Human Phosphatase CDC14A Is Recruited to the Cell Leading Edge to Regulate Cell Migration and Adhesion

Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion

Nan-Peng Chena,b, Borhan Uddina,b, Renate Voitc, and Elmar Schiebela,1

aZentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)-Zentrum für Molekulare Biologie (ZMBH) Allianz, 69120 Heidelberg, Germany; bHartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, Universität Heidelberg, 69120 Heidelberg, Germany; and cMolecular Biology of the Cell II, Deutsches Krebsforschungszentrum (DKFZ), 69120 Heidelberg, Germany

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved December 17, 2015 (received for review August 6, 2015) Cell adhesion and migration are highly dynamic biological pro- With mitotic exit, the proteins that were phosphorylated by cesses that play important roles in organ development and CDK1 are dephosphorylated, so that cells can return to the cancer metastasis. Their tight regulation by small GTPases and nonmitotic, interphase status (13). In Saccharomyces cerevisiae, protein phosphorylation make interrogation of these key pro- cell-division cycle 14 (Cdc14) is the major phosphatase that cesses of great importance. We now show that the conserved dual- counteracts Cdk1 activity after the metaphase–anaphase transi- specificity phosphatase human cell-division cycle 14A (hCDC14A) tion (14). The function of Cdc14 in budding yeast has been well associates with the actin cytoskeleton of human cells. To un- studied and two regulatory networks have been identified that derstand hCDC14A function at this location, we manipulated native promote the release of Cdc14 from the RENT complex in the PD loci to ablate hCDC14A phosphatase activity (hCDC14A )inun- nucleolus: “Cdc Fourteen Early Anaphase Release” (FEAR) and transformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines “Mitotic Exit Network” (MEN) (15–17). and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic Despite the conservation of the N-terminal catalytic phos- expression of hCDC14A induced stress fiber formation, whereas PD PD phatase domain and the ability of human CDC14B to comple- stress fibers were diminished in hCDC14A cells. hCDC14A cells ment the essential functions of budding yeast CDC14 (18), displayed faster cell migration and less adhesion than wild-type Cdc14 phosphatases play divergent roles in different organisms. controls. hCDC14A colocalized with the hCDC14A substrate kidney- Schizosaccharomyces pombe Cdc14/Flp1 primarily participates and brain-expressed protein (KIBRA) at the cell leading edge and CELL BIOLOGY in the regulation of the phosphatase Cdc25 and cytokinesis overexpression of KIBRA wasabletoreversethephenotypesof hCDC14APD cells. Finally, we show that ablation of hCDC14A ac- (19). Vertebrate CDC14s have been linked to diverse functions tivity increased the aggressive nature of cells in an in vitro tumor ranging from centrosome maturation and separation, DNA formation assay. Consistently, hCDC14A is down-regulated in many damage checkpoint control, DNA repair, and cytokinesis control – tumor tissues and reduced hCDC14A expression is correlated with (20 24). These studies have unraveled novel functions of mam- poorer survival of patients with cancer, to suggest that hCDC14A malian CDC14 phosphatases; however, they reveal striking may directly contribute to the metastatic potential of tumors. inadequacies in our understanding of this important phos- Thus, we have uncovered an unanticipated role for hCDC14A in phatase family. cell migration and adhesion that is clearly distinct from the mi- Here, we have addressed the function of hCDC14A (human totic and cytokinesis functions of Cdc14/Flp1 in budding and cell-division cycle 14A) using human genetically engineered fission yeast. hCDC14A phosphatase dead cell lines (PD). Mobility and spreading were both enhanced by ablation of hCDC14A, whereas cell–cell CDC14 | cell migration | cell adhesion | phosphatase adhesion was reduced. Moreover, ectopic hCDC14A expression

ell migration and adhesion play key roles in embryonic de- Significance Cvelopment, tissue remodeling and cancer metastasis (1). Many oncoproteins, such as Yes-associated protein 1 (YAP), Cell migration and adhesion play critical roles in animal de- STAT3, and K-RAS, regulate cancer metastasis by enhancing velopment and tumor metastasis and are regulated by protein cell migration and invasion (2–4). The dynamic behavior of the phosphorylation. Here we identified the human phosphatase actin cytoskeleton drives migration and invasion and is regulated hCDC14A (human cell-division cycle 14A) as F-actin binding by a combined impact of Rho GTPases, membrane phospho- protein at the leading edge that regulates both processes. Loss lipids, and protein phosphorylation (5). The switch of phos- of hCDC14A activity promotes cell mobility and reduces cell phorylation at the cell leading edge is crucial for rapid turnover adhesion. Importantly, cells devoid of hCDC14A activity were of actin filaments. For example, focal adhesion kinase (FAK) can more invasive in a colony-forming assay. Database analysis be activated by integrins and various growth factors. Once acti- indicates that hCDC14A expression is frequently down-regu- vated, FAK regulates actin polymerization, membrane pro- lated in cancer tissues and is associated with poor patient trusion and cell migration by promoting the phosphorylation of prognosis. Thus, the promigratory impact of hCDC14A inactiva- the actin cytoskeleton remodelers p130cas, GRB2/7, and WASP tion upon the F-actin cytoskeleton supports tumor proliferation (5, 6). The tyrosine phosphatase SHP2 increases cell mobility and metastasis. This hCDC14A function is clearly distinct from through activation of the SRC kinase family to promote tumor the mitotic functions of the budding and fission yeast orthologs metastasis (7). Conversely, the lipid phosphatase PTEN inhibits Cdc14/Flp1. tumor invasion by suppressing the activation of RAC GTPases Author contributions: N.