Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior Brandon C

Home , Amoeboid movement, Cell migration

Published OnlineFirst February 24, 2017; DOI: 10.1158/1541-7786.MCR-16-0411

Cell Death and Survival Molecular Cancer Research Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior Brandon C. Jones1, Laura C. Kelley2, Yuriy V. Loskutov2, Kristina M. Marinak2, Varvara K. Kozyreva2, Matthew B. Smolkin3, and Elena N. Pugacheva1,2

Abstract

Commonly upregulated in human cancers, the scaffolding tional amoeboid motility, indicating a role of NEDD9 in the protein NEDD9/HEF1 is a known regulator of mesenchymal regulation of downstream RhoA signaling effectors. Simulta- migration and cancer cell plasticity. However, the functional neous depletion of NEDD9 or inhibition of AURKA in com- role of NEDD9 as a regulator of different migration/invasion bination with inhibition of the amoeboid driver ROCK results modes in the context of breast cancer metastasis is currently in an additional decrease in cancer cell migration/invasion. unknown. Here, it is reported that NEDD9 is necessary for both Finally, we confirmed that a dual targeting strategy is a viable mesenchymal and amoeboid individual cell migration/inva- and efficient therapeutic approach to hinder the metastasis of sion in triple-negative breast cancer (TNBC). NEDD9 deficiency breast cancer in xenograft models, showcasing the important results in acquisition of the amoeboid morphology, but severe- need for further clinical evaluation of this regimen to impede ly limits all types of cell motility. Mechanistically, NEDD9 the spread of disease and improve patient survival. promotes mesenchymal migration via VAV2-dependent Rac1 activation, and depletion of VAV2 impairs the ability of Implications: This study provides new insight into the thera- NEDD9 to activate Rac1. In addition, NEDD9 supports a peutic benefit of combining NEDD9 depletion with ROCK mesenchymal phenotype through stimulating polymerization inhibition to reduce tumor cell dissemination and discovers a of actin via promoting CTTN phosphorylation in an AURKA- new regulatory role of NEDD9 in the modulation of VAV2- dependent manner. Interestingly, an increase in RhoA activity dependent activation of Rac1 and actin polymerization. Mol in NEDD9-depleted cells does not facilitate a switch to func- Cancer Res; 15(6); 670–82. 2017 AACR.

Introduction and amoeboid modes can be used interchangeably by the same cell population (3, 8). Initial nascent adhesions are highly present Scaffolding protein NEDD9/HEF1, commonly overexpressed in amoeboid cells, consisting of integrin, talin, and nonpho- in many human cancers, is a critical regulator of cancer cell sphorylated paxillin. The recruitment of vinculin and FAK/Src migration (1, 2). Cancer cells are capable of migrating either complex and activation/phosphorylation of paxillin and FAK is collectively or individually (3–5). In collective migration, cells indicative of focal adhesion (FA) maturation, which can culmi- retain most of their epithelial characteristics including cell–cell nate in formation of very elongated fibrillar adhesions (10), adhesions and invade as a sheet (6), thus enabling regional which are often found in mesenchymal cells. invasion and lymphatic-based metastasis. Alternatively, individ- Elevated expression of NEDD9 protein is required for individual migration is distinguished by a loss of cell–cell adhesions and ual mesenchymal migration of diverse tumor cells (1, 11, 12). the ability to invade via two different methods known as mes- Mesenchymal migration is characterized by an elongated cell enchymal and amoeboid (7–9), leading to blood-borne metas- morphology, multiple focal/3D adhesions, and the ability to tasis. Depending on the extracellular matrix (ECM) composition degrade ECM by matrix metalloproteinases (MMP; refs. 7, 13), and rigidity of the tumor microenvironment, the mesenchymal creating a path through the basement membrane/tissue. A master regulator of mesenchymal migration, Rac1 GTPase, is activated by 1Department of Biochemistry, West Virginia University School of Medicine, a number of guanine nucleotide exchange factors (GEF), includ- Morgantown, West Virginia. 2West Virginia University Cancer Institute, West ing melanoma-specific DOCK3 (14), which is recruited/activated Virginia University School of Medicine, Morgantown, West Virginia. 3Department by NEDD9 (11). Rac1 activates the WAVE-regulatory complex of Pathology, West Virginia University School of Medicine, Morgantown, West leading to polymerization of actin filaments (15). We have Virginia. previously shown that NEDD9 also activates and stabilizes Aurora Note: Supplementary data for this article are available at Molecular Cancer A kinase (AURKA; ref. 16), which phosphorylates and activates Research Online (http://mcr.aacrjournals.org/). histone deacetylase HDAC6 (17), leading to deacetylation and Corresponding Author: Elena N. Pugacheva, Department of Biochemistry and subsequent increased activity of the actin branching/stabilizing West Virginia University Cancer Institute, 1 Medical Center Drive, West Virginia protein cortactin (CTTN; ref. 18), which is necessary for lamelli- University School of Medicine, Morgantown, WV 26506. Phone: 304-293-5295; podia and invadopodia formation, structures that define mesen- Fax: 304-293-4667; E-mail: [email protected] chymal invasion. doi: 10.1158/1541-7786.MCR-16-0411 Rac1-driven mesenchymal migration runs counter to the cor- 2017 American Association for Cancer Research. responding RhoA GTPase and ROCKII kinase–driven amoeboid

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Targeting NEDD9/ROCK Inhibits TNBC Metastasis

