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The Eyes Absent (EYA) proteins combine transactivation, tyrosine phosphatase and threonine phosphatase activities in their role as part of a conserved regulatory cascade involved in embryonic organ development. EYA tyrosine phosphatase activity contributes to fly eye development, and vertebrate EYA is involved in promoting DNA damage repair subsequent to genotoxic stress. EYAs are known to be expressed at elevated levels in ovarian and breast cancers. Here we show that the tyrosine phosphatase activity of the EYAs promotes tumor cell migration, invasion, and transformation. These cellular effects are accompanied by alterations of the actin cytoskeleton and increased levels of active Rac and Cdc42. The invasiveness conferred by EYA is reflected in vivo by inhibition of metastasis seen when EYA3 expression is silenced in the invasive breast cancer cell line MDA-MB-231. Together our data directly associate the tyrosine phosphatase activity of the EYAs with the oncogenesis-associated cellular properties of motility and invasiveness.
The retinal determination pathway is an evolutionarily conserved developmental cascade known to be involved cell-fate determination in multiple contexts (eye, kidney, muscle, ear), and in species ranging from flies to humans (reviewed in (Hanson, 2001)). The components of this genetic network in vertebrates (and Drosophila) include the Pax (eyeless (ey) and twin of eyeless (toy)), Eyes Absent Eya (eya), Six (sine oculis (so) and optix), and Dachshund (dac) genes. Evidence is emerging that this network is aberrantly expressed in adult cancers, in a seemingly co-ordinated fashion where Eya and Six genes are up-regulated and Dach genes are down-regulated. There are reports of upregulation of Six1 in breast cancers (Coletta et al., 2004; Ford et al., 1998; Ford et al., 2000; Reichenberger et al., 2005), Wilms’ tumor (Li et al., 2002), and in rhabdomyosarcomas (Yu et al., 2006; Yu et al., 2004), Six3 in human extraskeletal myxoid sarcomas (Hisaoka et al., 2004; Laflamme et al., 2003), and Six5 in borderline ovarian tumors (Winchester et al., 2000). Eya2 levels are reportedly higher in breast and ovarian cancers (Zhang et al., 2005), and in desmoid tumors (Bacac et al., 2006), while Eya1 is over-expressed in Wilms’ tumor (Li et al., 2002). On the other hand Dach1 expression is lost in breast cancers with poor prognosis (Wu et al., 2008; Wu et al., 2006; Wu et al., 2007). Six1 and Eya4 are also up-regulated in malignant peripheral nerve sheath tumors while Dach1 is down-regulated (Miller et al., 2010). This coordinated dysregulation of the retinal determination genes is consistent with other examples of genetic programs involved in normal development being co-opted in the process of carcinogenesis.
Proteins encoded by the Pax, Six and Dach genes are transcription factors, while the Eyes Absent (EYA) genes encode dual-function transactivator-phosphatase proteins (Ikeda et al., 2002; Jemc and Rebay, 2007; Li et al., 2003; Okabe et al., 2009; Rayapureddi et al., 2003; Tootle et al., 2003). The biochemical activities of the EYA proteins reside in separable domains: the N-terminal transactivation and threonine phosphatase domain and the C-terminal tyrosine phosphatase domain (ED). In concert with the homeodomain DNA-binding SIX proteins, EYA is known to regulate the expression of several genes including c-Myc, Gdnf, muscle determination genes such as Myod, Mrf4 and Myogenin. Transcriptional targets of the Drosophila EYA-SO complex include lozenge, sine oculis itself, eyeless, hedgehog, and atonal (Pauli et al., 2005; Zhang et al., 2006). Experiments in Drosophila have demonstrated that the phosphatase activity of eya contributes to fly eye development (Rayapureddi et al., 2003; Tootle et al., 2003). While recent reports link the nuclear tyrosine phosphatase activity of EYA with DNA damage repair (Cook et al., 2009; Krishnan et al., 2009), the EYA tyrosine phosphatase activity has also been linked to a cytoplasmic cellular function (Xiong et al., 2009). We are interested in understanding how the biochemical activities of the EYA proteins might contribute to cellular properties characteristic of malignant tumor cells.
