Indispensable Role of STIL in the Regulation of Cancer Cell Motility Through the Lamellipodial Accumulation of ARHGEF7–PAK1 Complex

Oncogene (2020) 39:1931–1943 https://doi.org/10.1038/s41388-019-1115-9

ARTICLE

Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial accumulation of ARHGEF7–PAK1 complex

1 1 1 1 1 1 Hideaki Ito ● Takumi Tsunoda ● Miho Riku ● Shingo Inaguma ● Akihito Inoko ● Hideki Murakami ● 1 2 1 Hiroshi Ikeda ● Michiyuki Matsuda ● Kenji Kasai

Received: 14 January 2019 / Revised: 6 November 2019 / Accepted: 11 November 2019 / Published online: 21 November 2019 © The Author(s), under exclusive licence to Springer Nature Limited 2019

Abstract Cell motility is a tightly regulated phenomenon that supports the accurate formation of organ structure during development and homeostasis, including wound healing and inﬂammation. Meanwhile, cancer cells exhibit dysregulated motility, which causes spreading and invasion. The Dbl family RhoGEF ARHGEF7/β-PIX and its binding partner p21-activated kinase PAK1 are overexpressed in a variety of cancers and have been shown to be responsible for cancer cell migration. A key step in motility is the intracellular transport of ARHGEF7–PAK1 complex to the migrating front of cells, where lamellipodia protrusion and cytoskeletal remodeling efﬁciently occur. However, the molecular mechanisms of the intracellular transport

1234567890();,: 1234567890();,: of this complex are not fully understood. Here we revealed that SCL/TAL1-interrupting locus (STIL) is indispensable for the efficient migration of cancer cells. STIL forms a ternary complex with ARHGEF7 and PAK1 and accumulates with those proteins at the lamellipodia protrusion of motile cells. Knockdown of STIL impedes the accumulation of ARHGEF7–PAK1 complex within membrane ruffles and attenuates the phosphorylation of PAK1 substrates and cortical actin remodeling at the migrating front. Intriguingly, ARHGEF7 knockdown also diminishes STIL and PAK1 accumulation in membrane ruffles. Either STIL or ARHGEF7 knockdown impedes cell migration and Rac1 activity at the migrating front of cells. These results indicate that STIL is involved in the ARHGEF7-mediated positive-feedback activation of cytoskeletal remodeling through accumulating the ARHGEF7–PAK1 complex in lamellipodia. We conclude that its involvement is crucial for the polarized formation of Rac1-mediated leading edge, which supports the efficient migration of cancer cells.

Introduction behave inappropriately, as they spread and invade into the surrounding tissues, which causes patient death. Cell migration is a highly organized phenomenon that Experimentally, motile cells harbor characteristic pro- supports the accurate formation of tissue structure during trusions of the plasma membrane, lamellipodia, filopodia, development and is also key for the repair of wounded and blebs, at the migrating front of cells. The formation of tissues and biological defense against infection and these protrusions is controlled by attractive/repulsive cues inflammation. In neoplastic diseases, however, tumor cells (for instance, cytokines, extracellular matrix, and surrounding tissues) and internally controlled by the spatiotemporal activation of the Rho family of small guanosine triphosphatases (GTPases) (Rac1 for lamellipodia, CDC42 for filopodia, and RhoA for blebs formation) [1]. Among Supplementary information The online version of this article (https:// these, Rac1-mediated lamellipodia is believed to drive the doi.org/10.1038/s41388-019-1115-9) contains supplementary material, which is available to authorized users. directional migration of cells [2]. Rac1 is activated by switching from GDP-bound inactive * Kenji Kasai form to GTP-bound active form through its interaction with [email protected] Rho-specific guanine nucleotide exchange factors (GEFs). 1 Department of Pathology, Aichi Medical University School of Until now, ~70 members of the Dbl family GEFs and 11 Medicine, Nagakute, Japan members of the DOCK family GEFs have been identified [1], β 2 Department of Pathology and Biology of Diseases, Kyoto and among them the Dbl family GEF ARHGEF7/ -PIX (pak- University Graduate School of Medicine, Kyoto, Japan interacting exchange factor) has been most investigated: 1932 H. Ito et al.

