cleavage prediction algorithm (SignalP 4.1 - http://www.cbs.dtu.dk/services/SignalP/) predicts that both WNT5A precursors yield proteins of either 343 amino acids (start with QVVIEA…) or 338 amino acid (start with ANSWWS…). The latter predicted cleavage site has the highest cleavage score (Y-score of 0.625 for WNT5A-S [start at M21] and 0.246 for WNT5A-L). However, amino-terminal sequencing of mouse Wnt5a indicated that signal sequence cleavage occurred at a position 24 residues downstream from the predicted site [3]. Furthermore, since the 15 amino-acid difference in the N-terminal sequence of WNT5A-L and WNT5A-S precursors may influence the position of signal peptide cleavage, we experimentally determined the identity of the amino termini of the mature proteins.

To identify the position of signal peptide cleavage and the first amino acid of the mature proteins, we purified both WNT5A isoforms. Using a previously developed CHO expression system [30], we independently overexpressed each WNT5A isoform and purified them from the conditioned media (CM), according to published protocols [3,31]. Both WNT5A isoforms were secreted into the CM at equal levels (data not shown), and were purified to near homogeneity (~95% pure, as assessed by Coomassie staining, Figure 1C). Amino-terminal sequencing showed the purified WNT5A-L protein to start one amino acid downstream of the predicted signal peptide cleavage site, thus generating a 337 amino acid protein starting with the sequence NSWWS… (Figure 1B; Figure S1B and S1C). The secreted WNT5A-S protein lacked an additional amino-terminal 18 amino acid to generate a protein of 319 amino acid starting with the sequence IIGAQ… (Figure 1B; Figure S1B and S1C). Therefore, the two isoforms generate proteins with distinct amino terminal sequences. The WNT5A-S amino terminus matches that of mouse Wnt5a, which was previously identified by amino-terminal peptide sequencing [3]. Interestingly, the signal peptide cleavage algorithm did not predict this position as a likely site of signal sequence cleavage for either WNT5A-L or WNT5A-S (Y-score of 0.16 for WNT5A-S and 0.113 for WNT5A-L).

To confirm the distinct amino terminal composition of both mature WNT5A isoforms, we generated an antibody directed to the WNT5A-L-specific 18 amino acid (NSWWSLGMNNPVQMSEVY). In immuno-blots, the WNT5A-L-specific antibody only recognized WNT5A-L and not WNT5A-S (Figure 1D). This demonstrates that the exon 1- and exon 1β-initiated WNT5A transcripts produce distinct WNT5A proteins that differ in their amino termini. The mechanism by which these two precursors are differentially processed to yield distinct mature proteins is currently unclear, however, we speculate that differences in the amino termini influence the choice of the processing machinery to cleave the proteins at distinct positions. Further studies are needed to address these questions.

WNT proteins are highly hydrophobic, a property imparted by the covalent attachment of at least one lipid molecule to a conserved residue [31,32]. This modification is important for proper processing and secretion [32,33]. In addition, as revealed by the Wnt-Fzd co-crystal structure, the lipid moiety is critical for receptor binding [34]. To address whether the two WNT5A isoforms differed in their hydrophobic properties, and hence in their lipid modifications, we used the phase separation property of the detergent Triton X-114. We observed no difference between the two isoforms, as they both partitioned equally to the organic phase (Figure 1E), thus suggesting that both isoforms are similarly modified. To test whether the two isoforms differed in their stability, we incubated each protein at 37°C for up to 60 hours. Immuno-blot detection indicated that both proteins degraded at equal rates (Figure 1F). Therefore, the two WNT5A isoforms behave similarly with respect to their in vitro stability and hydrophobicity. It is, however, possible that each isoform has distinct biochemical properties in an in vivo setting, a possibility we have not addressed.

