EYA4 drives breast cancer progression and metastasis through its novel role in replication stress avoidance

The Eyes Absent (EYA) family of proteins is an atypical group of four dual-functioning protein phosphatases, which have been linked to many vital cellular processes and organogenesis pathways. Like the other isoforms, EYA4 possesses transcriptional activation and phosphatase functions, with serine/threonine and tyrosine phosphatase domains. EYA4 has been associated with several human cancers, with tumor-suppressing and tumor-promoting roles. However, EYA4 is the least wellcharacterized member of this unique family of phosphatases, with its biological functions and molecular mechanisms in cancer progression, particularly in breast cancer, still largely unknown. In the present study, we found that the over-expression of EYA4 in breast tissue leads to an aggressive and invasive breast cancer phenotype, while the inhibition of EYA4 reduced tumorigenic properties of breast cancer cells in vitro and in vivo. Cellular changes downstream of EYA4, including cell proliferation and migration, may explain the increased metastatic power of breast cancer cells over-expressing EYA4. Mechanistically, EYA4 prevents genome instability by inhibiting the accumulation of replication-associated DNA damage. Its depletion results in polyploidy as a consequence of endoreplication, a phenomenon that can occur in response to stress. The absence of EYA4 leads to spontaneous replication stress characterized by the activation of the ATR pathway, sensitivity to hydroxyurea, and accumulation of endogenous DNA damage as indicated by increased γH2AX levels. In addition, we show that EYA4, specifically its serine/threonine phosphatase domain, plays an important and so far, unexpected role in replication fork progression. This phosphatase activity is essential for breast cancer progression and metastasis. Taken together, our data indicate that EYA4 is a novel breast cancer oncogene that supports primary tumor growth and metastasis. Developing therapeutics aimed at the serine/threonine phosphatase activity of EYA4 represents a robust strategy for killing breast cancer cells, to limit metastasis and overcome chemotherapy resistance caused by endoreplication and genomic rearrangements.

response to stress. The absence of EYA4 leads to spontaneous replication stress characterized by the activation of the ATR pathway, sensitivity to hydroxyurea, and accumulation of endogenous DNA damage as indicated by increased γH2AX levels. In addition, we show that EYA4, speci cally its serine/threonine phosphatase domain, plays an important and so far, unexpected role in replication fork progression. This phosphatase activity is essential for breast cancer progression and metastasis. Taken together, our data indicate that EYA4 is a novel breast cancer oncogene that supports primary tumor growth and metastasis. Developing therapeutics aimed at the serine/threonine phosphatase activity of EYA4 represents a robust strategy for killing breast cancer cells, to limit metastasis and overcome chemotherapy resistance caused by endoreplication and genomic rearrangements.

Background
The Eyes Absent family (EYA1-4) is a unique group of dual-functioning protein phosphatases, which have been shown to promote cell proliferation, invasion, migration, and survival in a variety of cancers (1,2,3).
Members of the EYA family possess N-terminal transcriptional co-activation and threonine phosphatase activity, and C-terminal tyrosine phosphatase activity (4,5,6). The highly conserved C-terminal domain, also known as the EYA domain (ED), contains a haloacid dehalogenase (HAD) signature sequence, making them the only known HAD-family tyrosine phosphatases (Supp. Figure 1A) (4). As the founding members of a new class of non-thiol-based protein tyrosine phosphatases, EYAs have a unique active site, using aspartic acid rather than cysteine as the nucleophile, making these atypical phosphatases attractive targets for speci c inhibition with small molecules. However, the biological functions and cellular targets of these dual-phosphatases remain largely unknown, particularly for EYA4.