ARHGEF7 accumulates at the membrane ruffles of motile associates with the ARHGEF7–PAK1 complex and is cells, and the overexpression of ARHGEF7 induces mem- responsible for the accumulation of the complex within the brane ruffling and the formation of microvillous structures by membrane ruffles. Conversely, we also found that ARH- the activation of Rac1 and CDC42 [3]. Conversely, siRNA- GEF7 is crucial for the accumulation of STIL and PAK1 at mediated knockdown of ARHGEF7 impedes cell migration the membrane ruffles. Furthermore, either STIL knockdown in vitro, and knockout of arhgef7 gene disrupts the collective or ARHGEF7 knockdown attenuates Rac1 activity at the migration of anterior visceral endoderm cells at the early migrating front of cells. Intriguingly, the expression of STIL developmental stage of embryos [4], which indicates a key is much higher than that of CORONIN1A in cancer cell lines role of ARHGEF7 in cell motility. Then, how is ARHGEF7 we examined and in cancer tissues listed in the public regulated? database; our knockdown experiments revealed the pre- Many molecules that associate with ARHGEF7 have dominant role of STIL rather than CORONIN1A in the been identified. The family members of p21-activated control of cell migration and ARHGEF7-PAK1 transport in kinases (PAKs), PAK1, and PAK4, have been reported to cancer cells. This evidence reveals the indispensable role of be overexpressed in a variety of cancers and are responsible STIL in cancer cell migration through the positive-feedback for cell migration [5, 6]. PAK1 harbors the p21-binding activation of Rac1/ARHGEF7/PAK1 cascade. domain (PBD), the autoinhibitory domain, and a binding site for ARHGEF7. PAK1, when in the inactive status, forms a homodimer and associates with ARHGEF7 [7]. By Results binding to GTP-bound Rac1 via PBD, the homodimer of PAK1 is dissociated and the monomer PAK1 acquires its STIL accumulation at the leading edge of cells kinase activity to autophosphorylate itself and phosphor- ylate many substrates such as one of the essential proteins The upregulated expression of STIL has been reported in for cytoskeletal assembly (FILAMIN-A/FLNA) [5, 8]. In various types of cancers, such as lung, prostate, and ovarian addition, PAK1 enhances the interaction between ARH- cancers [19–21]. The Oncomine database also predicts that GEF7 and Rac1, which leads to further activation of Rac1 STIL expression is increased in cancer tissues of pancreas, and PAK1 [1, 9]. Thus, this Rac1/ARHGEF7/PAK1 cas- colon and breast (Fig.1a) [22–24]. It has been reported that cade participates in the positive-feedback activation of STIL participates in the regulation of the Hedgehog sig- cytoskeletal remodeling and the directional formation of naling and also in the centriole duplication during mitosis lamellipodia protrusion [2, 10]. In the above-mentioned [14, 18], but little is known about its roles in other cellular process of protrusion formation, a key step is the intracel- processes. lular transport of the ARHGEF7–PAK1 complex to the To obtain insight into the roles of STIL, we examined the migrating-front side of the plasma membrane. Actin- subcellular localization of STIL protein in cancer cell lines binding protein CORONIN1A (CORO1A/CLABP) was using epifluorescence microscope. In all types of cancer cell recently reported to participate in the complex transport to lines that we examined thus far, including Panc-1 (pan- the lamellipodia of COS1 cells and blood cells, while its creatic cancer), CaCo-2 (colorectal cancer), and MDA- expression is not high in epithelial cells, including cancer MB231 (breast cancer), we found that STIL was expressed cells [11]. Thus, little is known about how the subcellular in the lamellipodia-like ruffling of the plasma membrane at transport of the complex is regulated in cancer cells. the migrating front of motile cells, which was also marked SCL/TAL1-interrupting locus (STIL) is an indispensable with Cortactin (Fig. 1b). Cortactin is a cortical actin-binding protein for early embryonic development [12] and is protein and is well-known to accumulate in the actin-rich thought to participate in many aspects of cellular functions lamellipodia protrusion at the leading edge of motile cells [13]. We previously reported that STIL associates with [25, 26]. Therefore, we assumed that STIL is involved in suppressor-of-fused (SUFU), a negative regulator of the formation and maintenance of the leading edge, which Hedgehog signaling, in oncogenic KRAS signaling- regulates the motility of cells. dependent manner. The STIL-SUFU association releases GLI1, a key transcription factor of Hedgehog signaling, STIL knockdown impedes cancer cell motility from cytoplasmic tethering by SUFU, which leads to the nuclear accumulation and transcriptional activation of GLI1 To clarify the role of STIL in cancer cell motility, we in pancreatic cancer cells [14]. STIL is also known as a key examined the effects of STIL knockdown on cell migration molecule involved in centriole duplication in dividing cells (Fig. 2a). In the wound-healing assay, which estimates the [15–18], but the roles of STIL are still not fully understood. collective cell migration, we found that STIL knockdown In the present study, we demonstrate the indispensability impeded the migration of Panc-1 (Fig. 2b; right, the quan- of STIL in cancer cell migration: mechanistically, STIL titative presentation). In the cell-tracking analysis, which Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial. . . 1933

Fig. 1 STIL accumulation at the leading edge of cells. a Relative expression of STIL in normal and cancer tissues of pancreas (Pei Pancreas) [22], colon (Skrzypczak Colorectal2) [23], and breast (Richardson Breast2) [24], downloaded from the Oncomine database (www.oncomine.org). *p <0.01. b The subcellular localization of STIL and Cortactin in motile cancer cells. Scale bar 20 μm