pathway. Amoeboid migration is characterized by a round-cell Immunofluorescence morphology, small/weak adhesions, and increased cell con- The edges of 18-mm round glass coverslips (Fisher) were coated tractility (7, 19). This type of locomotion does not typically rely with a Super-HT-PAP-PEN hydrophobic slide marker and coated on matrix degradation, and instead slightly deforms the cell inside with 0.01% poly-L-lysine (Electron Microscopy Sciences). shape to squeeze through preexisting gaps in the ECM (20). For 3D matrix embedding, 2.5 104 cells suspended in 200 mLof Because of this, amoeboid movement is more suited for inva- 1.5 mg/mL collagen-I or 4 mg/mL Matrigel plug were loaded on sion through less dense and more elastic matrices such as the coverslips. Coverslips were placed into a 12-well plate and collagens. Mechanistically, RhoA binds and activates ROCKII incubated in a 37C humid chamber during polymerization. Full which phosphorylates myosin II light chain (MLC2), thus media were added, and the cells were allowed to acclimate to the increasing actomyosin contractility and amoeboid invasion. matrix for 48 hours. Fixation and immunofluorescence staining NEDD9 contributes to downregulation of the amoeboid path- was carried out as described previously (29). The list of used way through binding/activating Src kinase, leading to phos- antibodies and quantification details are provided in the Supple- phorylation/inhibition of ROCKII (11). mentary Materials and Methods. The impact of NEDD9 on collective tumor cell invasion has not been explored. However, collective migration of lymphocytes in Collagen gel contraction assay NEDD9 knockout mice was shown to be significantly reduced A total of 1 106 cells per sample were suspended in 1.5 mg/mL (21), and neuronal tube formation in mouse and chick embryos collagen and inserted into a well in a 12-well plate. Triplicate was inhibited upon NEDD9 depletion (22). We have shown that a wells were included per cell line and a well with no cells was decrease in NEDD9 expression leads to significant deficiency in included as control. Once polymerized, media were placed on top activation of MMPs (23, 24), suggesting a deficiency of these cells of the collagen plug. After 24 hours, the collagen plugs were to create tunnels through the matrix. detached from the walls and bottom of the well. Gels were imaged As individual tumor cells are capable of utilizing either one of (Syngene/G-Box) every 15 minutes for the first few hours followed these modes of movement, simultaneous targeting of both path- by hourly images up to 24 hours. Gel area was measured by ways may provide significant clinical benefit against metastatic manual outlining of the collagen plugs and analyzed in ImageJ. cancers (14, 25). In this current study, we define the molecular fi mechanisms of NEDD9-dependent Rac1 activation and the ef - Radioactive kinase assays cacy of combined inhibition of both types of cell movement to [g-32P]-ATP (2.5 mCi; Perkin Elmer) suspended in kinase buffer hinder the metastasis of triple-negative breast cancer (TNBC). (27) was added to recombinant AURKA and/or CTTN (25–50 ng) protein and incubated for 30 minutes at 30C. Samples were then Materials and Methods subjected to gel electrophoresis and membrane transfer followed by X-ray film exposure for 2 hours to visualize phosphorylation Cell lines, RNAs, and plasmids and Western blotting to visualize total protein level. TNBC cell lines MDA-MB-231LN (Caliper); Hs578T, HCC1143, BT549, HCC1395 (ATCC); and SUM159 (gift from Actin polymerization assays Dr. Boerner, Karmanos Cancer Institute, Rochester Hills, MI) were Actin polymerization assays were carried out according to the authenticated and propagated for less than 6 months. si/sh/ manufacturer's recommendations (Cytoskeleton). Recombinant sgRNAs and plasmids used in this study are described in detail AURKA (3 mmol/L), CTTN (75 nmol/L), Arp2/3 (50 nmol/L, in the Supplementary Materials and Methods. The plasmids/ Cytoskeleton), or WASP-VCA domain (5 mmol/L, Cytoskeleton) RNAs were introduced via nucleofection (Amaxa) according to suspended in magnesium/ATP cocktail were added to 5 mmol/L manufacturer's recommendation or virus infection (293T pack- pyrene-labeled G-actin in a 96-well plate and actin polymeriza- aging cells, Life Technologies) as previously described (23). tion was measured on the basis of the increase of fluorescence at excitation 365 nm/emission 407 nm every 30 seconds for 20 Recombinant protein production minutes at 30C using a GENios plate-reader (Tecan). Recombinant AURKA (18), wild-type and N/C-term CTTN (26), and GST and GST-PBD proteins were produced as described Cell kymography previously (27). Kymography analysis was carried out as described previously (18). In brief, cells were plated on glass bottom dishes (Fisher Cell morphology analysis Scientific) 48 hours after nucleofection with mCherry-LifeAct. 4 Cells (10 ) were plated on two-dimensional (2D) or in three- Cells were then treated overnight with 100 nmol/L AURKA dimensional (3D) 1.5 mg/mL collagen (Advanced BioMatrix) or 4 inhibitor MLN8237 (Selleckchem) and live-imaged the next day mg/mL Matrigel (Corning) as described previously (28). Cells every 45 seconds for 60 minutes. Kymograms were compiled from were treated with 50 or 100 mmol/L Y-27632 (ApexBio) where a perpendicular 1 pixel-width line from each frame on the same indicated. Cells were bright-field imaged on a Leica DM/IL micro- spot of the cell membrane. scope. Cell elongation was measured as a function of cell length divided by cell width (ImageJ software/NIH). Active Rac1 pulldown assays Cells were nucleofected with the indicated plasmids/siRNAs Western blotting and allowed to recover overnight, followed by suspension of Western blots were performed as described previously (23). 1.5 105 cells in 450 mLof1.5mg/mLcollagen-Ior4mg/mL Further details and a list of antibodies used can be found in the Matrigel. In indicated assays, cells were also treated overnight Supplementary Materials and Methods. with 4 mmol/L EHop-016 (ApexBio). After 24 hours, cells were