-P.C. and E.S. designed research; N.-P.C. and B.U. performed re- (8). There is also extensive evidence for control of cancer cell search; R.V. contributed new reagents/analytic tools; N.-P.C. and B.U. analyzed data; and migration and invasion through the phosphatase PP2A upon N.-P.C. and E.S. wrote the paper. Wnt/beta-catenin signaling, metal matrix proteases, and ERK The authors declare no conflict of interest. kinase (9–11). This article is a PNAS Direct Submission. At the G2/M transition, cyclin-dependent kinase 1 (CDK1) is 1To whom correspondence should be addressed. Email: [email protected]. activated to trigger mitotic entry and its kinase activity remains This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. high until metaphase to maintain the cell in a mitotic state (12). 1073/pnas.1515605113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1515605113 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 inhibited migration and the actin cytoskeleton was remodeled when hCDC14A activity was impaired. Consistent with these actin-modulating functions, a pool of hCDC14A associated with F-actin filaments at the leading edge where it colocalized with the Hippo pathway component kidney- and brain-expressed protein (KIBRA). KIBRA overproduction rescued the migration and adhesion defects in the hCDC14APD cells. Our study therefore reveals a previously unidentified function of hCDC14A. As hCDC14A expression is down-regulated in a variety of cancers, including colorectal, and this down-regulation is associated with poor prognosis, our results suggest that hCDC14A regulates tumor metastasis and is therefore of considerable clinical relevance. Results A Pool of hCDC14A Localizes to the Cell Leading Edge and F-Actin Fibers. Whether hCDC14 phosphatases impact upon actin-related functions is yet to be addressed. To this end, we monitored the distribution of a hCDC14A-YFP fusion protein stably integrated in the HeLa cell genome as a single copy via the Flp-in T-Rex system. The levels of ectopic hCDC14A expression achieved by induction were much lower than those arising from transient transfection (Fig. S1A) and did not generate obvious toxicity or ad- verse impact upon cell cycle progression based on the propidium iodide DNA staining (Fig. S1 B–D). Phosphohistone 3 serine 10 staining revealed that the ectopic expression of hCDC14A only slightly increased the mitotic index (Fig. S1 E and F). This was further confirmed by live cell imaging, which revealed only a mod- Fig. 1. hCDC14A colocalizes with F-actin at the cell leading edge. est increase in the duration of mitosis upon ectopic hCDC14A (A) hCDC14A-YFP colocalizes with F-actin at the leading edge of HeLa FRT T-Rex expression (Fig. S1 G and H). hCDC14A-YFP cells (Upper). (Scale bar: 10 μm.) (Lower) Regional magnifica- As in previous reports (22, 25), hCDC14A was found in the tion of the boxed area in the Upper panel. (Scale bar: 5 μm.) The white line cytoplasm, at centrosomes of interphase cells and at the midbody indicates the scanned segment in B.(B) Line scan of hCDC14A-YFP and during cytokinesis (Fig. S2A). In the Flp-in T-Rex cell line, we F-actin from A.(C) hCDC14A-YFP relocalizes with F-actin remnants after failed to detect the reported association of hCDC14A with mi- blebbistatin treatment. (Lower) Regional magnification of the area high- crotubules (18). However, strong overexpression of hCDC14A- lighted in the Upper panel. The white line indicates the scanned segment YFP through transient transfection generated a signal that that is shown in D as line scan. (D)LinescanofhCDC14A-YFPandF-actinfrom C.(E) Anti-hCDC14A antibodies show colocalization with F-actin at the leading showed partial overlap with microtubules (Fig. S2B). Fluores- edge of RPE1 WT cells. (Scale bar: 20 μm.) (Lower) Regional magnification of cence recovery after photobleaching (FRAP) analysis revealed the Upper panel. (Scale bar: 4 μm.) The white line indicates the scanned seg- different rates of recovery for hCDC14A at different locations. ment in F.(F) Line scan of the hCDC14A and F-actin signals from E. In general, hCDC14A was quite dynamic (half recovery time <10 s, Fig. S2C) with the exception of the late midbody pool that was relatively immobile (Fig. S2C). Thus, hCDC14A binds dynami- signals overlapped (Fig. S3H). Second, the F-actin associated cally to the centrosome and early midbody. signal of the anti-hCDC14A antibodies was not observed in Importantly, hCDC14A-YFP, but not YFP (Fig. S3A), lo- hTERT-immortalized retinal pigment epithelial (RPE1) calized to the protruding actin filaments at the leading edge hCDC14AKO cells (Fig. S3I) from which exon 2 had been re- of HeLa cells (Fig. 1A). Line scans of GFP nanobody and moved to introduce a frameshift that blocked the production of phalloidin stainings revealed extensive overlap between the hCDC14A protein (22). We note that the anti-hCDC14A anti- hCDC14A-YFP and F-actin signals in these protruding areas (Fig. bodies detected a weak nuclear signal that may arise from 1B). To exclude the possibility that this apparent colocalization was a the cross-reactivity with a nuclear protein in some RPE1 property bestowed by the YFP tag, we transfected myc-hCDC14A hCDC14AKO cells. The inability of the anti-hCDC14A anti- or flag-hCDC14A