Effects of WNT5A-L and WNT5A-S isoforms on signaling pathways

Several WNT signaling pathways have been described and proposed. The most commonly studied and best-established pathway, often referred to as the canonical WNT pathway, involves β-catenin as a central mediator. Other non-canonical pathways, which act independently of β-catenin, are poorly understood. At the very minimum, these two pathways are thought to share the following components: WNT ligands, FZD receptors and Dishevelled (DVL) signal transducers. Although WNT5A is predominantly associated with non-canonical WNT signaling, it has also been found to activate canonical β-catenin signaling in certain conditions [3]. We investigated if the WNT5A isoforms differentially impact the β-catenin/TCF signaling pathway. To this end, we generated two cell lines, HEK 293 and MDA-MB-231, carrying a WNT/β-catenin specific reporter system. This reporter system consists of a WNT responsive element comprised of 7 multimerized TCF binding sites (referred to as TOP for TCF Optimal Promoter [35,36]), which regulates the expression of a reporter gene, either Luciferase for HEK 293 cells (HEK293-TOP-Luciferase, Figure 2A; Figure S3A) or GFP for MDA-MB-231 cells (MDA-MB-231-TOP-GFP, Figure 2B; Figure S3B). These reporter cells are potently activated by Wnt3a (Figure 2A and B), a WNT protein commonly used to stimulate the canonical signaling pathway. In contrast, neither WNT5A isoform activated or consistently modulated the basal activity of these reporters (Figure S3A and S3B), indicating that these cellular systems do not provide the appropriate milieu to connect WNT5A signaling to the canonical β-catenin pathway.

Sensitive and robust cell-based assays for non-canonical WNT signaling are scarce. However, non-canonical WNT signaling has been shown to antagonize WNT/β-catenin signaling, an activity that can be assayed using the TOP-reporter system [3]. We found that both WNT5A isoforms inhibit WNT3A-induced reporter activation in a dose-dependent manner (Figure 2 A and B). These results show that, despite the difference in their amino-termini, both WNT5A proteins potently antagonize the WNT/β-catenin signaling. Similar inhibitory effects of WNT5A-L and WNT5A-S isoforms on the WNT/β-catenin-driven reporter were observed when cells were transfected with expression vectors (Figure 2C) rather than treated with purified WNT5A-L and WNT5A-S proteins. Therefore, in these assays, the WNT5A isoforms exhibit similar biological activities when delivered as purified proteins or transfected genes.

WNT signaling, canonical and non-canonical, leads to the hyperphosphorylation of Dvl proteins, signaling molecules that act immediately downstream of FZD receptors. This post-translational modification of Dvl proteins can be readily detected by an electrophoretic mobility shift [37-39]. We found that treatment of two cell lines (mouse L cells and the mouse myoblast C2C12 cell line) with either WNT5A isoform, as well as WNT3A, induces a mobility shift of Dvl proteins, as detected by immuno-blotting for Dvl1 and Dvl2 (Figure 2D). However, in agreement with the distinct activities of WNT3A and WNT5A isoforms on WNT/β-catenin signaling (Figures 2A, 2B; Figure S3A, S3B), only Wnt3a, but not WNT5A isoforms, induced accumulation of the β-catenin protein, as evidenced in L cells. This β-catenin protein accumulation is not apparent in C2C12 cells, which already express high levels of β-catenin (Figure 2D). Taken together, our data indicate that the two WNT5A isoforms exhibit identical biological activities in the established cell-based WNT signaling assays.