In this report we show that over-expression of Eya1, Eya2, or Eya3 results in increased proliferation, migration, invasion, and transformation of breast cancer cells. Interestingly, only the cellular functions of migration, invasion and transformation are dependent on the phosphatase activity. The role of Eya3 in promoting invasion is also supported by in vivo metastasis studies showing that silencing of EYA3 expression in MDA-MB-231 cells leads to reduced metastasis. Taken together, our data suggest that the over-expression of EYA proteins in mammary epithelial cells can promote tumorigenesis, and that their phosphatase activity can specifically contribute to increased invasiveness.
MCF-7 stable lines were analyzed for their proliferation rates using the GFP-MCF-7 line as a control and a WST-1 assay (Takamatsu, 1998).
Cell migration assays were performed using transwell inserts. Cell invasion was also measured using a transwell assay as above except that the filters were coated with basement membrane Matrigel (1:20 dilution).
Focus formation was measured at days 6 and 13 where a focus was defined as group of cells growing on top of each other in which a noticeable change in contrast was observed under Hoffman Optics. All experiments were conducted in triplicate.
Cells were plated on a cover-slip coated with 5 microgram/ml laminin, and fixed with 4% paraformaldehyde. Actin fibers were stained using Alexa-594 labeled phalloidin (Invitrogen).
Levels of GTP-bound RhoA, Rac1 and Cdc42 were measured in MCF7 cells stably expressing either GFP, Eya3-GFP, Eya3(D246N)-GFP, Eya2-GFP, or Eya2(D209N)-GFP after 15 minute serum-stimulation. Levels of total and active proteins were determined by Western blot analyses using anti-Rho antibody (Cell Biolabs), anti-Rac1 (Cell Biolabs) and anti-Cdc42 (Santa Cruz) antibodies.
To produce experimental lung metastasis, MDA-MB-231 cells stably expressing either scramble control RNA, EYA3 siRNA, EYA3 siRNA with mouse Eya3, or EYA3 siRNA with mouse Eya3(D246N) were transplanted into Female SCID Mice (5-6-week old) via lateral tail vein injection. All mice were sacrificed 7 weeks after injection, stained with India ink and metastatic foci were counted. Data are presented as a box-plot using SPSS software.
In order to examine the effect of Eyes Absent over-expression in cells we established stable MCF7 cell lines over-expressing Eya1, Eya2, or Eya3 as GFP fusions, as well as a cell line expressing the vector control GFP alone. We chose Eya3 and Eya2 for more extensive analysis in this study since Eya3 is mechanistically the best characterized of the mammalian Eyes Absent proteins (Rayapureddi and Hegde, 2006; Rayapureddi et al., 2005; Rayapureddi et al., 2003) and Eya2 has been reported to be elevated in ovarian and breast cancers (Zhang et al., 2005). To examine the effect of Eya3 dose on cellular functions, we also established two independent clones expressing different levels of Eya3-GFP. Over-expression resulted in significantly increased cell proliferation as measured in an WST assay (Fig. 1A). The dependence of the pro-proliferative effect on the tyrosine phosphatase activity of the Eya proteins was tested by over-expressing phosphatase-deficient forms of Eya1, Eya2, or Eya3 (Fig. 1A, B) and measuring proliferation. In each case the nucleophilic Aspartate was conservatively replaced with an Asparagine; such a substitution has been shown to significantly reduce the phosphatase activity of the Eyas (Rayapureddi et al., 2003). Proliferation of MCF7 cells stably over-expressing the mutant forms (Eya3(D246N), Eya2(D209N), or Eya1(D299N)) exhibited statistically insignificant differences in the levels of proliferation relative to over-expression of the wild-type proteins (Fig. 1A, B), suggesting that the tyrosine phosphatase activity does not contribute to cell proliferation.
