RESEARCH ARTICLE

Loss of CAMSAP3 promotes EMT via the modification ofmicrotubule–Akt machineryVarisa Pongrakhananon1,2,*, Onsurang Wattanathamsan2,3, Masatoshi Takeichi4, Paninee Chetprayoon5 andPithi Chanvorachote1,2

ABSTRACTEpithelial-to-mesenchymal transition (EMT) plays pivotal rolesin a variety of biological processes, including cancer invasion.Although EMT involves alterations of cytoskeletal proteins such asmicrotubules, the role of microtubules in EMT is not fully understood.Microtubule dynamics are regulated by microtubule-binding proteins,and one such protein is CAMSAP3, which binds the minus-end ofmicrotubules. Here, we show that CAMSAP3 is important to preservethe epithelial phenotypes in lung carcinoma cells. Deletion ofCAMSAP3 in human lung carcinoma-derived cell lines showed thatCAMSAP3-deficient cells acquired increasedmesenchymal features,mostly at the transcriptional level. Analysis of the mechanismsunderlying these changes demonstrated that tubulin acetylation wasdramatically increased following CAMSAP3 removal, leading to theupregulation of Akt proteins (also known as protein kinase B proteins,hereafter Akt) activity, which is known to promote EMT. Thesefindings suggest that CAMSAP3 functions to protect lung carcinomacells against EMT by suppressing Akt activity via microtubuleregulation and that CAMSAP3 loss promotes EMT in these cells.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: Calmodulin-regulated spectrin-associated protein 3,CAMSAP3, Cell migration, Epithelial-to-mesenchymal transition,EMT, Protein kinase B, Akt, Tubulin acetylation

INTRODUCTIONMetastasis remains a major obstacle for cancer therapy. Duringmetastasis, cancer cells invade nearby tissues, subsequentlydisseminating into the vascular system before invading secondaryorgans (Karlsson et al., 2017). The majority of cancer is derivedfrom epithelial organs, and the epithelial-to-mesenchymal transition(EMT) of cancer cells is thought to contribute to their invasion andmetastasis. The morphological change of epithelial cells to anelongated fibroblast-like shape, accompanied by looser cell–celladhesion and higher motility, favors their invasion of nearby tissues

(Godde et al., 2010; Lamouille et al., 2014). Over the past decade,an increasing number of studies have indicated that metastaticcancer cells acquire mesenchymal-like characteristics and that thischange is relevant to poor clinical outcomes (Aktas et al., 2009;Iwatsuki et al., 2010; Spaderna et al., 2006).

EMT involves reorganization of the cytoskeleton. Althoughthe role of actin and intermediate filaments in EMT is well-characterized (Haynes et al., 2011; Liu et al., 2015; Shankar andNabi, 2015; Velez-delValle et al., 2016), the possible functions ofmicrotubules in EMT or cancer behavior have only recently beenelucidated. Microtubules, major components of the cytoskeleton,have been shown to govern intracellular trafficking, molecularsignaling and directional cell movement through their dynamicproperties (Etienne-Manneville, 2013; Kaverina and Straube, 2011;Parker et al., 2014). Microtubules are composed of α- and β-tubulindimers, and display two ends, the minus- and plus-ends. The minus-end generally binds to microtubule-organizing centers (MTOCs),including the centrosome andGolgi complex, whereas themicrotubuleplus-end is involved in tubulin assembly or disassembly, causingdynamic instability (Dyachuk et al., 2016; Toya and Takeichi, 2016).These characteristics of microtubules are involved in the transitionalstages of microtubule growth and shrinkage, affecting the interactionof microtubules with other cytoskeletal and cytoplasmic elements.Microtubule dynamics have been linked with various cellularbehaviors and morphogenesis, both in tissue development andpathological diseases. For instance, an enhancement of microtubulestability in which tubulin acetylation becomes more enriched hasbeen shown to drive metastasis in breast cancers (Boggs et al., 2015;Matrone et al., 2010). Cooperation of microtubules with othercytoskeletal elements and signaling molecules also contributes to theactivation of downstream effector-related cell behaviors (Giustinianiet al., 2009; Jo et al., 2014; Wang et al., 2014).

In differentiated epithelial cells, themajority ofmicrotubules are notanchored to the centrosome, but instead theirminus-ends are stabilizedby binding to a family of proteins, including calmodulin-regulatedspectrin-associated proteins (CAMSAPs) in vertebrates, Patronin inDrosophila and PTRN-1 inCaenorhabditis elegans (Hendershott andVale, 2014; Nashchekin et al., 2016; Richardson et al., 2014). Thevertebrate CAMSAP family consists of CAMSAP1, CAMSAP2 (alsoknown as CAMSAP1L1) andCAMSAP3 (also known as Nezha), andeach plays roles in cell and tissue morphogenesis (Jiang et al., 2014;Nashchekin et al., 2016; Tanaka et al., 2012; Toya et al., 2015). Inintestinal epithelial cells, CAMSAP3 is required to maintain propermicrotubule organization and organelle polarization (Toya et al.,2015). The present study aimed to investigate the potential role ofCAMSAP3 in the behavior of carcinoma cells. Surprisingly, knockoutof CAMSAP3 in lung carcinoma lines upregulated variousmesenchymal markers at the transcriptional level in parallel withactivation of Akt proteins (also known as protein kinase B proteins,Akt hereafter), an EMT regulator. These findings suggested thatReceived 30 January 2018; Accepted 23 September 2018

1Cell-Based Drug and Health Product Development Research Unit, ChulalongkornUniversity, Bangkok 10330, Thailand. 2Department of Pharmacology andPhysiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University,Bangkok 10330, Thailand. 3Inter-department Program of Pharmacology, GraduateSchool, Chulalongkorn University, Bangkok 10330, Thailand. 4Laboratory for Celladhesion and Tissue Patterning, RIKEN Center for Developmental Biology andRIKEN Center for Biosystems Dynamics Research, Kobe 650-0047, Japan. 5NanoSafety and Risk Assessment Laboratory, National NanotechnologyCenter, NationalScience and Technology Development Agency, Pathum Thani 12120, Thailand.

*Author for correspondence (varisa.p@pharm.chula.ac.th)

V.P., 0000-0002-2220-0842

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CAMSAP3 functions to suppress EMT in lung carcinoma cells andthat loss of CAMSAP3 leads to greater cell migratory abilities.

RESULTSCAMSAP3 removal induces EMTCAMSAP3 genes were knocked out in H460 cells, a large-cell lungcarcinoma line, using the CRISPR/Cas9 system (Fig. S1A,B).In CAMSAP3-KO cells, termed H460/C3ko cells, no expressionof CAMSAP3 proteins was detected (Fig. 1A). Remarkably,mesenchymal markers, including N-cadherin, Slug and ZEB1, wereupregulated in these cells, whereas the epithelial marker,E-cadherin, was downregulated, suggesting that a type of EMTwas induced as a result of CAMSAP3 knockout. Changes to EMTmarker expression occurred at the transcriptional level, as theamounts of protein detected correlated with their mRNA levels,with the exception of Slug. The increase in Slug protein expressionmight have been through another mechanism (Fig. 1B). H460/C3kocells also showed a morphological EMT, exhibiting a mesenchymalshape with a dramatic increase in actin stress fibers anchored topaxillin, a focal adhesion protein (Fig. 1C,D). In contrast, vimentin,an intermediate filament protein that is known to be upregulatedafter EMT (Ahmad et al., 2012; Myong, 2012), was not increased inH460/C3ko cells (Fig. S1C), indicating that CAMSAP3 loss did notupregulate all mesenchymal markers.Wound-healing and transwell migration assays demonstrated that

cell migration was also enhanced in the absence of CAMSAP3(Fig. 1E,F). To test if cell motility increased in individual cells,time-lapse images were collected of cells seeded at low densities(Movies 1 and 2). Analysis of cell trajectories showed that H460/C3ko cells changed their positions more vigorously than controlcells (Fig. 1G), suggesting that individual cells acquired highermotility after CAMSAP3 knockout. Because EMT is thoughtto provide a survival mechanism for cancer cells in detachedenvironments (Guadamillas et al., 2011; Wu et al., 2016), acolony-forming assay in soft agar cultures was performed. H460/C3ko cells exhibited an enhanced growth and an increase in theformation of colonies (Fig. 1H), but also exhibited a slightlyslower growth rate than control cells in 2D cultures (Fig. S1D).To confirm if the behavior and properties of H460/C3ko cells

depended on the loss of CAMSAP3, rescue experiments wereconducted. H460/C3ko rescue cells (C3WT) were generated in whichCAMSAP3 was stably reintroduced (Fig. S2A). C3WT restored theoriginal epithelial morphology (Fig. S2B). Furthermore, the originalproportion of E-cadherin to N-cadherin was restored at both theprotein and mRNA levels (Fig. S2C,D), and cell motility was alsoreduced in C3WT cells (Fig. S2E). All these findings support thehypothesis that CAMSAP3 is important in maintaining epithelialphenotypes in lung carcinoma cells.No change in CAMSAP2 expression level was observed when