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lysed for 10 minutes on ice with 1 Rac1 lysis buffer as chamber slides (Thermo Fisher) coated with 150 mL4mg/mL described previously (28). Lysates were spun down at maxi- Matrigel. After 1 hour, the medium was removed and the cells mum speed for 12 minutes at 4C and supernatants were added covered with another 200 mL Matrigel, followed by full media to Glutathione-4FastFlow sepharose (GE Healthcare) loaded after Matrigel polymerization. Cells were live imaged in 5-mm with GST or GST-PBD protein, rotating at 4Cforatleast1 Z-steps away from the cell seeding position at 72 hours using a hour. Sepharose was then washed three times with lysis buffer, Nikon Sweptfield/Eclipse/TE2000-E confocal microscope. Inva- boiled in Laemmli buffer, and subjected to gel electrophoresis sion was quantified by total cell fluorescence intensity at the and Western blotting. furthest distance cells reached (100 mm). 3D invasion boxes were constructed using the Volume-Viewer plugin for ImageJ to Active Rac1 and RhoA G-LISA assays show cell fluorescence signal throughout the Matrigel from the G-LISA activation assays for Rac1 and RhoA were performed as seeding position. recommended by the manufacturer's protocol (Cytoskeleton). Mouse xenograft study Immunoprecipitations of GEF proteins Animals were housed in the West Virginia University (WVU) pcDNA3.1-mRFP-NEDD9 and Rac1 GEF-expressing plasmids Animal Facility under pathogen-free conditions according to an were cotransfected into 293T cells using TurboFect (Thermo approved Institutional Animal Care and Use Committee pro- Fisher) as per the manufacturer's protocol. After 24 hours, cells tocol. A total of 1 106 MDA-MB-231-luc2 cells expressing were lysed with PTY lysis buffer and processed as described doxycycline-inducible pTRIPZ-RFP-shControl or shNEDD9 previously (27). Lysates were precleared with protein-A/G-sephar- (24) were suspended in 4 mg/mL Matrigel (Trevigen) and ose (GE Healthcare) for 1 hour at 4C. Mouse IgG control/or anti- injected into the mammary fat pad of 6- to 8-week-old immu- NEDD9 (2G9) protein-A/G sepharose, or anti-HA/anti-FLAG nodeficient female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice EZview-Red antibody (Sigma) were added to precleared lysates (The Jackson Laboratory). Mice with mammary tumors (150– for 12 hours rotating at 4C. Beads were then washed three times 200 mm3) were pretreated in neoadjuvant settings with with PTY buffer. To elute proteins from the beads, 10% ammonia 10 mg/kg AURKA inhibitor MLN8237 via oral gavage for 1 week hydroxide was added for 15 seconds followed by speed-vacuum (4 days on, 3 days off), as described previously, to temporarily centrifuge to remove ammonia hydroxide. Protein was then halt metastasis, but continue mammary tumor growth (30). resuspended in Laemmli buffer for gel electrophoresis and West- shRNA expression in tumor cells was induced via introduction ern blotting. The immunoprecipitation (IP) of CTTN is detailed in of doxycycline-containing food (Bio-Serv) in combination with the Supplementary Materials and Methods. vehicle or 10 mg/kg Y-27632 which was administered by oral gavage for 3 weeks (4 days on, 3 days off). Tumor growth and Live-cell imaging of individual cell invasion dissemination was evaluated weekly through in vivo whole- Cells treated with si/sgRNAs against NEDD9 or RhoA and/or body bioluminescence imaging (BLI) using an IVIS/Lumina-II overexpressing CA-RhoA and/or treated with 50 mmol/L Y-27632 system as described previously (16). After 3 weeks, lungs, or 100 nmol/L MLN8237 for 24 hours were resuspended in 1.5 mammary tumors, and blood were collected for analysis. fi mg/mL collagen and placed into 3D-Chemotaxis m-slides (Ibidi) Paraf n-embedded lung sections were stained by hematoxylin according to the manufacturer's protocol. FBS was added to the andeosinandanalyzedforthenumberofmetastasesperlung left media reservoir to create a chemoattractant gradient. When area by a pathologist as described previously (16). Primary used, Y-27632 and/or MLN8237 were also added to both reser- tumors were analyzed for NEDD9 expression by Western blot voirs to keep the drugs present during the assay. Cells were imaged analysis. Ten mice per both shRNA groups (further separated every 15 minutes via DIC 10 objective on a Nikon Sweptfield/ into 5 per drug group for a total of 20 mice) were used based on Eclipse/TE2000-E confocal microscope for 23 hours. Time-lapse statistical analysis. images were imported into ImageJ software and combined to form video files. Quantification methods are detailed in the Quantification of circulating tumor cells Supplementary Materials and Methods. Submandibular mouse blood samples were collected into EDTA-coated tubes on ice to prevent clotting. Erythrocytes Live-cell imaging of collective cell invasion were lysed and removed from blood via incubation with RBC A total of 1 105 cells in full medium were plated on top of 4–5 lysis buffer (eBioscience) according to the manufacturer's mg/mL Matrigel-coated delta-T dishes (Bioptechs). After 1 hour, protocol. Cells were fixed in 2% paraformaldehyde for 10 min- the medium was removed and the cell layer was covered with utes, followed by centrifugation at 500 g for 5 minutes at more Matrigel. After polymerizing for 30 minutes at 37C, media 4 C, and resuspended in 1% BSA/PBS. FACS was performed was readded. Live-cell imaging was conducted every 10 minutes using a BD Biosciences/LSR Fortessa to count RFP-positive for 24 hours at 37C via Nikon Sweptfield/Eclipse/TE2000-E circulating tumor cells (CTC) in the blood samples. Final confocal microscope (20 objective). Time-lapse images were counts were normalized to initial primary tumor size at imported into ImageJ software and combined to form video files. week zero. Quantification methods are detailed in the Supplementary Mate- rials and Methods. Statistical analysis One-way ANOVA or Student t test statistical analyses were Fluorescent imaging of collective cell invasion sandwich assay performed with GraphPad Prism software where noted. P Cells were pretreated overnight with 50 mmol/L Y-27632 or 0.05 was considered to be significant (). Experimental values 100 nmol/L MLN8237, and 3 104 cells were plated in 8-well were reported as the mean SEM.