Defects in EYA4 have been linked to different developmental disorders including hearing loss (7) and cardiomyopathy (8). EYA4 has also been associated with cancer in various organs. In malignant peripheral nerve sheath tumors (MPNST) EYA4 is over-expressed (9), whilst it is down-regulated in esophageal adenocarcinoma (10,11), hepatocellular carcinoma (12), lung cancer (13) and colorectal cancer (14), where the EYA4 gene promoter has been found to be hypermethylated. Consistent with this, our group and collaborators identi ed EYA4 as a potential novel breast cancer gene (15). Speci cally, our observation that EYA4 is hypermethylated in the rst intron-exon junction particularly in triple-negative breast cancer patients when compared to matched normal samples, led us to pursue its role in carcinogenesis and its cellular functions. To do this, we inactivated or over-expressed EYA4 in a variety of cell lines and investigated the resulting phenotypes, including cell cycle progression and DNA replication e ciency.
Here, we show that over-expression of EYA4 increases proliferation and migration in breast cancer cells, features that are linked with aggressive breast cancer in vivo. The function of EYA4 in promoting breast cancer growth and metastasis is also supported by in vivo xenograft studies showing that silencing of EYA4 expression in MDA-MB-231 cells leads to reduced cancer burden and distant metastasis.
Interestingly, we found that the serine/threonine phosphatase activity of EYA4, but not its tyrosine phosphatase, is essential for breast cancer progression and metastasis.
In cells, we uncovered that EYA4 depletion promotes endoreplication and consequently polyploidy, a phenomenon that can occur in response to stress (16, 17) and can cause drug resistance (18). The absence of EYA4 leads to spontaneous replication stress characterized by activation of key cell cycle checkpoints (pChk1 and pChk2), sensitivity to hydroxyurea, and accumulation of endogenous DNA damage, as indicated by increased γH2AX levels. Upon induction of replication stress by hydroxyurea in EYA4-depleted cells, enhanced levels of unresolved DNA breaks are observed, suggesting that EYA4 plays a crucial role in the repair of replication-associated DNA damage.
Taken together, our data indicate that EYA4 is a novel oncogene in breast cancer and could play a role in cell cycle maintenance. This makes EYA4 an attractive druggable target in cancer treatments, especially in triple-negative breast cancer, to limit metastasis and overcome chemotherapy resistance.
RNA extraction and quantitative reverse transcription PCR (qRT-PCR). Total RNA was isolated from transfected or transduced cells by phenol-chloroform extraction (TRIzol; Invitrogen) followed by nucleic acid precipitation. The GoScript Reverse Transcription System (Promega) was used to generate rststrand cDNA. Quantitative PCR was performed using TaqMan probes spanning across exons for human EYA4 (Invitrogen Hs01012406_mH) and human 18S (Invitrogen Hs99999901_s1) to amplify 70 bp and 187 bp fragments, respectively. The relative expression of EYA4 was determined using the 2 -ΔΔCt method with 18S as an endogenous control for normalization.
Cell proliferation assay. Cells were seeded in a 96-well plate at 2.0 x 10 3 cells/well. Phase contrast images of cells were acquired every 2 h using an IncuCyte Zoom (Essen BioScience) live imaging system. Proliferation was measured as a percentage of con uency.
In vitro migration assay. Cells were cultured in a 96-well plate for 24 h to achieve 100% con uency. An IncuCyte Woundmaker was used to make a scratch in the cell monolayer. Cells were then incubated in serum-free media and automatically imaged every 2 h using an IncuCyte Zoom (Essen BioScience) live imaging system. The scratch gap width and con uence were measured at each time point and compared between groups.
Double thymidine block and cell cycle progression ( ow cytometry). HeLa cells were synchronized in early S-phase by a double thymidine block. Brie y, cells were blocked with 2 mM thymidine for 18 h, released for 9 h, and blocked again with 2 mM thymidine for 17 h. After the second block, cells (asynchronized and synchronized) were released and collected according to time points, then xed in ice-cold 70% ethanol at -20°C for at least 24 h. DNA was stained with 38 mM trisodium citrate, 100 µg/mL RNase A and 150 µg/mL propidium iodide (PI) for 1 h at RT. A DNA control PI (trout erythrocytes) was used as an internal control to normalize the cell cycle. Data were collected using a CytoFLEX Flow Cytometer (Beckman Coulter) and cell cycle pro les were analyzed with FlowJo to determine the percentage of cells in G1, S and G2/M. 10,000 events were collected, and aggregated cells were gated out.