CAMSAP3 was removed, suggesting that CAMSAP2 was notinvolved in CAMSAP3 KO-mediated EMT (Fig. S1E). Takentogether, these observations suggest that CAMSAP3 is important tomaintain the epithelial phenotypes of H460 cells. Consistent withthis notion, in the lungs of fetal mice, CAMSAP3 was expressed inepithelial cells but not in mesenchymal cells, and CAMSAP2 wasexpressed in both cell types (Fig. S1F).

CAMSAP3-sensitive EMT is conserved in another lungcarcinoma lineTo test whether the CAMSAP3 deletion-dependent EMT can beobserved in other lung carcinoma cells, we used A549 cells, a lungadenocarcinoma line. We depleted CAMSAP3 using specific

siRNAs in these cells, and also in H460 cells for comparison.As a result of CAMSAP3 knockdown, A549 cells displayed EMT,assessed by changes in cell morphology (Fig. S3A), EMT markers(Fig. S3B), and cell migration speed (Fig. S3C), as observed inH460 cells, suggesting that the requirement of CAMSAP3 forepithelial phenotypes is conserved in other lung carcinoma cells.

Additionally, the changes induced by CAMSAP3 depletion werecompared to those induced by TGF-β treatment, an establishedmethod to induce EMT (Fig. S3A). Upregulation of Slug and ZEB1,as well as downregulation of E-cadherin, were similarly observedin both CAMSAP3-depleted and TGF-β-treated A549 cells(Fig. S3B). However, CAMSAP3 was not reduced in TGF-β-treatedcells, suggesting that EMT was induced through distinct signalingpathways between the CAMSAP3-depleted and TGF-β-treated cells.We did not use H460 cells for the TGF-β experiment because this cellline is not responsive to TGF-β (Finger et al., 2008; Yang et al., 2015).

CAMSAP3 loss overactivates AktAkt is a key player in EMT (Suman et al., 2014; Xu et al., 2015;Yan et al., 2012).We therefore assessed possible involvement ofAkt inCAMSAP3 loss-mediatedEMT.Western blot analysis showed that thephosphorylated form of Akt (p-Akt) greatly increased in H460/C3kocells but that the total Akt level was not altered (Fig. 2A), suggestingthat CAMSAP3 removal enhanced Akt activity. p-Akt upregulationwas also observed in CAMSAP3-depleted A549 cells (Fig. S4A).Immunostaining showed that p-Akt exhibited punctate distributions inthe cytoplasm of control cells and that p-Akt puncta substantiallyincreased after CAMSAP3 knockout, resulting in coverage oflamellipodial edges of the cells (Fig. 2B). These findings suggestedthat CAMSAP3 normally acts to suppress Akt activity.

To confirm if CAMSAP3 knockout-dependent Akt overactivationis responsible for EMT, H460/C3ko cells were treated withLY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K),which activates Akt through phosphorylation of its serine 473 andthreonine 308 residues. The expression levels of mesenchymalmarkers were reduced in LY294002-treated cells (Fig. 2C). Wound-scratch assays also showed that attenuation of Akt activation resultedin a reduction of cell migration rate (Fig. 2D). Furthermore, Aktknockdown using specific siRNAs downregulated mesenchymalmarkers and suppressed cell movement in CAMSAP3-depleted cells(Fig. 2E, Fig. S4B). Thus, these findings demonstrate that CAMSAP3knockout promotes EMT via p-Akt upregulation.

Microtubules are required for Akt activationThe mechanism of the effect of CAMSAP3 on Akt activity wereinvestigated by initially testing the possibility that CAMSAP3might physically interact with Akt. His-tagged CAMSAP3 plasmidswere introduced into H460 cells due to the lack of antibodiesfor endogenous CAMSAP3 useful for this study, and cellswere immunostained for both endogenous Akt and His-taggedCAMSAP3. The data showed no overlapping between localization ofthese two proteins (Fig. S4C). In addition, immunoprecipitationexperiments indicated no coprecipitation of CAMSAP3 with eitherAkt or p-Akt (Fig. S4D). These findings suggest that CAMSAP3affects Akt activity indirectly.

As CAMSAP3 is a microtubule-binding protein, we nexthypothesized that CAMSAP3 might regulate Akt via microtubules.Importantly, it has been reported that localization of Akt on stable oracetylated microtubules is important for sustaining Akt activity(Giustiniani et al., 2009; Jo et al., 2014). We therefore testedwhether Akt indeed interacts with microtubules and, if so, whetherthis interaction is important for Akt activity in H460 cells.

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Microtubule sedimentation assays showed that at least a fraction ofp-Akt or Akt coprecipitated with microtubules, and p-Akt in thepellet fraction was decreased in nocodazole-treated cells(Fig. S5A). Furthermore, nocodazole treatment reduced even thetotal level of p-Akt (Fig. S5B), and a similar reduction was alsoobserved in nocodazole-treated H460/C3ko cells as revealed by

immunoblot and immunofluorescence experiments (Fig. S5C,D).This reduction of p-Akt was rescued by adding Taxol, whichstabilizes microtubules, to the nocodazole-treatment medium(Fig. S5C,D). These results confirmed that microtubules arerequired to maintain proper p-Akt levels and for CAMSAP3loss-dependent upregulation of p-Akt.

Fig. 1. CAMSAP3 depletion induces epithelial-to-mesenchymal transition in H460 cells. (A) Western blots for CAMSAP3 and EMT markers in control(Ctrl) and CAMSAP3-deleted (C3ko) cells. Graph shows mean±s.e.m. band intensity in H460/C3ko relative to H460/Ctrl cells. **P<0.01, n=3. In allimmunoblotting quantifications, band intensities were normalized to the intensity of GAPDH bands. (B) Mean±s.e.m. levels of CAMSAP3 and EMT markermRNA in H460/C3ko relative to H460/Ctrl cells, quantified by real-time qPCR. **P<0.01, n=3. (C) Phase-contrast images of H460/Ctrl and H460/C3ko cells.(D) Staining for paxillin (red), actin (green) and DNA (blue) in H460/Ctrl and H460/C3ko cells. (E) Wound-healing assay for H460/Ctrl and H460/C3ko cells.Graph shows mean±s.e.m. wound areas of H460/C3ko relative to H460/Ctrl cells at the indicated time. *P<0.05, **P<0.01 vs time 0 h; #P<0.05, ##P<0.01 vsH460/Ctrl cells; n=3. (F) Transwell migration assay for H460/Ctrl and H460/C3ko cells. The image is representative of 5 fields per sample from triplicateexperiments. Graph shows mean±s.e.m. cells per field of H460/C3ko and H460/Ctrl cells. *P<0.05, n=3. (G) Trajectories of H460/Ctrl and H460/C3ko cellsover 12 h. Lower graph shows mean±s.e.m. (n>180) displacement of cells every 16 min. *P<0.01. (H) Colony growth of H460/C3ko and H460/Ctrl cells insoft agar. Graph shows mean±s.e.m. number of colonies in H460/C3ko relative to H460/Ctrl cells. *P<0.01, n=3. Scale bars: 100 µm in C, 10 µm in D. C3,CAMSAP3; E-cad, E-cadherin; N-cad, N-cadherin.