672 Mol Cancer Res; 15(6) June 2017 Molecular Cancer Research

Targeting NEDD9/ROCK Inhibits TNBC Metastasis

Results boid NEDD9-depleted cells may not possess all of the mechanistic properties of amoeboid cells. The inability of NEDD9- TNBC cell morphology changes upon NEDD9 and ROCK deficient cells to translate the increased pMLC2 signaling to inhibition matrix deformation/contraction may be indicative of impaired Metastatic TNBC cell lines with primarily mesenchymal adhesion or actin dynamics connecting the internal and exter- morphology (MDA-MB-231, BT549, HCC1143, HCC1395, nal forces of the cell. Hs578T, and SUM159) were used to assess the role of NEDD9 in switching cells from mesenchymal to amoeboid morphology. NEDD9 was depleted using previously characterized NEDD9 depletion leads to a decrease in mature focal adhesion siRNAs (refs. 16, 31; Fig. 1A) in a SMARTpool, and the changes dynamics in individual cell morphology were monitored using live cell Mature, large focal adhesions have been associated with the imaging microscopy when plated in 2D or suspended in 3D mesenchymal phenotype, whereas a decrease in size and matu- matrix (collagen I or Matrigel). rity has been linked to amoeboid morphology (35, 36). Stain- In 2D, control (siCon) cells have well-defined elongated ing for phospho-FAK (pFAKY397) or phospho-paxillin (pPax- morphology, while NEDD9-depleted (siNEDD9) cells show up illinY31) has been commonly used to label mature focal adhe- to a 40% decrease in cell elongation (Supplementary Fig. S1A sions (36, 37). Immunofluorescenceanalysisshowsuptoa2- and S1B) reflected by a decrease in the ratio of cell length over fold decrease in the number of adhesions positive for pFAKY397 cell width. In 3D matrix, the pore size is a critical determinant and pPaxillinY31 upon NEDD9 depletion (Fig. 2G–J; Supple- of cell morphology and migration type (19). The average pore mentary Fig. S1G–S1J), indicating a critical role of NEDD9 in size of the collagen I matrix used was about 15 mm2 (Supple- adhesion maturation. The decrease in FAK and paxillin phos- mentary Fig. S1C and S1D), which supports both amoeboid phorylation was further confirmed by Western blot analysis in and mesenchymal movement (32). Alternatively, the tightly multiple breast cancer cell lines (Fig. 2K–M; Supplementary Fig. packed fibers that make up Matrigel are not conducive for S2). NEDD9 deficiency trended toward a decrease in the num- amoeboid migration (<1 mm) and are permissive primarily for ber of adhesions containing themoremature/stablefocal matrix-degrading mesenchymal movement. Interestingly, when adhesion marker vinculin (38), which would be concurrent seeded in either of these 3D scaffolds, siNEDD9 cells show with amoeboid phenotype; however, the length of many of acquisition of rounded amoeboid morphology and up to a these adhesions was increased compared with control, suggest- 50% decrease in cell elongation (Fig. 1B and C), which is ing the stabilization of fibrillar adhesions (Supplementary Fig. similar to 2D results. S3). Nevertheless, NEDD9-depleted cells also had an increase Knockout of NEDD9 using sgRNAs and CRISPR/Cas9 technol- in the total number of small adhesions that contained talin ogy led to similar changes in morphology with less variability in (Supplementary Fig. S4), which is one of the first proteins cell elongation and showing an elongation ratio closer to 1, recruited to focal contacts/adhesions (38). This indicates that indicating that they are more circular with similar cell length and there is an increase in small, weak nascent adhesions and a width (Supplementary Fig. S1E and S1F). This phenotype was decrease in mature adhesions, which is concurrent with amoe- similar to cells overexpressing constitutively active RhoA GTPase boid phenotype (39). This may suggest that NEDD9 is not (Fig. 1D–F), suggesting RhoA activity may be high in NEDD9- needed for adhesion initiation but is required for fibrillary deficient cells. Importantly, almost all TNBC cells demonstrated adhesion disassembly. Such duality may potentially affect both significant plasticity in their ability to not only be able to switch to mesenchymal and amoeboid adhesion dynamics. amoeboid morphology, but also be able to increase mesenchymal morphology, which was confirmed as control cells became further NEDD9 regulates AURKA-driven phosphorylation of CTTN and elongated upon application of the well-characterized Rho kinase stability of actin filaments inhibitor, Y-27632 (Fig. 1G and H). Amoeboid morphology is characterized by a decrease in filamentous actin polymerization while the mesenchymal elongated NEDD9 depletion leads to increased myosin light-chain shape relies on an increase in filamentous actin (7). Previously, we phosphorylation but decreased contractility have shown that NEDD9 binds and activates AURKA and the Next, we tested whether the acquisition of amoeboid mor- actin-stabilizing protein CTTN, which results in an increase in phology by NEDD9-deficient TNBC cells is accompanied by a actin polymerization and stabilization of lamellar protrusions switch to amoeboid migration, which could potentially pro- and invadopodia (18). Here we show that AURKA directly phos- mote intra/extravasation during metastasis of breast cancer. phorylates CTTN (Fig. 3A) at the (C-terminus 350–546 aa; Fig. 3B; Actomyosin contractility and force-mediated remodeling of the Supplementary Fig. S5). To assess the impact of this phosphor- matrix is a signature of the amoeboid type of cell migration ylation on actin dynamics, an in vitro actin polymerization assay (33). A key driver of actomyosin contractility is the Rho kinase was carried out using recombinant AURKA and CTTN proteins. ROCK, which phosphorylates myosin II light chain (MLC2; Incubation of AURKA with CTTN resulted in a significant increase ref.34).InbothcollagenandMatrigel,NEDD9deficiency in the actin polymerization rate (Fig. 3C) compared with CTTN resulted in up to a threefold increase in pMLC2 (Fig. 2A–D; without AURKA. AURKA alone or in combination with Arp2/3 Supplementary Fig. S1J), which colocalized with cortical actin and WASP did not significantly affect actin polymerization, indi- around the cell membrane. cating that CTTN is the major substrate of AURKA in this assay. Interestingly, despite this increase in pMLC2 in NEDD9- Moreover, kymography analysis of mCherry-LifeAct–expressing depleted cells, there was a significant decrease in the ability cells shows that the inhibition of AURKA results in a complete of the collective cell population to contract floating collagen blockade of lamellar protrusions (Fig. 3D–F), indicating that gels (Fig. 2E and F), indicating that the morphologically amoe- AURKA has a key role in cellular actin dynamics.

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A MDA-MB-231 HCC1143 Hs578T siCon siNEDD9 siCon siNEDD9 siCon siNEDD9 #1 #2 #1 #2 #1 #2 -115 -115 -115 NEDD9 NEDD9 NEDD9 -80 -80 -80 -40 -40 -40 GAPDH GAPDH GAPDH -35 -35 -35 B 3D Collagen C siCon MDA-MB-231 HCC1143 Hs578T siNEDD9 P = 0.03 4 P = 0.03 P = 0.04 P = 0.03 < 0.0001 3 < 0.0001 P * P * 2 * * * * 1 Cell elongation

(cell length/width) 0 Col Mat Col Mat Col Mat MDA-MB-231 HCC1143 Hs578T E 33 P < 0.0001 P < 0.0001

siNEDD9 siCon P < 0.0001 * * * BT549 HCC1395 SUM159 2 * * * siCon D siNEDD9 1 CA-RhoA Cell elongation

(cell length/width) 0 BT549 HCC1395 SUM159 siCon

BT549 HCC1395 SUM159 F CA-RhoA: – + – + – + -50 RhoA -35 -65

siNEDD9 α-Tubulin -50 siNEDD9: – + – + – + -115 NEDD9 -85 -65 α-Tubulin

CA-RhoA -50

G MDA-MB-231 HCC1143 Hs578T BT549 HCC1395 SUM159 Vehicle Y-27632 100 μmol/L

H P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 Vehicle 6 * P < 0.0001 * 50 μmol/L Y-27632 5 * * * 100 μmol/L Y-27632 * ns * * 4 * * 3 2

Cell elongation 1 (cell length/width) 0 MDA-MB-231 HCC1143 Hs578T BT549 HCC1395 SUM159

Figure 1. TNBC cell morphology changes upon NEDD9 and ROCK inhibition. A, Western blot analysis of NEDD9 expression in MDA-MB-231, HCC1143, and Hs578T treated with multiple siRNAs. Bright-field images of MDA-MB-231, HCC1143, and Hs578T cells treated with siCon or siNEDD9 in 3D collagen I (B) and cell elongation quantified as cell length/width (40 cells/group; C). Bright-field images of BT549, HCC1395, and SUM159 cells expressing siCon, siNEDD9, or CA-RhoA (D), cell elongation quantified as cell length/width (100 cells/group; E), and Western blots of NEDD9 knockdown and CA-RhoA expression (F). Bright-field images of TNBC cells, vehicle, or Y-27632 treatment (G) and cell elongation quantified as cell length/width (40 cells/group; H). ns, not significant; , P < 0.05, Student t test versus control.

