An Integrin-Linked Machinery of Cytoskeletal Regulation that

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Cancer Cell

Article

An Integrin-Linked Machinery of Cytoskeletal Regulation that Enables Experimental Tumor Initiation and Metastatic Colonization

Tsukasa Shibue,1,2Mary W.Brooks,1,2and Robert A.Weinberg1,2,3,*

1Whitehead Institute for Biomedical Research,Cambridge,MA02142,USA

2MIT Ludwig Center for Molecular Oncology,Cambridge,MA02139,USA

3Department of Biology,Massachusetts Institute of Technology,Cambridge,MA02139,USA

*Correspondence:weinberg@229631f5af45b307e97197d7

229631f5af45b307e97197d7/10.1016/229631f5af45b307e97197d7r.2013.08.012

SUMMARY

Recently extravasated metastatic cancer cells use the Rif/mDia2actin-nucleating/polymerizing machinery in order to extend integrin b1-containing,?lopodium-like protrusions(FLPs),which enable them to interact pro-ductively with the surrounding extracellular matrix;this process governs the initial proliferation of these cancer cells.Here,we identify the signaling pathway governing FLP lifetime,which involves integrin-linked kinase(ILK)and b-parvin,two integrin:actin-bridging proteins that block co?lin-mediated actin-?lament severing.Notably,the combined actions of Rif/mDia2and ILK/b-parvin/co?lin pathways on FLPs are required not only for metastatic outgrowth but also for primary tumor formation following experimental implantation.This provides one mechanistic explanation for how the epithelial-mesenchymal transition (EMT)program imparts tumor-initiating powers to carcinoma cells,since it enhances FLP formation through the activation of ILK/b-parvin/co?lin pathway.

INTRODUCTION

The great majority of disseminated cancer cells fail to survive and proliferate after landing in a foreign tissue(Chambers et al., 2002).This explains why only a small minority of disseminated cancer cells succeeds,via the process of colonization,in gener-ating the macroscopic metastases that are responsible for more than90%of cancer-associated deaths(Fidler,2003).This highlights the need to elucidate the mechanisms that allow metastasized cells to survive and proliferate after settling in the parenchyma of foreign tissues.

We and others previously studied a set of three mouse mam-mary carcinoma cell lines—D2.0R,D2.1,and D2A1(hereinafter collectively referred to as D2cells)—with differing metastatic potentials(Barkan et al.,2008;Shibue and Weinberg,2009). Thus,after being introduced into mice via the tail vein,these three cell populations extravasate into the lung parenchyma with equal ef?ciency and exhibit comparable rates of initial survival;however,while the colonization-competent D2A1cells subsequently proliferate rapidly,the colonization-de?cient D2.0R and D2.1cells fail to do so(see Figure S1A available online).Hence,these three D2cell populations provide a model system to study the mechanisms governing the proliferation of recently extravasated cancer cells in the lung parenchyma. These studies led us to discover that focal adhesion kinase (FAK)signaling governs the postextravasation proliferation of the aggressive D2A1cells in the lungs,doing so by controlling the activity of the extracellular-signal regulated kinases(ERKs) (Shibue and Weinberg,2009;Shibue et al.,2012).FAK activation in these D2A1cells appeared to depend,in turn,on the interac-tions of these cells with components of the extracellular matrix (ECM)in the lung parenchyma,which are mediated speci?cally by the formation of elongated,integrin b1-containing adhesion plaques.We found that the development of such plaques

require

Cancer Cell24,481–498,October14,2013a2013Elsevier Inc.481

talin1vinculin paxillin α-actinin

ILK α-parvin β-parvin

β-actin

D 2.0R

D 2.1

D 2A 1

Matrigel on-top,12 hr

zyxin

i n t e g r i n :a c t i n -l i n k i n g p r o t e i n s

F L P s /c e l l

D2A1 cells

s h s c r a m b l e d s h β-p a r v i n J s h β-p a r v i n L s h α-p a r v i n D s h α-p a r v i n E s h I L K C s h I L K E s h t e n s i n 1 A s h t e n s i n 1 C s h z y x i n A s h z y x i n B

sh scrambled

sh β-parvin L

D2A1 cells

r e l a t i v e m R N A e x p r e s s i o n u n i t s

P I N C H 1

P I N C H 2

α-p a r v i β-p a r v i z y x i fi l a m i n m e r l i e z r i r a d i x i m o e s i p a x i l l i v i n c u l i t a l i n

t e n s i n α-a c t i n i n α-a c t i n i n C r p 130C a s

I L K

K i n d l i n -K i n d l i n -K i n d l i n -mRNAs encoding integrin:actin-linking proteins

D2.1FLP initiation/hr/cell/plane

D2A1

D2.0R time (min)

% o f s u r v i v i n g F L P s

time (min)

% o f s u r v i v i n g F L P s

F-actin DAPI

F L P i n i t i a t i o n /h r /c e l l /p l a n e

s h s c r a m b l e d s h β-p a r v i n J

s h β-p a r v i n L D2A1 cells

D 2.1

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D 2.0R

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406080100

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m e t a s t a t i c

time (min)

% o f s u r v i v i n g F L P s

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100

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100150F L P i n i t i a t i o n /h r /c e l l /p l a n e

B T 474

S K -B R -3

T 47D

Z R -75-1

S U M 159B T 549

M D A -M B -231

S U M 1315

nonmetastatic metastatic

135 min 175 min 305 min 355 min 0

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100

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D2.0R

SUM159BT474BT549

SK-BR-3MDA-MB-231T47D SUM1315

ZR-75-1

1234

50.1

1101001000

m o c k α-p a r v i n

F L P s /c e l l

234

5s h s c r a m b l e d s h β-p a r v i n J s h β-p a r v i n L

s h s c r a m b l e d s h β-p a r v i n L

s h s c r a m b l e d s h β-p a r v i n L T S /A

B 1

6F 1

T R

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B16F10

TRAMP-C2

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fi l a m i n Figure 1.b -Parvin as a Key Regulator of FLP Formation

(A)Kinetics of FLP assembly and disassembly.The periods of FLP persistence (top left)and the rate of de novo FLP formation (bottom left)were plotted.Three different D2cell populations (expressing an actin marker lifeact-YPet)were analyzed by time-lapse microscopy (right).The appearance of new FLPs and the retraction of previously present FLPs are marked by blue and open white arrowheads,respectively.(*),p <0.002by log-rank test versus D2A1.(ns),p >0.1.

(B and C)mRNA (B)and protein (C)expression for various integrin-actin linkers.

(legend continued on next page)

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the prior assembly of integrin b 1-containing,?lopodium-like pro-trusions (FLPs)—actin-rich protrusions morphologically resem-bling ?lopodia formed by cells growing in monolayer culture.In contrast,the slowly proliferating D2.0R and D2.1cells develop very few FLPs and elongated adhesion plaques in the lung parenchyma and display low levels of FAK and ERK activation (Shibue et al.,2012;Figure S1A).

By testing various breast cancer cell lines that exhibit differing metastatic powers in mice,we also found that a perse array of colonization-competent cells assemble such FLPs in far greater numbers than do their colonization-de?cient counterparts (Shi-bue et al.,2012).This suggested that the ability to extend abun-dant FLPs critically determines the competence of these breast cancer cells to colonize foreign tissues.In the present study,we undertook to identify the key regulators of FLP formation,with the anticipation that these regulators also serve as molecular determinants of colonization competence.RESULTS

Differing Expression Levels of b -Parvin in Colonization-Competent and -De?cient Cells

In an attempt to elucidate the mechanism(s)governing FLP for-mation,we exploited a three-dimensional (3D)culture model,termed ‘‘Matrigel on-top’’(MoT),in which cells are plated above a layer of 100%Matrigel and then covered with culture medium containing 2%Matrigel (Debnath et al.,2003).When propagated in this MoT model,the aggressive D2A1cells displayed abun-dant FLPs,while the nonaggressive D2.0R and D2.1cells failed to do so;this mirrored the in vivo behaviors of these various cell types in the lung parenchyma (Shibue et al.,2012;Figures S1A and S1B).