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CAMSAP3 loss promotes tubulin acetylationThe ability of CAMSAP3 to control interactions between p-Aktand microtubules was next investigated. Previous observationshave reported that CAMSAP3 depletion causes changes inthe posttranscriptional modification (PTM) of microtubules,such as detyrosination and acetylation (Nagae et al., 2013;

Tanaka et al., 2012). Therefore, the effect of CAMSAP3 knockouton PTMwas investigated. Tubulin acetylation was greatly enhancedin H460/C3ko cells (Fig. 3A,B) and in CAMSAP3-depletedA549 cells (Fig. S4A). In contrast, the levels of total tubulinand detyrosinated tubulin were not significantly altered (Fig. 3B).C3WT cells were used to confirm if the increased tubulin

Fig. 2. See next page for legend.

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acetylation depended on CAMSAP3 deletion, and we found thatacetylated tubulin level was decreased in correlation with p-Aktreduction in C3WT cells (Fig. 3C,D). Next, we closely observedthe distribution of p-Akt and acetylated microtubules bycoimmunostaining in mock-transfected H460 (H460/Ctrl) andH460/C3ko cells, finding that at least a fraction of p-Akt punctaoverlapped with acetylated microtubules (Fig. 3E), which wasconsistent with the observation that p-Akt co-precipitated withmicrotubules (Fig. S5A). Quantitative analysis of the imagesindicated a small increase in the colocalization of p-Akt andacetylated tubulin in CAMSAP3-deleted cells (Fig. 3E). Moreover,the p-Akt reduction in nocodazole-treated H460/C3ko cellscoincided with a reduction of acetylated tubulin (Fig. S5C).

CAMSAP3 loss promotes Akt-dependent EMT by increasingtubulin acetylationTo investigate if enhanced tubulin acetylation plays a role in Aktactivation and EMT, we knocked down α-tubulin acetyltransferase 1(αTAT1, encoded by ATAT1) using specific siRNAs (si-αTAT1) inH460/C3ko cells, as αTAT1 is the sole enzyme responsible fortubulin acetylation in mammals (Akella et al., 2010; Chien et al.,2016; Kalebic et al., 2013; Shida et al., 2010). Although the αTAT1protein level could not be determined due to a lack of appropriateantibodies for αTAT1, ATAT1 mRNA levels were reduced inresponse to RNAi (Fig. 4A). Western blot analysis also confirmedthat acetylated tubulin levels were decreased following si-αTAT1transfection (Fig. 4B). Importantly, p-Akt level was also reduced inthe αTAT1 knockdown cells, suggesting that Akt activation ispromoted by tubulin acetylation. Furthermore, αTAT1 depletionalso suppressed the enhanced cell migration and mesenchymalmarker expression in H460/3ko cells (Fig. 4C,D). These resultssuggest that tubulin acetylation is required for CAMSAP3 loss-mediated EMT.To confirm whether enhanced tubulin acetylation is sufficient to

promote EMT, we examined the effect of GFP-tagged αTAT1overexpression in wild-type H460 cells on p-Akt activation andmesenchymal marker expression. Because transfection efficiencywas relatively low (30–40%), we decided to observe p-Akt and EMTmarkers in individual transfected cells using immunocytochemistryrather than immunoblotting of whole cell lysates. Under thisexperimental setup, only Slug was detectable by immunostainingas an EMT marker. As expected, overexpression of αTAT1,but not its mutant D517N αTAT1 with no enzymatic activity

(Shida et al., 2010), increased tubulin acetylation (Fig. 5A).In the same way, p-Akt and Slug levels were increased in cellsoverexpressing αTAT1, but not D517N αTAT1 (Fig. 5B),confirming that tubulin acetylation is important for Akt activationand the resultant EMT. We also examined whether Taxol treatmentwas sufficient to induce EMT. However, cells treated with Taxol didnot survive for the periods required for the EMT assays (24 h), andtherefore we could not determine whether or not simple stabilizationof microtubules is sufficient for EMT induction. Overall, theseresults suggest that CAMSAP3 loss-induced p-Akt upregulationand EMT promotion are mediated by elevated tubulin acetylation.

Finally, the mechanism by which acetylated microtubules promoteAkt activation was examined. A previous study has reported thatdynactin p150 (also known as DCTN1, dynactin hereafter), a cofactorof the microtubule dynein complex, mediates the interaction betweenAkt and acetylated (stable) microtubules to sustain Akt activation(Jo et al., 2014). Therefore, the involvement of dynactin inCAMSAP3-mediated activation of Akt was examined. Dynactinp150 was shown to be increased in H460/C3ko cells (Fig. 6A).When dynactin was depleted in H460/C3ko cells using siRNA,the p-Akt level was reduced, which was consistent with a previousreport (Jo et al., 2014). Furthermore, dynactin depletion substantiallyattenuated the expression of mesenchymal markers in these cells(Fig. 6B), as well as their wound-healing rate (Fig. 6C). Inaddition, the interaction of dynactin with microtubules waspromoted by CAMSAP3 knockout (Fig. 6D). These findingssuggested that dynactin is involved in the CAMSAP3-dependentAkt-activation process.

DISCUSSIONEMT is a process that is thought to promote cancer invasion(Fenouille et al., 2012; Polireddy et al., 2016). The present studysuggests that lung carcinoma cells have an intrinsic mechanism tosuppress EMT. CAMSAP3 loss caused EMT-like changes alongwith upregulation of mesenchymal markers in these cells. Studies ofmechanisms underlying this phenomenon showed that removal ofCAMSAP3 promoted tubulin acetylation, which was consistentwith previous studies (Tanaka et al., 2012). Concomitantly,CAMSAP3 knockout cells had a higher level of active Akt thanwild-type cells. We showed that inhibition or removal of Aktabrogated CAMSAP3 loss-mediated EMT, and tubulin acetylationwas required for Akt activation. Based on these observations, wepropose that CAMSAP3 normally suppresses Akt activity bycontrolling tubulin acetylation and that this process is important tomaintain epithelial phenotypes in lung carcinoma cells. Since thecell lines used in this study already expressed a certain level ofmesenchymal markers, it is likely that CAMSAP3 loss altered cellbehavior by promoting EMT rather than triggering it.

Microtubules are a major cytoskeletal component of cells, andtheir dynamic properties are regulated by a number of mechanisms,including PTM (Al-Bassam and Chang, 2011; Kadavath et al.,2015; Song and Brady, 2015; Zhang et al., 2015). Acetylation ofα-tubulin, a type of PTM, occurs in stable or long-livedmicrotubules (Janke and Chloë Bulinski, 2011; Portran et al.,2017). Tubulin acetylation is mediated by αTAT1 (Akella et al.,2010; Chien et al., 2016; Kalebic et al., 2013; Shida et al., 2010),and the present study confirmed that this enzyme was required forCAMSAP3 loss-mediated upregulation of acetylation. The questionof how CAMSAP3 suppresses αTAT1-dependent tubulin acetylationremains to be clarified. Our recent study suggested that CAMSAP3maintains dynamic microtubules, and thereby preventing an increasein acetylation (Pongrakhananon et al., 2018). On the other hand,

Fig. 2. CAMSAP3 removal causes Akt overactivation in H460 cells.(A) Western blots for phosphorylated Akt (p-Akt) and total Akt in H460/Ctrl andH460/C3ko cells. Graph shows mean±s.e.m. band intensity in the blots ofH460/C3ko relative to H460/Ctrl cells. *P<0.01, n=3. (B) Immunostaining forp-Akt (green) and DNA (blue) in H460/Ctrl and H460/C3ko cells. Scale bar:10 µm. Box plot shows fluorescence signals of p-Akt relative to α-tubulin(images not shown), using 25 cells. Box represents the 25–75th percentilesof relative fluorescence intensity, and the median is indicated. The whiskersshow the range. *P<0.01. (C) H460/C3ko cells were treated with LY294002(0–20 µM) for 14 h, followed by immunoblotting detection of p-Akt, Akt andEMT markers. Each band was normalized to GAPDH. Graph shows mean±s.e.m. band intensity relative to untreated cells. *P<0.01, n=3. (D) H460/C3kocells were incubated with or without LY294002 (10 µM) for 24 h, and thensubjected to wound healing migration assay. Graph shows mean±s.e.m.wound area relative to the initial time point. *P<0.05 vs time 0 h, #P<0.05 vsuntreated cells; n=3. (E) H460/C3ko cells were transfected with siRNA againstAkt (siAkt) or control siRNA (siCtrl) for 72 h. Expression of p-Akt, Akt andmesenchymal markers were analyzed by immunoblot assay. Graph showsmean±s.e.m. band intensity in the blots of siAkt cells relative to siCtrl cells.*P<0.05, **P<0.01, n=3.