into HeLa cells. These hCDC14A constructs body to detect the protein in immunoblots suggests that it exhibited similar colocalization with F-actin structures as shown recognizes a conformation-specific antigen. by hCDC14-YFP (Fig. S3 B and C). To determine whether Taken together, our data indicate that a fraction of hCDC14A hCDC14A localization changes upon perturbation of the F-actin associates with F-actin along the leading edge of cells. cytoskeleton, we treated cells with blebbistatin, a myosin II inhibitor that inhibits F-actin formation. Blebbistatin treatment hCDC14APD Cell Lines. Studies that relied upon siRNA depletion of prompted all three tagged versions of hCDC14A to join hCDC14A/B have revealed a number of functions of these F-actin remnants in relocalizing to peripheral regions of the cell phosphatases but a coherent picture as to hCDC14 functions is (Fig. 1 C and D and Fig. S3 E and F), whereas unfused YFP yet to emerge (20–22, 24, 25, 28). We therefore turned to ge- did not colocalize with these collapsed F-actin structures nome editing as a strategy for hCDC14A inactivation. We pre- (Fig. S3D). viously reported the construction of an RPE1 hCDC14AKO cell We next monitored the distribution of endogenous hCDC14A line through two rounds of adenovirus-mediated gene knockout with polyclonal antibodies that had been raised against the di- (22). However, for confident interpretation of phenotypic anal- vergent C terminus of hCDC14A (26, 27). These anti-hCDC14A ysis, it was important to study independent gene knockout clones antibodies costained the cell leading edge and the nearby actin to exclude the possibility of clonal variation. In addition, we filaments (Fig. 1 E and F and Fig. S3G). Two lines of evidence wanted to analyze hCDC14KO phenotypes in cell lines of dif- suggest that this staining was specific. First, this antibody rec- ferent origins. We therefore constructed independent cell lines ognized overexpressed myc-hCDC14A as the myc and hCDC14A that lacked hCDC14A activity by using zinc finger nuclease

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1515605113 Chen et al. Downloaded by guest on September 26, 2021 (ZFN)-induced homolog recombination (29). We chose RPE1 and human colon cancer (HCT116) cells because they each have a stable genotype (30). Phosphatase activity of hCDC14A criti- cally depends on three amino acid residues, of which D251 and C278 are encoded by exon 9 (31). Mutant yeast Cdc14 and human CDC14 proteins that harbor a mutation at C278 are catalytically inactive and nonfunctional (20, 32, 33). We therefore chose to interfere with hCDC14A’s phosphatase activity by tar- geting exon 9. A ZFN cut in exon 9 together in the presence of a homologous neomycin resistance donor cassette (Fig. S4A) induced homologous recombination. Southern blot analysis indicated that exon 9 had been successfully targeted by our strategy and that the donor was integrated into the ZFN cut site (Fig. S4B). To further confirm disruption of exon 9, we carried out RT-PCR to amplify the mRNA of hCDC14A from the wild-type (WT) cells and putative hCDC14A-KO (knockout) clones of both cell lines. Surprisingly, in clones from either cell line the hCDC14A RT- PCR detected a band of 216 bp in hCDC14A-KO cells, whereas the PCR product was 447 bp in wild-type cells (Fig. S4C). Se- quence analysis indicated that exon 9 together with the neomycin gene was spliced out in the mRNA of the hCDC14A-KO cells (Fig. S4D). The exon 9-skipped mRNA lacked exon 9 of 231 nucleotides, and so encoded a protein with an internal deletion of 77 amino acids. This truncated hCDC14A no longer carried the two essential D251 and C278 residues and so should lack phosphatase activity. This was confirmed by an in vitro phosphatase Fig. 2. hCDC14A regulates cell mobility. (A) Representative view of Trans- Δ CELL BIOLOGY assay using immunoprecipitated hCDC14A- exon9 from transwell membranes. HeLa FRT T-Rex hCDC14A inducible cells cultured for 24 h fected HEK 293T cells (Fig. S4 E and F). We therefore named with or without doxycycline in a Transwell chamber. Cells on the transside of PD these cell lines hCDC14A (phosphatase dead). Analysis of the chamber were fixed and stained with crystal violet. (B) Quantification of these cells by indirect immunofluorescence confirmed the A. The cell number was counted (Left graphs) or the crystal violet was eluted presence of the hCDC14APD mutant protein (Fig. S4G). with 30% acidic acid and then measured at 570 nm (Right graphs). n = 3. Consistent with previous reports of hCDC14KO cells (22, 28), Error bars are ±SEM of three independent experiments. (C) Representative PD the hCDC14APD cells were viable. Flow cytometry (FACS) view of the Transwell membrane. RPE1 WT and RPE1 hCDC14A clones 4 comparisons with WT cells revealed no significant differences in and 6 were used in this experiment. (D) Quantification of C using crystal PD violet as in B. n = 3. Error bars are ±SEM between three independent ex- the distribution of cell cycle phases in hCDC14A (Fig. S5A). periments. (E) Plot graphs of cell tracking experiments. HCT116 WT cells, Moreover, centrosome number and centrosome separation dis- HCT116 hCDC14APD clones 2 and 4, and HCT116 hCDC14APD clone 4 trans- PD tances of HCT116 hCDC14A cells were indistinguishable fected with hCDC14A-YFP (PD4-rescue) were seeded onto the live-imaging from the behavior of WT hCDC14A cells (Fig. S5 B and C). We chamber and tracked for 4 h. (F) Quantification of E. HCT116 hCDC14APD therefore concluded that hCDC14A is not essential for cell vi- clones 2 and 4 migrate faster than the HCT116 WT cells. This enhanced mi- ability, centrosome