Expression of WNT5A isoforms in cancer cell lines and normal tissues

Previous studies of WNT5A expression have not discriminated between the two isoforms described here. Therefore, we measured their transcript levels in cancer cell lines and in normal tissues by performing quantitative RT-PCR (qRT-PCR) with isoform-specific primers (see Table S1 and Figure S1). This analysis showed that expression of these isoforms is highly variable between individual cancer cell lines. For example, while MDA-MB-231 (derived from a breast carcinoma) and SH-SY5Y (derived from a neuroblastoma) express predominantly WNT5A-L, HeLa cells (derived from a cervix carcinoma) express predominantly WNT5A-S (Figure 3A). Other cell lines, including MCF-7 (derived from a breast carcinoma) or IMR-32 (derived from a neuroblastoma) express extremely low levels of either WNT5A isoform (Figure S4A). Although less quantitative, gel analysis of end point RT-PCR products obtained from representative cell lines confirmed the qRT-PCR data (Figure S4A). We next analyzed the transcript levels of WNT5A isoforms in normal adult tissues. We detected WNT5A-L expression in nearly all tissues, except adipose tissue. In contrast, we detected WNT5A-S expression only in placenta, and, to a lesser extent, in trachea and small intestine (Figure S4B).

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Figure 3. Effects of WNT5A isoform expression on cancer cell line proliferation.

A. WNT5A isoform expression in three cancer cell lines. Transcript levels of WNT5A isoforms were determined in MDA-MB-231 (breast carcinoma), HeLa (cervix carcinoma), and SH-SY5Y (neuroblastoma) by qRT-PCR and normalized by EF1-α mRNA (normalization by GAPDH mRNA and 18S rRNA produced similar results). Mean ratios (WNT5A isoforms/ EF1-α) ± SEM from independent measurements are shown. B. Effects of ectopic expression of WNT5A-L and WNT5A-S isoforms on proliferation. The indicated cell lines were transduced with expression vectors encoding WNT5A-L (blue line), WNT5A-S (red line) or no WNT (Black line) and cell numbers were determined at 3 and 6 days. WNT5A-S increases while WNT5A-L decreases proliferative rates. Data (mean ± SEM from triplicate determinations) from a representative experiment are shown. Each experiment was performed at least three independent times. C. Isoform specific siRNAs reduce expression of each WNT5A isoform. The efficiency of WNT5A-L and WNT5A-S isoform knockdown (KD) was evaluated in MDA-MB-231, HeLa and SH-SY5Y transfected with control siRNA and WNT5A isoform-specific siRNA (KD). WNT5A isoforms transcript levels were measured by quantitative RT-PCR (qRT-PCR) and normalized by EF1-α mRNA (normalization by GAPDH mRNA and 18S rRNA produced similar results). D. Effects of siRNA-mediated knockdown (KD) of WNT5A-L and WNT5A-S isoforms on proliferation. Cells were transfected with siRNAs specific to WNT5A-L (blue line) or WNT5A-S (red line) and cell numbers were determined at 3 and 6 days. A scrambled siRNA served as control (black line). Co-transduction of cells with a WNT5A-S expression vector rescues the effect of the WNT5A-S-specific siRNA (dashed red line). Data (mean ± SEM from triplicate determinations) from a representative experiment are shown. Each experiment was performed at least three independent times. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001. E. Differential regulation of AXIN2 and CDK8 expression by isoform-specific knockdown of WNT5A. Transcript levels of AXIN2 and CDK8 were determined by quantitative RT-PCR analysis (normalized by EF1-α mRNA) in control MDA-MB-231 (breast carcinoma), HeLa (cervix carcinoma) and SH-SY5Y (neuroblastoma) cells, and in response to siRNA-mediated knock-down (KD) of WNT5A-L or WNT5A-S. Mean ratios ± SEM from independent measurements are shown.

https://doi.org/10.1371/journal.pone.0080526.g003

WNT5A-S promotes and WNT5A-L suppresses growth of cancer cell lines

To study the functions of the WNT5A isoforms, we manipulated their expression in several cell lines, including MDA-MB-231, HeLa and SH-SY5Y, and analyzed the phenotypic consequences (Figure 3B-D; Figure S5). Since signaling activities of purified WNT proteins are short-lived [30] and biological assays generally require long periods of stimulation, we utilized transfection of WNT genes rather than application of purified proteins. Ectopic expression of WNT5A-L substantially reduced cell numbers in these cell lines (Figure 3B). No evidence of increased cell death was observed (data not shown), indicating that WNT5A-L acts to inhibit cell proliferation. In contrast, ectopic expression of WNT5A-S promoted proliferation of MDA-MB-231, HeLa and SH-SY5Y cells (Figure 3B).