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CAMSAP2 expression level did not change in correlation withEMT, suggesting that CAMSAP2 and CAMSAP3 have distinctbiochemical or physiological functions.The Akt signaling pathway is of paramount importance in several

sporadic cancers, and it is thus a potential target for anticancer drug

development (Altomare and Testa, 2005; Banerji et al., 2017;Hyman et al., 2017). Akt is known to have multiple targets, andEMT regulatory proteins are a target of the Akt signalingpathway, which controls them at both the transcriptional andposttranscriptional level (Suman et al., 2014; Xu et al., 2015).

Fig. 3. See next page for legend.

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Phosphorylation of Akt at serine 473 and threonine 308 by upstreamPI3K initiates its activity; however, its activated state needs to besustained. Emerging evidence has shown that there is an association

between microtubules and Akt activation, and, importantly, it hasbeen suggested that acetylated microtubules are required forprolonged Akt stimulation (Giustiniani et al., 2009; Jo et al., 2014).The present data demonstrates that an increase in acetylated tubulinmediated by CAMSAP3 depletion leads to the upregulation of Aktphosphorylation, despite the lack of cytological colocalization ofCAMSAP3 and Akt. This aberrant increase in Akt phosphorylationwas diminished by treating cells with either αTAT1 siRNA ormicrotubule-destabilizing agents, indicating a noticeable influenceon Akt function of tubulin acetylation induced by CAMSAP3 loss.Furthermore, αTAT1 overexpression was sufficient for Aktactivation and mesenchymal marker upregulation. In addition,dynactin was involved in CAMSAP3 knockout-mediated processesfor Akt activation and EMT induction, which agreed with theprevious finding that microtubule-dependent Akt activation ismediated by dynactin or its associated proteins (Jo et al., 2014;Kunoh et al., 2010). These observations indicate that Akt activity iselevated via CAMSAP3 knockout-induced increased tubulinacetylation, thus prompting cells to undergo EMT.

EMT is mediated by the binding of various stimuli, such as TGF-β, to specific receptors, which initiates intracellular signaling thatregulates the transcription of genes involved in cell–cell adhesion,

Fig. 3. Tubulin acetylation increases in CAMSAP3-deleted H460 cells.(A) Staining for acetylated tubulin (Ace-tub, green) and total α-tubulin (Tubulin,red) in H460/Ctrl and H460/C3ko cells. Graph shows individual data points,with mean±s.e.m. fluorescence intensity of acetylated tubulin relative to α-tubulin. *P<0.01. (B) Western blots for acetylated, detyrosinated (Detyro-tub)and total (Tub) tubulin in H460/Ctrl and H460/C3ko cells. Each band wasnormalized to GAPDH. Graph shows mean±s.e.m. band intensity relative toH460/Ctrl cells. *P<0.01, n=3. (C) Western blots to detect p-Akt, Akt andacetylated tubulin in H460/C3ko cells that were transfected with C3WT ormock-transfected. Each band was normalized to GAPDH. Graph shows mean±s.e.m. band intensity relative to mock-transfected cells. *P<0.05, **P<0.01;n=3. (D) Staining for p-Akt (green), acetylated tubulin (red), α-tubulin (gray)and DNA (blue) in H460/C3ko cells transfected with C3WT or mock-transfected. Graph shows individual data points, with mean±s.e.m.fluorescence intensity of p-Akt and acetylated tubulin relative to α-tubulin.*P<0.01. (E) Staining for p-Akt (green), acetylated tubulin (red), tubulin (gray)and DNA (blue) in H460/Ctrl and H460/C3ko cells. The boxed areas areenlarged at the right. Arrows indicate examples of p-Akt puncta overlapping withmicrotubules. Graph shows individual data points, with mean±s.e.m.colocalization of p-Akt and acetylated tubulin, calculated asManders’ coefficient.*P<0.01. Scale bars: 10 µm.

Fig. 4. Depletion of α-tubulin acetyltransferase 1 suppresses EMT in H460/C3ko cells. H460/C3ko cells were transfected with two different siRNAoligos (#1 and #2) that target α-tubulin acetyltransferase 1 (si-αTAT1), or with control siRNA (siCtrl). (A) Mean±s.e.m. ATAT1 mRNA levels were quantifiedby real-time qPCR. *P<0.01, n=3. (B) Acetylated tubulin, p-Akt and Akt were analyzed by immunoblotting. Graph shows mean±s.e.m. band intensity relativeto siCtrl cells. *P<0.05, **P<0.01, n=3. (C) Wound-healing assay in H460/C3ko cells transfected with si-αTAT1 or siCtrl. Graph shows mean±s.e.m. wound areasrelative the initial time point. *P<0.05 vs time 0 h, #P<0.05 vs siCtrl-transfected cells; n=3. (D) EMTmarkers were analyzed in si-αTAT1 (siRNA#1)- or siCtrl-treatedH460/C3ko cells by immunoblotting. Graph shows mean±s.e.m. band intensity relative to siCtrl-transfected cells. **P<0.01, n=3.

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reorganization of the cytoskeleton, and survival mechanisms tosupport metastasis (Heerboth et al., 2015; Lotz-Jenne et al., 2016).However, our results suggest that distinct signaling pathways areused for EMT in CAMSAP3-deficient and TGF-β-treated cells.Because these cells share upregulation of some EMT markers, it islikely that both pathways merge in some signaling steps, but furtherstudies are required to understand these mechanisms. Of note,changes of EMT markers in CAMSAP3-deficient cells occurred atthe transcriptional level, suggesting that CAMSAP3 loss-dependentEMT is not induced by simple reorganization of the cytoskeletalsystem but instead involves complex gene regulation that maydepend on signaling events downstream of Akt. In the present study,we assayed EMT using 2D-culture dishes, but it is possible for cellsto migrate through 3D environments in vivo. Different types of cellattachment and migration may affect EMT signaling, and thereforeit would be important to test the role of CAMSAP3 in EMT under3D conditions in future studies.The present study suggests that if CAMSAP3 is mutated in vivo

during cancer progression, it would lead to overactivation of Akt,thereby promoting EMT. In contrast, a recent study has shown thatalthough CAMSAP3 mutation or depletion interferes with theintracellular architecture in mouse intestinal cells (Toya et al., 2015),it does not induce EMT in these cells. It is possible that carcinoma

cells are more susceptible to CAMSAP3 loss or EMT-inducingsignals than normal epithelial cells. In conclusion, the presentfindings provide evidence for the remarkable function ofCAMSAP3 in Akt signaling via a tubulin acetylation-dependentmechanism, highlighting a cooperation between these proteins thatinfluences cancer cell behavior.