In order to identify the mechanistic basis of differing FLP abun-dance observed in the MoT cultures,we tested the kinetics of FLP assembly and disassembly by time-lapse imaging.We found that the rate of de novo FLP formation was not noticeably different among these three D2cell types (Figure 1A).In contrast,they exhibited a profound difference in the lifetime of FLPs:more than 60%of FLPs observed in the aggressive D2A1cells per-sisted for more than 6hr,while the majority (>75%)of FLPs formed in the nonaggressive D2.0R and D2.1cells persisted less than 90min (Figure 1A;Movies S1,S2,and S3).This indi-cated that the difference in FLP abundance between these cell types could be attributed largely to the differing lifetimes of FLPs.We proceeded to identify the molecular machinery governing FLP lifetime.In previous work,we found that the FLPs formed by the aggressive D2A1cells in MoT culture displayed the b 1subunit of integrins along the lengths of their shafts (Shibue et al.,2012).We also noted that others had demonstrated the critical role of integrin-ECM ligation in controlling local actin dy-namics (Geiger et al.,2001).Together,these observations led us to speculate that the engagement of b 1-subunit-containing in-tegrins with their ECM ligands along the lengths of FLP shafts governs the persistence of actin ?bers that structurally support FLPs,thereby controlling FLP lifetime.Consistent with this spec-ulation,the knockdown of integrin b 1expression in the D2A1cells signi?cantly reduced the lifetime,as well as the steady-state numbers,of FLPs formed by these cells (Figure S1C).In contrast,the knockdown of integrin b 1expression in the poorly FLP-displaying D2.0R and D2.1cells did not noticeably reduce either the abundance or lifetime of FLPs (Figure S1D).

Nonetheless,the short-lived FLPs that are naturally formed by these two indolent cell types,like those extended by the aggres-sive D2A1cells,harbored integrin b 1along their shafts,as demonstrated by use of an active-conformation speci?c anti-body 9EG7(Lenter et al.,1993;Figure S1E).We concluded that integrin b 1was actively involved in the adhesions to ECM occurring along the shafts of FLPs in all the three types of D2cells,regardless of the persistence time of their FLPs.It appeared,therefore,that integrin b 1-mediated adhesions contributed critically to the prolonged lifetime of FLPs in the aggressive D2A1cells,doing so by increasing the persistence of actin ?laments that formed the core of these protrusions,whereas formation of these adhesions did not appear to affect the stability of the FLPs in the indolent D2.0R and D2.1cells.Based on this thinking,we examined the mechanisms con-necting integrin b 1-mediated adhesions with the actin ?bers that structurally support FLPs.In particular,we addressed the roles of integrin:actin-linking proteins,which are thought to govern the coordination between the integrin-mediated adhe-sions and the control of actin organization in the vicinity of these adhesions (Geiger et al.,2001).To begin,we measured the abun-dance of messenger RNAs (mRNAs)encoding 23known linker proteins.Among these,the mRNA encoding b -parvin stood out,since its levels were approximately 100-fold higher in the aggressive D2A1cells than in the other two indolent D2cell types.In contrast,none of the other 22mRNAs surveyed ex-hibited more than a 2.2-fold difference in expression levels in the D2A1cells relative to the mean expression levels in the indo-lent D2.0R and D2.1cells (Figure 1B).

As expected from mRNA expression,the expression of b -parvin protein could be detected by immunoblotting only in the aggressive D2A1cells (Figure 1C).Moreover,the knockdown of b -parvin expression by 87%–94%in the D2A1cells,achieved

(D)Role of ILK/b -parvin in FLP formation.The numbers of FLPs per cell were plotted (left).D2A1cells with knockdowns for various integrin-actin linkers or overexpression of a -parvin were propagated in MoT cultures and stained with phalloidin (F-actin;green)and DAPI (nuclei;blue)(right).Knocking down the expression of b -parvin and ILK,but not the overexpression of a -parvin,reduced FLP abundance.Hence,the expression level of b -parvin,but not that of a -parvin,has a critical effect on FLP abundance.(*),p <0.01versus sh scrambled/mock.(ns),p >0.3.

(E)Contribution of b -parvin to the extended FLP lifetime.The D2A1cells manipulated as indicated (also expressing lifeact-YPet)were analyzed as in (A).(*),p <0.002by log-rank test versus sh scrambled.(ns),p >0.1.

(F)Persistence of FLPs formed by human breast cancer cells.Various human breast cancer cell lines (expressing lifeact-YPet)were analyzed by time-lapse microscopy for FLP persistence and the rate of de novo FLP formation.

(G)b -parvin-dependent FLP formation in various metastatic cell types.Three metastatic mouse cell types were analyzed for FLP formation in MoT cultures.(*),p <0.01versus sh scrambled/mock.

Values are means ±SD (n =3in B)or means ±SEM (n z 20in A,E,and F;n =100in D and G).Bars,10m m.See also Figure S1and Movies S1,S2,S3,S4,and S5.

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by either of two shRNAs targeting b-parvin(sh b-parvin J and L), caused a51%–56%decrease in FLP abundance,as well as a signi?cant reduction of FLP lifetime(Figures1D and1E;Fig-ure S1F;Movies S4and S5).In addition,the knockdown of the expression of integrin-linked kinase(ILK),an essential linker between b-parvin and integrin b chains(Yamaji et al.,2001;Fig-ure2A),also reduced the number of FLPs in the D2A1cells(Fig-ure1D).Conversely,ectopic b-parvin expression in the more indolent D2.0R and D2.1cells extended FLP lifetime and increased the steady-state number of these protrusions,an effect that could be blunted by concomitant ILK knockdown(Fig-ures S1F–S1H;Movies S6and S7).Hence,the differences in the lifetime(and,thus,abundance of FLPs observed in the various D2cell types)were attributable,at least in part,to the differing expression levels of b-parvin,which appeared to control FLP life-time in an ILK-dependent manner.

b PIX/Cdc42/PAK Axis as an Effector of ILK/b-Parvin in Controlling FLP Formation

Given the key role played by b-parvin in the regulation of FLPs, we sought to uncover the role of other proteins beyond ILK that might collaborate with b-parvin in this process.In fact,in addition to serving as a physical link between integrins and the actin cytoskeleton,b-parvin is known to play a regulatory role in the cytoskeleton by interacting with a PIX and b PIX(PAK-interacting exchange factors)(Rosenberger et al.,2003).Both PIX proteins recruit and then activate Cdc42and Rac1GTPases, which,in turn,proceed to activate the Group I class of p21-activated kinases(PAKs),i.e.,PAK1-3,thereby regulating cyto-skeletal organization(Figure2A;Bokoch,2003).

We therefore determined whether PIX/Cdc42(Rac)/PAK signaling contributes to the b-parvin-dependent process of FLP regulation.Immunoprecipitation analysis revealed that b-parvin expressed in the aggressive D2A1cells interacted physically with b PIX,the only PIX isoform expressed at a detect-able level in these cells(Figures2B and S2F).Moreover,the knockdown of b PIX expression(by98%to99%)resulted in a 46%–66%decrease in the number of FLPs formed by the MoT-cultured D2A1cells(Figures2C and S2A),indicating that b-parvin cooperates with b PIX in regulating FLP abundance. We proceeded to examine the role of b-parvin in driving the signaling events downstream of b PIX,namely,the sequential activation of Cdc42(Rac1)and PAK1-3.The knockdown of b-parvin expression in the D2A1cells grown in MoT culture reduced the levels of GTP-bound,active Cdc42and Rac1by 70%–88%and57%–78%,respectively(Figures2D and S2B). This was accompanied by the reduced phosphorylation of PAK1at residues critical to its kinase activity—threonine423 (T423)within its catalytic domain as well as serines199and204 (S199/204)within its autoinhibitory domain(Figure2E).Conversely, b-parvin overexpression in the MoT-cultured,otherwise indolent D2.0R and D2.1cells elevated the levels of active Cdc42and Rac1and augmented phosphorylation of PAK1on T423and S199/204(Figures2D and2E;Figure S2B).Hence,in these D2 carcinoma cell types,b-parvin expression was both necessary and suf?cient for activating Cdc42,Rac1,and PAKs when these cells were growing in MoT cultures.