duplication, and centrosome linker assembly gratory phenotype was reversed to wild-type levels by the introduction of PD in either HCT116 or RPE1 cells. hCDC14A-YFP into the HCT116 hCDC14A cells (PD4-rescue). n = 25–30. Error bars are ±SEM of individual cells. hCDC14A Regulates Cell Mobility. Given the colocalization of hCDC14A with F-actin at the leading edge of cells, we asked Cell tracking revealed that both of the HCT116 hCDC14APD whether hCDC14A has a function in cell migration. We used ∼ four different assays to address this question. First, we used clones migrated 60% more rapidly than corresponding WT cells (Fig. 2 E and F). Importantly, transfection of hCDC14A- Transwell chambers to test the migratory behavior of Flp-in PD T-Rex HeLa cells following doxycycline-induced expression of YFP into the hCDC14A cell lines reversed this enhanced mi- hCDC14A. hCDC14A expression led to a 1.8-fold decrease in gratory phenotype to wild-type levels (Fig. 2 E and F). Finally, we measured directed cell migration using a wound-healing assay. cell migration in the Transwell assay (Fig. 2 A and B). We then PD monitored the RPE1 hCDC14APD cell line in the same assay. As observed with the other assay systems, hCDC14A HCT116 The fibroblast-like morphology of RPE1 cells made it difficult to cells migrated faster than WT HCT116 cells (Fig. S5 F and G). score cell number on the chamber membrane. To circumvent this Taken together, our data show that the phosphatase activity of problem, we stained the cells with crystal purple before eluting hCDC14A plays an important role in restraining migration of the dye with acetic acid from the attached cells. Compared with diverse cell types. WT cells, both independent hCDC14APD clones 4 and 6 dis- PD played patterns that were indicative of enhanced cell migration hCDC14A Cells Display Altered Adhesion Behavior and Form More (Fig. 2 C and D). Enhanced migration was also seen in the RPE1 Invasive Microcolonies. The localization of hCDC14A and the PD hCDC14AKO cell line (Fig. S5 D and E). We therefore con- altered migration of hCDC14A cells indicate that hCDC14A clude that hCDC14A normally restrains migration of HeLa and regulates properties of the actin cytoskeleton. To further test this RPE1 cells. notion, we analyzed cell spreading, cell–cell contact formation, PD We next asked whether the observations made in HeLa and and the formation of focal adhesions in WT and hCDC14A RPE1 cells also applied to HCT116 cells. We measured the cells. To analyze cell spreading, HCT116 cells were first trypsi- mobility of adherent WT and hCDC14APD HCT116 cells by live nized and then allowed to settle on the fibronectin-coated cell imaging. The WT and two independent hCDC14APD clones, chamber for 30 min (t = 0). The spreading process was then clones 2 and 4, were seeded onto the fibronectin-coated surface recorded by light microscopy. After 140 min, all hCDC14APD and allowed to attach for 24 h before measuring cell migration. cells had spread on the chamber surface and achieved an average

Chen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 surface area of 409 μm2. In contrast, HCT116 WT control cells the lack of hCDC14A activity inhibits the formation of cell– spread more slowly and occupied a much smaller average surface cell contacts. area of 265 μm2 after 140 min (Fig. 3 A and B). A similar ac- We next analyzed focal adhesion formation on fibronectin celeration in the cell spreading of hCDC14APD cells was ob- surfaces using the focal adhesion marker paxillin. Fibronectin- served when HCT116 cells exited mitosis and reattached to the attached hCDC14APD cells had fewer focal adhesions than WT extracellular matrix (Fig. S6A). After 24 h of seeding onto the cells (Fig. 3 E and F). Because hCDC14A was not detected at surface, ∼30–40% of the hCDC14APD cells had adopted an focal adhesions (Fig. 1), this defect is most likely to arise as elongated morphology, whereas only ∼15% of the WT controls an indirect consequence of the altered actin cytoskeleton of PD had done so (Fig. 3C). Thus, hCDC14APD cells form cell-to- hCDC14A cells. surface adhesion faster and acquire a mature morphology earlier hCDC14A mRNA levels are significantly down-regulated in a range of cancers (34–37) (Fig. S6 C–E). When considered than WT cells. PD After 48 h, both wild-type and hCDC14APD HCT116 cells alongside the faster cell migration of hCDC14A cells, this formed microcolonies. hCDC14APD microcolonies were less reduction in mRNA levels raises the possibility that hCDC14A may contribute to the aggressive metastatic behavior of tumor densely packed than the WT control colonies (Fig. S6B). Con- – sistently, the E-cadherin signal of WT cells concentrated at the cells. Importantly, Kaplan Meier survival analysis from 278 pa- cell–cell adhesion regions, whereas it was dispersed in hCDC14APD tients with colon cancer indicated that down-regulation of hCDC14A expression contributes to poor prognosis (Fig. 3G). cells even when they were touching (Fig. 3D). We conclude that Similar results were found in neuroblastoma (Fig. S6F). Indeed, in a soft agar colony formation assay, most of the HCT116 WT cells formed compact cell masses with a smooth edge (Fig. 3 H and I). However, the HCT116 hCDC14APD tumors had loose cell–cell contacts with rougher, less clearly defined edges (Fig. 3 H and I). Cancer cells at the periphery were prone to escape from the local mass. The heterogeneous morphology and more aggressive migratory behavior are highly reminiscent of metastatic migration. These data therefore suggest that the lack of hCDC14A increases the tumor aggressiveness by reducing cell adhesion and promoting migration.