MATERIALS AND METHODSCells and reagentsNCI-H460 and A549 cells were purchased from American Type CultureCollection (ATCC; Manassas, VA, USA). The cells were not authenticated,but they were tested for contamination. H460 cells were cultured in RPMImedium, and A549 cells in DMEM, both incubated at 37°C with 5% CO2.Both media were supplemented with 10% fetal bovine serum, 1%L-glutamine and 1%penicillin/streptomycin. Cell culture media, supplementsand Phalloidin were obtained from Invitrogen. Taxol, nocodazole, LY294002and DAPI were purchased from Sigma. The antibody against CAMSAP3wasgenerated as previously described (Tanaka et al., 2012) and used at 1:500.Other antibodies were purchased as follows: rabbit anti-CAMSAP2 (1:1000for immunoblotting; Proteintech, 17880-1-AP), rabbit anti-Slug (1:1000for immunoblotting, 1:200 for immunofluorescence; Cell SignalingTechnology, 9585), rabbit anti-ZEB1 (1:1000 for immunoblotting;Cell Signaling Technology, 3396), rabbit anti-E-cadherin (1:1000 forimmunoblotting; Cell Signaling Technology, 3195), rabbit anti-N-cadherin

Fig. 5. αTAT1 overexpression leadsto an increase of p-Akt and Sluglevels. (A) H460 cells were transfectedwith wild-type (wt) αTAT1–GFP orD517N αTAT1–GFP plasmids, andimmunostained for GFP, acetylatedtubulin (red), α-tubulin (gray) and DNA(blue). Box plot shows ratio ofacetylated tubulin intensity in GFP-positive to GFP-negative cells.*P<0.001, n=20 cells. (B) H460 cellstransfected with wt αTAT1–GFP orD517N αTAT1–GFP plasmids wereimmunostained for GFP and p-Akt(red) or Slug (red). Asterisks indicatecells expressing the plasmids atvarious levels. Box plots show ratio ofp-Akt or Slug intensity in GFP-positiveto GFP-negative cells. *P<0.005, n=18cells. Boxes represents the 25–75thpercentiles in all box plots, and themedian is indicated. The whiskersshow the range. Scale bars: 10 µm.

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(1:1000 for immunoblotting; Cell Signaling Technology, 13116), rabbitanti-phosphorylated Akt (S437; 1:1000 for immunoblotting, 1:25 forimmunofluorescence; Cell Signaling Technology, 9271), rabbit anti-Akt (1:1000 for immunoblotting, 1:200 for immunofluorescence;Cell Signaling Technology, 9272), mouse anti-GAPDH (1:1000;Cell Signaling Technology, 97166), mouse anti-tubulin (1:5000 forimmunoblotting, 1:1000 for immunofluorescence; Sigma, T6199),mouse anti-acetylated tubulin (1:5000 for immunoblotting, 1:1000 forimmunofluorescence; Sigma, T7451), mouse anti-paxillin (1:500 forimmunofluorescence; BD Biosciences, 610051), mouse anti-His (1:1000for immunoblotting, 1:500 for immunofluorescence; MBL, D291-3),mouse anti-dynactin (1:2000 for immunoblotting; BD Biosciences,610473), rat anti-ECCD2 (1:400 for immunofluorescence; Shirayoshiet al., 1986) and rat anti-α-tubulin (1:5000 for immunoblotting, 1:1000 forimmunofluorescence; Millipore, MAB1864). The secondary antibodiesused were as follows: goat AlexaFluor 488-, 568-, 555- and 647-conjugatedanti-mouse, -rabbit or -rat IgG (1:000 for immunofluorescence; Invitrogen),sheep HRP-conjugated anti-mouse and anti-rabbit IgG (1:5000 forimmunoblotting; Cell Signaling Technology).

Plasmids and transfectionsgRNA sequences were designed to target CAMSAP3 exon 1 by an onlinetool (http://crispr.mit.edu/) as follows: sgRNA#1, 5′-CACCGACTAGAA-AGGTCCTCCGCAG-3′; and sgRNA#2, 5′-CACCGAGCCCAGCCCAG-TCCGAGCG-3′.

The CRISPR/Cas9 plasmid was constructed as described previouslywith some modifications (Ran et al., 2013). Plasmids expressing sgRNA

were cloned by annealing each DNA oligo and ligating into pSpcas9-2A-puro (Addgene #48139, Cambridge, MA, USA) at the BsbI site. ThesgRNA sequencing of vectors was conducted using the U6F primer.Transfection was performed using Lipofectamine 2000 (Invitrogen)according to the manufacturer’s instructions. Briefly, 2 μg of eachplasmid in optiMEM was mixed with 6 μl of Lipofectamine 2000. After30 min, the mixture was added to cells in culture, and cells were incubatedat 37°C for 6 h. The addition of puromycin antibiotics was performedthe next day. The expression of CAMSAP3 was confirmed byimmunoblotting and real-time qPCR. The CAMSAP3 knockout cellswere designated H460/C3ko, and the control mock transfectant cells weredesignated H460/Ctrl.

For construction of the CAMSAP3-expressing plasmid, full-lengthCAMSAP3 cDNA with a His-tag sequence on its 3′ end was cloned intopCANw. To generate αTAT1 plasmids, αTAT1 wild-type and D157NcDNA were amplified from pEF5B-FRT-GFP-αTAT1 and pEF5B-FRT-GFP-αTAT1-D157N (Addgene), respectively. A Not1 site and Kozaksequence at the 5′ terminus as well as a Sal1 site at the 3′ terminus wereadded to the cDNAs, and cDNAs were then inserted into the pCA-Sal-GFPvector. Transfection was performed using Lipofectamine 2000 according tothe manufacturer’s instructions. Briefly, 3 μg of plasmid in optiMEMmediawas mixed with 6 μl of Lipofectamine 2000. After 30 min, the mixture wasadded to cells in culture, and cells were incubated at 37°C for 6 h. Stabletransfectants were isolated by culturing cells in a medium supplementedwith antibiotic G418 (400 µg/ml) for at least 7 days followed by subsequentcloning. Expression of exogenousDNAwas confirmedby immunoblotting orimmunofluorescence assay.

Fig. 6. CAMSAP3 depletion leads to an increase of dynactin-mediated Akt activation. (A) Dynactin expression in H460/Ctrl and H460/C3ko cells wasanalyzed by immunoblotting. Graph showsmean±s.e.m. band intensity relative toH460/Ctrl cells. *P<0.01, n=3. (B) H460/C3ko cells were transfectedwith dynactin-specific siRNA (siDyn) or siCtrl. After 72 h, cells were analyzed for dynactin, p-Akt, Akt and EMT markers by immunoblotting. Graph shows mean±s.e.m. bandintensity relative to siCtrl-transfected cells. *P<0.05, **P<0.01; n=3. (C) Wound-healing assay of H460/C3ko cells transfected with siDyn or siCtrl. Graph showsmean±s.e.m. wound area relative to the initial time point. *P<0.05, **P<0.01 vs time 0 h; #P<0.05 vs siCtrl-transfected cells; n=3. (D) H460/Ctrl and H460/C3kocells were treated with 1 µM taxol for 30 min at 37°C. Each of their lysates was then separated into a soluble (S) and pellet (P) fraction using a microtubulesedimentation protocol, and analyzed for dynactin and α-tubulin by immunoblotting. Graph shows mean±s.e.m. ratio of the pellet to total fraction. *P<0.05, n=3.

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siRNA transfectionCells were transfected with siRNAs specific for target proteins byLipofectamine RNAiMAX according to the manufacturer’s protocol(Invitrogen). Stealth RNAis targeting CAMSAP3 or α-TAT1 with thefollowing sequences and control siRNAwere purchased from Invitrogen:siCAMSAP3, 5′-ACAGUGGCAGCAGUUCUCCUGUCUU-3′; siα-TAT1#1, 5′-ACCGCACCAACTGGCAATTGA-3′; and siα-TAT1#2,5′-GAGCCAUUAUUGGUUUCCUCAAAGU-3′.

For Akt and dynactin knockdown experiments, the following siRNAstargeting Akt and dynactin were synthesized and annealed: siAkt, sense5′-GGAGAUCAUGCAGCAUCGC-3′ and anti-sense 5′-GCGAUGCUG-CAUGAUCUCC-3′; si-mismatch control, sense 5′-GGGAAUCAUAA-AGCAUUUC-3′ and anti-sense 5′-CCGGGGCUGCAUAAACUUC-3′;siDynactin, sense 5′-GACUUCACCCCUUGAUUAA-3′ and anti-sense5′-UUAAUCAAGGGGUGAAGUC-3′; and si-mismatch control, sense5′-GCUACUUCGUCCAAUCAUA-3′ and anti-sense 5′-UAUGAUUGG-ACGAAGUAGC-3′.

Briefly, 100 nM siRNAs in optiMEM were incubated withLipofectamine RNAiMAX mixture for 15 min at room temperature andthen added to cells, and cells were incubated at 37°C for 6 h. At 72 h aftertransfection, cells were subjected to western blot analysis orimmunofluorescence assay.