Cell migration was measured using transwell assays and the MCF7 cell lines described above. MCF7 cells over-expressing Eya3 (Fig. 2A), Eya1 or Eya2 (Fig. 2B) each exhibited significantly higher cell migration relative to MCF7 cells expressing the vector control. A similar effect was seen upon over-expressing Eya3 in MDA-MB-231 cells (Fig. 2A). This effect on cell motility was consistently attenuated when the tyrosine phosphatase-dead forms of the Eyas (Eya3(D246N), Eya2(D209N) and Eya1(D299N)) were over-expressed (Figure 2A, B). Similar results were obtained with another tyrosine-phosphatase-dead mutant Eya3(D248N) in which the Asp248 which acts as a general acid in the catalytic reaction (Rayapureddi et al., 2003) is replaced by an Asn, and using differently tagged Eya3 and in COS7 cells (Supplementary Figure 2). These observations suggest that the tyrosine phosphatase activity of the Eyas positively impacts cell motility. Cell motility was also increased upon over-expression of just the C-terminal tyrosine phosphatase domain of Eya3 (Eya3(ED)), but not the inactive mutant Eya3(ED)(D248N) (Figure 2A). However in MCF7 cells expressing Eya3 targeted to the nucleus by the addition of the nuclear localization signal (NLS) from SV40 large T-antigen (PKKKRKV) at the C-terminus of the protein, no increase in motility above that of the parental MCF7 cells was measured (Figure 2A). Together these data support a role in cell migration for the tyrosine phosphatase activity residing in the C-terminal ED domain of Eya.
These results were mirrored when the invasive potential of MCF7 and MDA-MB-231 cells expressing the forms of Eya3 or Eya2 described above was measured using transwells coated with matrigel (Fig. 2C). In each case there was a clear increase in invasiveness upon Eya3 over-expression that was significantly lower when Eya3(D246N) was over-expressed, supporting a positive role for the Eya phosphatase activity in promoting cell invasion. When EYA3 mRNA was silenced using siRNA in MDA-MB-231 cells, both migration and invasion were reduced relative to the same cells expressing a scramble control (Fig. 2D). Cell motility was rescued upon re-expression of Eya3, but not Eya3(D246N), in the siEYA3 containing cells.
The ability of the Eyas to transform cells was measured using focus formation assays (Fig. 3A, B). Eya1, 2 or 3 over-expression resulted in a significant increase in transformation as measured by the number of foci. In all cases the effect was dependent on the tyrosine phosphatase activity as judged by the fewer foci seen in cells expressing the phosphatase-dead mutant forms of the proteins. As a positive control in these experiments we used MCF7 cells stably expressing constitutively active Ras(Q61L). Notably, Eya over-expression not only increased the number of foci but also accelerated the rate of focus formation relative to MCF7-Ras(Q61L) (Figure 3C), supporting a role for the Eyas as potent mediators of cellular transformation.
In further investigations of the cellular role(s) of the EYA phosphatase activity we focused on Eya3 and Eya2. We first examined how the EYAs affect the actin cytoskeleton and found that MCF7 cells over-expressing either Eya3, Eya2, Eya2(D209N) or Eya3(D246N) were morphologically distinct from the parental MCF7 cells or those expressing the vector control (Fig. 4A). The Eya3 and Eya2 over-expressing cells were more rounded and phalloidin staining indicated that they had fewer stress fibers. Cortical actin and many filopodia and lamellipodia were present. In contrast, the Eya3(D246N) or Eya2(D209N) over-expressing cells were relatively flat and spread, and had more prominent stress fibers.