hCDC14A Activity Influences Stress Fiber Formation. Stress fibers are bundles of ∼20–30 actin filaments that assemble with the help of alpha-actinin. In animal cells, stress fibers are major contractile structures that influence cell movement, the establishment of focal adhesions, and cell–cell attachments (38). Based on the observation that hCDC14A localized to actin fibers and its role in cell migration and adhesion (Figs. 1, 2, and 3), we asked whether hCDC14A affects stress fiber formation. We transfected HeLa cells with active myc-hCDC14A or the phosphatase dead version myc-hCDC14ACS in which the active cysteine at position 278 had been replaced by an alanine residue and stained the cells with phalloidin to visualize the F-actin network. Myc-hCDC14A– transfected cells harbored an average of ∼16 stress fibers, whereas their nontransfected counterparts displayed only ∼6(Fig.4A–C). Importantly, this increase in stress fiber formation was not found when cells had been transfected with the catalytically inactive myc-hCDC14ACS (Fig. 4 A–C), indicating that hCDC14A phosphatase activity was required for this transformation of the actin cytoskeleton. Furthermore, hCDC14A stress fiber induction Fig. 3. hCDC14APD cells show altered adhesion behavior and form more relied upon rho-associated protein kinase (ROCK) kinase ac- invasive microcolonies. (A) Time course recording of the HCT116 WT and tivity as it was blocked by the inhibitor Y27632 (Fig. 4 A–C). HCT116 hCDC14APD clone 4 in the spreading assay. (Scale bar: 10 μm.) (B) hCDC14A expression did not induce apoptosis as indicated by Quantification of A. n = 10 cells for each cell type. Error bars are ±SEM. the absence of C-PAPR1 staining (Fig. 4A). (C) Representative views of spread, elongated, and rounded cells stained with μ We next compared the F-actin networks of HCT116 WT and phalloidin. (Scale bar: 10 m.) (Right) Quantification of the cell morphology hCDC14APD cells. Although 3D structured illumination micros- distribution 24 h after seeding of the indicated cells. (n = WT, 196; PD2, 270; – copy (SIM) detected robust and well-organized F-actin fibers in and PD4, 246). (D) E-cadherin is not enriched at cell cell contact regions of PD hCDC14APD cells. Indicated cells were seeded onto the fibronectin-coated WT cells, it failed to detect any fibers at all in the hCDC14A coverslips for 48 h. Cells were fixed with paraformaldehyde (PFA) and cells (Fig. 4D). Thus, hCDC14A activity promotes stress stained with phalloidin (red) and E-cadherin antibodies (green). (Scale bar: fiber formation. 20 μm.) (E) Representative view of fully spread HCT116 WT and HCT116 hCDC14APD cells stained with phalloidin and antipaxillin antibodies. (Scale Overexpression of KIBRA Rescues the Migration and Adhesion bar: 10 μm.) (F) Quantification of the paxillin foci count from E. n = 100. Phenotypes of the HCT116 hCDC14APD Cells. The WW domain Error bars are ±SEM of individual cells. **P < 0.01. (G) Poor prognosis of pa- protein KIBRA has been described as hCDC14A substrate (39). tients with colon cancer correlated with low level of hCDC14A expression. – – This upstream regulator of Hippo pathway (40 42) controls the Kaplan Meier survival analysis was generated from R2: Genomics Analysis and activity of the transcriptional coactivator YAP through its in- Visualization Platform (r2.amc.nl). (n = high, 142; low, 136. P = 0.032.) (H) Tumor morphology of HCT116 WT and HCT116 hCDC14APD cells. (Scale bar: 500 μm.) teraction with large-tumor suppressor kinase 1 (LATS1) and (Lower) Fivefold regional magnification of the Upper panel. (I) Percentage of LATS2 kinases (42). Alongside these characterized functions rough colonies of HCT116 WT and HCT116 hCDC14APD tumors. n = 200. Error in Hippo signaling, recent studies have revealed a role for KIBRA bars are ±SEM between three independent experiments. **P < 0.01. in cell migration and polarization (43–45). Overexpression of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1515605113 Chen et al. Downloaded by guest on September 26, 2021 mCherry and GFP-KIBRA upon the migration and spreading behavior of hCDC14APD cells was monitored (Fig. 4 E–G and Fig. S7 C and D). A slight downshift in the migration of the KIBRA band on SDS/PAGE was observed in the immunoblot, indicating the dephosphorylation of KIBRA by hCDC14A (39) (Fig. 4G). Single transfection of hCDC14A-mCherry or GFP- KIBRA resulted in a similar reduction of migration and spreading as seen with cotransfection of both constructs (Fig. 4 E and F and Fig. S7 C and D). The nonadditive nature of this behavior suggests that hCDC14A and KIBRA function in the same pathway. Discussion Unlike budding yeast CDC14, neither human hCDC14A nor hCDC14B are essential for either cell viability or mitotic exit (22, 28). Rather, hCDC14A has functions outside cell cycle regulation. The combination of localization analyses, overexpression, and hCDC14A knockout approaches used here reveals an important role for hCDC14A in the regulation of cell adhesion and cell migration. We suggest that hCDC14A executes these important functions through the dephosphorylation of F-actin associated proteins. Thus, our work reveals an unanticipated and unidentified function for hCDC14A in modulating properties of actin filaments. The reduction in the number of stress fibers in hCDC14APD cells and their induction following hCDC14A overexpression indicate that hCDC14A activity stabilizes actin filaments. Stress