FUCCI. HeLa FUCCI cells stably transfected with empty vector or EYA4 shRNAs were seeded in a 96-well plate (100 cells/well). Phase contrast and green/orange images were acquired every 2 h to monitor cell cycle progression using an IncuCyte SX5 (Sartorius) live-cell imaging system. Images were analyzed using cell-by-cell analysis software and population subsets were classi ed based on green and red uorescence. G1 phase (red), G1-S transition (green + red), S/G2/M phase (green) and M-G1 transition (non-uorescent) (19).
Nuclear foci quanti cation was performed using CellPro ler.
Twenty-four hours after seeding, increasing concentrations of ATR inhibitor (AZ20) or hydroxyurea were added to the culture (24 h pulse). Cell cytotoxicity was measured after 96 h following manufacturer's protocol (Abcam ab211091). Brie y, 50 µL serum-free media (no phenol red) and 50 µL MTT reagent was added to each well and incubated at 37°C for 3 h. MTT media was replaced with 150 µL of MTT solvent and incubated with agitation for 15 min. Absorbance was measured at 590 nm. The cell viability was calculated using the following equation: OD treated and OD control represented the absorbance of sampled and control, respectively. EdU incorporation. HeLa control and EYA4 knockdown cells (4.0 x 10 4 cells/well) were seeded in 12-well plates with coverslips for 24 h. 5-ethynyl-2'-deoxyuridine (EdU) incorporation was performed according to

Results
EYA4 is a novel breast cancer gene. We investigated whether EYA4 is expressed in speci c breast cancer subtypes using real-time quantitative PCR and immunoblotting in several breast cancer cell lines (Supp. Figure 1B-C). The expression of EYA4 varied greatly across cell lines, however, the triple-negative breast cancer cell line MDA-MB-231 showed the highest endogenous expression of EYA4. In most mouse strains, knockout of EYA4 is lethal shortly after birth (22) and is toxic in several lung cancer cell lines (23) and other cell lines that we tested. Using short-hairpin RNAs (shRNAs), EYA4 expression could be signi cantly decreased in MDA-MB-231 cells (Supp. Figure 1D) or in HeLa cells (Fig. 3A). The most e cient hairpin, shRNA3, induces cell death in MDA-MB-231, indicating that EYA4 is essential in these cells. In parallel, we over-expressed EYA4 using two different vectors (Supp. Figure 1E) in the ER + /PR + breast cancer cell line, MCF-7, which expresses low or no detectable endogenous EYA4 (Supp. Figure 1B-C and Cancer Cell Line Encyclopedia, https://sites.broadinstitute.org/ccle). We assessed the effects of EYA4 deregulation on primary cancer growth and metastasis in vivo using luciferase-expressing cell lines. A human tumor xenograft model was established using NOD scid gamma mice. MCF-7/Luc wild type (WT) and EYA4 over-expressing cells were injected subcutaneously into the left mammary fat pad (MFP) of female mice supplemented with 17β-estradiol and monitored by caliper measurement and in vivo imaging for 8 weeks.
Following an intraperitoneal injection with D-luciferin (150 mg/kg), the re y luciferase enzyme catalyzes this substrate, which results in light photons that are captured by a charge-coupled device (CCD) camera mounted within an IVIS® Spectrum Imaging System (24). As shown in Fig. 1A-B, the bioluminescence intensity (BLI) signal measurement con rmed tumor engraftment for all mice. Primary tumors show a signi cant increase in volume when EYA4 is over-expressed. BLI signal correlated with caliper measurements as observed in Fig. 1C, and with tumor volume and weight ( Fig. 1E and F) once surgically removed postmortem (Fig. 1D). EYA4 over-expression leads to a more aggressive breast cancer, as observed by immunohistochemistry (IHC) staining (Fig. 1G). Our observations correspond with previous reports that in MPNST, EYA4 is dramatically upregulated in cells and primary tumors, and its depletion leads to reduced cell adhesion and migration in vitro and has an inhibitory effect in tumorigenesis in vivo (9).