674 Mol Cancer Res; 15(6) June 2017 Molecular Cancer Research

Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. www.aacrjournals.org ﬂ pFAK for stained ( hours I 24 ( collagen over analysis cells 20 blot sgCon/sgNEDD9 bar, Western CRISPR area. MDA-MB-231 Scale cell of dye. the assay to paxillin. DNA normalized and Hoechst was FAK and Intensity cells/group. actin, of 30 pMLC2, phosphorylation for decreases stained and I MLC2 collagen of phosphorylation increases depletion NEDD9 2. Figure quanti uorescence, Downloaded from ﬁ aino A ( FAK of cation n ¼ G D A L H ,a es 0clsgop( cells/group 20 least at 3, Band intensity RFI (% to con) Band intensity siCon 100 150 200 (% to con) 100 (% to con) siNEDD9 50 L 20 40 60 80 siNEDD9 siCon 100 150 200 250 0 50 0 n ailnpopoyain( phosphorylation paxillin and ) Published OnlineFirstFebruary24,2017;DOI:10.1158/1541-7786.MCR-16-0411 0 siCon io siNEDD9 siCon pMLC2 io siNEDD9 siCon Y397 pFAK pFAK pMLC2 pFAK mcr.aacrjournals.org rpPaxillin or siNEDD9 P =0.0002 P =0.0003 P <0.01 * Y397 * * Time:0123.524h Y31 E I M ). n ocs N y.Saebr 20 bar, Scale dye. DNA Hoechst and I Merge/DNA

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pPaxillin Gel area (% to control) A, GAPDH 100 Paxillin NEDD9 75 50 25 n C GAPDH 0 D-B21sCnadsND9clsin cells siNEDD9 and siCon MDA-MB-231 pFAK pMLC2 NEDD9 ¼ 0 FAK MLC2 3( 123456 F ). siCon G, Y397 sgNEDD9 sgCon siCon D-B21sCnadsND9clsin cells siNEDD9 and siCon MDA-MB-231 ﬂ oecneitniy(RFI), intensity uorescence Hours ﬂ o acrRs 56 ue2017 June 15(6) Res; Cancer Mol oecne( uorescence siNEDD9 n siNEDD9 ¼ K, 3( etr ltaayi and analysis blot Western D .Clae e contraction gel Collagen ). 3252423 -80 -40 -115 -115 -65 -65 -115 -50 -50 P <0.001 -35 -40 -15 -25 -15 -25 -80 -115 H n pPaxillin and ) * n ¼ Y31 ,20 3, – 675 Published OnlineFirst February 24, 2017; DOI: 10.1158/1541-7786.MCR-16-0411

Jones et al.

A B C-term CTTN: - - - + + CTTN: - + + N-term CTTN: - + + - - AURKA: + - + AURKA: + - + - + -65 CTTN -80 N-CTTN -65 -50 -40 Figure 3. AURKA -50 C-CTTN -35 NEDD9 regulates AURKA-driven CTTN -80 -65 phosphorylation of CTTN and stability P32 -65 AURKA -50 of actin filaments. A, Radioactive in vitro AURKA -50 kinase assay of recombinant AURKA -65 and full-length wild-type (WT) CTTN AURKA -50 n P32 proteins using radiolabeled P32-ATP, C-CTTN -40 ¼ 3. Western blot analysis shows total -35 protein (top and middle) while autoradiograph shows P32- C phosphorylated protein (bottom). 600 in vitro P < 0.0001 B, Radioactive kinase assay of 500 * recombinant AURKA and N-term Buffer Only (1–350 aa) and C-term (350–546 aa) 400 CTTN proteins using radiolabeled G-Actin Only 300 P32-ATP, n ¼ 3. Western blot analysis AURKA shows total protein (top and middle) 200 Arp2/3, WASP, AURKA while autoradiograph shows P32- Arp2/3, WASP, CTTN phosphorylated protein (bottom, Fluorescence (AFU) 100 Arp2/3, WASP, CTTN, AURKA 24-hour exposure). C, Pyrene-actin 0 polymerization assay of AURKA and 0 5 10 15 20 CTTN, n ¼ 4. Curves were fit with Time (min) Boltzmann sigmoidal analysis, , P < 0.05. D, Kymographs of cell D Vehicle MLN8237 E F 20 6 membrane of MDA-MB-231 cells treated with vehicle or MLN8237 and 15 quantifications of protrusion velocity 4

Time (E) and maximum membrane extension 10 (F). n.d., not detected. 2

(nm/sec) 5 n.d. n.d.

Protrusion velocity 0

0 Max extension (μm) Veh MLN Veh MLN Membrane protrusion 8237 8237

NEDD9 depletion decreases Rac1 activity in breast cancer different GEFs and their downstream effectors, several potential cells Rac1 GEF candidates were examined for their ability to bind Rac1 GTPase has been shown to be one of the key drivers of NEDD9 in coimmunoprecipitation experiments including mesenchymal migration/morphology. To measure Rac1 activ- DOCK4, DOCK180, VAV2, Tiam1, GEF-H1, ARHGEF2, ARH- ity in cells grown in either 3D collagen or Matrigel, GST-tagged GEF4, DEF6, ECT2, and DBL (Supplementary Table S1). Some PBD (PAK p21–binding domain) protein was used to specif- GEFs (such as DEF6, Supplementary Fig. S6E) were coimmu- ically pull down Rac1-GTP. Cells overexpressing dominant- noprecipitated with NEDD9 using anti-NEDD9–specific mono- negative Rac1 (DN) or constitutively active Rac1 (CA) were clonal (2G9) antibody; however, only VAV2 was found to also used as controls (Supplementary Fig. S6A). NEDD9 depletion reciprocally pull down NEDD9 (Fig. 4D; Supplementary Fig. resulted in a significant decrease in the amount of active Rac1 in S6F) when immunoprecipitating VAV2. To confirm that VAV2 is cells grown in both scaffolds (Fig. 4A and B). This was consis- required for Rac1 activation in breast cancer cells, active Rac1 tent when repeated in two other mesenchymal breast cancer cell pulldowns were performed on cells treated with siCon or siRNA lines as well, Hs578T and HCC1143 (Supplementary Fig. S6B targeting VAV2 (Fig. 4E). VAV2 depletion resulted in a 40%– and S6C). As a complementary approach, Rac1 G-LISA assays 60% reduction in the activity of Rac1 (Fig. 4F), supporting the (40) were used to evaluate the effect of NEDD9 depletion. notion that VAV2 is a critical downstream effector of NEDD9- Similarly to the active Rac1 pulldown assay, Rac1 G-LISA shows dependent Rac1 activation. Importantly, treatment of siCon a decrease in the amount of active Rac1 (Fig. 4B; Supplementary cells with EHop-016 (41), a small molecular compound which Fig. S6D). In addition, the RhoA G-LISA shows an increase in inhibits Rac1 activation by VAV2 through interaction with the amount of active RhoA, which is concurrent with the Rac1's VAV2-binding site, resulted in a reduction in Rac1 activity amoeboid phenotype (Fig. 4C). that was similar to the effect caused by siNEDD9 (Supplemen- tary Fig. S6G and S6H). This inhibition effect by EHop-016 was NEDD9 drives Rac1 activity through interaction with GEF not found to be additive when combined with NEDD9 deple- VAV2 tion. In addition, an increase in Rac1 activity through NEDD9 Previously, it was shown in melanoma cells that NEDD9 overexpression was able to be blocked by VAV2 knockdown, activates Rac1 via interaction with the Rac1-GEF DOCK3 (11). further supporting that NEDD9-induced Rac1 activity is VAV2 On the basis of the breast cancer cell expression profiles of dependent (Fig. 4G).