Using isoform-specific siRNAs (see Figure S1; Table S2), we disrupted expression of each WNT5A isoform (Figure 3C). The siRNAs were able to specifically knockdown their targets up to 80% as determined by qRT-PCR (Figure 3C). Importantly, each WNT5A isoform was selectively inhibited by the corresponding siRNA without affecting levels of the other isoform. Consistent with the proliferation promoting effects of WNT5A-S ectopic expression, knockdown of WNT5A-S reduced cell numbers (Figure 3D). Even in cells expressing low WNT5A-S levels, such as MDA-MB-231 (Figure 3A), knockdown of the WNT5A-S isoform produced a strong inhibitory effect (Figure 3D). The cell inhibition resulting from WNT5A-S siRNA was rescued in all tested cell lines by co-transduction with a WNT5A-S vector devoid of the siRNA target sequence (Figure 3D, KD WNT5A-S + rescue). These rescuing data demonstrate that the effects of this siRNA are due to WNT5A-S knock-down rather than non-specific off-target effects. Furthermore, WNT5A-S knockdown increased cell death in the three cell lines (Figure S5A) and induced caspase activity in MDA-MB-231 and HeLa (Figure S5B). However, the ≥3-fold reduction in cell numbers (Figure 3D) exceeded the observed modest increase in cell death (Figure S5A), suggesting that WNT5A-S knockdown mainly impaired proliferation. In contrast, knockdown of WNT5A-L had no effect on cell proliferation (Figure 3D), cell death or caspase activity in the 3 cell lines (Figure 3D; Figure S5A, B). Taken together, these experiments demonstrate that endogenously and ectopically expressed WNT5A isoforms exert distinct effects in breast, cervix and neuroblastoma tumor cell lines, with WNT5A-S promoting and WNT5A-L inhibiting cell proliferation. Since we did not observe distinct signaling activities for WNT5A-L and WNT5A-S in WNT/β-catenin and DVL phosphorylation assays (Figure 2), we speculate that these growth promoting and inhibiting activities of WNT5A are independent of the canonical WNT signaling pathway.

In an attempt to identify a mechanism by which WNT5A isoforms exert differential effects on proliferation, we screened the expression of several known WNT regulated genes. We found that expression of two genes, AXIN2 and CDK8, both of which have been implicated in various aspects of WNT signaling [7,40,41], were differentially regulated by isoform-specific knockdown of WNT5A. Knockdown of WNT5A-L, but not WNT5A-S, led to a decrease in AXIN2 expression in 3 tested cell lines (MDA-MB-231, HeLa and SH-SY5Y, Figure 3E). In contrast, knockdown of WNT5A-S, but not WNT5A-L, led to an increase in CDK8 expression in 2 out of 3 cell lines (MDA-MB-231 and HeLa) and a decrease in SH-SY5Y (Figure 3E). This data offers a potential molecular link between the observed differential effects of WNT5A isoforms on proliferation and gene regulation and indicates that each WNT5A isoform impinges on gene expression in a distinct manner. Further exhaustive studies, such as transcriptome analysis, will be needed to identify and define pathways that mediate these differential effects.