Estrogen receptor alpha (ER-α) co-stain was used to validate human cells. Interestingly, cells expressing high levels of EYA4 also showed high expression of ER-α, the proliferation-related antigen Ki-67, and γH2AX, a marker of DNA damage (Fig. 1G-H). ER-α has a well-established role in supporting estrogendependent breast tumor growth through its association with aberrant proliferation (up-regulating Ki-67), which can result in the accumulation of random DNA mutations (marked by γH2AX), and when highly expressed it is associated with poor prognosis in breast cancer (25,26), which can explain the aggressive breast cancer subtype observed when EYA4 is over-expressed.
Since breast cancer subtypes are associated with unique patterns of metastatic spread, we assessed metastatic capacity utilizing MDA-MB-231 stably expressing re y luciferase. MDA-MB-231/Luciferase WT cells and cells in which EYA4 was stably knocked down (shRNA1 and shRNA2) were injected into the tail vein and monitored by in vivo imaging over 5 weeks. While WT and EYA4-depleted cells colonized the lungs as expected following systemic injection, we observed a decrease in BLI signal in mice injected with EYA4-depleted cells compared to the control ( Fig. 2A). This was directly linked to a lesser number and a decrease in the area of metastatic foci observed in livers as revealed by histological analyses (Fig. 2B-E).
Importantly, these IHC analyses also showed signi cant areas of central necrosis with in ammatory cells and blood vessel congestion (left panel) and scant brosis (right panel) was observed in the control group but not in the EYA4 knockdown mice (Fig. 2F). This particular observation could be due to the role that EYA4 plays in innate immune system regulation by enhancing the expression of IFN-β and CXCL10, in response to DNA stimulation (5). In cancer cells, the cGAS-STING pathway is constitutively activated, inducing chronic IFN-β expression, triggered by the accumulation of DNA damage due to replication fork collapse or reactive oxygen species (ROS) that leads to the presence of DNA in the cytoplasm (27).
Altogether, our data suggest that EYA4 is a driver of breast cancer and that decreasing its expression reduces tumor and metastatic burdens.
The S/T phosphatase domain of EYA4 contributes to breast cancer development. EYA4 possesses both serine/threonine (S/T) and tyrosine (Y) phosphatase activities (Supp. Figure 1A) (6). To investigate the relevance of these activities on tumor growth, MDA-MB-231/Luc cells expressing either EYA4 mutated in the S/T domain (Y281F, Y284F, Y285F; henceforth, 3YF281) or the pY dead combination mutant (D375N, D377N, T548A, E606Q, E607Q, E608Q) were injected into the tail-vein and monitored by in vivo imaging for 4 weeks using the luciferase reporter. The phosphatase mutants (3YF281 and pY dead) caused even more signi cant outcomes that EYA4 depletion, especially the 3YF281 mutant. Both EYA4 phosphatase mutants did not complement EYA4 depletion with shRNA1, as observed by both BLI signal (Fig. 2G) and by metastatic foci observed in livers (Fig. 2H-K). However, the serine/threonine phosphatase activity of EYA4 (3YF281) is the one that shows more signi cant outcomes, as observed not only by decreased tumor burden to lungs (Supp. Figure 2C-E), but also by a lesser number of metastatic foci to the liver, with an average of 2 foci for 3YF281, compared to 6 for EYA4 shRNA2 and 7 for pY dead (Fig. 2J). In addition, as observed by IHC staining, when a metastatic site is observed (marked by H&E) in mouse injected with 3YF281 cells, there is no stain by Ki-67 or γH2AX (Supp. Figure 2F). For γH2AX, only a background level (mouse cells stained), can be observed. Notably, all mice injected with 3YF281 cells showed liver enlargement and hyperplasia (Fig. 2H-I), which could be driven by an increased hepatocyte number, prompting further investigation. These data suggest that the serine/threonine phosphatase activity of EYA4 is essential for breast cancer progression and metastasis.