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A B PD: Active Rac1 C G-LISA: Active RhoA GST siCon siNEDD9 3D Collagen 3D Matrigel 3D Collagen 3D Matrigel -25 Active 0.15- PD: 100 100 2.0 * Rac1 P < 0.05 * -15 80 P = 0.02 80 1.5 P = 0.027 -25 0.10- P = 0.036 Total 60 * * 60 1.0 Rac1 -15 40 40 0.05- -115 (490 nm) 0.5

20 20 Absorbance (% to control) NEDD9 Rac1 Activity -80 0 0 0- 0 siConsiNEDD9 siCon siNEDD9 siCon siNEDD9 siConsiNEDD9

α-Tub. -50 WCL

D IP E GST siCon siVAV2 F -25 100 Active P < 0.05 WCL IgG NEDD9 IgG VAV2 PD: Rac1 80 -115 -15 60 * VAV2 -25 -80 Total 40 Rac1 -140 -15 20 Rac1 Activity NEDD9 -115 (% to control) -115 0 VAV2 siCon siVAV2 -80 -40 GFP-NEDD9 GAPDH overexpression -25 WCL G GST siCon siCon siVAV2

Active -25 PD: Rac1 -15

Total -25 Rac1 -15 -140 Exog.

NEDD9 -115 Endog.

-115 VAV2 -80 WCL

α-Tub. -50

Figure 4. NEDD9 drives Rac1 activity through interaction with GEF VAV2. A, Western blot analysis of active-Rac1 pulldown (PD) from MDA-MB-231 siCon/siNEDD9 cells in 3D Matrigel using GST-PBD–conjugated beads. Lane 1, GST-empty beads control. WCL, whole cell lysate. B, Quantification of Rac1 activity in 3D collagen and Matrigel (active/total Rac1) by Western blot analysis. C, Quantification of RhoA activity in 3D collagen and Matrigel by G-LISA. D, Coimmunoprecipitation of HA-VAV2 and RFP-NEDD9 through IP of each protein. IgG, mouse IgG control. Western blot analysis of active-Rac1 pulldown from MDA-MB-231 siCon/siVAV2 cells (E) and quantification of Rac1 activity (F). G, Western blot analysis of active-Rac1 pulldown from GFP-NEDD9–overexpressing MDA-MB-231 siCon/siVAV2 cells. Lane 1, GST-empty beads control. Endog, endogenous; Exog, exogenous; WCL, whole cell lysate. For all experiments: n ¼ 3. , P < 0.05, Student t test versus control.

Simultaneous targeting of NEDD9 and ROCK/RhoA hinders cally decreased invasion (Fig. 5A–E; Supplementary Videos S1 and breast cancer cell motility/invasion in vitro S2; Supplementary Fig. S7A–S7D), similar to the overexpression As NEDD9 depletion supports the ROCK/RhoA pathway, of constitutively active RhoA (Supplementary Fig. S7F–S7J; Sup- knockdown of NEDD9 was combined with the ROCK-targeting plementary Video S3). Combination of Y-27632 þ siNEDD9 led compound Y-27632 to test for an additional therapeutic beneﬁtin to an increase in leading edge speed (extension/retraction), 3D invasion assays. NEDD9 depletion alone (via siRNA or but decreased the cell body speed, thus rendering the elongated sgRNA) led to an increase in amoeboid morphology and drasti- cell immobile (Fig. 5C). The directionality and elongation of

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A B siCon siNEDD9 siCon siNEDD9

100 100 50 50 μm -250-200-150-100 -50 50 -250-200-150-100 -50 50 -50 -50

-100 -100

100 100

50 50 μm -250-200-150-100 -50 50 -250-200-150-100-50 50 -50 -50 Y-27632 Vehicle Y-27632 Vehicle CHEMOATTRACTANT -100 -100

μm μm C P = 0.0001 P = 0.0003 P < 0.0001 D E P = 0.0005 P = 0.03 P = 0.02 P < 0.0001 P = 0.005 P < 0.0001 1.0 0.5 P = 0.01 P = 0.008 *** 10 * 0.8 * * 8 *** 0.4 * * ns 6 m/min) 0.6

μ 0.3 0.4 4 0.2 2

0.2 Cell elongation 0.1 (cell length/width) 0 Cell body directionality 0 (Euclidean/total distance)

Cell speed ( - - 0 siNEDD9: - + - + siNEDD9: + + - - LE CB LE CB LE CB LE CB Y-27632: - - + + Y-27632: + + siNEDD9: - + - + Y-27632: - - + +

0.8 F shCon shNEDD9 G H 1.0 P < 0.0001 GFP GFP 0.8 0.6 * P < 0.0001 0.6 0.4 * 0.4 0.2 0.2

0 Cell body directionality 0 (Euclidean/total distance) Cell body speed ( μ m/min) shCon shNEDD9 shCon shNEDD9

I shCon shNEDD9 J 100 P = 0.006 75

50 *

Invasion (% to con) 0 shCon shNEDD9

K shCon shNEDD9 -115 NEDD9 -80 -40 Invasion Invasion GAPDH -35

Figure 5. Simultaneous targeting of NEDD9 and ROCK/RhoA hinders breast cancer cell motility/invasion in vitro. Bright-field outlined cell morphology images (12 hours; A), individual cell tracking movement plots toward FBS chemoattractant (left; B), box and whisker plots of CB and LE speed (C), cell body directionality (D), and cell elongation of MDA-MB-231 cells with siCon/siNEDD9 þ Vehicle/Y-27632 treatment in 3D collagen Ibidi chemotactic invasion movies (E). Scale bar, 20 mm. n ¼ 3, 30–40 cells per group; ns, not significant; , P < 0.05, one-way ANOVA. GFP immunofluorescence images and box and whisker plots (F) of cell body speed (G) and cell body directionality of MDA-MB-231 pGIPZ-GFP shControl and shNEDD9 cells after 24 hours being densely seeded in a 3D Matrigel sandwich assay (H). Scale bar, 20 mm. n ¼ 3, at least 60 cells per group; , P < 0.05, Student t test versus control. 3D projection boxes (I) and collective invasion quantification (J) of the amount of MDA-MB-231 pGIPZ-GFP shControl/shNEDD9 cells reaching 100-mm invasion distance after 72 hours, being densely seeded in a 3D Matrigel sandwich assay. , P < 0.05, Student t test versus control. K, Western blot analysis of shNEDD9 knockdown in a collective invasion assay.