estimates individual cell migration, we also found that STIL It is well-known that PAK1 associates with ARHGFE7 and knockdown reduced the migration distance of the cells (Fig. functions as a modulator and effector for Rac1 by phos- 2c; right, the quantitative presentation). phorylation of downstream cytoskeletal remodeling signals Intriguingly, in contrast to control siRNA (siControl)- [5]. Therefore, here we focused on these proteins to transfected cells, STIL-knockdown cells harbored enlarged examine the mechanistic role of STIL in cell motility. and flattened cytoplasm. Furthermore, siControl-transfected We first examined the precise localization of these pro- cells extended and projected their cytoplasm in a relatively teins using laser scanning confocal microscope. We found persistent direction and moved quickly, whereas STIL- that STIL co-localized with ARHGEF7 as well as PAK1 at knockdown cells hurriedly and inconsistently extended their the leading edge of individually migrating cells (Fig. 3a edge in random directions and moved slowly (Fig. 2d left; upper panels; Fig. 3b, line profiles along white line of Fig. Supplementary Movie S1 and S2). As expected from the 3a), and also in the membrane ruffles facing the wound movies, the velocity of STIL-knockdown cells, even with during collective migration (Fig. 3a lower panels). their hurriedly ruffling membrane, was significantly lower The subcellular localization of ARHGEF7 is regulated than that of siControl-transfected cells (Fig. 2d right). It is through its interaction with many types of molecules [1]. known that the speed and directional persistence of motile Thus, we next examined whether STIL associates with cells are controlled by cascades of cytoskeletal rearrange- ARHGEF7. Through an immunoprecipitation assay using ment [2]. Importantly, Rac1 activation was reported to total lysates of Panc-1 cells, we found that STIL protein co- enhance the directional formation of membrane ruffles, precipitated with ARHGEF7 (Fig. 4a). We constructed the while CDC42 activation functions in random formation expression vectors for wild-type and deletion mutants of [27]. Therefore, we assumed that STIL knockdown would ARHGEF7 and STIL (Fig. 4b) and then transfected affect the Rac1 cascades, which would cause the impaired HEK293T cells with the indicated vectors and used them in polarity of leading edge during migration. the immunoprecipitation assay (Fig. 4c–e). We found that wild STIL associated with wild ARHGEF7, while mutant STIL associates with the ARHGEF7–PAK1 complex STIL harboring a deletion in the coiled-coil region did not (Fig. 4c). In addition, wild STIL did not associated with As of today, many molecules have been reported to control ARHGEF7 containing a deletion of the carboxyl terminal the polarity of migrating cells. Among them, ARHGEF7/ region including the coiled-coil domain (Fig. 4d). These β-PIX, a member of the Dbl family of RhoGEF, is well- results indicate the necessity of coiled-coil domains in STIL known to accumulate at the leading edge where it controls and ARHGEF7 for their association, which is in agreement the Rac1-mediated migration of cells. ARHGEF7-knock- with a previous report showing that the ARHGEF7 accu- down cells reportedly lose the polarity of protrusions and mulation and formation of membrane ruffles depend on its exhibit randomly directed protrusions and shortened coiled-coil region [3]. migration distance [4], which is similar to that of STIL In contrast, we faintly detected PAK1 precipitation with knockdown (Fig. 2c, d; Supplementary Movie S1 and S2). STIL (Fig. 4e). It was previously reported that PAK1 1934 H. Ito et al.

Fig. 2 STIL knockdown impedes cancer cell motility. a Immunoblot Diagrams representing the migrating trajectories of each cell examined analysis of Panc-1 cells transiently transfected with either human for 10 h (left). Moving distance of each cell was plotted as mean + STIL-speciﬁc siRNAs (siSTIL-1 and siSTIL-2) or their control siRNA SEM (right). *p < 0.01. d Images of siRNA-transfected Panc-1 cells at (siControl). b Wound-healing assay. Images at 0 h and 24 h after every 20 min (left). Scale bar 20 μm. Movies of those images are making wounds were photographed (left) and the wound-healing rates supplemented as Supplementary Movie S1 and S2. The velocity of were calculated. The results represent as mean + SEM based on three each cells was plotted as mean + SEM (right). *p < 0.01. Open col- experiments performed in triplicate (n = 9) (right). *p < 0.01. c Cell- umn, siControl-transfectants; gray column, siSTIL-1-transfectants; tracking analysis. Fifteen motile cells were tracked using IncuCyte. ﬁlled column, siSTIL-2-transfectants (b, c, d)

Fig. 3 STIL co-localizes with ARHGEF7 and PAK1 at the membrane ruffles. a Confocal images of the subcellular localization of STIL, ARHGEF7, and PAK1 in an individually migrating Panc-1 cell (upper panels) and collectively migrating Panc-1 cells facing the wound (lower panels; arrows indicate the direction of migration). Scale bar 20 μm. b Line profiles of the fluorescent intensity along white line of Fig. 3a. Arrows indicate the membrane ruffles Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial. . . 1935

Fig. 4 STIL associates with the ARHGEF7–PAK1 complex. a Immunoprecipitation analysis of Panc-1 cells. b Schema of the expression vectors of STIL and ARHGEF7. SH3 and Dbl homology (SH3-DH) domain, pleckstrin homology (PH) domain, polyproline (PxxP) motif, GIT-binding domain (GBD), and coiled-coil (CC) domain. c–e Immunoprecipitation analyses of HEK293T cells transiently transfected with indicated vectors. Immunoprecipitation (IP) and immunoblot (IB) were done with indicated antibodies. f Proximity ligation assay (PLA) of Panc-1 cells was done using indicated antibody. PLA signals were represented as green dots and merged with phalloidin staining (red). Note that PLA signals were not observed in negative controls (anti-PAK1 antibody alone and anti-STIL antibody alone). Scale bar 20 μm