EYA4 promotes cell proliferation and migration. One simple explanation for variations in primary tumor sizes is the accumulation of larger cells (28, 29) or increased proliferation rates. Uncontrolled and unlimited cell proliferation is a hallmark of cancer (30) and another member of the Eyes Absent family, EYA2, has been shown to increase cell proliferation in lung cancer (31). We generated stable knockdowns in HeLa cells, using three independent short-hairpin RNAs, and a signi cant decrease in EYA4 protein levels was achieved (Fig. 3A). We followed growth rates by live-cell imaging. In both, HeLa (Fig. 3B) and MDA-MB-231 (Supp. Figure 3A) cells, depletion of EYA4 led to lower proliferation rates compared to control. On the contrary, the over-expression of EYA4 in MCF-7 leads to higher proliferation rates when compared to control (Supp. Figure 3C), suggesting that EYA4 promotes cell proliferation. In addition, we investigated the effect of EYA4 on cell migration by comparing the number of control, EYA4 knocked down and EYA4 over-expressing cells at the scratch wound at different time points by live-cell imaging.
HeLa (Fig. 3C) and MDA-MB-231 (Supp. Figure 3B) cells depleted for EYA4, exhibited signi cantly lower migratory capacity relative to cells expressing the empty vector (EV) control, whilst EYA4 over-expression in MCF-7 (Supp. Figure 3D) primes the migration capacity of cells, indicating that EYA4 plays a role in driving cell migration. EYA4 phosphatase mutants, specially 3YF281, display a phenotype comparable, or even more dramatic, than EYA4 depleted cells when tested for proliferation and migration capacities in HeLa cells (Supp. Figure 3E-G), showing a signi cant decrease for both. However, we did not observe the same phenotype in MDA-MD-231 cells (Supp. Figure 3H-J), suggesting that the role in cell migration might be cell line dependent. As we cannot exclude that apparent slower proliferation is caused by cell death, we followed HeLa control and EYA4 knockdown cells after the addition of the apoptosis marker, annexin V. Compared to HeLa control cells, EYA4 shRNA3 showed a slight increase in apoptosis in normal growth conditions (Fig. 3D and Supp. Figure 3K), which could explain, at least partially, the slower proliferation rate observed for shRNA3. The increase in apoptosis observed in HeLa EYA4 shRNA3 cells re ects the fact that this hairpin could not be used in MDA-MB-231 cells, as severe knockdown of EYA4 is incompatible with cell viability.
EYA4 perturbs cell cycle progression. Cell cycle is tightly regulated via checkpoints that are activated by DNA damage, low nutrient content, or other endogenous and external stresses. Aberrant cell cycle progression tends to result in genome instability and contributes to cancer progression. To determine how EYA4 might affect cell cycle progression, ow cytometry was used to pro le asynchronous populations of either control or EYA4-depleted cells (Fig. 4A). We observed a slight increase (2-3%) in S-phase when EYA4 is silenced and a signi cant increase (8%) in the G2/M population for shRNA1 (Fig. 4A), when compared to empty vector control, which suggested a delay in cell cycle progression upon EYA4 depletion. However, shRNA3 does not show a signi cant increase in G2/M, which could be explained due to its characteristic phenotype (enlarged, at and multinucleated cells, Supp. Figure 3L), and this subpopulation could have been gated out by ow cytometry (raw data in Supp. Figure 4). We used the FUCCI system (19) and live-cell imaging ( Fig. 4B and Supp. Figure 5A-B) to overcome these technical issues and pro le single cells. We observed a subtly different behavior for EYA4 shRNA3, especially when it comes to cells in S-G2-M (Fig. 4B). This correlates with cells depleted for EYA4 (especially with shRNA3) undergoing endoreplication (Fig. 4C). Endoreplication refers to a cell cycle variant that only consists of the G and S phases, during which cells replicate their DNA content without dividing, thus giving rise to polyploid cells (18, 32). The result is either a cell that maintains separate nuclei and remains multinucleated, due to a process called endomitosis, or a cell with an enlarged-single nucleus containing all the DNA, derived from a process called endocycling (18). As described above, shRNA3 cells tend to be enlarged and multinucleated, which is characteristic of endomitosis, a major form of endoreplication in which mitosis is initiated but not completed (green/non-uorescent/green; white arrowhead; Fig. 4C). The endoreplication and consequent polyploidy observed, which can occur in response to stress, is a phenomenon that has been linked to cancer progression and chemotherapy resistance (33).