We also compared the roles of Cdc42and Rac1as down-stream effectors of the b-parvin/b PIX complex and found that Cdc42knockdown reduced both PAK1phosphorylation and FLP abundance far more ef?ciently than did Rac1knockdown (Figures2E,S2C,and S2D;Shibue et al.,2012).This indicated that Cdc42,rather than Rac1,serves as a key intermediary in the b-parvin/b PIX-dependent PAK activation.

Finally,we tested the involvement of PAKs in FLP regulation. The inhibition of PAK activity by overexpressing a dominant-negative PAK1-AID fragment,which inhibits all the three of PAK1-3(Zhao et al.,1998),resulted in a47%reduction in the number of FLPs formed by the D2A1cells in MoT culture(Fig-ure3A).Conversely,overexpression of a constitutively active PAK1mutant(PAK1L107F)in the D2.0R and D2.1cells increased the number of FLPs by3.2-and3.0-fold,respectively (Figure3A).Together,these observations demonstrated the critical role of the ILK/b-parvin/b PIX/Cdc42/PAK signaling axis in supporting abundant FLP display(see Figure3B).

LIMK/Co?lin Axis as a Downstream Mediator of the Effect of PAKs on FLPs

The work described earlier did not reveal how PAKs control FLP abundance.However,we noted that earlier studies had impli-cated the presence of multiple effector pathways contributing to the PAK-dependent control of cytoskeletal organization (Bokoch,2003;Figure3B).Thus,PAKs activate LIM domain kinases(LIMKs),which,in turn,inactivate the ADF/co?lin family of proteins(i.e.,ADF,co?lin1,and co?lin2;hereinafter referred to collectively as co?lin),the central regulators of actin?lament severing.Independent of this,PAKs also impair actomyosin contractility by inactivating myosin light-chain kinase(MLCK), thereby reducing the phosphorylation level of the regulatory myosin light chain(rMLC)(see Figure2A).

We undertook to specify the role(s)of PAKs in FLP regula-tion.In the MoT-cultured,aggressive D2A1cells,the inhibition of PAK activity,achieved either by b-parvin knockdown or by PAK1-AID overexpression,impaired the phosphorylation of co?lin1on serine3(S3),an inhibitory modi?cation usually catalyzed by the PAK-effector LIMKs(Yang et al.,1998;Fig-ure2F).In contrast,neither of these manipulations noticeably affected rMLC phosphorylation on serine19,which is cata-lyzed by another PAK-effector,MLCK(Figure2F).Hence, between the two effector pathways of PAKs(Figure2A),the LIMK/co?lin pathway,but not the MLCK/rMLC pathway,was controlled by the ILK/b-parvin/b PIX/Cdc42/PAK signaling in these cells.

Consistent with this notion,FLP formation in the MoT-cultured D2A1cells was impaired by overexpression of a constitutively active co?lin1mutant(co?lin1S3A;Moriyama et al.,1996;Fig-ure3A).Moreover,the overexpression of either b-parvin or the constitutively active mutant of either PAK1or LIMK1(PAK1 L107F and LIMK1-Kd3,respectively)enabled the nonaggressive D2.1cells to extend far more FLPs(a2.4-to2.7-fold increase in FLP number),while failing to do so when the constitutively active co?lin1S3A mutant was expressed concomitantly(Figure3C). Collectively,these observations indicated that ILK/b-parvin/ b PIX/Cdc42/PAK signaling contributes to the display of abun-dant FLPs largely,if not entirely,by causing LIMK-dependent co?lin inactivation,which protects the actin spine of FLPs from co?lin-mediated cleavage,thereby resulting in the increased persistence of FLPs(Figure3B).

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484Cancer Cell24,481–498,October14,2013a2013Elsevier Inc.

FLAG

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0.380.41 1.00 1.000.260.061.000.320.20 1.000.050.051.000.060.05 1.000.280.411.000.080.18 1.00 2.191.00 2.39 1.00 1.73

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Figure 2.ILK/b -Parvin/b PIX/Cdc42/PAK/LIMK/Co?lin Signaling in FLP Formation

(A)Integrin-actin coupling by ILK/a -parvin and ILK/b -parvin complexes.a -parvin and b -parvin bind to ILK,each providing a link between integrins and actin cytoskeleton while having different effects on cell behaviors.

(B)b -parvin/b PIX interactions.Lysates from FLAG-b -parvin-,FLAG-a -parvin-or FLAG-b PIX-expressing cells were subjected to anti-FLAG immunoprecipitation and analyzed by immunoblotting.b PIX interacts with b -parvin but not with a -parvin.Asterisk indicates nonspeci?c bands.

(C)Role of b PIX in FLP regulation.Knocking down the expression of b PIX,but not that of dysferlin (a transmembrane protein that interacts with b -parvin;Matsuda et al.,2005),reduced FLP abundance in the D2A1cells.Values are means ±SEM (n =100).(*),p <0.001.(ns),p >0.3.

(D)b -parvin expression and Cdc42activation.Values represent the intensities of the active Cdc42bands relative to that of corresponding total Cdc42band.(E)b -parvin/Cdc42/b PIX signaling in PAK phosphorylation.Here and in (F),values represent the intensities of pPAK1(phospho-co?lin1/phospho-rMLC)bands relative to that of the corresponding total PAK1(co?lin1/rMLC)band.The blots are representative of multiple independent experiments.(F)Co?lin and rMLC phosphorylation in the downstream of b -parvin/PAK signaling.See also Figure S2.

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Figure 3.Cooperation of Rif/mDia2and ILK/b -Parvin/Co?lin Pathways in FLP Regulation

(A)Role of PAK/LIMK/co?lin axis in FLP regulation.Cells manipulated as indicated to alter the activity of PAK/LIMK/co?lin signaling were analyzed for FLP formation in MoT cultures.Bar,10m m.

(B)Cooperation of the two signaling axes,Rif/mDia2and ILK/b -parvin/co?lin,for abundant FLP display.

(C)Differential requirement for co?lin inactivation between Rif/mDia2and ILK/b -parvin/co?lin signaling pathways.The control (mock)and co?lin1S3A-expressing D2.1cells were further engineered to activate either of Rif/mDia2or ILK/b -parvin/co?lin signaling pathways and analyzed for FLP formation.

(D)Requirement for basal Rif/mDia2activity in b -parvin/PAK-driven FLP formation.The D2.1cells with enforced activation of ILK/b -parvin/co?lin signaling were further engineered to knockdown Rif or mDia2expression (left),while those with enforced Rif/mDia2activation were additionally engineered to knockdown b PIX or to overexpress PAK1-AID (right),before the FLP formation by these cells was analyzed.

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Two Signaling Pathways that Cooperatively Govern FLP Formation

The ILK/b -parvin/b PIX/Cdc42/PAK/LIMK/co?lin (hereinafter referred to as ‘‘ILK/b -parvin/co?lin’’)signaling axis characterized earlier was not the only determinant of FLP abundance.In earlier work,we had uncovered the essential contribution of the Rif/mDia2actin-nucleating/polymerizing machinery to FLP forma-tion (Shibue et al.,2012).Indeed,others had shown that Rif and mDia2cooperatively induce the nucleation of actin mono-mers and subsequent elongation of actin ?laments (Mellor,2010),which constitute the structural core of FLPs.These earlier observations,together with the presently demonstrated contri-bution of co?lin-inactivating signaling pathway to FLP abun-dance,suggested that there are actually two distinct signaling pathways—Rif/mDia2signaling on the one hand and ILK/b -parvin/co?lin signaling on the other—that collaborate in the induction and maintenance of FLPs (Figure 3B).Thus,the ?rst pathway causes the formation of actin ?bers that drives the initial extension of FLPs,while the second pathway ensures the stabi-lization of these FLPs once they are formed.