directly forms a complex with ARHGEF7 and is recruited STIL-knockdown cells. We found that STIL knockdown to the focal adhesion and lamellipodia [9]. Therefore, to nearly abolished the accumulation of ARHGEF7 and PAK1 examine the involvement of ARHGEF7, we transfected at the membrane ruffles (Fig. 5b upper panels). Co- HEK293T cells with the STIL, PAK1, and ARHGEF7 transfection of FLAG-tagged mouse STIL, the expression vectors and subjected those cells to immunoprecipitation. of which was not inhibited by human STIL-specific siRNAs, We found that the co-transfection of ARHGEF7 increased rescued the ARHGEF7 accumulation at the edge of siSTIL- the amount of PAK1 precipitation with STIL, which indi- transfected cells (Fig. 5b lower panels). The PLA using ant- cates that STIL indirectly associates with PAK1 via ARH- ARHGEF7 and anti-PAK1 antibodies detected the PLA GEF7 (Fig. 4e). Consistently, the proximity ligation assay signals (the ARHGEF7–PAK1 complex) at the membrane (PLA) using both of anti-STIL and anti-PAK1 antibodies ruffles as well as in the cell body of siControl-transfected detected the PLA signals (the complex including STIL and Panc-1 cells. In contrast, STIL knockdown diminished the PAK1) at the membrane ruffles as well as in the cell body of PLA signals at the membrane ruffles, while the signals of Panc-1 cells, supporting that STIL associates with the cell body were maintained (Fig. 5c upper panels; lower, ARHGEF7–PAK1 complex (Fig. 4f). quantitative presentation of PLA signal at the membrane ruffles). These evidences indicate that STIL controls the STIL regulates ARHGEF7–PAK1 accumulation at accumulation of the ARHGEF7–PAK1 complex within the membrane ruffles membrane ruffles. As seen in Fig. 2d and Supplementary Movie S1 and S2, Concordant with the immunoprecipitation analyses, we we observed that STIL-knockdown cells harbored enlarged found that the overexpressed STIL co-localized with and flattened cytoplasm compared with siControl- endogenous ARHGEF7 at the membrane ruffles of COS7 transfected cells (Fig. 5b, c). It was previously reported cells (Fig. 5a). Next, we examined whether STIL controls that ARHGEF7 controls the intracellular transport of PAK1 the subcellular localization of ARHGEF7 and PAK1 using to the membrane ruffles [9] and that the knockdown of 1936 H. Ito et al.

Fig. 5 STIL regulates the ARHGEF7–PAK1 accumulation at mem- ARHGEF7–PAK1 complex. After PLA detection, the samples were brane ruffles. a Confocal images of the subcellular localization of dried and followed by phalloidin staining. PLA signals (green dots) endogenous ARHGEF7 and FLAG-tagged STIL in COS7 cells. Note were demonstrated as merged images with phalloidin staining (red). that FLAG-STIL accumulated at the membrane ruffles and was Scale bar 20 μm. Note that STIL knockdown significantly diminished overlapped with endogenous ARHGEF7. Scale bar 20 μm. b Confocal PLA signals at the membrane ruffles, while those of cell body were images of Panc-1 cells transfected with siRNAs alone (upper panels) maintained (lower statistical analysis, *p < 0.01). d Immunoblot ana- or siSTIL-1 plus FLAG-tagged mouse STIL expression vecor (lower lysis of Panc-1 cells transiently transfected with either human panels). Note that siSTIL-1 transfection abolished the accumulation of ARHGEF7-specific siRNAs (siARHGEF7-1 and siARHGEF7-2) or ARHGEF7 and PAK1 at the membrane ruffles (arrowheads). Scale their control siRNA (siControl) (upper panels). Confocal images of bar 20 μm. c Proximity ligation assay (PLA) of Panc-1 cells transfected siRNA-transfected Panc-1 cells (lower images). Note that with either siControl or siSTIL siRNAs. PLA was done using anti- siARHGEF7-1 transfection abolished the accumulation of STIL and ARHGEF7 and anti-PAK1 antibodies to demonstrate the PAK1 at the membrane ruffles (arrowheads). Scale bar 20 μm