fibers are important as a major contractile structure for cell CELL BIOLOGY morphology, migration, and mechanical signal sensing. Under many conditions, stress fibers restrict cell mobility because their turnover is relatively slow (47). Indeed, many highly motile cells, including invasive cancer cells, lack stress fibers. How hCDC14A Fig. 4. hCDC14A levels affect the actin cytoskeleton and hCDC14A and regulates the stress fiber dynamics process and how hCDC14A KIBRA function in the same pathway to regulate cell migration. (A) Transient transfection of myc-hCDC14A triggers the formation of F-actin stress fibers. itself is recruited to F-actin filaments remain open questions. HeLa cells were transfected with the indicated constructs or treated with the One possibility is that hCDC14A dephosphorylates actin mod- ROCK inhibitor Y-27632. Fixed cells were stained with the myc and PARP1 ulators such as the guanine nucleotide exchange factors (GEFs) antibodies. F-actin was visualized with phalloidin. DNA was stained with and GTPase-activating proteins (GAPs) that regulate the activity DAPI. The red lines indicate the scanned segments in B. (Scale bar: 20 μm.) (B) of actin controlling small GTPases. Indeed, components of Rho Representative results of line-scan profiles from A. The peaks represented GTPase pathways such as the nucleotide exchange factor GEF- the F-actin filaments. (C) Quantification of B. n = 50 per cell type. Error bars H1, MyoGEF, RacGAP, and mDia3 are phosphorylated by the are ±SEM of individual cells. (D) The 3D-SIM pictures of the HCT116 WT and – PD mitotic kinases CDK1, Polo kinase 1, and Aurora kinases (48 HCT116 hCDC14A clone 4. In the WT cells, F-actin fibers were marked with 52). Because hCDC14A is a proline-directed phosphatase (31), it arrows. (Scale bar: 5 μm.) (E) Plot graphs of cell tracking experiments. HCT116 hCDC14APD clone 4 was transfected with GFP-KIBRA or hCDC14A- is plausible that it counteracts a proline-directed kinase such mCherry, respectively, or cotransfected with GFP-KIBRA and hCDC14A- as CDK1. mCherry. The transfected cells were seeded onto fibronectin-coated live cell It has been shown that budding yeast Cdc14 plays critical roles imaging chambers and tracked for 4 h. (F) Quantification of E. Single in cytokinesis (53), a process that involves rapid actin cytoskel- transfection of GFP-KIBRA or hCDC14A-mCherry had a similar effect on cell eton rearrangement (54). Yeast cells that lack CDC14 function migration as cotransfection (combine). This was shown by velocity and ac- fail to form an actomyosin ring, whereas it forms prematurely cumulated distance. n = 25–30. Error bars are ±SEM of individual cells. **P < after overexpression of CDC14 (55). Cdc14 regulates F-actin PD 0.01. (G) Immunoblot of the transfected HCT116 hCDC14A clone 4 cell dynamics through dephosphorylation of actin-interacting pro- lysate using GFP and mCherry antibodies. The star shows the slight down- teins such as Iqg1 and Aip1 (55, 56). Consistent with these data shift of GFP-KIBRA in the presence of ectopic hCDC14A-mCherry. from yeast, hCDC14A also regulates actin dynamics, however, in interphase. Thus, the actin-remodeling function of CDC14 may KIBRA can inhibit migration of MCF10A cells (44). In contrast, be conserved through evolution. KIBRA knockdown in breast cancer cells induced the epithelial- How does hCDC14A regulate actin function? The protein to-mesenchymal transition (46). KIBRA is an upstream regulator of Hippo pathway that is crit- We transfected flag-KIBRA into the hCDC14A-YFP sta- ical for the control of contact inhibition and organ size (40). KIBRA was previously reported to be a hCDC14A substrate that ble HeLa cells and found that flag-KIBRA colocalized with also has functions in cell migration (39). We now show that hCDC14A at the leading edge (Fig. S7A). In addition, we found KIBRA colocalizes with hCDC14A at the leading edge and colocalization of endogenous KIBRA with hCDC14A at the that transient expression of KIBRA bypassed the hCDC14APD cell leading edge (Fig. S7B). Overexpression of KIBRA could abro- migration defects. Given that KIBRA is a hCDC14A substrate, gate the enhanced migration (Fig. 4 E and F) and spreading these data suggest a function of KIBRA downstream of hCDC14A. phenotypes (Fig. S7 C and D) arising from the loss of hCDC14A Moreover, the nonadditive effect of cotransfection of both genes activity in HCT116 hCDC14APD cells, suggesting that hCDC14A is most consistent with a function of KIBRA and hCDC14A in may regulate cell mobility and adhesion through KIBRA. one linear pathway. Interestingly, the S. cerevisiae MEN and the To determine whether hCDC14A and KIBRA function in metazoa Hippo pathway have a conserved kinase module core the same pathway, the impact of cotransfection of hCDC14A- structure in common (the Cdc15/Hippo/MST1-Dbf2/LATS1-Mob1

Chen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 module) (23). Budding yeast Cdc14 feeds back on the MEN by in many tumors makes a significant contribution to the propagation dephosphorylating Cdc15 and Mob1 (53, 57). hCDC14A may of cancers by decreasing cell adhesion and enhancing cell migration regulate the Hippo pathway component KIBRA. Thus, regulation (34–37). of the MEN and Hippo pathways by CDC14 phosphatases may be a conserved principal. Materials and Methods Down-regulation of hCDC14A expression is associated with Details are in the SI Materials and Methods including experimental proce- poor prognosis in colon cancer and neuroblastoma to reveal an dures and reagents. important involvement of hCDC14A in restraining tumor metastasis. Excitingly, the in vitro “tumor formation activity” ACKNOWLEDGMENTS. We thank Ilaria Lucibello for helpful discussion; of HCT116 cells in soft agar assays was enhanced by the Dr. Jixin Dong (University of Nebraska) for KIBRA constructs; and Dr. Ulrike PD Engel from Nikon Imaging Center (University of Heidelberg) for help with hCDC14A inactivating mutation hCDC14A . We therefore the 3D-SIM imaging. This study is funded by the Deutsche Forschungsge- propose that the down-regulation of hCDC14A mRNA seen meinschaft Project DFG Schi 295/3-2.