Down-regulation of WNT5A-L and overexpression of the WNT5A-S isoform in cancers

To determine whether the respective pro- and anti-proliferative functions of the WNT5A-S and WNT5A-L isoforms are relevant to oncogenesis, we analyzed their expression levels by qRT-PCR in primary tumor samples from breast and cervix carcinomas and neuroblastomas. As compared to normal breast tissues, WNT5A-L was down-regulated ≥3-fold in 46% (14 of 30) of breast carcinomas (P= 0.04) (Figure 4A). WNT5A-S was undetectable in normal breast tissue and overexpressed in a small subset of breast cancers (4 of 30, 13%, n. s.) (Figure 4A). In normal uterine cervix epithelium, both WNT5A-L and WNT5A-S were expressed, WNT5A-L being the predominant isoform (Figure 4B). In comparison with normal cervix, WNT5A-L was down-regulated ≥3-fold in 53% (8 of 15) of cervix carcinomas (P= 0.047). In contrast, WNT5A-S was up-regulated ≥4-fold in 40% (6 of 15) of these tumors (P= 0.045) (Figure 4B). Neuroblastomas, pediatric cancers of the sympathetic nervous system, can be classified into low and high risk tumors with 90% and 30% of survival, respectively. Half of the high risk neuroblastomas are fatal within 2 years from diagnosis [42]. High risk neuroblastomas showed low WNT5A-L expression more frequently than low risk tumors (55% -22 of 40- versus 21% -8 of 37-, P= 0.004), thus suggesting that WNT5A-L down-regulation correlates with high risk neuroblastoma. Furthermore, elevated WNT5A-S expression was associated more frequently with high risk than with low risk neuroblastomas (17% -7 of 40- versus 5% -2 of 37-, n. s.) (Figure 4C). In our cohort, 5% (2 of 40) of patients with low risk neuroblastomas died over a 10-year follow-up period, in contrast with 70% (half of them within 2 years from diagnosis) in the high risk group. In this latter group of the most aggressive tumors, low expression of WNT5A-L or high expression of WNT5A-S were both associated with shorter survival (mean= 26.05 versus 37.1 months for WNT5A-L and 27.56 versus 36.2 months for WNT5A-S). However, this association was not statistically significant. High risk neuroblastomas carry several genetic alterations, each of high prognostic value [42]. Therefore, only analysis of larger cohorts of patients will determine whether the expression status of WNT5A-L or WNT5A-S isoforms is of further relevance to clinical outcome.

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Figure 4. Altered expression of WNT5A isoforms in cancers.

Transcript levels of WNT5A-L (left panels) and WNT5A-S (right panels) isoforms were measured in different types of cancers by qRT-PCR and normalized by EF1-α mRNA (normalization by GAPDH mRNA and 18S rRNA produced similar results). Symbols represent mean ratios (WNT5A isoforms/ EF1-α) from at least two independent measurements for each sample. Horizontal bars correspond to the mean expression value of WNT5A isoforms in each subgroup of samples. For breast (A) and uterine cervix (B), expression in carcinomas was compared to that in normal tissues. For neuroblastomas (NB, C), expression was compared between low risk tumors with good prognosis (Stages 1, 2, 3: localized or locally invasive NB; Stage 4S: spontaneously regressing metastatic NB; Stage 4 < 1 year, metastatic NB of infants) and high risk tumors with poor prognosis (Stage 4 > 1 year: metastatic NB of older children). n.s. = not significant.

https://doi.org/10.1371/journal.pone.0080526.g004

As WNT5A inactivation is frequent in hematological malignancies [17,19], we also analyzed the expression status of its isoforms in these cancers and corresponding normal tissue. Normal blood cells exclusively express the WNT5A-L isoform, which was strongly down-regulated in most of the myeloid and B-cell lymphoid leukemia as well as T-cell lymphoma samples (Figure S6). The WNT5A-S isoform was only marginally expressed in one B-cell leukemia and one T-cell lymphoma, indicating that this isoform is not significantly implicated in hematological cancers. These results are consistent with previous observations [17,19], however, these previous studies did not distinguish between the two WNT5A isoforms. Thus, in these selected human tumors, the WNT5A-L and WNT5A-S isoforms behave as tumor suppressive and oncogenic factors, respectively.