We undertook to study in detail the cooperative actions of these two signaling axes.We found that changes in FLP abun-dance induced by the enforced activation of Rif/mDia2signaling were not associated with noticeable alterations in the co?lin1phosphorylation on S 3,the site critical to the regulation of co?lin1activity (Figures 2F and 3C).This contrasted sharply with the regulation of FLPs by ILK/b -parvin/co?lin signaling,which involved and depended critically on changes in co?lin activity (Figures 2F and 3C).Together,these observations indicated the independent,complementary actions of these two signaling pathways in regulating FLP abundance (Figure 3B).

We also analyzed the effect of simultaneously manipulating both pathways.Here,we found that the enhanced display of FLPs in the naturally poorly FLP-forming D2.1cells,which could be achieved by the enforced activation of ILK/b -parvin/co?lin signaling,was reversed by the concomitant knockdown of either Rif or mDia2expression (Figure 3D).This indicated that the tonic activity of Rif/mDia2signaling is naturally maintained in the D2.1cells and suggested that the inability of these cells to display abundant FLPs could be ascribed largely to their ineffective acti-vation of ILK/b -parvin/co?lin signaling,which resulted,in turn,from their failure to synthesize signi?cant levels of b -parvin pro-tein (Figure 1C).

We proceeded further to examine the kinetics of FLP assembly and disassembly by time-lapse imaging.Consistent with the effects of b -parvin in extending the lifetime of FLPs (Figures 1E and S1G),inhibition of ILK/b -parvin/co?lin signaling in the aggressive D2A1cells,achieved by the expression of either dominant-negative PAK1-AID fragment or constitutively active co?lin1S3A mutant,reduced the persistence period of FLPs,while the enforced activation of this signaling in the indolent D2.1cells increased this persistence (Figures 3E and 3F).How-ever,none of these manipulations noticeably affected the rate of

de novo FLP formation.In contrast,the enforced elevation of Rif/mDia2signaling activity in the indolent D2.1cells resulted in a signi?cant (R 1.9-fold)increase in the rate of FLP initiation and a modest extension of FLP lifetime (Figure 3F;Movies S6and S8).

These observations reinforced the notion that Rif/mDia2signaling contributes primarily to the de novo formation of FLPs by stimulating the nucleation/polymerization of the actin ?bers that structurally support FLPs,while ILK/b -parvin/co?lin signaling speci?cally helps to maintain the resulting FLPs by sup-pressing the co?lin-dependent cleavage of such actin ?bers.Together,these two signaling collaborate to enable cells to display large numbers of FLPs (see Figure 3B).

ILK/b -Parvin/Co?lin Signaling and In Vitro Cell Behavior As mentioned earlier,we had found that the formation of FLPs contributes to the assembly of integrin b 1-containing,mature adhesion plaques of elongated morphology in cells grown in MoT culture,doing so by fostering the nucleation of protein complexes that constitute the core of these plaques (Figure 4A;Shibue et al.,2012).Consistent with this earlier observation,the inhibition of ILK/b -parvin/co?lin signaling—the signaling axis critical to the extended lifetime and,thus,to the display of abun-dant FLPs—reduced the number of integrin b 1-containing adhe-sion plaques in the D2A1cells growing in MoT culture (Figures 4B and S4A).In addition,the inhibition of ILK/b -parvin/co?lin signaling reduced the levels of activation-associated phosphor-ylation of FAK and ERKs and attenuated proliferation when the D2A1cells were growing under MoT culture conditions,while not noticeably affecting their proliferation in monolayer culture (Figures 4C–4E and S4B–S4D).This reinforced the role of FLP formation as a critical trigger for the establishment of cell-matrix adhesions and rapid cell proliferation in cells grown in MoT cul-ture (see Figure 4A).

We proceeded to test whether the contribution of ILK/b -parvin/co?lin signaling to these processes was generalizable to other cancer cell types.In fact,our previous work had demon-strated that,among 18different lines of human breast cancer cells,those that were competent to form metastatic colonies developed far more abundant FLPs than did their colonization-de?cient counterparts (Shibue et al.,2012).The subsequently pursued time-lapse observations of such cells growing in MoT culture revealed that the FLPs formed in the colonization-competent BT549,MDA-MB-231,and SUM1315cells persisted far longer than those extended by the colonization-de?cient BT474,SK-BR-3,T47D,and ZR-75-1cells (Figure 1F).Consis-tently,these three colonization-competent cell types exhibited elevated activity of the FLP-stabilizing ILK/b -parvin/co?lin signaling,as determined by examining the levels of co?lin1phos-phorylation on S 3—the endpoint of this signaling pathway (see Figure 3B)—relative to the four colonization-de?cient cell types tested here (Figures S2E and S2F).Together,these obser-vations supported the notion that the display of abundant FLPs,

(E and F)Effects of signaling manipulation on FLP dynamics.D2A1(E)and D2.1(F)cells (expressing lifeact-YPet)were engineered to block and stimulate FLP formation,respectively,and analyzed by time-lapse microscopy.

Values are means ±SEM (n =100in A,C,and D;n z 20in E and F).(*),p <0.0001.(**),p <0.05.(***),p <0.0001(versus mock;by log-rank test).(ns),p >0.2(by Student’s t test).

See also Figure S3and Movies S6,S7,and S8.

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pFAK [Y ]

861

total FAK pY397/total FAK

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t o t a l c e l l l y s a t e

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sh β-parvin L i n t e g r i n β1

i n t e g r i n β1

F -a c t i n D A P I

406080100s h β-p a r v i n J s h β-p a r v i n L s h s c r a m b l e d s h βP I X E s h βP I X G m o c k P A K 1-A I D % o f c e l l s w i t h ≥ 5e l o n g a t e d a d h e s i o n p l a q u e s

D2A1 cells/Matrigel on-top, 5 days

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P A K 1-A I D c o f i l i n 1 S 3A

s h s c r a m b l e d m o c k s h I L K C s h I L K E s h α-p a r v i n D s h α-p a r v i n E s h β-p a r v i n J s h β-p a r v i n L s h βP I X E s h βP I X G

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Figure 4.In Vitro Effects of ILK/b -Parvin/Co?lin Signaling Manipulation

(A)Cell-biological and biochemical events that drive cell proliferation in MoT culture and within the lung parenchyma.

(B and C)Role of ILK/b -parvin/co?lin signaling in adhesion plaque assembly and proliferation.The D2A1cells were manipulated as indicated,with which the rates of mature adhesion plaque assembly in MoT cultures (B)and the cell numbers after 10days of monolayer or MoT cultures (C)were determined.Bar,10m m.(D and E)b -parvin/PAK signaling and FAK/ERK activation.Values represent the intensities of pFAK (pERK)bands relative to that of the corresponding total FAK (ERK)band.

(F)ILK/b -parvin/co?lin signaling and proliferation in various cell types.Indicated cell types were manipulated to block ILK/b -parvin/co?lin signaling,with which the cell numbers after 10(15for MDA-MB-231)days of monolayer/MoT cultures were determined.

Values are means ±SD (n =3)in (B),(C),and (F).(*),p <0.02.(ns),p >0.1versus sh scrambled/mock.See also Figure S4.