Since regulation of the actin cytoskeleton is closely linked with the activation states of the Rho family of GTPases we compared the levels of active Rho, Rac and Cdc42 in MCF7 cells over-expressing Eya2 or Eya2(D209N) relative to cells expressing the vector control. GST-Rhotekin (for RhoA) or GST-PAK (for Rac and Cdc42) was used for the selective pulldown of the GTP-bound forms. The data indicate increase in Rac and Cdc42 activation in cells expressing Eya2 or Eya3 (Figure 4B). GTP-Rac promotes the formation of lamellipodia at the leading edge of a moving cell, while GTP-Cdc42 is associated with formation of filopodia, cell polarization and directional cell movement (Ridley et al., 2003). In contrast, the level of GTP-RhoA was increased in MCF7-Eya2(D209N) and Eya3(D246N) cells, consistent with the observed increase in stress fibers (Figure 4A). These observations suggest that the EYA tyrosine phosphatase activity can produce opposing effects on Rho and Rac/Cdc42 activities, which could in turn influence cell motility and the actin cytoskeleton.
We next used an in vivo assay to establish whether EYA3 plays a role in the metastatic activity of MDA-MB-231 cells. MDA-MB-231 expresses EYA3 and produces lung metastases (Richert et al., 2005). Thus, it was a good candidate for RNAi knockdown-mediated assessment of EYA3 function in metastasis. MDA-MB-231 cells stably expressing either a scramble control RNA, EYA3 siRNA, EYA3 siRNA along with mouse Eya3, or EYA3 siRNA along with mouse Eya3(D246N) were injected into the tail vein of immunodeficient SCID mice at 5-6 weeks of age and lung metastases visualized 7 weeks later. Metastases, appearing as white regions of tissue against a black background of the ink-filled lung were counted and a statistical analysis performed. This showed that parental MDA-MB-231 cells produced significantly more metastases than those expressing EYA3 siRNA (Fig. 5). MDA-MB-231 EYA3 siRNA cells also produced smaller metastases than control MDA-MB-231 cells. In the rescue experiments, re-expression of wild-type Eya3 lead to near-control level metastasis, while re-expression of the tyrosine-phosphatase dead mutant Eya3(D246N) did not. These results indicate that, consistent with the in vitro observations presented earlier, EYA3 is a determinant of metastatic behavior in these tumor cells, and that this function needs the tyrosine phosphatase activity.
Phosphatases play important roles in regulating cell migration by altering the phosphorylation state of proteins involved in focal adhesion and extra-cellular matrix interactions. This in turn can alter the actin cytoskeleton via the Rho family GTPases (reviewed in (Larsen et al., 2003; Ostman et al., 2006)). Protein tyrosine phosphatases that both promote (PRL PTPs (Fiordalisi et al., 2006), PTP1B (Liang et al., 2005)), and inhibit (HD-PTP (Castiglioni et al., 2007; Mariotti et al., 2009), DEP-1 (Jandt et al., 2003)) cell motility and invasiveness have been reported. Our results provide evidence that the Eyes Absent family of tyrosine phosphatases promote cell migration, invasion and transformation, and that these functions require the phosphatase activity. This observation has important implications for the role of the EYAs in cell-fate determination during embryonic development, as well as possibly in cancer metastasis.
A link between the EYA phosphatase activity and cell motility/invasiveness complements a recent report suggesting that phosphorylation of Drosophila Eya by Abl shuttles it to the cytoplasm where it carries out an, as yet undefined, essential cellular function (Xiong et al., 2009). The effects of Eya on cell motility and the actin cytoskeleton reported here are consistent with a cytoplasmic function of the Eya phosphatase, distinct from the recently reported nuclear dephosphorylation of H2AX (Cook et al., 2009; Krishnan et al., 2009). The cytoplasmic substrate(s) of Eya remain to be elucidated, but a direct link with a cellular function such as cell motility could provide a critical readout for validating such a target once it is identified.