ARHGEF7 enlarges the size of the cytoplasm. In agreement CORONIN1A was recently reported to associate with the with these studies, we observed that ARHGEF7-knockdown ARHFEG7–PAK1 complex and transport it to lamellipodia cells harbored the enlarged and flattened cytoplasm (Fig. 5d of COS1 cells and T-cell leukemia Jurkat cells [11]. How- upper, immunoblot analysis of ARHGEF7 knockdown; ever, our qRT-PCR analyses revealed much higher lower, the representative images of ARHGEF7 knock- expression of STIL than that of CORONIN1A in cancer cell down). Importantly, we found that STIL accumulation lines of pancreas (Panc-1 and KP-4), breast (MDA-MB231 within the membrane ruffles was abolished in ARHGEF7- and Hs578T), and colon (CaCo-2) (Supplementary Table1). knockdown cells, which was concordant with PAK1 abol- The wound-healing analyses revealed that the migration of ishment (Fig. 5d). This evidence indicates that STIL parti- these cell lines was more strongly inhibited by STIL cipates in the accumulation of the ARHGEF7–PAK1 knockdown than by CORONIN1A knockdown (Supple- complex within the membrane ruffles and that ARHGEF7 mentary Fig. S1a, the immunoblot analysis of CORONIN1A concurrently supports STIL accumulation in the ruffles. knockdown; Supplementary Fig. S1b, the wound-healing Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial. . . 1937 rates of knockdown cells), while double knockdown of cascades. Therefore, it has been suggested that the accu- STIL and CORONIN1A impeded Panc-1 migration more mulation of the ARHGEF7–PAK1 complex at the plasma than each of single knockdowns (Supplementary Fig. S1c). membrane triggers the positive-feedback activation of In addition, CORONIN1A knockdown did not result in any cytoskeletal remodeling, which is crucial for the persistence obvious change in the shape and size of Panc-1 cells, which of cell polarity during migration [2, 10]. still maintained the accumulation of ARHGEF7, PAK1, and Given that STIL knockdown diminishes the accumula- STIL at the membrane ruffles (Supplementary Fig. S1d). tion of the ARHGEF7–PAK1 complex at the membrane It was reported that CORONIN1A is not ubiquitously ruffles, we assumed that STIL knockdown blocks the expressed [28] and the Oncomine database predicts that positive-feedback activation of cytoskeletal remodeling. To the expression of CORONIN1A is high in leukemia and test this possibility, we analyzed the phosphorylation of lymphoma cells, whereas it is much lower than STIL FLNA as a substrate of PAK1 [5, 8]. In the immunoblot expression in many types of solid tumors [29] (Supple- analyses, we found that STIL knockdown mildly reduced mentary Fig. S1e). Therefore, we concluded that the sub- the amounts of phosphorylated FLNA as well as phos- cellular localization of the ARHGEF7–PAK1 complex in phorylated PAK1 (Fig. 6a). In siControl-transfected cells, solid tumors is predominantly regulated by STIL rather than phosphorylated FLNA accumulated at the leading edge and CORONIN1A. overlapped with the F-actin marker phalloidin. In STIL- knockdown cells, however, phosphorylated FLNA and STIL knockdown impedes the activity of PAK1 phalloidin signals were aberrantly expressed around the nuclei. The signals at the plasma membrane existed beyond PAK1 is key for cell motility regulation, the mechanism of the detectable level under the confocal microscope, and which depends on its subcellular localization and catalytic their intensities were significantly less in STIL-knockdown activity. PAK1 self-associates and dimerizes in vivo, and cells than in control cells (Fig. 6b). These findings sug- ARHGEF7 binds to the dimerized form of PAK1, which gested that STIL knockdown reduces PAK1 activity at the does not exhibit an obvious kinase activity [7]. Upon an plasma membrane. initial activation of Rac1, the GTP-bound form of Rac1 Next, we questioned whether overexpressed PAK1 might interacts with the PBD of PAK1, which leads to the dis- rescue cytoskeletal remodeling and cell motility in STIL- sociation of the ARHGEF7–PAK1 complex at the plasma knockdown cells. To examine this, we established a stable membrane. The dissociated monomer PAK1 then autop- cell line from Panc-1 cells in which HA-tagged PAK1T423E, hosphorylates its own residues and in turn activates its the active mutant form of PAK1, was induced by the kinase activity to induce the phosphorylation of substrates removal of doxycycline (DOX), an analog of tetracycline, involved in cytoskeletal remodeling cascades such as FLNA from the culture medium (Fig. 7a). As seen in the immu- [5, 8]. In contrast, ARHGEF7 at the plasma membrane nofluorescence analysis, PAK1T423E induction (DOX stimulates the exchange of a bound GDP nucleotide for minus) along with siControl transfection increased phos- GTP of Rac1, which leads to the dissociation of the phorylated FLNA as well as phalloidin staining in the entire ARHGEF7–PAK1 complex, the further activation of PAK1 cell body including the cell edge. With siSTIL-1 transfec- and the phosphorylation of downstream cytoskeletal tion, however, phosphorylated FLNA was not detected at

Fig. 6 STIL knockdown impedes the activity of PAK1. a Immunoblot phosphorylated FLNA and phalloidin at the membrane rufﬂes analysis of Panc-1 cells transiently transfected with siRNAs. Note that (arrowheads). Scale bar 20 μm. Note that phalloidin signal was sig- STIL knockdown reduces the amount of phosphorylated forms of niﬁcantly reduced in STIL-knockdown cells but still existed beyond FLNA and PAK1. b Confocal images of siRNA-transfected Panc- the detectable level in observing under the laser confocal microscope 1 cells. Note that STIL knockdown reduced the accumulation of 1938 H. Ito et al.