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observed speci?cally in the colonization-competent cells,is attributable,in part,to the elevated activity of the ILK/b-parvin/ co?lin signaling and the resulting prolonged lifetime of FLPs. We also blocked the activity of ILK/b-parvin/co?lin signaling in the colonization-competent SUM159and MDA-MB-231human breast cancer cells.This resulted in impaired FLP formation and reduced proliferation rate in MoT cultures while minimally affecting their proliferation in monolayer cultures(Figures4F and S3A).Hence,ILK/b-parvin/co?lin signaling contributed crit-ically to the abundant FLP display and rapid cell proliferation in the3D MoT cultures of multiple colonization-competent carci-noma cell types.

ILK/b-Parvin/Co?lin Signaling and Metastatic Aggressiveness In Vivo

The MoT culture used in the experiments cited earlier was designed to approximate the microenvironment surrounding cancer cells that have recently extravasated into the lung paren-chyma(Barkan et al.,2008;Shibue and Weinberg,2009).We wanted to extend the?ndings of these experiments by deter-mining whether the ILK/b-parvin/co?lin signaling axis also regu-lates metastatic cell behaviors in the lungs.In fact,various strategies to block this signaling pathway all reduced(by2.3-to9.0-fold)the number of macroscopic lung metastases formed 24days after the tail-vein injection of the D2A1cells;this was associated with impaired proliferation within the lung tissue as measured7days after the injection(Figure5A;Figure S5A).In contrast,none of these manipulations noticeably affected D2A1cell proliferation in monolayer culture(Figure4C).

We also examined in detail lung sections prepared10days after tail-vein injection of the D2A1cells.Here again,the inhibi-tion of ILK/b-parvin/co?lin signaling in the D2A1cells decreased (2.3-to4.1-fold)the numbers of large metastatic colonies(those with>20cells per colony),while the numbers of small colonies (%20cells per colony),which consisted largely of viable but nonproliferative cells,were actually increased(1.3-to2.4-fold; Figures5B and S5B).This echoed the previously observed effect of knocking down Rif or mDia2expression in the D2A1 cells(Shibue et al.,2012).Together,these observations led us to conclude that blocking FLP formation in the recently extravasated D2A1cells constrains these cells to reside in the lung parenchyma as viable,weakly proliferating micrometastatic cells.

We undertook to analyze how the inhibition of ILK/b-parvin/ co?lin signaling affects the initial steps of extravasation and postextravasation processes of metastasis.In fact,the blockade of this signaling axis in the aggressive D2A1cells did not notice-ably affect the ef?ciency of extravasation into the lung paren-chyma while being effective in reducing the abundance of FLPs and mature adhesion plaques formed by the extravasated cells;this was accompanied by a reduced level of FAK activation relative to that of the control cells(Figures5C,5D,and S5C–S5E).Hence,the action of ILK/b-parvin/co?lin signaling was crit-ical,following extravasation into the lung parenchyma,to the FLP-dependent establishment of productive cell-matrix interac-tions by the D2A1cells,which,in turn,governed their subse-quent proliferation(see Figure4A).

We asked whether other types of colonization-competent cells also depend on the activity of ILK/b-parvin/co?lin signaling in order to colonize the lung tissue.Accordingly,we tested three colonization-competent mouse cell types,namely,TS/A mammary carcinoma cells,B16F10melanoma cells,and TRAMP-C2prostate cancer cells,all of which exhibited b-parvin-dependent FLP formation in MoT cultures(Figures1G and S1I). We found that,in all three cases,the knockdown of b-parvin expression reduced the number of macroscopic lung metasta-ses formed after tail-vein injection(Figures5E and S5F).Simi-larly,blockade of ILK/b-parvin/co?lin signaling in the human breast cancer cell lines SUM159and MDA-MB-231also impaired formation of lung macrometastases by these cells(Fig-ure5F).Notably,none of these manipulations discernibly affected the proliferation of these cells in monolayer cultures (Figures4F and S4E).These observations con?rmed the role of ILK/b-parvin/co?lin signaling as a critical controller of metastatic aggressiveness in multiple cancer cell types.

FLP Formation and Tumorigenicity of Orthotopically Implanted Cells

These various observations caused us to test the involvement of ILK/b-parvin/co?lin signaling in earlier steps of experimental tumor formation.Thus,we speculated that the adaptations that experimentally implanted cancer cells must initially undergo in sites of engraftment resemble those that are required for the establishment of disseminated cancer cells as founders in sites of metastatic colonization.Based on this thinking,we implanted various cancer cell types into either orthotopic(mammary fat pads in the case of mammary carcinomas)or ectopic subcu-taneous sites in murine hosts,doing so with or without manipu-lating these cells by blocking ILK/b-parvin/co?lin signaling.This revealed that some of these manipulations reduced the inci-dence of primary tumor formation:for example,the knockdown of b PIX expression reduced tumor incidence following the implantation of the D2A1cells in the mammary fat pad from 96%to42%–64%(Figures S6A–S6C).This echoed our previous observation that inhibition of Rif/mDia2signaling in the D2A1 cells reduced the rate of primary tumor formation after the ortho-topic implantation(Shibue et al.,2012).Together,these observa-tions suggested that the ability of cells to display abundant FLPs contributes to the establishment of primary tumors at sites of implantation.

We pursued these effects further by measuring tumor-initi-ating frequency of two cancer cell types,namely,D2A1and MDA-MB-231cells,after implanting them at limiting dilutions. We found that blocking FLP formation,achieved by inhibiting either the Rif/mDia2or the ILK/b-parvin/co?lin pathway,signi?-cantly reduced the estimated frequency of tumor-initiating cells (TICs)in both cell populations(Figure6A;Figure S6D).This led us speculate that the formation of FLPs,which enables rapid proliferation of cancer cells following extravasation into the lung parenchyma(see Figure4A),also governs the initial prolifer-ation of cancer cells at sites of implantation,thereby critically affecting the ef?ciency with which these cells establish primary tumors in host mice.

Indeed,shortly after implantation into mammary fat pads,the D2A1cells displayed FLPs,which resembled those formed by these cells following extravasation into the lung parenchyma (Figures6B and6C).Moreover,the number of FLPs formed 2days after the implantation of the D2A1cells was decreased

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macrometastases/left upper lobe

cofilin1 S3A

pFAK [Y ]

861

total FAK

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pY397/total FAK s h s c r a m b l e d

pY861/total FAK

pFAK [Y ]

397

s h β-p a

r v i n J

s h β-p a r v i n L 1.000.310.211.00

0.560.39

I P : a n t i -H A

m o c k

P A K 1-A I D

1.000.231.00

0.47

D2A1 cells/in the lungs, 5 days

mock

PAK1-AID

cofilin S3A

MDA-MB-231 cells

macrometastases/whole lung

sh scrambled

sh β-parvin L

B16F10 cells

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l i f e a c t -T a g R F P -T i n t e g r i n α5-Y P e t

sh scrambled sh β-parvin L D2A1 cells/in the lungs, 10 days

sh βPIX E M

t d T o m a t o -m e m b r a n e H o e c h s t 33342

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metastases/unit lung area (a.u.)

D 2A 1 c e l l s

s m a l l m e t a s t a s e s (≤ 20 c e l l s )sh scrambled sh βPIX E sh β-parvin J sh β-parvin L sh βPIX G

mock PAK1-AID cofilin1 S3A

(ns)(**)(ns)(**)

(ns)(**)

l a r g e m e t a s t a s e s (> 20 c e l l s )

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mock PAK1-AID cofilin1 S3A (**)

(**)

% of cells with ≥ 5 elongated plaques

(5 days after injection)

010********(**)

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123

FLPs/cell

(2 days after injection)

sh scrambled mock PAK1-AID

D 2A 1 c e l l s

sh β-parvin J sh β-parvin L

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(*)sh scrambled sh β-parvin L

macrometastases/left upper lobe

B 16F 10c e l l s

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M D A -M B -231c e l l s

macrometastases/whole lung

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S U M 159c e l l s

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by blocking either the Rif/mDia2or ILK/b -parvin/co?lin signaling pathway,which was accompanied by the signi?cant reduction of the proliferation rate that was measured 3days later (Figures 6D and 6E).Hence,the cooperative actions of Rif/mDia2and ILK/b -parvin/co?lin signaling pathways and the resulting,abundant formation of FLPs (see Figure 3B)contributed critically to the active proliferation of recently implanted D2A1cells within mam-mary fat pads.