EYA4 induces cell cycle arrest. The most common change leading from a mitotic to an endoreplication cycle is a switch in activation/inactivation of cyclins and cyclin-dependent kinases (CDKs), key regulators of cell cycle progression (34). To investigate if EYA4 expression impacts individual phases of the cell cycle, cells were arrested in early S-phase with a double thymidine block (Fig. 4D) and assessed for cell cycle progression. Propidium iodide (PI) staining of the DNA and ow cytometry in HeLa cells showed that EYA4 decrease (shRNA1) leads to a delay in S-phase restart compared to control (Fig. 4D). Upon release, 74.9% of control cells entered G2/M after 6 h, compared to 49.43% of EYA4-depleted cells (raw data can be found in Supp. Figure 4). EYA4-depleted cells resumed/ nished S with a 2 h delay, and 78.3% of depleted cells entered G2/M 8 h post-release, showing that EYA4 depletion extends S-phase and delays cell division. The most logical explanations for such observations are defects in DNA replication and aberrant checkpoint signals. Since EYA4 depletion appears to halt the cell cycle in the transition between S-phase and G2, we evaluated the activation of several proteins involved in G1 checkpoint (G1/S transition) and G2 checkpoint (schematic in Supp. Figure 5C). We examined the G1/S transition to assess if the cells can initiate DNA replication. For this, we determined the expression of cyclin E1, its partner CDK2, and its corresponding CDK inhibitors, p21 WAF1/CIP1 and p27 KIP1 (Fig. 4E). After double thymidine block (G1/S transition), synchronized EYA4 depleted cells appeared to accumulate p21 WAF1/CIP1 and p27 KIP1 , especially EYA4 depleted with shRNA3. However, CDK2 does not seem to be affected by the CDK inhibitors, since the level of expression appears to be similar between control and EYA4 silenced cells. Cyclin E1 levels increase sharply in late G1, where it interacts and activates CDK2 allowing G1/S transition, then decrease in S-phase (35), as observed in control cells, but not in EYA4-depleted cells. This correlates with the accumulation of cells in G1-S at 6 h observed in cells depleted for EYA4 (Fig. 4D).
Cyclin E1/CDK2 is an important part of the G1 checkpoint and deregulation in the G1/S transition may impair normal DNA replication, causing replication stress and DNA damage (36). Nevertheless, EYA4 silenced cells appear to be able to overcome the G1 checkpoint and initiate DNA replication with little or no delay. Upon release from the thymidine block, we observed that EYA4-depleted cells, especially shRNA1, exhibit a notable delay in S-phase compared to the EV control (Fig. 4D). This was con rmed by the accumulation of cyclin A (highly expressed in S-phase, decreasing in G2) for up to 10 hours postrelease (Fig. 4E). Altogether, these data indicate that in the absence of EYA4, S-phase and its subsequent transition into G2 become prolonged. These effects could stem from faulty DNA replication and/or the accumulation of DNA damage during S-phase.