Fig. 7 PAK1-overexpression experiments and FRET analyses. a distance of each cell examined for 10 h was plotted as mean + SEM. Immunoblot analysis of the stable Panc-1 cells harboring a *p < 0.01. Open column, siControl-transfectants; gray column, siS- tetracycline-regulated induction of HA-tagged active mutant of PAK1 TIL-1-transfectants; filled column, siSTIL-2-transfectants. d FRET (PAK1T423E). Note that phosphorylated FLNA was increased in the analysis. The Rac1 biosensor (Raichu-Rac1)-expressing Panc-1 cells Doxycycline (DOX)-minus condition. b Confocal images of the were transfected with indicated siRNAs for 3 days and plated on expression of phosphorylated FLNA (p-FLNA) and phalloidin in the collagen type I-coated glass bottom. Represents are the ratio images of PAK1T423E-inducible Panc-1 cells. In siControl-transfectants, the YFP-to-CFP created in intensity-modulated display mode (IMD) using DOX-minus condition increased p-FLNA and phalloidin at the macro program of MetaMorph software. In siControl cell, strong membrane ruffles (arrows vs star marks), while it did not increase FRET signal was observed at the leading edge, while either siSTIL or those at the cell edge of siSTIL-1-transfectants (arrowheads). Note siARHGEF7 transfection reduced FRET signals at the plasma mem- that in siSTIL-1 transfection the staining intensity of p-FLNA was brane (upper left images). For the estimate of FRET activity at the increased in the cell body by the DOX minus. And note that phalloidin membrane, the intensity of FRET signals of the two red-circled areas signal was much stronger in siControl/Dox(-) cells than in siControl/ at either side of the white lines (the movement direction) (lower left Dox(+) or siSTIL-1 cells. For images of phalloidin, the gain, and images) were statistically analyzed (right). *p < 0.01. e Correlation offset of laser confocal microscope were adjusted for siControl/Dox between relative expression of STIL and metastasis of breast cancer (−) cells so that the images not be oversaturated; phalloidin signals of (Schmidt Breast) [35] or recurrence of colon cancer (Jorissen Colon3) the other cells were barely seen in the figure but still existed beyond [36], downloaded from the Oncomine database (www.oncomine.org). the detectable level in observing under the laser confocal microscope. STIL expression was demonstrated as a log2 median-centered inten- Scale bar 20 μm. c Cell-tracking analysis of PAK1T423E-inducible sity. **p < 0.05 Panc-1 cells. Fifteen motile cells were tracked using IncuCyte. Moving Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial. . . 1939 the cell edge, while it was increased around the nuclei upon ARHGEF7 knockdowns (Fig. 7d right). Given that the PAK1T423E induction (Fig. 7b). In the cell-tracking analysis, membrane translocation of the ARHGEF7–PAK1 complex we found that PAK1T423E induction slightly increased the participates in the positive-feedback activation of Rac1 migration distance of siRNA-transfected cells, while it did [2, 10], our findings indicate that the role of STIL for the not significantly rescue the migration distance reduced by motility is to accumulate the ARHGEF7–PAK1 complex at siSTIL transfection (Fig. 7c). This evidence indicates that a part of plasma membrane, where Rac1 activity con- STIL-mediated PAK1 transport to the membrane ruffles is centrates, leading to the polarized formation of leading edge. crucial for the proper formation of leading edge and cell Cell migration supports the fundamental activity for the motility. spreading of cancer cells. As expected, the Oncomine database shows the increased expression of STIL is statis- STIL knockdown impedes Rac1 activation tically associated with metastasis of breast cancer [35] and with recurrence of colon cancer [36] (Fig. 7e), indicating Considering that the ARHGEF7–PAK1 complex did not that STIL could be a potential target to control cancers. reach to the plasma membrane in STIL-knockdown cells (Fig. 5c), we thought the possibility that STIL participates in the spatiotemporal regulation of Rac1 activity at the Discussion plasma membrane. In migrating cells, Rac1 accumulates at leading edge in which cascades of cytoskeletal rearrange- The STIL gene was originally identified as a deleted locus in ment proceed. In agreement with this, we observed that in T-cell acute lymphoblastic leukemia [37]. Later, knockout siControl-transfected Panc-1 cells, the endogenous Rac1 of the stil gene revealed its roles in the axial development was strongly accumulated at the membrane ruffles, which and the left–right specification of embryos [12], which were marked with phalloidin (Supplementary Fig. S2a). suggests its involvement in Hedgehog signaling. Indeed, we However, we found that, whereas the endogenous Rac1 was clarified that STIL associates with SUFU in oncogenic distributed along the broad plasma membrane in STIL- KRAS-dependent manner and functions to dissociate GLI1, knockdown cells, the phalloidin signal at the membrane a key transcriptional factor of Hedgehog signaling, from was, though detectable, significantly reduced (Supplemen- SUFU-mediated tethering in the cytoplasm, which enhances tary Fig. S2a). It was well-demonstrated that cells injected the nuclear accumulation and transcriptional activity of with the plasmid coding a dominant-active mutant of Rac1 GLI1 [14]. (V12Rac1) harbor a pancake-like shape [30, 31]. Con- In the present study, we revealed STIL participation in cordant with the morphological similarity with STIL- the regulation of cancer cell motility. Mechanistically, STIL knockdown cells, the GST-PBD pull-down assay revealed associated with ARHGEF7 via their coiled-coil domains, that the activity of Rac1 in STIL-knockdown cells was and this complex then formed a ternary complex together higher at 2 h after plating on dishes. Unlike siControl- with PAK1. Knockdown of STIL impeded the accumulation transfection, which showed an enormous increase in the of the ARHGEF7–PAK1 complex at the membrane ruffles. Rac1 activity from 2 to 6 h after plating, STIL-knockdown Consistently, STIL knockdown reduced the phosphorylation