We also found that both Rif-and b -parvin-knockdown impaired FAK activation in the orthotopically implanted D2A1cells (Figure 6F).This attenuation of FAK signaling was likely to account for the reduced TIC frequency caused by these knock-downs,since the concomitant expression of the constitutively active CD2-FAK fusion protein partially restored this frequency (Figure 6G).In addition,the knockdown of FAK expression impaired ERK activation in the mammary fat-pad-implanted D2A1cells (Figure 6F).Together,these observations indicated that the proliferation of the recently implanted cancer cells in the mammary fat pads is governed,in part,by the mechanisms involving FLP extension and the resulting activation of FAK/ERK signaling,which mirrored the regulation on the proliferation of recently extravasated cancer cells in the lungs (see Figure 4A).Correlation between ILK/b -Parvin/Co?lin Signaling Activity and Tumorigenic Potential

We examined in greater detail the behavior of the D2A1cells that had been recently implanted into the mammary fat pads.This revealed that the numbers of FLPs formed by the inpidual cells within the D2A1cell population were highly variable from one cell to another (Figure S6E),which led us to speculate that the cells displaying more abundant FLPs had a higher tumor-initiating potential,i.e.,an ability to seed tumors in host mice following implantation,than did those forming only small numbers of these protrusions.To test this,we fractionated the D2A1cells into different subpopulations in order to examine whether the FLP-forming ability of the cells in these various sub-populations correlated with their content of TICs.

Others have separated mouse mammary carcinoma cells based on the expression of CD29and CD24cell surface markers and found that the subpopulation of cells with CD29high /CD24high (29H/24H)pro?le exhibited a signi?cant enrichment of TICs (Zhang et al.,2008).Consistent with this observation,we noted that the 29H/24H fraction of the D2A1cells exhibited a TIC fre-

quency 4.1-and 27-fold higher than that of the CD29high /CD24low (29H/24L)and CD29low (29L)groups,respectively (Figures 7A and 7B).We also found that cells of the aggressive 29H/24H sub-population extended a larger number of FLPs than did the cells of the other two subpopulations both in MoT culture and within the mammary fat pads (Figures 7C,7D,and S7A).Hence,the observed difference in the TIC frequency of these various D2A1cell subpopulations correlated with the FLP-forming abili-ties of their constituent cells.

Wishing to extend these observations to other cell types,we studied the HMLER transformed human mammary epithelial cells (Elenbaas et al.,2001).As reported previously,these HMLER cells can be sorted,according to the expression pro?le of the CD44and CD24markers,into two subpopulations:a TIC-enriched CD44high /CD24low (44H/24L)subpopulation and a TIC-depleted CD44low /CD24high (44L/24H)subpopulation (Mani et al.,2008;Figure 7E).When propagated in MoT culture,cells of the 44H/24L subpopulation displayed a far larger (13.43)number of FLPs than did cells of the 44L/24H subpopulation (Figure 7F).Hence,in both the D2A1and HMLER cultures,the cells from the TIC-enriched subpopulations extended FLPs more abundantly than did the cells from the TIC-depleted subpopulations.

We undertook to identify the determinants of differing FLP abundance between the cells of these various subpopulations.Time-lapse observation of these cells growing in the MoT culture revealed that FLPs formed by cells of the TIC-enriched subpop-ulations (i.e.,29H/24H in the D2A1cells and 44H/24L in the HMLER cells)exhibited a signi?cantly longer lifetime than those extending from cells of the corresponding other subpopulations (Figures S7C and S7D).Consistent with this observation,cells of these TIC-enriched subpopulations exhibited a higher level of the co?lin1-inactivating S 3phosphorylation—the endpoint of the signaling pathway that governs FLP lifetime,i.e.,ILK/b -parvin/co?lin signaling (see Figure 3B)—than did the cells of the other subpopulations (Figure 7G).Moreover,FLP formation by cells of the HMLER 44H/24L subpopulation was impaired by expressing the constitutively active co?lin1S3A mutant in these cells,which ultimately reduced the TIC frequency in this subpopulation (Figures 7H,S7E,and S7F).Collectively,these various observations supported the notion that the elevated TIC frequency—observed in the 29H/24H subpopulation of the D2A1cells and the 44H/24L subpopulation of the HMLER

Figure 5.In Vivo Effects of ILK/b -Parvin/Co?lin Signaling Manipulation

(A and B)ILK/b -parvin/co?lin signaling and metastatic colonization.The D2A1cells expressing ?uorescent markers—GFP or tdTomato in (A)and tdTomato-membrane in (B)—were manipulated as indicated and tail-vein injected.In (A),representative lung images (left)and the numbers of macrometastases (right)24days after injection,as well as the phospho-histone H3positivity of the cells residing in the lungs 7days after injection (middle),were presented.Here and in (E)and (F),the red bar represents the mean value within each sample group.In (B),relative numbers of small (%20cells)and large metastases (>20cells)were quanti?ed on the lung sections prepared 10days after injection.M,large metastases.

(C and D)b -parvin/PAK axis and in vivo cell-matrix adhesions.In (C),the D2A1cells expressing integrin a 5-YPet (green)and lifeact-Tag-RFP-T (red),further engineered as indicated,were tail-vein injected.FLP formation was analyzed on the lung sections,where blood vessels (PECAM-1;white)and nuclei (Hoechst 33342;blue)were also visualized (left and middle).The formation of elongated adhesion plaques was scored similarly,except for using a -actinin-Tag-RFP-T fusion protein instead of lifeact-Tag-RFP-T (right).In (D),the D2A1cells engineered as indicated,also expressing FAK-HA,were tail-vein injected.Five days later,FAK-HA was immunoprecipitated from the lung lysate and analyzed by immunoblotting.

(E and F)Role of ILK/b -parvin/co?lin signaling in lung colonization by various cell types.The control and manipulated B16F10,TRAMP-C2,SUM159,and MDA-MB-231cells,also expressing GFP (E)or tdTomato (F),were tail-vein injected,and subsequent formation of lung metastases was analyzed.

Values are means ±SD (n =3in (A,middle),(B),and (C,right)]and means ±SEM (n =150in C,left).Bars,2mm in (A),(E),and (F);100m m in (B);and 10m m in (C).(*),p <0.005.(**),p <0.05.(ns),p >0.05versus sh scrambled/mock.See also Figure S5.

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pFAK [Y ]

861

total FAK

(FAK-HA)

pY397/total FAK

s h s c r a m b l e d

pY861/total FAK

pFAK [Y ]

397

s h R i f F

s h β-p a r v i n L

sh scrambled

sh Rif F

sh β-parvin L

mock

cofilin1 S3A

D2A1 cells/fat pad, 2 days

sh scrambled

sh Rif F

sh β-parvin L

mock

cofilin1 S3A

D2A1 cells/fat pad, 5 days

K i 67H o e c h s t 33342

c l e a v e

d c a s p a s

e -3H o e c h s t 33342

lifeact-TagRFP-T integrin α5-YPet Hoechst 33342

% o f c e l l s w i t h ≥ 5e l o n g a t e d a d h e s i o n p l a q u e s

time (days)

s h s c r a m b l e d s h R i f F s h β-p a r v i n

m o c k c o f

i l i n 1 S 3A

P A K 1-

I D s h m D i a 2 A s h βP I X E D2A1 cells

F L P s /c e l l

% K i 67-p o s i t i v e

0/62/64/44/4

F L P s /c e l l

time (hr)

0D2A1 cells/fat pad, 5 days

integrin α5-YPet α-actinin-TagRFP-T

Hoechst 33342

s h s c r a m b l e s h R i f s h β-p a r v i n m o c c o f i l i n 1 S 3P A K 1-A I s h m D i a 2 s h βP I X % c l e a v e d c a s p -3-p o s i t i v e

s h s c r a m b l e s h R i f s h β-p a r v i n m o c c o f i l i n 1 S 3P A K 1-A I s h m D i a 2 s h βP I X 0

12340

1234

1.000.440.28

1.000.260.28

lifeact-TagRFP-T integrin α5-YPet Hoechst 33342

D2A1 cells/fat pad, 2 days

D2A1 cells

s h s c r a m b l e d

D2A1 cells/fat pad, 5 days

s h F A K B

I P : a n t i -F L A G

s h F A K C total ERK1

(FLAG-ERK1)

pERK1/totalERK1

1.000.270.20

pERK1 [T /Y ]

202204

I P : a n t i -H A

D2A1 cells/fat pad, 5 days

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cells—is attributable,in part,to the enhanced FLP-stabilizing ability of their constituent cells,which results,in turn,from the elevated activity in these cells of the ILK/b -parvin/co?lin signaling.