Over-expression of the Eyas also promotes cell proliferation. However, our data suggest that cell proliferation is not dependent on the phosphatase activity, but rather that the transactivation function of Eya may be the more important contributor. In keeping with our observations, Kriebel et al recently reported that over-expression of Xeya3 in Xenopus results in an expansion of the neural plate, and dramatically enlarged brain structures and eyes at the tailbud stage (Kriebel et al., 2007). Further they have shown that phosphatase-deficient forms of Xeya3 enhance the observed neural hyperplasia above that seen with wild-type Xeya3. eya over-expression also leads to increased tissue overgrowth in Drosophila (Bonini et al., 1997). Commensurate with a role for Eya in cell proliferation are previous reports that cell cycle regulatory genes cyclin D1 and cyclin A1 (Coletta et al., 2004; Yu et al., 2006), and the proto-oncogene c-Myc (Li et al., 2003) are transcriptional targets of the mammalian Eya - Six protein complex. Although proliferation is not tyrosine phosphatase-activity dependent, the reduced contact-inhibition of cell growth seen in the transformation assays is dependent on the phosphatase activity. While the mechanisms underlying contact inhibition are incompletely understood, there is precedent for the mediation of cell transformation by tyrosine phosphatases (Agazie et al., 2003).
The retinal determination genes EYA, SIX and DACH appear to be coordinately de-regulated in a variety of malignant conditions including ovarian and breast tumors, and malignant peripheral nerve sheath tumors. The recurring trend is that EYA and SIX expression is increased and DACH expression reduced. In this context it is interesting to note that the pro-proliferation and pro-migratory effects of Eyes Absent over-expression reported here are mirrored when Dachshund levels are lowered in breast cancer cells (Wu et al., 2008; Wu et al., 2006). Furthermore, increased proliferation has been noted upon SIX1 over-expression in breast cancer (Christensen et al., 2007; Coletta et al., 2004). Taken together, these observations suggest that mis-deployment of this cell-fate determination cascade could contribute to increased tumor malignancy and poorer clinical outcomes.
The ability of EYA2 to promote tumor growth in a xenograft model was previously described (Zhang et al., 2005). Interestingly, despite the in vivo evidence linking EYA2 over-expressing cells with high proliferative rates, there appeared to be no effect of EYA2 over-expression on the cell cycle or on cell proliferation in vitro using several ovarian cancer cell lines and MDA-MB-468 (Zhang et al., 2005). In contrast our data show a clear and positive correlation between Eya over-expression and cell proliferation in MCF7 cells. Furthermore, the positive correlation between EYA levels and cell motility is consistently seen both in vitro (cell migration) and in vivo (tumor metastasis). Together, these data link each of the biochemical activities of the EYA proteins, transactivation and tyrosine phosphatase, with cancer phenotypes.
Finally, the association of the tyrosine phosphatase function of EYA with a cancer-associated cellular phenotype raises the distinct possibility that inhibition of the EYA phosphatases could be an attractive new mode of targeted anti-cancer therapy. As targets for inhibitor design the EYAs possess a distinct advantage; they are mechanistically and structurally distinct from the large family of classical thiol-based PTPs thus increasing the feasibility of selective inhibition.
Supplementary Figure 1. qRT-PCR was performed on the stable cell lines MCF7-GFP, MCF7-Eya3-GFP#1 and MCF7-Eya3-GFP#2. The level of mRNA in the Eya3 expressing cell lines is shown relative to the vector control. A Western blot performed on the two Eya3 over-expressing cell lines and probed using anti-GFP antibody (Miltenyi Biotec) is inset.
Supplementary Figure 2. Cell migration was measured using transwell assays performed on MCF7 or COS7 cells either stably or transiently transfected with the indicated constructs. The number of cells migrating to the bottom side of the transwell insert are indicated.
We thank Dr. Steve Danzer for help with confocal microscopy. Viral vectors were produced by the Viral Vector Core at the Translational Core Laboratories, Cincinnati Children’s Hospital Research Foundation. This study was supported by NIH grants RO1-EY014648 and R21-EY19125 to RSH.