Cell migration assayFor the wound-healing assay, 1.5×104 cells were seeded into each well of a24-well culture plate. After cells had reached confluence, wound scratcheswere generated using a pipette tip, and detached cells were removed bywashing with PBS. Images were acquired at indicated time points, andwound area was quantified by ImageJ software. For transwell migrationassays, 5×104 cells in a serum-free medium were placed in the upperchamber with a 0.8 μmpore-sized membrane, and complete culture mediumwas added into the lower chamber. After 24 h, cells on the upper side of themembrane were swabbed out, and those on the lower side of the membranewere fixed with cold methanol at −20°C for 5 min followed by incubationwith DAPI for 10 min. Migrating cells were imaged randomly using anOlympus IX51 fluorescence microscope.

Time-lapse imaging of cell migrationCells were cultured in chamber slides (Lab-Tek II, Thermo Fisher Scientific)overnight. Images were captured every 16 min by an Olympus FluoViewFV10i confocal laser-scanning microscope, with 5% CO2 at 37°C for6:40 (h:min). The displacement of individual cells was tracked byFiji software using the TractMate plug-in and analyzed by MicrosoftExcel-executable DiPer program (Gorelik and Gautreau, 2014). The totaltrajectories of cell migration from the start to the end position were plottedusing Plot At Origin macro.

Cell proliferation assayTwo-thousand cells were seeded into each well of 96-well plates with at leastfive replicates. After the indicated times, medium was replaced with theMTT solution (0.5 mg/ml), and plates were incubated at 37°C for 4 h.Dimethyl sulfoxide (DMSO; 100 μl) was added to dissolve formazanproducts, and the absorbance was measured at 570 nm using a microplatereader. Cell proliferation was calculated relative to the initial time point.

Soft agar colony formation assayA 24-well plate was coated with 0.3% agarose in complete medium as thebottom layer. After solidification, 103 cells were suspended in 0.5% agarose inmedium and seeded onto the bottom layer. Cells were incubated for 14 days,andmediumwas added every 2 days to prevent dryness. Cellswere then stainedwith 0.01% crystal violet in 10% ethanol for 30 min at room temperature. Afterwashing several times with deionized water, colonies were collected andcounted, and the colony number was presented as a relative number to thecontrol cells using ImageJ software with the particle analysis plugin.

Western blot analysisCells were suspended in TMN lysis buffer (20 mM Tris-HCl, pH 7.5; 1 mMMgCl2; 150 mM NaCl; 20 mM NaF; 0.5% sodium deoxycholate; 1%

nonidet-40; 0.1 mM phenylmethylsulfonyl fluoride; and protease inhibitorcocktail, Roche) for 45 min on ice. The protein content was measured byBCA Protein Assay Reagent Kit (Thermo Scientific). Proteins wereseparated by SDS-PAGE and transferred to PVDF membranes. Blots wereblocked with 5% skim milk in TBST (Tris buffer saline with 0.075%Tween-20), incubated with a specific primary antibody overnight at 4°C andincubated with a corresponding secondary antibody for 2 h. The proteinexpression levels were visualized by the enhanced chemiluminescencesystem using SuperSignal West Pico (Thermo Scientific) and ImmobilonWestern (Millipore).

Microtubule sedimentation assayCells were treated with 1 µM Taxol for 30 min at 37°C and lysed by amicrotubule stabilizing buffer (MTB) containing 80 mM PIPES, 80 mMK-1,4-piperazinediethanesulfonic acid (pH 6.8), 1 mM EGTA, 1 mMMgCl2, 0.5% (vol/vol) nonidet P-40, 20 mM NaF, 0.5% sodiumdeoxycholate, 10 mM Taxol, 0.1 mM phenylmethylsulfonyl fluoride, andprotease inhibitor cocktail (Roche) for 5 min at 37°C in dark. The lysate wasfractionated into pellet and supernatant by centrifugation at 17,400 g for15 min at 30°C. The pellet was washed with MTB lacking detergent andresuspended with MTB in an equal volume of supernatant. Both pelletand supernatant fractions were boiled at 95°C with sampling buffer andsubjected to western blot analysis.

RNA extraction and quantitative real-time polymerasechain reactionTotal RNAwas isolated from cells using GENEzol reagent (Geneaid Biotech,Shijr, New Taipei, Taiwan). RNA (1 μg) was reverse transcribed to cDNAusing ProtoScript II Reverse transcriptase (NewEnglandBioLabs) as describedby the manufacturer’s instructions. mRNA expression levels of CAMSAP3,E-cadherin, N-cadherin, Snail, Slug and ZEB1 were measured by a Bio-RadT100 Thermal Cycler using 2× iTaq Universal SYBR Green Supermix(Bio-Rad). The primer pairs used are indicated in Table S1. The thermocyclingconditions were as follows: 95°C for 10 min; and 35 cycles at 95°C for 30 sand 60°C for 30 s. The data were calculated using the ΔΔCt method (Livakand Schmittgen, 2001). Each sample was performed in triplicate.

Immunofluorescence assayCells were fixed with 4% paraformaldehyde in PBS for 20 min at roomtemperature in the dark and permeabilized in 0.1% Triton X-100 in PBS for10 min. For microtubule staining, cells were fixed with cold methanol for5 min at −20°C. Nonspecific signals were blocked by treatment with 3%BSA for 30 min or longer followed by incubation with primary antibodiesovernight at 4°C. Cells werewashed with PBS and incubated with secondaryantibody for 2 h at room temperature in the dark. After washing withPBS containing DAPI, coverslips were washed with deionized water andsubsequently mounted using FluorSave (EMDMillipore). Confocal imageswere acquired by either a Zeiss LSM880 through a Plan-Apochromat63×/1.40 N.A. or a Leica TCS SP8 with 100× oil immersion objective lens.Image preparation and analysis were performed using ImageJ software.Colocalization of p-Akt and acetylated tubulin was quantified using ImageJwith JaCoP plugin, and presented as Manders’ colocalization coefficient(Bolte and Cordelieres, 2006; Bravo-Sagua et al., 2016).

ImmunohistochemistryThe mouse experiment was performed in accordance with the protocol(s)approved by the Institutional Animal Care and Use Committee of RIKENKobe Branch. For lung tissue preparation, lungs were removed fromembryonic day 18.5 C57BL/6N mice and fixed with 2% paraformaldehydein PEM buffer with 75 mM sorbitol for 1 h at room temperature. Tissueswere frozen in OTC solution (Sakura Finetek) and sectioned at a 7 µmthickness prior to immunostaining. Sections were incubated in HistoVTOne(Nacalai) at 95°C for 5 min to retrieve antigens and then incubated in 3%BSA and 3% FCS in PBST for blocking. Sections were then incubated in theblocking solution containing primary antibody at 4°C overnight. Sectionswere then incubated in blocking solution containing a fluorochrome-conjugated secondary antibody for 2 h at room temperature. Samples weremounted on MAS-coated glass slides (Matsunami) using FluorSave reagent

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(Calbiochem). Images were acquired by a Zeiss Axioplan2 through aPlan-Apochromat 63×/1.40 N.A. Image preparation and analysis wereperformed using AxioVision software (Zeiss).

Immunoprecipitation assayCells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5; 1 mM MgCl2;150 mM NaCl; 20 mM NaF; 10 mM EGTA; 0.5% sodium deoxycholate;1% nonidet-40; 0.1 mM phenylmethylsulfonyl fluoride; and proteaseinhibitor cocktail, Roche) for 45 min on ice. The supernatant was collectedby centrifugation at 20,000× g for 20 min at 4°C, precleared with proteinG-conjugated Sepharose beads (GE Healthcare) for 1 h, and incubated withspecific antibody or IgG as control overnight. Protein complexes were pulleddown by incubationwith Protein G-conjugated Sepharose beads for 1 h at 4°Cand then washed five times with lysis buffer. Precipitates were boiled withsample buffer at 95°C for 5 min and analyzed by immunoblotting.

Statistical analysisAll data are presented as the mean±s.e.m. obtained from at least fourindependent experiments. Statistical analysis was performed using unpairedStudent’s t-test or Mann-Whitney U-test using Prism 7 (GraphPad). P-values less than 0.05 were considered statistically significant.