ILK/b -Parvin/Co?lin Signaling and the Process of Epithelial-Mesenchymal Transition

Cancer cells can often develop a higher tumor-initiating potential during the course of tumor progression (Pece et al.,2010).In the case of carcinomas,this acquisition of malignant phenotypes is often achieved by the passage through the cell-biological pro-gram termed the epithelial-mesenchymal transition (EMT);more-over,as demonstrated previously,the forced induction of an EMT in cancer cells endows them with a greatly increased tumor-initiating potential (Mani et al.,2008;Morel et al.,2008).These earlier observations,together with our present demon-stration of the critical role of FLPs in the process of tumor initia-tion,prompted us to ask whether the EMT-dependent induction of the tumor-initiating potential involves and depends on the enhancement of FLP formation.Accordingly,we ectopically ex-pressed the Twist transcription factor,an inducer of the EMT program (Yang et al.,2004),in the naturally epithelial HMLER cells (Figures 8A and 8B;Figure S8A).Consistent with previous observations (Mani et al.,2008),Twist-induced EMT in these cells was accompanied by an enhanced tumor-initiating poten-tial in the mammary fat pads (a 132-fold increase in TIC frequency)and an increased power of metastasis formation (Figures 8C and 8D).

Of relevance here,Twist-induced EMT in the HMLER cells stimulated their FLP-forming ability both in MoT culture and following orthotopic implantation,which was accompanied by marked increases in the expression levels of multiple compo-nents of the ILK/b -parvin/co?lin signaling,namely ILK (4.73),b -parvin (13.63),and LIMK1(3.23)(Figures 8B,8E,and 8F).Moreover,both the elevated expression of these three compo-nents of the ILK/b -parvin/co?lin signaling pathway and the enhanced display of FLPs were reproduced by other strategies of EMT induction,speci?cally,the overexpression of Snail tran-scription factor and the knockdown of E-cadherin adhesion pro-tein (Figures 8B and 8E).

We proceeded to examine the functional role of the elevated b -parvin expression on the behavior of Twist-expressing HMLER cells.This revealed that the knockdown of b -parvin expression partially reversed multiple properties conferred on these cells

by the expression of Twist,including increased FLP abundance and elevated phosphorylation levels of PAK1(on T 423and S 199/204)and co?lin1(on S 3),all of which were observed in MoT culture (Figures 8B,8E,8F,and S8B).Moreover,b -parvin knock-down also reduced the aggressiveness of HMLER-Twist cells in vivo:both tumor initiation and metastasis formation were impaired signi?cantly by this knockdown (Figures 8C and 8D).We concluded that the elevated tumor-initiating and metas-tasis-forming powers imparted to the HMLER cells by Twist-induced EMT,and presumably by the EMTs induced by other strategies,depended on enhanced FLP display;this was enabled,in turn,by the elevated expression levels of several components of ILK/b -parvin/co?lin signaling pathway,including b -parvin.

We also tested whether the connection between FLP forma-tion and EMT process could be extended to other cell types.Accordingly,we induced a mesenchymal-epithelial transition (MET)—the reverse process of an EMT—in the naturally mesenchymal D2A1cells,doing so by the combination of Snail knockdown and E-cadherin overexpression (Figures 8B and S8C–S8F).As anticipated,the D2A1cells that underwent an MET exhibited a profound loss of tumor-initiating and metas-tasis-forming powers (Figures 8C and 8D).Moreover,this MET induction was associated with the 2.7-fold decrease in the number of FLPs formed by these cells in MoT culture (Fig-ure S8G).In addition,the D2A1cells that underwent an MET also exhibited decreased expression of several components of the ILK/b -parvin/co?lin signaling pathway—namely,ILK,b -parvin,and LIMK1—all of which contrasted to the effects of EMT in the HMLER cells (Figures 8B and S8H).Hence,in both HMLER cells and the D2A1cells,the transition between the epithelial and mesenchymal states involved changes in the expression levels of multiple components of the ILK/b -parvin/co?lin signaling pathway.This altered,in turn,their ability to form FLPs and ultimately contributed to changes in tumor-initiating and metastasis-forming powers exhibited by the cells undergoing this cell state transition.DISCUSSION

While the role of integrin-mediated cell-matrix adhesions in enabling the outgrowth of metastases had been recognized (Aguirre Ghiso et al.,1999;Barkan et al.,2008),the manner by which extravasated cancer cells interact with the ECM

Figure 6.FLP Formation and Tumorigenesis of Experimentally Implanted Cells

(A)Role of FLP-regulating proteins in primary tumor formation.The D2A1cells were engineered as indicated and implanted into the mammary fat pads.Twenty-eight days later,the formation of palpable tumors was scored,from which TIC frequency was calculated.

(B and C)FLPs and elongated adhesion plaques formed by the mammary fat-pad-implanted cells.The D2A1cells expressing integrin a 5-YPet (green)and either of the lifeact-Tag-RFP-T (red in B)or a -actinin-Tag-RFP-T (red in C)were implanted together with nonlabeled D2A1cells.The formation of FLPs (blue arrowheads in B)and elongated adhesion plaques (pink arrowheads in C)was analyzed on the sections of the fat pads.

(D and E)Effect of blocking FLP formation in the mammary fat-pad-implanted cells.In (D),?uorescent-labeled and nonlabeled D2A1cells,further manipulated as indicated,were mixed and implanted to analyze FLP formation within the mammary fat pads.In (E),D2A1cells were engineered as indicated and implanted.Proliferation and apoptosis of implanted cells were analyzed by staining the sections of the fat pads for Ki67(red)and cleaved caspase-3(green),respectively.(F)FAK/ERK activation in the mammary fat-pad-implanted cells.The D2A1cells expressing either of FAK-HA or FLAG-ERK1were further manipulated as indicated and implanted.Subsequently,FAK-HA or FLAG-ERK1was immunoprecipitated from the lysate of the fat pads and analyzed.

(G)Restoring tumor-initiating ability by enforced FAK activation.The D2A1cells with Rif or b -parvin knockdown were further manipulated to express the constitutively active CD2-FAK.Primary tumor formation by these and the control cells was analyzed.

Values are means ±SEM (n =100in B and D)and means ±SD (n =3in C and E).Bars,10m m in (B)through (D)and 100m m in (E).(*),p <0.01,(ns),p >0.1.See also Figure S6.