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Y-27632–treated cells was significantly increased regardless of Discussion NEDD9 depletion (Fig. 5D and E). As a complementary approach, NEDD9 protein is found consistently upregulated in TNBCs RhoA depletion was performed and resulted in comparable trends and highly correlates with invasive/metastatic spread (1, 16). Our (Supplementary Fig. S8; Supplementary Video S4). Effects on findings provide a mechanistic explanation for NEDD9-driven invasion were also evaluated using Hs578T and HCC1143 cells invasion processes and strongly advocates for the combination of treated with siNEDD9, confirming similar results (Supplementary anti-NEDD9/AURKA and anti-ROCK–targeting compounds to Fig. S9; Supplementary Videos S5 and S6). In addition, the inhibit these movement signaling cascades. In this study, we AURKA inhibitor MLN8237 was tested as a substitute for report that deficiency in NEDD9 signaling itself leads to inhibi- siNEDD9 in combination treatments. Cell invasion distance was tion of key aspects of both mesenchymal and amoeboid migra- found to be decreased with MLN8237/Y-27632 treatment, and tion in TNBC cells, resulting in substantial hindrance on cell was similar to the efficacy of siNEDD9/Y-27632 treatment (Sup- invasion and metastasis. Similar to results in melanoma (14), plementary Fig. S10A and S10B; Supplementary Video S7). This NEDD9 deficiency in TNBC cells results in rounded/amoeboid suggests that MLN8237/Y-27632 could potentially be a viable morphology along with a decrease in the total number of mature combination regimen as well. (pFAK/pPaxillin positive) adhesions and an increase in the num- NEDD9 is required for collective migration in 3D matrix ber of nascent adhesions (39). However, these cells also saw an fi To test the impact of NEDD9 depletion on collective tumor cell increase in long brillar adhesions (Supplementary Fig. S3D). fi invasion, cells were seeded in high density while sandwiched in These ndings suggest that NEDD9 is also required for the fi between two Matrigel layers and time-lapse microscopy was disassembly of brillar adhesions similar to vinculin (42), which performed. Control cells invaded as strand-like collective streams regulates the recruitment and release of focal adhesion proteins in ("highways"), similar to previous observations (ref. 9; Fig. 5F; a force-dependent manner (43). The role of NEDD9/HEF1 as a Supplementary Video S8). Formation of these streams is dimin- sensor of altered adhesion states has been previously reported ished following NEDD9 knockdown, which suggests the cells (44). A decrease in disassembly could be due to matrix metallo- have minimal matrix degradation and/or rearrangement capacity. protease inhibition (45), as previously reported by our group This deficiency in path-generating activity is required for leading (23). Hindered disassembly of FAs would be in disagreement with cells during collective invasion. In addition, cell speed and direc- a previously noted increase in adhesion turnover of NEDD9-KO fi tionality were both significantly decreased in NEDD9-depleted or mutant NEDD9 (Y189A) expressing broblasts in 2D cultures cells (Fig. 5G and H), which corroborates with a significant (46, 47); however the structure, composition, and dynamics of fi reduction in their invasion distance (Fig. 5I–K). Furthermore, adhesions in 2D broblasts may be different from 3D tumor cells. fi inhibition of AURKA yielded a similar decrease in collective cell NEDD9 de ciency led to an increase in myosin-phosphorylat- invasion compared with NEDD9 depletion (Supplementary Fig. ed amoeboid-like cells, but this increase in pMLC2 did not S10C and S10D), again suggesting that AURKA could be a viable translate to increased collective-cell contractility or invasion in therapeutic target. 3D matrix. This disconnect may suggest a potential decoupling between the actin filaments and myosin motors during cell body contraction (48). Alternatively, the inability of NEDD9-deficient Combination of NEDD9 knockdown and Y-27632 treatment cells to disassemble adhesions as we have suggested would limit hinders breast cancer cell invasion and metastasis in vivo amoeboid movement, which is in agreement with our previous MDA-MB-231-luc2 pTRIPZ-RFP-shControl or shNEDD9 cells reports on integrin dynamics (31). were injected into the mammary fat pad of NSG mice. After 2.5 Consistent with past findings, we found that NEDD9 deficiency weeks, mice were treated with the AURKA inhibitor MLN8237 (4 resulted in over a 40% decrease in active Rac1, suggesting that days on, 3 days off) to prevent pulmonary metastases outgrowth NEDD9 is critical for Rac1 activation to occur in TNBC cells. We without any effect on the mammary tumor growth as we reported discovered that an interaction between NEDD9 and the Rac1-GEF previously (16). Seventy-two hours post-MLN8237 pretreatment, protein VAV2 is required for Rac1 activation. Similar to NEDD9 mice with tumors were subjected to doxycycline-induced shCon depletion, loss of VAV2 yields a comparable decrease in Rac1 or shNEDD9 expression and administration of Y-27632 com- activity. Furthermore, the small-molecule compound EHop-016, pound for 3 weeks (Fig. 6A). Primary tumor growth was measured which efficiently blocks the interaction of Rac1 with VAV2 (41), weekly and was significantly reduced in shNEDD9/Y-27632 com- did not cause an additive effect on Rac1 inhibition when given to bination-treated animals compared with vehicle (Fig. 6B and C). siNEDD9 cells, suggesting that NEDD9 and VAV2 share the same At the end of the study, NEDD9 depletion was confirmed in the pathway to Rac1 activation. The potential clinical application of mammary tumors by Western blot analysis (Fig. 6D and E). To this compound has yet to be determined; however, another VAV- measure the metastatic ability of the tumor cells, terminal blood family–targeting drug, the purine analogue azathioprine, was samples were collected and analyzed after euthanization for recently found to inhibit pancreatic cancer metastasis (49). In CTCs using flow cytometry. Nearly a 50% reduction in the addition, overexpression of NEDD9 increased Rac1 activity, number of CTCs was documented with shNEDD9 induction, which was abrogated upon depletion of VAV2, supporting that which decreased even further with the addition of Y-27632 NEDD9's influence on Rac1 activation is VAV2-dependent. Select- (Fig. 6F). A significant abatement in the number of metastases ing NEDD9 as a therapeutic target could potentially be more per lung area was seen with NEDD9 knockdown, and an even beneficial than targeting VAV2 due to its upstream placement in more pronounced inhibition was seen in combination with the mesenchymal pathway and the multiple downstream Y-27632 (Fig. 6G), supporting our hypothesis that dual targeting branches that it links to including VAV2/Rac1 and AURKA/CTTN. of both the mesenchymal and amoeboid pathways via NEDD9 The effect of AURKA inhibition on actin dynamics in TNBCs and and ROCK/RhoA can be a promising therapy against breast cancer its ability to regulate filamentous actin polymerization via metastasis (Fig. 6H).