AcknowledgementsWe thank Hiroko Saito for lung tissue preparation.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: V.P.; Methodology: V.P., O.W.; Formal analysis: V.P.;Investigation: V.P., P. Chetprayoon; Writing - original draft: V.P.; Writing - review &editing: M.T., P. Chanvorachote; Supervision: M.T., P. Chanvorachote; Projectadministration: V.P.; Funding acquisition: V.P.

FundingThis work was supported by the Thailand Research Fund (MRG5980021) to V.P.,and also by the Japan Society for Promotion of Science program Grant-in-Aid forScientific Research (S) (25221104) to M.T.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.216168.supplemental

ReferencesAhmad, A., Sarkar, S. H., Bitar, B., Ali, S., Aboukameel, A., Sethi, S., Li, Y.,Bao, B., Kong, D., Banerjee, S. et al. (2012). Garcinol regulates EMT and Wntsignaling pathways in vitro and in vivo, leading to anticancer activity againstbreast cancer cells. Mol. Cancer Ther. 11, 2193-2201.

Akella, J. S., Wloga, D., Kim, J., Starostina, N. G., Lyons-Abbott, S.,Morrissette, N. S., Dougan, S. T., Kipreos, E. T. and Gaertig, J. (2010).MEC-17 is an α-tubulin acetyltransferase. Nature 467, 218-222.

Aktas, B., Tewes, M., Fehm, T., Hauch, S., Kimmig, R. and Kasimir-Bauer, S.(2009). Stem cell and epithelial-mesenchymal transition markers are frequentlyoverexpressed in circulating tumor cells of metastatic breast cancer patients.Breast Cancer Res. 11, R46.

Al-Bassam, J. and Chang, F. (2011). Regulation of microtubule dynamics byTOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol. 21, 604-614.

Altomare, D. A. and Testa, J. R. (2005). Perturbations of the AKT signaling pathwayin human cancer. Oncogene 24, 7455-7464.

Banerji, U., Dean, E. J., Perez-Fidalgo, J. A., Batist, G., Bedard, P. L., You, B.,Westin, S. N., Kabos, P., Garrett, M. D., Tall, M. et al. (2017). A Phase Iopen-label study to identify a dosing regimen of the pan-AKT inhibitor AZD5363for evaluation in solid tumors and in PIK3CA-mutated breast and gynecologiccancers. Clin. Cancer Res. 24, 2050-2059.

Boggs, A. E., Vitolo, M. I., Whipple, R. A., Charpentier, M. S., Goloubeva, O. G.,Ioffe, O. B., Tuttle, K. C., Slovic, J., Lu, Y., Mills, G. B. et al. (2015). α-Tubulinacetylation elevated in metastatic and basal-like breast cancer cells promotesmicrotentacle formation, adhesion, and invasive migration. Cancer Res. 75,203-215.

Bolte, S. and Cordelieres, F. P. (2006). A guided tour into subcellular colocalizationanalysis in light microscopy. J. Microsc. 224, 213-232.

Bravo-Sagua, R., Lopez-Crisosto, C., Parra, V., Rodriguez-Pen a, M.,Rothermel, B. A., Quest, A. F. G. and Lavandero, S. (2016). mTORC1

inhibitor rapamycin and ER stressor tunicamycin induce differential patterns ofER-mitochondria coupling. Sci. Rep. 6, 36394.

Chien, J.-Y., Tsen, S.-D., Chien, C.-C., Liu, H.-W., Tung, C.-Y. and Lin, C.-H.(2016). αTAT1 downregulation induces mitotic catastrophe in HeLa and A549cells. Cell Death Discov. 2, 16006.

Dyachuk, V., Bierkamp, C. and Merdes, A. (2016). Non-centrosomal microtubuleorganization in differentiated cells. In The Microtubule Cytoskeleton (ed. J.Luders), pp. 27-41. Vienna: Springer.

Etienne-Manneville, S. (2013). Microtubules in cell migration. Annu. Rev. Cell Dev.Biol. 29, 471-499.

Fenouille, N., Tichet, M., Dufies, M., Pottier, A., Mogha, A., Soo, J. K., Rocchi, S.,Mallavialle, A., Galibert, M.-D., Khammari, A. et al. (2012). The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstreamtarget of SPARC and AKT in promoting melanoma cell invasion. PLoS ONE7, e40378.

Finger, E. C., Turley, R. S., Dong, M., How, T., Fields, T. A. and Blobe, G. C.(2008). TβRIII suppresses non-small cell lung cancer invasiveness andtumorigenicity. Carcinogenesis 29, 528-535.

Giustiniani, J., Daire, V., Cantaloube, I., Durand, G., Pous, C., Perdiz, D. andBaillet, A. (2009). Tubulin acetylation favors Hsp90 recruitment to microtubulesand stimulates the signaling function of the Hsp90 clients Akt/PKB and p53.Cell. Signal. 21, 529-539.

Godde, N. J., Galea, R. C., Elsum, I. A. and Humbert, P. O. (2010). Cell polarity inmotion: redefining mammary tissue organization through EMT and cell polaritytransitions. J. Mammary Gland Biol. Neoplasia 15, 149-168.

Gorelik, R. and Gautreau, A. (2014). Quantitative and unbiased analysis ofdirectional persistence in cell migration. Nat. Protoc. 9, 1931-1943.

Guadamillas, M. C., Cerezo, A. and Del Pozo, M. A. (2011). Overcoming anoikis –pathways to anchorage-independent growth in cancer. J. Cell Sci. 124,3189-3197.

Haynes, J., Srivastava, J., Madson, N., Wittmann, T. and Barber, D. L. (2011).Dynamic actin remodeling during epithelial-mesenchymal transition depends onincreased moesin expression. Mol. Biol. Cell 22, 4750-4764.

Heerboth, S., Housman, G., Leary, M., Longacre, M., Byler, S., Lapinska, K.,Willbanks, A. and Sarkar, S. (2015). EMT and tumor metastasis. Clin. Transl.Med. 4, 6.

Hendershott, M. C. and Vale, R. D. (2014). Regulation of microtubule minus-enddynamics by CAMSAPs and Patronin. Proc. Natl. Acad. Sci. USA 111,5860-5865.

Hyman, D. M., Smyth, L. M., Donoghue, M. T. A., Westin, S. N., Bedard, P. L.,Dean, E. J., Bando, H., El-Khoueiry, A. B., Perez-Fidalgo, J. A., Mita, A. et al.(2017). AKT inhibition in solid tumors with AKT mutations. J. Clin. Oncol.35, 2251-2259.

Iwatsuki, M., Mimori, K., Yokobori, T., Ishi, H., Beppu, T., Nakamori, S., Baba, H.and Mori, M. (2010). Epithelial-mesenchymal transition in cancer developmentand its clinical significance. Cancer Sci. 101, 293-299.

Janke, C. and Chloe Bulinski, J. (2011). Post-translational regulation of themicrotubule cytoskeleton: mechanisms and functions. Nat. Rev. Mol. Cell Biol.12, 773-786.

Jiang, K., Hua, S., Mohan, R., Grigoriev, I., Yau, K. W., Liu, Q., Katrukha, E. A.,Altelaar, A. F. M., Heck, A. J. R., Hoogenraad, C. C. et al. (2014). Microtubuleminus-end stabilization by polymerization-driven CAMSAP deposition. Dev. Cell28, 295-309.

Jo, H., Loison, F. and Luo, H. R. (2014). Microtubule dynamics regulates Aktsignaling via dynactin p150. Cell. Signal. 26, 1707-1716.

Kadavath, H., Hofele, R. V., Biernat, J., Kumar, S., Tepper, K., Urlaub, H.,Mandelkow, E. and Zweckstetter, M. (2015). Tau stabilizes microtubules bybinding at the interface between tubulin heterodimers. Proc. Natl. Acad. Sci. USA112, 7501-7506.

Kalebic, N., Sorrentino, S., Perlas, E., Bolasco, G., Martinez, C. andHeppenstall, P. A. (2013). αTAT1 is the major α-tubulin acetyltransferase inmice. Nat. Commun. 4, 1962.