cells barely increased the Rac1 activity during the same of PAK1 substrates and the F-actin formation at the cell period of time, suggesting that STIL is involved in the edge, which indicates the role of STIL in PAK1-mediated activation process of Rac1 (Supplementary Fig. S2b). cytoskeletal remodeling. Furthermore, the FRET experi- To further examine the spatiotemporal regulation of ments revealed the implication of STIL in the polarized RAC1 activity, we transduced the Förster resonance energy concentration of active Rac1 at a part of membrane ruffles. transfer (FRET) biosensor of Rac1 (Raichu-Rac1) [32–34] Intriguingly, ARHGEF7 knockdown impeded the accumu- and then siRNAs into Panc-1 cells (Fig. 7d). With either lation of STIL and PAK1 at the membrane ruffles, atte- siControl, siSTIL, or siARHGEF7 transfection, the FRET nuated Rac1 activity and led to changes in the cell shape signals were observed primarily at the plasma membrane; in and motility, which mimicked STIL knockdown. This evi- siControl-transfected cells, the high-Rac1 activity was dence indicates that STIL is involved in the positive- confined at the membrane ruffles localized at the leading feedback regulation of the Rac1/ARHGEF7/PAK1 cascade edge of migrating cells, which was consistent with the of cytoskeletal remodeling, which is crucial for the polar- previous report [33]. In contrast, such a steep gradient of ized formation of leading edge that supports the efficient Rac1 activity was not observed in cells transfected with cell migration. either siSTIL or siARHGEF7 (Fig. 7d left). The quantifi- It was previously reported that CORONIN1A binds cation of Rac1 activity also confirmed that the Rac1 activity ARHGEF7 and regulates the subcellular localization of the was, even distributed along the broad membrane, less con- ARHGEF7–PAK1 complex in COS1 cells and Jurkat cells centrated at the putative leading edge in either STIL or [11]. As expected from the coiled-coil region-mediated 1940 H. Ito et al. interaction of STIL and ARHGEF7, we found a short STIL is responsible for the recruitment of the coiled-coil region at the carboxyl terminus of mouse Cor- ARHGEF7–PAK1 complex to the centrosome and for onin1A protein using the Multicoil program (http://cb.csail. PAK1-mediated Aurora-A activation. However, it would be mit.edu/cb/multicoil/cgi-bin/multicoil.cgi) and the COILS an attractive possibility that STIL broadly participates in the program (https://embnet.vital-it.ch/software/COILS_form. proper progression of cell cycle from the centriole dupli- html). However, in our experiments, CORONIN1A knock- cation of G1/S-phase to the mitotic spindle formation of M- down did not result in any obvious changes either in the phase. ARHGEF7–PAK1 accumulation at the membrane ruffles or Importantly, besides centriole duplication, PLK4 has also in the size and shape of Panc-1 cells. The Oncomine data- been reported to regulate cell spreading and motility. base predicts that CORONIN1A expression is not as high as Plk4+/− MEFs showed a reduction in cell spreading and STIL expression in solid-type tumors such as breast, col- motility, which was rescued by the overexpression of PLK4 orectal, and pancreatic cancers. Consistently, we revealed [43]. The autophosphorylated PLK4 at Ser305 accumulated that cancer cell lines we examined in this study expressed at the lamellipodia and filopodia protrusions of motile cells, more STIL than CORONIN1A and that the migration of which indicates the presence of activated PLK4 at the these cells were more strongly inhibited by STIL knock- protrusions [43]. In addition, the overexpressed PLK4 down than by CORONIN1A knockdown. Therefore, we phosphorylates the actin-regulator complex Arp2/3, which believe that ARHGEF7–PAK1 complex-mediated motility induces actin remodeling at the leading edge of motile cells is predominantly regulated by STIL in cancer cells. [44]. In the present study, we found that the active form of Reportedly, the scaffold protein Scrib, a human homolog of PAK1 failed to completely rescue the impeded motility of Drosophila Scribble protein, binds to ARHGEF7 through a STIL-knockdown cells. One possible reason is that STIL coiled-coil region and is responsible for the heregulin- knockdown affects not only the PAK1 pathway but also the induced accumulation of the ARHGEF7–PAK1 complex at PLK4-mediated pathway at the leading edge of cells, which the membrane ruffles of mouse embryonic fibroblasts might not be recused solely by PAK1 induction. This (MEFs) and T47D human breast cancer cells [38]. How- possibility of STIL–PLK4 interaction at the lamellipodia ever, again, the expression of Scrib is not as high as STIL should be investigated in the future. expression according to data in the Oncomine database. In summary, here, we revealed unexpected roles of STIL Therefore, as the case of CORONIN1A, we suspected that in the regulation of cancer cell motility. Preliminarily, we Scrib might participate to a lesser extent than STIL in Rac1- found that STIL knockdown impaired the invasion of Panc- mediated lamellipodia-driven migration of cancer cells. 1 cells as well as the formation of invadopodia (data not STIL and its orthologues, Drosophila melanogaster shown). These experimental data are concordant with the Ana2 and Caenorhabditis elegans SAS-5, have been iden- clinical evidences that the increased STIL expression sta- tified as indispensable proteins for the duplication of cen- tistically linked to the metastasis or recurrence of cancer. trioles during cell division [13, 15, 39]. Centrioles have a Taken together with its role in cell cycle regulation, STIL microtubule-based cylindrical structure with a ninefold might be a hopeful therapeutic target in preventing cancer symmetric array of microtubule triplets. During centriole cell progression. duplication, a protein called SASS6 is involved in the cartwheel structure of procentriole and determines the dia- meter and symmetry of a newly made centriole. Until now, Materials and methods STIL/Ana2/SAS-5 and SASS6 are thought to be mutually dependent in terms of their accumulation in the