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29H /24H

29H /24L

29L

D2A1cells pS3/total cofilin1total cofilin1

phospho-cofilin1 [S ]

pT423/total PAK1pS199/204/total PAK1total PAK1

pPAK1 [T ]

423

pPAK1 [S ]

199/204

44H /24L

44L /24H

HMLER cells Matrigel on-top, 12 hr

total cofilin1/α-tubulin t u m o r s p h e r e s /1000 c e l l s

CD24

C D 29

injected cell number

D2A1 (orthotopic)

estimated TIC frequency

29H/24L

2 x 10

1 x 10

5 x 10

2.5 x 10

29H /24H

29H /24L

29L

D2A1 cells/Matrigel on-top

29H/24H

29H/24L

29L

D2A1 cells/fat pad, 2 days

29H/24H

29H/24L

29L

12 h r

5 d a y s

D2A1 cells

F-actin DAPI integrin β1

DAPI

lifeact-YPet Hoechst33342

F L P s /c e l l

D2A1 cells 12 hr 29H /24H

29H /24L

29L

% o f c e l l s w i t h ≥ 5 e l o n g a t e d a d h e s i o n p l a q u e s

D2A1 cells 5 days

29H /24H

29H /24L

29L

1° sphere

2° sphere 3° sphere

29H/24H 29H/24L 29L

D2A1 cells/sphere culture (2°), 10 days

29L

29H/24H

29H/24L

91/63/64/44/41/12,552E

44L/24H

44H/24L

204060

800

3060

90120injected cell number

HMLER (orthotopic)

estimated TIC frequency

cof S3A: 44H/24L

1 x 10

1/61/63/4

1/53,17744L/24H

44H/24L

F L P s /c e l l

HMLER 12 hr

44H /24L

44L /24H

HMLER cells/Matrigel on-top

D2A1 cells

HMLER cells

0.5

11.522.5α-tubulin

integrin β1

LIMK1β-parvin ILK

βPIX Cdc42

LIMK2PAK1-3mDia2F-actin DAPI

integrin β1

DAPI

% o f c e l l s w i t h ≥ 5 e l o n g a t e d a d h e s i o n p l a q u e s

12 h r

5 d a y s

44H /24L

44L /24H

HMLER 5 days

CD24

D 44

Rif

1.000.630.06

1.000.970.351.00

2.28

3.82

1.000.430.14

1.000.53

1.000.25

1.000.36

1.000.79

(**)

(*)

(*)(*)

(*)

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components of the parenchyma of their host tissue remained to be elucidated.We previously reported that the ability of cancer cells to form abundant FLPs following extravasation into the parenchyma of foreign tissues contributes critically to the estab-lishment by these cells of productive interactions with the ECM of their host tissue (Shibue et al.,2012).We also demonstrated the essential role of Rif/mDia2actin-nucleating/polymerizing machinery in the formation of FLPs.Here,we have described two ?ndings that together provide an important extension to our understanding of the role of FLPs in controlling cancer cell behaviors.

To begin,we identi?ed a signaling mechanism that yields an extended lifetime of FLPs and thereby contributes to the abun-dance of these protrusions in the colonization-competent cancer cells.Thus,the activation of the ILK/b -parvin/co?lin signaling axis at sites of FLP formation enables the persistence of these protrusions once they are assembled.As is the case with the Rif/mDia2signaling (Shibue et al.,2012),the action of ILK/b -parvin/co?lin signaling is critical to the metastatic colony-forming ability of multiple aggressive cancer cell types.

Of note,the enforced activation of this signaling pathway,on its own,does not always suf?ce to confer metastatic powers on otherwise indolent cancer cells.For example,in the nonag-gressive D2.1cells,which do not express b -parvin at a detect-able level,ectopic b -parvin expression suf?ced to enable these cells to display abundant FLPs in MoT culture.However,these b -parvin-expressing D2.1cells did not subsequently succeed in developing mature adhesion plaques and in proliferating rapidly under the MoT conditions,nor did they form a large num-ber of macroscopic metastases in the lungs following tail-vein injection (Figure S3).Hence,these D2.1cells appeared to suffer at least one additional defect beyond the lack of b -parvin expres-sion that precluded their aggressive behavior both in MoT culture in vitro and within the lung parenchyma in vivo.

While this was not addressed directly by the present work,we suggest that the dynamics of FLP formation and the contribution of FLPs to metastatic outgrowth will prove to be relevant to the colonization process of various target tissues.Indeed,we previ-ously demonstrated that blocking FLP formation by Rif knock-down in the B16F10melanoma cells diminishes the ability of these cells to colonize multiple organs,such as the lungs,liver,and bone marrow,following intracardiac injection into syngeneic mice (Shibue et al.,2012).This prompts us to suggest that FLP formation and resulting establishment of productive cell-matrix interactions represent a common prerequisite to the metastatic colonization of many types of target organs.

The second lesson of the present study relates to the role of FLPs in the establishment of primary tumors by experimentally implanted cancer cells.Thus,as shown here,multiple cancer cell types depend on the FLP-regulating signaling pathways for ef?ciently establishing primary tumors following experimental implantation in murine hosts.In support of this ?nding,we also presented examples where the increased tumor-initiating poten-tial of cancer cells was correlated closely with their enhanced ability of FLP formation.More speci?cally,cells of the TIC-enriched subpopulations of both D2A1and HMLER cells dis-played FLPs far more abundantly than the remaining cells in these populations.

Recent studies have revealed that many types of solid tumors contain both cells that can ef?ciently seed tumors upon trans-plantation into mice and those that are unable to do so;these are often referred to as cancer stem cells (CSCs)and nonstem cancer cells (non-CSCs),respectively (Clevers,2011).The ef?-cient tumor seeding by many,if not all,types of carcinoma CSCs is likely to be supported by the EMT program.Indeed,the induction of EMT suf?ces to confer on cancer cells not only an increased tumor-initiating potential but also many other attri-butes of CSCs,including enhanced resistance to chemothera-peutic agents and a slower rate of proliferation (Gupta et al.,2009).However,the speci?c mechanism(s)by which the EMT program potentiates the tumor-initiating powers of carcinoma cells has remained elusive.The present observations point to the contribution of the EMT program to increasing the expres-sion of proteins that are critical for FLP formation and to the role of FLP formation in governing tumor-initiating potential.Together,they provide a mechanistic explanation of how the EMT program can contribute to the elevated tumor-initiating ability of CSCs.

To summarize,we propose that in certain,and perhaps many,types of cancer cells,their initial proliferation following both experimental implantation and metastatic dissemination is governed,in part,by the common regulatory mechanism involving FLP formation and the resulting assembly of mature adhesion plaques.Clearly,other factors,such as cytokines,

Figure 7.Display of Abundant FLPs by Cells of the TIC-Enriched Subpopulation

(A)Sorting of the D2A1cells by CD29/CD24expression.The cells of each subpopulation were implanted into mammary fad pads to score primary tumor for-mation (right).

(B)Tumor sphere formation by the sorted D2A1cells.Cells were sequentially passaged for the three rounds of 10-day culture.Representative images of cells after the second round of culture are presented (top).The numbers of tumorspheres after each round of culture were scored (bottom).

(C and D)Formation of FLPs and elongated adhesion plaques by the sorted D2A1cells.In C,sorted D2A1cells were propagated in MoT cultures to analyze FLP/elongated adhesion plaque formation.In D,the D2A1cells that did and did not express the ?uorescent actin marker lifeact-YPet (green)were sorted,mixed,and implanted.The formation of FLPs (gray arrowheads)by these cells was analyzed on the sections of the fat pads.(E)Sorting of the HMLER cells by CD44/CD24expression.

(F)Formation of FLPs and elongated adhesion plaques by the sorted HMLER cells in MoT cultures.

(G)Expression of FLP-regulating proteins in cells of the various D2A1and HMLER subpopulations.Cells of these subpopulations were propagated in MoT cultures and analyzed by immunoblotting.

(H)Blocking ILK/b -parvin/co?lin signaling in the 44H/24L subpopulation of HMLER cells.HMLER cells were manipulated to express constitutively active co?lin1S3A.These and the control cells were sorted,and the 44H/24L subpopulation obtained from each cell type was implanted to score primary tumor formation.Values are means ±SD (n =3in B,C right,and F right)and means ±SEM (n =100in C left and F left).Bars,200m m in (B)and 10m m in (C),(D),and (F).(*),p <0.005.(**),p <0.02.