1. Ridley AJ, et al. (2003) Cell migration: Integrating signals from front to back. Science 30. Stingele S, et al. (2012) Global analysis of genome, transcriptome and proteome re- 302(5651):1704–1709. veals the response to aneuploidy in human cells. Mol Syst Biol 8:608. 2. Rokavec M, et al. (2014) IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated 31. Gray CH, Good VM, Tonks NK, Barford D (2003) The structure of the cell cycle protein colorectal cancer invasion and metastasis. J Clin Invest 124(4):1853–1867. Cdc14 reveals a proline-directed protein phosphatase. EMBO J 22(14):3524–3535. 3. Whittle MC, et al. (2015) RUNX3 controls a metastatic switch in pancreatic ductal 32. Wolfe BA, McDonald WH, Yates JR, 3rd, Gould KL (2006) Phospho-regulation of the adenocarcinoma. Cell 161(6):1345–1360. Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe. Dev Cell 11(3): 4. Zhang L, et al. (2015) The hippo pathway effector YAP regulates motility, invasion, and 423–430. castration-resistant growth of prostate cancer cells. MolCellBiol35(8):1350–1362. 33. Sullivan M, Higuchi T, Katis VL, Uhlmann F (2004) Cdc14 phosphatase induces rDNA 5. Ridley AJ (2011) Life at the leading edge. Cell 145(7):1012–1022. condensation and resolves cohesin-independent cohesion during budding yeast 6. Zhao X, Guan JL (2011) Focal adhesion kinase and its signaling pathways in cell mi- anaphase. Cell 117(4):471–482. gration and angiogenesis. Adv Drug Deliv Rev 63(8):610–615. 34. Piccaluga PP, et al. (2007) Gene expression analysis of peripheral T cell lymphoma, 7. Hartman ZR, Schaller MD, Agazie YM (2013) The tyrosine phosphatase SHP2 regulates unspecified, reveals distinct profiles and new potential therapeutic targets. J Clin focal adhesion kinase to promote EGF-induced lamellipodia persistence and cell mi- Invest 117(3):823–834. – gration. Mol Cancer Res 11(6):651 664. 35. Hong Y, Downey T, Eu KW, Koh PK, Cheah PY (2010) A ‘metastasis-prone’ signature 8. Mense SM, et al. (2015) PTEN inhibits PREX2-catalyzed activation of RAC1 to restrain for early-stage mismatch-repair proficient sporadic colorectal cancer patients and its tumor cell invasion. Sci Signal 8(370):ra32. implications for possible therapeutics. Clin Exp Metastasis 27(2):83–90. 9. Basu S, Ray NT, Atkinson SJ, Broxmeyer HE (2007) Protein phosphatase 2A plays an 36. Frierson HF, Jr, et al. (2002) Large scale molecular analysis identifies genes with al- important role in stromal cell-derived factor-1/CXC chemokine ligand 12-mediated tered expression in salivary adenoid cystic carcinoma. Am J Pathol 161(4):1315–1323. + – migration and adhesion of CD34 cells. J Immunol 179(5):3075 3085. 37. Calvisi DF, et al. (2009) SKP2 and CKS1 promote degradation of cell cycle regulators 10. Nakada N, Kuroda K, Kawahara E (2005) Protein phosphatase 2A regulatory subunit and are associated with hepatocellular carcinoma prognosis. Gastroenterology Bbeta promotes MAP kinase-mediated migration of A431 cells. Cell Physiol Biochem 137(5):1816–1826.e1–10. – 15(1-4):19 28. 38. Hotulainen P, Lappalainen P (2006) Stress fibers are generated by two distinct actin 11. Xu L, Deng X (2006) Suppression of cancer cell migration and invasion by protein assembly mechanisms in motile cells. J Cell Biol 173(3):383–394. phosphatase 2A through dephosphorylation of mu- and m-calpains. J Biol Chem 39. Ji M, et al. (2012) Phospho-regulation of KIBRA by CDK1 and CDC14 phosphatase 281(46):35567–35575. controls cell-cycle progression. Biochem J 447(1):93–102. 12. Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: A changing paradigm. 40. Baumgartner R, Poernbacher I, Buser N, Hafen E, Stocker H (2010) The WW domain Nat Rev Cancer 9(3):153–166. protein Kibra acts upstream of Hippo in Drosophila. Dev Cell 18(2):309–316. 13. Bembenek J, Yu H (2003) Regulation of CDC14: Pathways and checkpoints of mitotic 41. Genevet A, Wehr MC, Brain R, Thompson BJ, Tapon N (2010) Kibra is a regulator of exit. Front Biosci 8:d1275–d1287. the Salvador/Warts/Hippo signaling network. Dev Cell 18(2):300–308. 14. Visintin R, et al. (1998) The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk- 42. Xiao L, Chen Y, Ji M, Dong J (2011) KIBRA regulates Hippo signaling activity via independent phosphorylation. Mol Cell 2(6):709–718. teractions with large tumor suppressor kinases. J Biol Chem 286(10):7788–7796. 