Karlsson, M. C., Gonzalez, S. F., Welin, J. and Fuxe, J. (2017). Epithelial-mesenchymal transition in cancer metastasis through the lymphatic system.Mol. Oncol. 11, 781-791.

Kaverina, I. and Straube, A. (2011). Regulation of cell migration by dynamicmicrotubules. Semin. Cell Dev. Biol. 22, 968-974.

Kunoh, T., Noda, T., Koseki, K., Sekigawa, M., Takagi, M. and Shin-ya, K. (2010).A novel human dynactin-associated protein, dynAP, promotes activation of Akt,and ergosterol-related compounds induce dynAP-dependent apoptosis of humancancer cells. Mol. Cancer Ther. 9, 2934-2943.

Lamouille, S., Xu, J. and Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178-196.

Liu, C.-Y., Lin, H.-H., Tang, M.-J. and Wang, Y.-K. (2015). Vimentin contributesto epithelial-mesenchymal transition cancer cell mechanics by mediatingcytoskeletal organization and focal adhesion maturation. Oncotarget 6,15966-15983.

Livak, K. and Schmittgen, T. (2001). Analysis of relative gene expressiondata using real-time quantitative PCR and the 2−ΔΔCT method. Methods25, 402-408.

11

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216168. doi:10.1242/jcs.216168

Journal

ofCe

llScience

Lotz-Jenne, C., Luthi, U., Ackerknecht, S., Lehembre, F., Fink, T., Stritt, M.,Wirth, M., Pavan, S., Bill, R., Regenass, U. et al. (2016). A high-content EMTscreen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβreceptor. Oncotarget 7, 25983-26002.

Matrone, M. A., Whipple, R. A., Thompson, K., Cho, E. H., Vitolo, M. I., Balzer,E. M., Yoon, J. R., Ioffe, O. B., Tuttle, K. C., Tan, M. et al. (2010). Metastaticbreast tumors express increased tau, which promotes microtentacle formationand the reattachment of detached breast tumor cells. Oncogene 29, 3217-3227.

Myong, N.-H. (2012). Loss of E-cadherin and acquisition of vimentin in epithelial-mesenchymal transition are noble indicators of uterine cervix cancer progression.Korean J. Pathol. 46, 341-348.

Nagae, S., Meng, W. and Takeichi, M. (2013). Non-centrosomal microtubulesregulate F-actin organization through the suppression of GEF-H1 activity. GenesCells 18, 387-396.

Nashchekin, D., Fernandes, A. R. and St Johnston, D. (2016). Patronin/Shotcortical foci assemble the noncentrosomal microtubule array that specifies thedrosophila anterior-posterior axis. Dev. Cell 38, 61-72.

Parker, A. L., Kavallaris, M. and McCarroll, J. A. (2014). Microtubules and theirrole in cellular stress in cancer. Front. Oncol. 4, 153.

Polireddy, K., Dong, R., McDonald, P. R., Wang, T., Luke, B., Chen, P.,Broward, M., Roy, A. and Chen, Q. (2016). Targeting epithelial-mesenchymaltransition for identification of inhibitors for pancreatic cancer cell invasion andtumor spheres formation. PLoS ONE 11, e0164811.

Pongrakhananon, V., Saito, H., Hiver, S., Abe, T., Shioi, G., Meng, W. andTakeichi, M. (2018). CAMSAP3 maintains neuronal polarity through regulation ofmicrotubule stability. Proc. Natl. Acad. Sci. USA 115, 9750-9755.

Portran, D., Schaedel, L., Xu, Z., Thery, M. and Nachury, M. V. (2017). Tubulinacetylation protects long-lived microtubules against mechanical ageing. Nat. CellBiol. 19, 391-398.

Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang, F. (2013).Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 8, 2281-2308.

Richardson, C. E., Spilker, K. A., Cueva, J. G., Perrino, J., Goodman, M. B. andShen, K. (2014). PTRN-1, a microtubule minus end-binding CAMSAP homolog,promotes microtubule function in Caenorhabditis elegans neurons. eLife 3,e01498.

Shankar, J. and Nabi, I. R. (2015). Actin cytoskeleton regulation of epithelialmesenchymal transition in metastatic cancer cells. PLoS ONE 10, e0119954.

Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B. and Nachury, M. V. (2010). Themajor α-tubulin K40 acetyltransferase αTAT1 promotes rapid ciliogenesis andefficient mechanosensation. Proc. Natl. Acad. Sci. USA 107, 21517-21522.

Shirayoshi, Y., Nose, A., Iwasaki, K. and Takeichi, M. (1986). N-linkedoligosaccharides are not involved in the function of a cell-cell bindingglycoprotein E-cadherin. Cell Struct. Funct. 11, 245-252.

Song, Y. and Brady, S. T. (2015). Post-translational modifications of tubulin:pathways to functional diversity of microtubules. Trends Cell Biol. 25, 125-136.

Spaderna, S., Schmalhofer, O., Hlubek, F., Berx, G., Eger, A., Merkel, S., Jung,A., Kirchner, T. and Brabletz, T. (2006). A transient, EMT-linked loss ofbasement membranes indicates metastasis and poor survival in colorectalcancer. Gastroenterology 131, 830-840.

Suman, S., Kurisetty, V., Das, T. P., Vadodkar, A., Ramos, G.,Lakshmanaswamy, R. and Damodaran, C. (2014). Activation of AKTsignaling promotes epithelial-mesenchymal transition and tumor growth incolorectal cancer cells. Mol. Carcinog. 53, E151-E160.

Tanaka, N., Meng, W., Nagae, S. and Takeichi, M. (2012). Nezha/CAMSAP3 andCAMSAP2 cooperate in epithelial-specific organization of noncentrosomalmicrotubules. Proc. Natl. Acad. Sci. USA 109, 20029-20034.

Toya,M. and Takeichi, M. (2016). Organization of non-centrosomal microtubules inepithelial cells. Cell Struct. Funct. 41, 127-135.

Toya, M., Kobayashi, S., Kawasaki, M., Shioi, G., Kaneko, M., Ishiuchi, T. andMisaki, K. (2015). CAMSAP3 orients the apical-to-basal polarity of microtubulearrays in epithelial cells. Proc. Natl. Acad. Sci. USA 113, 332-337.

Velez-delValle, C., Marsch-Moreno, M., Castro-Mun ozledo, F., Galvan-Mendoza, I. J. and Kuri-Harcuch, W. (2016). Epithelial cell migration requiresthe interaction between the vimentin and keratin intermediate filaments. Sci. Rep.6, 24389.

Wang, B., Rao, Y.-H., Inoue, M., Hao, R., Lai, C.-H., Chen, D., McDonald, S. L.,Choi, M.-C., Wang, Q., Shinohara, M. L. et al. (2014). Microtubule acetylationamplifies p38 kinase signalling and anti-inflammatory IL-10 production. Nat.Commun. 5, 3479.

Wu, F., Jordan, A., Kluz, T., Shen, S., Sun, H., Cartularo, L. A. and Costa, M.(2016). SATB2 expression increased anchorage-independent growth and cellmigration in human bronchial epithelial cells. Toxicol. Appl. Pharmacol. 293,30-36.

Xu,W., Yang, Z. and Lu, N. (2015). A new role for the PI3K/Akt signaling pathway inthe epithelial-mesenchymal transition. Cell Adh. Migr. 9, 317-324.

Yan, D., Avtanski, D., Saxena, N. K. and Sharma, D. (2012). Leptin-inducedepithelial-mesenchymal transition in breast cancer cells requires β-cateninactivation via Akt/GSK3- and MTA1/Wnt1 protein-dependent pathways. J. Biol.Chem. 287, 8598-8612.

Yang, H., Wang, L., Zhao, J., Chen, Y., Lei, Z., Liu, X., Xia, W., Guo, L. andZhang, H.-T. (2015). TGF-β-activated SMAD3/4 complex transcriptionallyupregulates N-cadherin expression in non-small cell lung cancer. Lung Cancer87, 249-257.

Zhang, R., Alushin, G. M., Brown, A. andNogales, E. (2015). Mechanistic origin ofmicrotubule dynamic instability and its modulation by EB proteins. Cell162, 849-859.

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RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216168. doi:10.1242/jcs.216168

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