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A Week: –3.5 –1 0 1 2 3

Inject cancer cells MLN8237 10 mg/kg Vehicle/Y-27632 10 mg/kg treatment 4 days on, 3 days off + Doxycycline food (shCon/shNEDD9 induction)

B Time of treatment (weeks) C shCon+Vehicle 0123 shNEDD9+Vehicle shCon+Y-27632 shNEDD9+Y-27632 /sr, 150

shCon 2 + Vehicle 8 100 Radiance (×10 Radiance * Figure 6. 50

% to week 0) Simultaneous targeting of NEDD9 and shNEDD9 0 ROCK hinders breast cancer cell (photons/sec/cm + Vehicle 6 Primary tumor growth 123 invasion and metastasis in vivo. A, MDA-

8 Time of treatment (weeks)

, p , MB-231-luc2 TZ RFP-shCon and RFP-

hotons/sec/cm shNEDD9 cells injected into the D mammary fat pad of NSG mice were 4 E grown for approximately 3.5 weeks shCon shNEDD9: – + – + + Y-27632 100 before starting treatment (time 0) for Y-27632: – – + + P = 0.006 3 weeks with 10 mg/kg Y-27632 and 2 -115 /sr) shRNA induction. B, Weekly images of 2 NEDD9 50 -80 * tumor cells in vivo via BLI signal which (% to con) was quantified at the primary tumor site α-Tub. -50 shNEDD9 NEDD9 Expression 0 (C). Western blot analysis (D)and + Y-27632 0 shCon shNEDD9 quantification (E) of NEDD9 knockdown in final primary tumors. H Mesenchymal Amoeboid F, Quantification of number of RFP- F positive circulating tumor cells in mouse blood by FACS analysis. 125 G, Quantification of number of visible 100 metastases per area of lung tissue. P < 0.0001 75 P < 0.0001 * NEDD9 RHOA H, Proposed schematic of 50 VAV2 AURKA mesenchymal and amoeboid pathways * in breast cancer. n ¼ 5 mice/group; 25 # CTCs (% to con) , P < 0.05, one-way ANOVA. 0 ROCK shNEDD9: – + – + RAC1 Y-27632: – – + +

G P P 100 * * WASP Arp2/3 CTTN MLC 2 75 P = 0.009 P = 0.002 50 Actin polymerization and Actomyosin

(% to con) * # mets/mm P = 0.04 contractility 25 lamellipodia/invadopodia formation 0 shNEDD9: – + – + Y-27632: – – + +

phosphorylation of CTTN suggests a potential mechanistic expla- The substantially impeded movement of combination-treated nation for the mesenchymal to amoeboid morphology changes cells could be related to the large difference in speed observed observed upon NEDD9 depletion and provides additional venues between the cell body (CB) and leading edge (LE). LE speed to explore CTTN/AURKA signaling in TNBC metastasis. remains high, continuing to protrude and retract, but the cell is In collagen invasion assays, both NEDD9-deficient and incompetent to degrade matrix and/or make anchor points, due to MLN8237-treated cells underwent a drastic reduction in cell NEDD9 deficiency. Meanwhile, the cell rear cannot efficiently invasion. It was anticipated that amoeboid cells could potentially contract due to inhibition of ROCK/RhoA, leaving the CB more move faster with a more erratic cell trajectory. While a small immobile. This combination therefore results in a severe gap decrease in directionality was seen upon NEDD9 depletion, it was between a stagnant, immotile CB along with a quickly protrud- not significant. Inhibition of ROCK or RhoA in NEDD9-deficient ing/retracting, yet functionally impaired LE. These results support TNBC cells reverted them back to an elongated cell shape, but not the notion that simultaneous inhibition of both the NEDD9/ their original migration proficiency. These combination-treated AURKA and ROCK/RhoA pathways could be an efficient antimi- cells also had a significant increase in cell directionality—this is gratory/metastasis treatment option. These conclusions were fur- likely due to their significant increase in cell length which was even ther supported by our findings in an in vivo breast cancer xenograft higher than control cells. mouse model, demonstrating a significant reduction in the

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number of circulating tumor cells and pulmonary metastases of Acquisition of data (provided animals, acquired and managed patients, mice that received combination treatment. These findings are also provided facilities, etc.): B.C. Jones, L.C. Kelley, Y.V. Loskutov, K.M. Marinak, in agreement with previously published reports of NEDD9 deple- V.K. Kozyreva, E.N. Pugacheva Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tion showing a decrease in the invasion and dissemination of computational analysis): B.C. Jones, V.K. Kozyreva, M.B. Smolkin, E.N. diverse cancer types in vivo (1, 2). Pugacheva In conclusion, while NEDD9-deficient cells may have many Writing, review, and/or revision of the manuscript: B.C. Jones, Y.V. Loskutov, hallmarks of amoeboid phenotype, we have shown that amoe- E.N. Pugacheva boid movement appears defective in some respects such as Administrative, technical, or material support (i.e., reporting or organizing decreased cell contractility. Nonetheless, depletion of RhoA or data, constructing databases): B.C. Jones, E.N. Pugacheva fi Study supervision: E.N. Pugacheva addition of ROCK inhibitor to NEDD9-de cient cells provided Other (pathology/histologic analysis of tissue samples): M.B. Smolkin additional benefit in further hindrance of cell invasion/metastasis. Through concurrent targeting of these pathways, this approach Acknowledgments merits further evaluation as a clinical option to augment breast The authors would like to thank and acknowledge the WVU Shared Research cancer patient survival. Facilities and the MBRCC and HSC core facilities including the WVU Microscope Imaging Facility and the WVU Flow Cytometry Core Facility. fl Disclosure of Potential Con icts of Interest Grant Support No potential conflicts of interest were disclosed. This research was supported by grants CA148671 (to E.N. Pugacheva) and KG100539 (to E.N. Pugacheva) from the NIH/NCI and Susan G. Komen for Disclaimer the Cure foundation, and in part by a NIH/NCRR5 P20-RR016440-09. TheWVUHSCCoreFacilitiesweresupportedbytheNIHgrantsP30- This manuscript contains original work only and has not been published or RR032138/GM103488, S10-RR026378, S10-RR020866, S10-OD016165, submitted elsewhere. All of the authors have directly participated in the and P20GM103434. planning, execution and/or analysis of this study and have approved the submitted version of this manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: B.C. Jones, L.C. Kelley, E.N. Pugacheva Received November 12, 2016; revised December 14, 2016; accepted February Development of methodology: B.C. Jones, L.C. Kelley, Y.V. Loskutov 4, 2017; published OnlineFirst February 24, 2017.

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682 Mol Cancer Res; 15(6) June 2017 Molecular Cancer Research

Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior

Brandon C. Jones, Laura C. Kelley, Yuriy V. Loskutov, et al.

Mol Cancer Res 2017;15:670-682. Published OnlineFirst February 24, 2017.

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