The Eyes Absent phosphatase-transactivator proteins promote proliferation, transformation, migration, and invasion of tumor cells

Abstract

The Eyes Absent (EYA) proteins combine transactivation, tyrosine phosphatase, and threonine phosphatase activities in their function 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.

Figure 1

Over-expression of Eya1, Eya2 or Eya3 results in increased cell proliferation. A) Cell proliferation was measured using the WST assay with MCF7 cells stably expressing the constructs listed on the x-axis. The vector control is GFP, and all over-expression constructs were made as GFP-fusions. Eya3#1 and Eya3#2 indicate separate clones expressing increasing levels of Eya3 as measured by qRT-PCR and by fluorescence intensity (from the GFP tag) (see Supplementary Figure 1). The phosphatase-dead mutant Eya3(D246N) is indicated by the gray bar and Eya3(D246N)-GFP mRNA level is comparable to that of Eya3#2. Bars indicate fold-increase in number of viable cells 48 hours after the start of the experiment (mean ±SD of six experiments; *** p< 0.001, ** p< 0.01, * p < 0.05). B) Cell proliferation measured as in (A) for MCF7 cells over-expressing either the vector control GFP, Eya1-GFP, or Eya2-GFP. Proliferation of MCF7 cells expressing phosphatase-deficient mutants Eya1(D299N)-GFP or Eya2(D209N)-GFP are indicated by the gray bars.

Figure 2

The tyrosine phosphatase activity of Eya promotes cell migration and invasion. A) Cell migration was measured using transwell assays performed on MCF7 or MDA-MB-231 cells over-expressing either GFP, Eya3#2 or the phosphatase-deficient Eya3(D246N) (gray bar). The number of cells migrating to the bottom of the transwell insert are indicated. B) Cell migration measured and presented as in 2A for MCF7 cells over-expressing either the vector control GFP, Eya1-GFP, Eya1(D299N)-GFP, Eya2-GFP or Eya2(D209N)-GFP. C) Matrigel invasion assays were performed as in A & B above, except that the transwells were coated with basement membrane Matrigel (1:20 dilution) and cells migrating to the bottom side of the insert were measured 48 hours after the start of the experiment. D) Cell migration and invasion were measured for MDA-MB-231 cells stably transfected with either a scramble control RNA or siRNA to EYA3. Rescue cell lines expressing either mouse Eya3 or mouse Eya3(D246N) in addition to EYA3 siRNA or scramble control RNA were also assayed. The reduction in EYA3 mRNA is indicated in the inset graph. All data represent the mean of three experiments ± SD, *** p< 0.001, ** p< 0.01, * p< 0.05, NS not significant.

Figure 3

Eya1, Eya2 and Eya3 promote transformation of MCF7 cells. A) Cells stably expressing the indicated constructs were assessed for focus-formation. Photographs of plates 13 days post-plating are shown. Activated Ras(Q61L) expressing MCF7 cells were used as a positive control. The number of foci/field were quantified and the results are shown in (B). Each bar is the mean of three experiments ± SD, *** p< 0.001, ** p< 0.01. C) The number of foci measured at day 6 with each bar being the mean of three experiments ± SD.

Figure 4

Eyes Absent affects the actin cytoskeleton and levels of active Rho-family GTPases. A) Eya3 or Eya2 over-expression induces the formation of filopodia and lamellipodia while the mutants Eya3(D246N) and Eya2(D209N) lead to cell flattening and stress fiber formation. MCF7 cells stably expressing GFP, Eya3-GFP, Eya2-GFP, Eya2(D209N)-GFP or Eya3(D246N)-GFP were plated on laminin-coated slides. Cells were fixed, stained for F-actin (red) and DAPI (blue) and photographed after 1 hour. The lower panel shows images taken on a Leica TCS-SP5 confocal microscope. B) MCF7 cells expressing either the vector control, Eya3-GFP, Eya3(D246N)-GFP, Eya2-GFP or Eya2(D209N)-GFP were serum-starved and then stimulated with 10% serum for 15 minutes. Whole cell lysates were subject to GST-RBD or GST-PAK pull-down and immunoblotted for RhoA, Rac1 or Cdc42. The GTP-bound forms of RhoA, Rac1 and Cdc42 were normalized to loading control and the average relative activation is shown in the bar graph (average of three experiments).

Figure 5

Reduced expression of Eya3 by siRNA-mediated knockdown in MDA-MB-231 cells decreases the number of lung metastasis, and this is rescued by re-expression of wild-type Eya3, but not Eya3(D246N). MDA-MB-231 cells stably expressing the indicated constructs were injected into the tail vein of SCID mice. Mice were sacrificed 7 weeks after injection. Lungs were removed, bleached and examined for surface metastasis after India ink injection. The number of metastatic foci in the largest left lobe examined were counted and shown as a box plot. The horizontal black line is the median value, the box represents the inter-quartile range (25% to 75% of the distribution) and the line bars the minimum and maximum values.