Ethics Statement

According to the French Public Health and Bioethical Law, our study was considered by our Research Direction Lawyer (Direction de la Recherche Clinique, Centre Hospitalier Universitaire de Bordeaux, France) as a non interventional study without need for ethics committee approval (Article L1121-1 and Article R1121-3). The need for written informed consent from the participants was not waived, and, according to that law, all patients (or their legal tutors for pediatric tumors, herein neuroblastomas) received oral and written information concerning research on their material with possibility to refuse the use of their clinical data and biological samples. None of the patients whose material was analyzed in this study refused such use and all patients signed an informed consent form. The present study complied with the Declaration of Helsinki Principles and was approved by the medical and ethics committee of the Centre Hospitalier Universitaire de Bordeaux (Bordeaux, France) and Institut de Cancérologie Gustave Roussy (Villejuif, France).

Patient tissues and cell collection

Normal and cancerous tissues and cells were collected and stored frozen at -80°C prior to nucleic acid extraction. Samples with estimated cancer cells content of more than 70% on the basis of cyto-pathological review were retained for analysis. RNA from a series of normal adult tissues were purchased from Ambion.

Plasmids and antibodies

The sequences encoding WNT5A-Long and WNT5A-Short isoforms were separately amplified by polymerase chain reaction (PCR) using cDNA from HeLa cells as a template. PCR products were cloned in the pCDNA3.1(+)Neo expression vector (Invitrogen) and constructs were verified by sequencing. For the WNT5A-S producing CHO line we engineered a construct lacking the sequence for the WNT5A-L specific peptide (NSWWSLGMNNPVQMSEVY). Generation of CHO cell lines have been described elsewhere [30]. The SuperTOPFLASH luciferase reporter was kindly provided by the laboratory of Dr. R. Moon. The TOP-GFP construct was obtained from Addgene (Plasmid #24305, described in Fuerer and Nusse 2010 PLoS One[51]). The WNT5A antibody has been described elsewhere [3]. The WNT5A-L specific antibody was generated by immunizing two rabbits with the peptide specific to the long isoform (NSWWSLGMNNPVQMSEVY). Immunizations, serum collections and exsanguination were performed by Epitomics, Inc. Antisera were tested by immuno-blotting of WNT5A-L and WNT5A-S. The antisera were affinity purified against the peptide (NSWWSLGMNNPVQMSEVY) immobilized to Sepharose beads. The Dvl2 antibody was generated by immunizing two rabbits with a GST fusion protein to the 86 carboxy terminal amino acids of mouse Dvl2. Antisera were affinity purified against the immunogen. The rabbit anti-Dvl1 antibody was provided by Epitomics, and the mouse anti-β-catenin antibody was purchased from Sigma-Aldrich.

Cell culture

MDA-MB-231, MCF-7, T-47D, SK-Br-3, HeLa, SH-SY5Y, SK-N-AS, IMR-32, HEK-293, L cells and C2C12, all obtained from the ATCC (American Tissue Culture Collection), IGR-N-91[52], IGR-NB8 [53], LAN-1 [54], LAN-5 (Life Technologies) and CLB-Pe [55] cell lines were grown in DMEM supplemented with 10% fetal bovine serum. BT-474 cells (ATCC), were grown in RPMI 1640 supplemented with 10% fetal bovine serum. CHO cells (ATCC) were cultured in DMEM supplemented with 10% bovine serum, 1% non essential amino acids (Gibco), 400µg/ml of G418, 10ng/ml Doxycycline and 2ug/ml Blasticidin.

RNA expression analysis

RNA was isolated from freshly frozen tumor or cell samples and matched normal counterparts using the "RNeasy mini" kits (QIAGEN). For gene expression analysis, cDNA synthesis was performed from total RNA using the SuperScript II reverse transcriptase kit (Invitrogen). For quantitative reverse transcription-PCR (qRT–PCR), we used the SYBR Green detection system (Applied Biosystems) for housekeeping genes and a TaqMan probe (Applied Biosystems) for WNT5A transcripts. Sequences of qPCR primers and probe are given in Table S1.