centriole. A Cells proposed model suggests that the centriolar kinase polo-like kinase 4 (PLK4) binds and phosphorylates STIL, which The human cell lines were obtained from the American leads to the phosphorylated STIL-mediated association and Type Culture Collection and JCRB. Stable cell line Panc-1 centriolar accumulation of SSAS6; this is key for the proper that was able to induce T423E mutant PAK1 in the formation of procentriole [40]. tetracycline-off manner was established using Tet-system Intriguingly, it was reported that ARHGEF7 and PAK1 from TAKARA/Clontech according to the manufacturer’s as well as GIT1 accumulate at the centrosome and that in protocol. the centrosome, GIT1 activates the kinase activity of PAK1 [41]. Once activated, PAK1 phosphorylates and activates siRNA, plasmid, and transfection the key centrosomal kinase Aurora-A, which controls mitotic spindle formation [41]. In addition, PAK1 also The 21-nucleotide duplex siRNAs for human STIL (siSTIL- phosphorylates histone H3, which enhances chromosome 1, siSTIL-2), ARHGEF7 (siARHGEF7-1, siARHGEF7-2), condensation [42]. Currently, we do not know whether CORONIN1A (siCORONIN1A-1, siCORONIN1A-2), and Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial. . . 1941 negative control (siControl) were synthesized as follows: ARHGEF7 antibody 07-1450-I (Millipore) and sc-393184 siSTIL-1, 5′-GACCAUCCGACUUGCUUAUTT-3′ and 5′- (Santa Cruz Biotechnology), anti-PAK1 antibody ab168439 AUAAGCAAGUCGGAUGGUCTT-3′;siSTIL-2,5′-GCCC (Abcam) and #2602 (Cell Signaling), anti-phospho-PAK1 UUCAAAAGCAUUCAUTT-3′ and 5′-AUGAAUGCUUU (Ser144) /PAK2 (Ser141) antibody #2606 (Cell Signaling), UGAAGGGCTT-3′;siARHGEF7-1,5′-AUAUCUUUCAU anti-FILAMIN A antibody MAB1680 (Millipore), anti- UCUUUUCCTT-3′ and 5′-GGAAAAGAAUGAAAGAUA phospho-FILAMIN A (Ser2152) antibody #4761 (Cell UTT-3′;siARHGEF7-2,5′-UUUCAAAUGCAUUUCUAU Signaling), anti-CORONIN1A antibody ab56820 (Abcam), GTT-3′ and 5′-CAUAGAAAUGCAUUUGAAATT-3′; anti-GAPDH antibody sc-32233 (Santa Cruz Biotechnol- siCORONIN1A-1, 5′-UCAAAGUACCGGAUUGAGCTT- ogy), anti-DYKDDDDK (FLAG) antibody 2H8 (Trans- 3′ and 5′-GCUCAAUCCGGUACUUUGATT-3′; siCOR- Genic), anti-HA antibody (Cell Signaling), anti-Myc ONIN1A-2, 5′-UUUUAUUAUGAUUUUUUUCTT-3′ and antibody (Cell Signaling), anti-mouse IgG H&L Alexa 5′-GAAAAAAAUCAUAAUAAAATT-3′; siControl, 5′-CG Fluor 488 conjugated ab150109 (Abcam), anti-rabbit IgG UACGCGGAAUACAACGATT-3′ and 5′-UCGUUGUAU H&L Alexa Fluor 555 conjugated ab150074 (Abcam). Line UCCGCGUACGTT-3′. The expression plasmid vector of profiles of the fluorescence intensity (ARHGEF7, PAK1, mouse stil was previously described [14]. The expression STIL, and Hoechst 33342) (Fig. 3b) were generated by vectors of human ARHGEF7 and PAK1 were constructed ImageJ software (NIH). from pCMVTNT vector (Promega). Transient transfection was carried out using Lipofectamine PLUS reagent (Invitro- Wound-healing and cell-tracking analysis gen) for plasmids and Lipofectamine RNAiMax reagent (Invitrogen) for siRNAs according to the manufacturer’s For wound-healing analyses, cells were seeded onto mul- protocol. tiwell plates precoated with collagen type I (Sigma). After scratching, the wounds were photographed using IncuCyte Quantitative RT-PCR live cell analysis system (Essen BioScience). For cell- tracking analyses, cells were seeded onto 96-well Image- Quantitative RT-PCR (qRT-PCR) was performed using Lock plates (Essen BioScience) precoated with collagen StepOnePlus real-time PCR system (Applied Biosystems) type I (Sigma). Images of every time point were taken using in conjunction with probes for TaqMan Gene Expression IncuCyte. The trajectories of highly motile cells were Assays (Applied Biosystems) according to the manu- tracked manually using ImageJ (NIH) along with plugin for facturer’s protocol. the manual tracking (Fabrice Cordelieres, Institut Curie, Orsay, France). The trajectory of each cell was plotted and Immunofluorescent staining and antibodies analyzed using R 3.4.1 (R Foundation for Statistical Com- puting, http://www.R-project.org). Cells were grown on collagen type I-coated cover slips (Corning) in D-MEM medium supplemented with 5% FBS. Proximity ligation assay (PLA) After a brief fixation in 4% paraformaldehyde/PBS, cells were treated with 0.1% Triton X/PBS for 10 min, 10% PLA was performed according to the manufacturer’s pro- normal donkey serum (Abcam) for 30 min, and then fol- tocol (Duolink in situ PLA, Sigma-Aldrich). After per- lowed by the incubation with antibodies. Nuclear staining forming PLA, the samples were dried and followed by was done using Hoechst 33342 (Thermo Fisher Scientific) phalloidin staining. These signals were detected using laser and F-actin staining using Phalloidin–iFluor488 (Abcam) confocal microscope (LSM710, Carl Zeiss). The authors and Rhodamine-phalloidin (Thermo Fisher Scientific). experienced that the drying steps increased the background Images of stained cells were obtained by epifluorescence of phalloidin staining but the difference of the intensities microscope (IX71, Olympus) or laser confocal microscope between control and STIL-knockdown cells was still (LSM710, Carl Zeiss). For images of F-actin, the imaging maintained (Fig. 5c). parameters (gain and offset) of laser confocal microscope were adjusted for control cells, which have the high inten- FRET analysis sity of phalloidin staining, so that the images not be oversaturated; the same parameters were applied for STIL- FRET analysis was previously described [32]. Briefly, the knockdown cells, which expressed the very low yet lentivirus vector harboring Rac1 biosensor Raichu-Rac1 detectable level of F-actin when observed under the con- was produced from HEK293T cells by co-transfection of focal microscope (Figs. 6b, 7b and Supplementary Fig. pCSIIbsr-2279×, pCMV-VSV-G-RSV-Rev, and psPAX2 S2a). Anti-STIL antibody was previously described [14]. using polyethyleneimine Max (Polysciences), and applied The other antibodies were purchased as follows: anti- to Panc-1 cells with polybrene. After the selection with 1942 H. Ito et al.

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