Spontaneous replication stress is observed in the absence of EYA4. Since EYA4-depleted cells transition through G1/S and enter DNA replication, but S-phase appears to be longer and the S-G2 transition halted, we decided to evaluate the level of expression of pChk1 (S345) and pChk2 (T68) by immunoblotting, to assess if the cells have accumulated spontaneous damaged DNA. To do so, cells were arrested in early Sphase with a double thymidine block. Checkpoint kinase 1 (Chk1) is a key player of DNA-damageactivated checkpoint response that acts downstream of ATR (Ataxia Telangiectasia and Rad3 related) kinase, in response to the formation of single-stranded DNA due to DNA damage of blocked replication forks (Fig. 5A). It is activated by all known forms of DNA damage, particularly triggering the intra-S-and G2/M-phase checkpoints (37). Chk2 is a stable protein expressed throughout the cell cycle. In response to DNA double-strand breaks, Chk2 becomes rapidly phosphorylated at threonine 68 by ATM (Ataxia Telangiectasia Mutated) (Fig. 5A). The kinase activity of Chk2 depends on the severity of DNA damage (38). Under normal conditions, EYA4-depleted cells accumulated pChk1 (S345) up to 8 h after release (Fig. 5A), but not the control, implying that replication fork stalling occurs in the absence of EYA4, and its resolution becomes delayed. Additionally, pChk2 (T68) is highly expressed in the absence of EYA4 (Fig. 5A), which suggests the accumulation of double-stranded breaks (DSBs) that might be a consequence of replication fork collapse. Spontaneous accumulation of DNA damage was con rmed by evaluating the expression of the phosphorylated histone variant H2AX (S319, γH2AX) in early S-phase. Accumulation of γH2AX was observed in EYA4-depleted cells (Fig. 5A), but not in the control, indicating the presence of replication stress, which probably triggers the phosphorylation of H2AX on S139 by ATR (Fig. 5A). In accordance with these results, cells depleted for EYA4 were also found sensitive to AZ20, an ATR inhibitor (Fig. 5B). Since longer S-phase and halted cell cycle observed in the absence of EYA4 might be due to accumulation of replication stress, we sought to assess if the cells are able to progress throughout the cell cycle upon DNA damage induction. We followed the formation of CENP-F foci after 4 Gy γ-irradiation, to identify S-phase and G2/M. CENP-F gradually accumulates during the cell cycle until it reaches peak levels in G2/M phases (weakly positive in S-phase), where it rst associates with kinetochores in late G2 (39). Control cells accumulate in S-G2/M after γ-irradiation, indicating that the cell cycle is halted (1 h after 4 Gy) but they progress once DNA damage is resolved. Nevertheless, in the absence of EYA4, accumulation of CENP-F was observed up to 4 h after irradiation, indicating that the cells are taking longer to resolve DNA damage (Fig. 5C).
EYA4 contributes to HU resistance. To address the potential role of EYA4 in the cellular response to replication stress, we examined the effects of knocking down EYA4 on the sensitivity to hydroxyurea (HU), which causes replication stress by depleting the intracellular pool of deoxynucleotides (40). In accordance with the accumulation of replication stress and checkpoint activation, cells depleted for EYA4 were found to be sensitive to hydroxyurea in an MTT assay (Fig. 6A). In order to monitor DNA synthesis, we treated cells with 4 mM hydroxyurea for 2 h and then measure 5-ethynyl-2'-deoxyuridine (EdU) incorporation after the removal of HU. Under these conditions, silencing EYA4 resulted in a slightly increased rate of EdU incorporation (Fig. 6B), indicating that EYA4 might be involved in maintaining replication fork stability since EYA4-depleted cells appear to overcome the HU blockage and resume synthesis.
EYA4 depletion results in increased and unresolved levels of HU-induced DSBs. Replication fork collapse resulting from chronic HU exposure generates double-stranded breaks (40), which are rapidly marked by γH2AX. To examine the possible role of EYA4 in the repair of HU-induced DSBs, HeLa cells were incubated with 4 mM HU for 2 h and then allowed to recover for 2 h in the absence of the drug. Even though EYA4depleted cells have high levels of endogenous DNA damage, an increase in HU-induced DSBs was observed in the absence of EYA4 (Fig. 6C). Next, HeLa cells were incubated with 4 mM HU for 16 h and then released for 18 h, to assess for unresolved DNA damage in the absence of EYA4. Although residual γH2AX foci were present in HeLa control cells after recovery from HU exposure, ~ 10% more cells with > 10 γH2AX foci per cell (Fig. 6D) were observed in the absence of EYA4, implying that these cells have a diminished ability to resolve HU-induced DNA damage. Together, our results suggest that EYA4 contributes to replication-associated DNA damage repair.