Transfections and WNT activity assays

Short interfering RNA (siRNA, sequences in Table S2) were transfected using Oligofectamine (Invitrogen). Plasmids were transfected using Lipofectamine LTX (Invitrogen). Co-transfection of siRNA and plasmids were performed using Lipofectamine 2000 (Invitrogen). For RNA analyses, cells were collected after 3 days. For proliferation and viability assays, cells were harvested after 3 and 6 days. In case of transfection with plasmids, cells were selected for 6 days using 1 mg/ml of G418. Resistant cells were replated in equal number and grown for up to 6 days before counting. For luciferase reporter assays, stable HEK293 transfectants carrying a TOP-Luciferase reporter element (Addgene plasmid 12456) were plated in 96 well plates and collected after 24h of WNT treatment and assayed using the Luciferase Assay kit (Promega) on a luminometer (Perkin Elmer Envision). For GFP reporter assays, stable MDA-MB-231 transfectants carrying a TOP-GFP reporter element (Addgene plasmid 35489) were collected after 48h of WNT treatment and regulation of TOP-GFP was monitored by flow cytometry (BD FACS Canto). For the Dvl phosphorylation assay, L1 and C2C12 cells were grown to ~80% confluence, treated with WNT proteins at 10 μM for 2 hours, lysed on ice in 1% Triton X-100, 150 mM NaCl, 50 mM Tris-Cl, pH7.5, and protease inhibitors Leupeptin (1 μg/ml), Aprotinin (10 μg/ml), and Pefabloc (1 mM) and the phosphatase inhibitor β-Glycerophosphate (10 mM). 20 μg total soluble whole cell lysate was resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and immuno-blotted with the indicated antibodies.

Proliferation and viability

Cells transfected with plasmids and/or siRNA were harvested by trypsin treatment after 3 and 6 days and assessed for proliferation and viability using Trypan Blue exclusion and counting with a ViCell counter (Beckman).

Statistical analysis

The Student’s t-test was used to compare the expression levels of WNT5A isoforms in two groups of samples and the proliferation of control and genetically engineered cells. All graphs represent mean values ± s.e.m. Clinical outcome was analyzed in neuroblastoma patients as a function of WNT5A isoforms expression levels. Kaplan-Meier curves were used to assess overall survival, and differences between groups were compared by the log-rank test. * : P < 0.05; ** : P < 0.01; *** : P < 0.005; **** : P < 0.001 (when P values are not indicated on the figure).

WNT5A protein expression, purification and characterization

WNT5A isoforms were expressed in CHO cells [30] and conditioned media (CM) were collected the day after confluence was reached. WNT proteins were purified from 6 liters of CHO CM. CM was complemented with 1% Triton X-100 (v/v), 20 mM Tris-Cl pH 7.5 and 0.01% NaN₃. The purification method consists of four consecutive steps performed on an Äkta Purifier (GE Healthcare) using the method previously described [56]. WNT yields were determined by Coomassie staining of SDS-polyacrylamide gels. Yields for WNT5A isoforms averaged at 40µg per liter of CM. Stability of WNT5A isoforms was tested by spiking HEK 293 growth media with equal amounts of purified WNT5A isoforms, and incubating at 37°C for the indicated time points. Hydrophobicity of WNT5A proteins was assessed by Triton X-114 phase separation. 500µl of CM was mixed with 500µl Triton buffer (2.2% Triton X-114; 300mM NaCl; 20mM Tris-HCl, pH7.5). The mixture was incubated 5min on ice, then 4min at 31°C and subsequently centrifuged at 2000xg for 3min at 31°C. The two phases (top = aqueous, bottom = hydrophobic/organic) were separated and assayed for protein by immuno-blot.