15. Torres-Rosell J, Machín F, Aragón L (2005) Cdc14 and the temporal coordination 43. Duning K, et al. (2008) KIBRA modulates directional migration of podocytes. J Am Soc between mitotic exit and chromosome segregation. Cell Cycle 4(1):109–112. Nephrol 19(10):1891–1903. 16. Visintin R, Hwang ES, Amon A (1999) Cfi1 prevents premature exit from mitosis by 44. Wilson KE, et al. (2014) PTPN14 forms a complex with Kibra and LATS1 proteins and anchoring Cdc14 phosphatase in the nucleolus. Nature 398(6730):818–823. negatively regulates the YAP oncogenic function. J Biol Chem 289(34):23693–23700. 17. Shou W, et al. (1999) Exit from mitosis is triggered by Tem1-dependent release of the 45. Yang S, et al. (2014) Phosphorylation of KIBRA by the extracellular signal-regulated protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97(2):233–244. kinase (ERK)-ribosomal S6 kinase (RSK) cascade modulates cell proliferation and mi- 18. Cho HP, et al. (2005) The dual-specificity phosphatase CDC14B bundles and stabilizes gration. Cell Signal 26(2):343–351. microtubules. Mol Cell Biol 25(11):4541–4551. 46. Moleirinho S, et al. (2013) KIBRA exhibits MST-independent functional regulation of 19. Clifford DM, et al. (2008) The Clp1/Cdc14 phosphatase contributes to the robustness – of cytokinesis by association with anillin-related Mid1. J Cell Biol 181(1):79–88. the Hippo signaling pathway in mammals. Oncogene 32(14):1821 1830. 20. Mailand N, et al. (2002) Deregulated human Cdc14A phosphatase disrupts centro- 47. Tojkander S, Gateva G, Lappalainen P (2012) Actin stress fibers: Assembly, dynamics – some separation and chromosome segregation. Nat Cell Biol 4(4):317–322. and biological roles. J Cell Sci 125(Pt 8):1855 1864. 21. Bassermann F, et al. (2008) The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage- 48. David M, Petit D, Bertoglio J (2012) Cell cycle regulation of Rho signaling pathways. – response checkpoint. Cell 134(2):256–267. Cell Cycle 11(16):3003 3010. 22. Mocciaro A, et al. (2010) Vertebrate cells genetically deficient for Cdc14A or Cdc14B 49. Birkenfeld J, et al. (2007) GEF-H1 modulates localized RhoA activation during cyto- – retain DNA damage checkpoint proficiency but are impaired in DNA repair. J Cell Biol kinesis under the control of mitotic kinases. Dev Cell 12(5):699 712. 189(4):631–639. 50. Asiedu M, Wu D, Matsumura F, Wei Q (2008) Phosphorylation of MyoGEF on Thr-574 23. Mocciaro A, Schiebel E (2010) Cdc14: A highly conserved family of phosphatases with by Plk1 promotes MyoGEF localization to the central spindle. J Biol Chem 283(42): – non-conserved functions? J Cell Sci 123(Pt 17):2867–2876. 28392 28400. 24. Wei Z, et al. (2011) Early-onset aging and defective DNA damage response in Cdc14b- 51. Touré A, et al. (2008) Phosphoregulation of MgcRacGAP in mitosis involves Aurora B deficient mice. Mol Cell Biol 31(7):1470–1477. and Cdk1 protein kinases and the PP2A phosphatase. FEBS Lett 582(8):1182–1188. 25. Kaiser BK, Zimmerman ZA, Charbonneau H, Jackson PK (2002) Disruption of centro- 52. Cheng L, et al. (2011) Aurora B regulates formin mDia3 in achieving metaphase some structure, chromosome segregation, and cytokinesis by misexpression of human chromosome alignment. Dev Cell 20(3):342–352. Cdc14A phosphatase. Mol Biol Cell 13(7):2289–2300. 53. König C, Maekawa H, Schiebel E (2010) Mutual regulation of cyclin-dependent kinase 26. Ovejero S, Ayala P, Bueno A, Sacristán MP (2012) Human Cdc14A regulates Wee1 and the mitotic exit network. J Cell Biol 188(3):351–368. stability by counteracting CDK-mediated phosphorylation. Mol Biol Cell 23(23): 54. Meitinger F, Palani S, Pereira G (2012) The power of MEN in cytokinesis. Cell Cycle 4515–4525. 11(2):219–228. 27. Schindler K, Schultz RM (2009) The CDC14A phosphatase regulates oocyte maturation 55. Miller DP, et al. (2015) Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in in mouse. Cell Cycle 8(7):1090–1098. budding yeast. Mol Biol Cell 26(16):2913–2926. 28. Berdougo E, Nachury MV, Jackson PK, Jallepalli PV (2008) The nucleolar phosphatase 56. Kuilman T, et al. (2015) Identification of Cdk targets that control cytokinesis. EMBO J Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells. 34(1):81–96. Cell Cycle 7(9):1184–1190. 57. Rock JM, Amon A (2011) Cdc15 integrates Tem1 GTPase-mediated spatial signals with 29. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with Polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev 25(18): engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646. 1943–1954.

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