EYA4 impacts replication fork speed. To investigate the functional role of EYA4 in DNA replication, we utilized single-molecule analysis of replicated DNA bers to test if the increased DSBs in EYA4-de cient cells affected replication fork (RF) progression and speed. We found that the replication tracts are much shorter in EYA4 de cient cells compared to control cells, under normal conditions (Fig. 6E), demonstrating that genome-wide RF progression is strongly impaired by EYA4 depletion. Interestingly, the fork slowing observed was even more dramatic in the 3YF281 mutant cells, but not in the pY dead cells, showing that the serine/threonine phosphatase activity of EYA4 is essential for replication fork progression.

Discussion
EYA protein phosphatases have been associated with cancer pathologies, and they exhibit characteristics of oncogenic and tumor-suppressive activities depending on the tissue of origin. Because EYA are protein phosphatases, it is expected that lack of phosphorylation would impact a variety of cellular pathways depending on the protein substrates expressed and targeted in speci c tissues. In this study, we sought to gain a better understanding of the cellular processes impacted by EYA4 deregulation in cancer, and speci cally understand the possible role of EYA4 in breast carcinogenesis.
Together with collaborators, we have reported that the EYA4 gene is hypermethylated in the rst intronexon junction (15), and possibly over-expressed in triple-negative breast cancer patients, which correlates with publicly available TCGA dataset that shows ampli cation as the most common alteration in breast cancer patients. In this study, we used HeLa and breast cancer cell lines to investigate the proliferation rates of cells knocked down or over-expressing EYA4, ex vivo and as xenografts in small animals. While EYA4 is often not expressed in normal breast, we found that MDA-MB-231 over-express EYA4, and are depending on its expression for survival. We show that over-expression of EYA4 drives the growth of ER + primary tumors, and promotes metastasis to distant organs such as lungs and livers. In triple-negative breast cancer xenografts, the knockdown of EYA4 was able to e ciently limit the spread of metastasis and the overall cancer burden. These two xenograft studies suggest that EYA4 therapeutic targeting is an interesting avenue that should be pursued for anti-breast cancer drug development. Over-expressing EYA4 in cancer could be used to predict patient outcomes and drug response.
EYA4 level is inversely correlated with ER status, with high expression largely found in triple-negative breast cancer, while ER + tumors and cell lines express little or no EYA4. This warrants further investigation to fully understand the connection between Eyes Absent phosphatases and the hormonal status of cancerous tissues. In breast cancer, it is well-established that estrogen is a major driver of breast tumor growth through its role in cell proliferation, as well as an effective therapeutic target. It has been proposed that in MCF-7 cells, ER-α induces cell proliferation by regulating the cell cycle by stimulating the Data availability: All materials underlying this study are available upon request. All data generated or analyzed during this study are included in the main text or the supplementary information.     formation. Representative images (scale bar 10 mm), quanti cation (mean ± SEM; n ≥ 350), and more than 10 foci per nucleus are shown for 4 mM HU treatment for 2 hours followed by 2 hours release (C) or 16 hours HU treatment followed by 18 hours release (D). (E) A representative DNA ber image is shown for each genetic condition. Replication fork speed (kb/min) is shown for empty vector control, EYA4depleted cells and EYA4 phosphatase mutant cells (mean ± SEM; a minimum of 150 forks was scored in two independent experiments yielding similar results. Statistical analysis: unpaired t-test). For all panels *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (F) Model: Over-expression of EYA4 leads to an aggressive and invasive breast cancer phenotype. EYA4 has a protective role in cells against replication stress, triggering the activation of the ATR pathway and cell cycle arrest.

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