Commentary 1195

IntroductionIn the complex extracellular matrix (ECM) microenvironment cellsencounter a multitude of coordinated, simultaneously active stimulithat are biochemical, structural and mechanical in nature (Fig. 1A).In this context, mechanical forces have a role in regulating thedevelopment and correct function of virtually every tissuein the human body. In fact, the local mechanical interaction betweenthe cell and its microenvironment is emerging as a unifyingprinciple that connects micro- and macro-scale tissue architecturewith cell shape, organization and differentiation (for reviews, seeDischer et al., 2005; Vogel and Sheetz, 2006; Wang, N. et al.,2009; Wozniak and Chen, 2009). For instance, cell and ECMmechanics (Box 1) are known to be motivating factors in celldifferentiation (Engler et al., 2008; Engler et al., 2006), migration(Gardel et al., 2008; Hadjipanayi et al., 2009; Lo et al., 2000),morphogenesis (Paszek et al., 2005; Wozniak et al., 2003) andproliferation (Provenzano et al., 2009; Ulrich et al., 2009; Wanget al., 2000). Furthermore, mechanics and associatedmechanotransduction (the mechanisms by which cells transducemechanical stimuli into a biochemical response), is known to havea substantial role in numerous human diseases (e.g. Grinnell, 2003;Hahn and Schwartz, 2009; Robling et al., 2006), including tumorformation and progression (Paszek et al., 2005; Provenzano et al.,2009; Ulrich et al., 2009; Wozniak et al., 2003).

Mechanical stress and associated cell strain, can arise fromexternally imposed, ‘outside–in’ mechanical stimuli (Fig. 1B),which is common during force transmission to cells via the ECMin load-bearing tissues such as bone (Robling et al., 2006).Alternatively, or in coordination with outside–in stimuli, anchoredcells also probe their microenvironment to sense and respond to

the stiffness of the microenvironment by pulling on the ECM, i.e.‘inside–out’ mechanical stimuli. Such processes are dependent onECM adhesions that act as a bridge to transmit force between theECM and the cellular cytoskeleton, in which myosin-basedcontractility acts as a primary regulator of cellular traction(contractile) forces (Geiger et al., 2001). Indeed, these intracellularforces have a role in regulating signaling pathways that are involvedin fundamental cell processes that determine cell phenotype. Forinstance, myosin-based contractility has a role in regulating stemcell differentiation (Engler et al., 2006), epithelial morphogenesis(Alcaraz et al., 2008; Gehler et al., 2009; Sahai and Marshall,2002; Wozniak et al., 2003), branching morphogenesis of epithelialand endothelial cells (Fischer et al., 2009; Moore et al., 2005), andthe cancer-associated invasive phenotype that is induced inmammary epithelial cells by stiff 3D matrices (Provenzano et al.,2009; Provenzano et al., 2008b).

Importantly, cells possess the ability to modulate their forcegeneration in response to local stiffness of 2D substrates or 3Dmicroenvironments. Increasing substrate stiffness results inincreased traction force in fibroblasts (Lo et al., 2000; Paszek etal., 2005) and epithelial cells (Saez et al., 2005). Moreover,fibroblasts adjust their internal cell stiffness to match substratestiffness (Solon et al., 2007). Thus, feedback loops exist by whichcells sense the stiffness of the microenvironment and exertcontractile force at a magnitude that scales with this stiffness (Fig.2), a characteristic that is a crucial determinant of cell behavior. Ifthe stiffness of the microenvironment is abnormally low or thereis no substrate, cells will experience anchorage-independentconditions under which normally adherent non-transformed cellswill not survive (Ma et al., 2008). However, under normal

SummaryThe notion that cell shape and spreading can regulate cell proliferation has evolved over several years, but only recently has this beenlinked to forces from within and upon the cell. This emerging area of mechanical signaling is proving to be wide-spread and importantfor all cell types. The microenvironment that surrounds cells provides a complex spectrum of different, simultaneously active,biochemical, structural and mechanical stimuli. In this milieu, cells probe the stiffness of their microenvironment by pulling on theextracellular matrix (ECM) and/or adjacent cells. This process is dependent on transcellular cell–ECM or cell–cell adhesions, as wellas cell contractility mediated by Rho GTPases, to provide a functional linkage through which forces are transmitted through thecytoskeleton by intracellular force-generating proteins. This Commentary covers recent advances in the underlying mechanisms thatcontrol cell proliferation by mechanical signaling, with an emphasis on the role of 3D microenvironments and in vivo extracellularmatrices. Moreover, as there is much recent interest in the tumor–stromal interaction, we will pay particular attention to exciting newdata describing the role of mechanical signaling in the progression of breast cancer.

Key words: Cancer, Cell proliferation, Cytoskeleton, Focal adhesion, Mechanotransduction, Rho GTPAse

Journal of Cell Science 124, 1195-1205 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.067009

Mechanical signaling through the cytoskeletonregulates cell proliferation by coordinated focaladhesion and Rho GTPase signalingPaolo P. Provenzano1,2,* and Patricia J. Keely2,3

1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA2Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI 53706, USA3Laboratory of Molecular Biology and Department of Pharmacology, University of Wisconsin, Madison, WI, 53706, USA*Author for correspondence (ppproven@fhcrc.org)

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physiological conditions, a dynamic force balance – termedtensional homeostasis – occurs between the cell and themicroenvironment, and the cell will proliferate at normal levels(Klein et al., 2009). By contrast, if matrix stiffness isuncharacteristically elevated, an abnormally elevated tensionalhomeostasis arises that can result in aberrant cellular behaviors.For example, increased matrix stiffness associated with theincreased deposition, altered composition and alignment of thecollagenous stroma that accompanies breast tumor progression canpromote disruption of epithelial architecture (Paszek et al., 2005;Provenzano et al., 2008a; Wozniak et al., 2003). Cells in thisenvironment hyperactivate a mechanically regulated signaling loopthat results in increased expression of a conserved set of carcinoma-associated proliferation genes, such as genes that encode cyclins,aurora kinases, cell division cycle regulators and E2F transcriptionfactors, which are important for cell proliferation (Provenzano etal., 2009). Hence, in addition to the cellular microenvironmentbeing an intricate system that includes a complex chemical milieusurrounding the cell, it is now clear that the stiffness of the ECMand the corresponding mechanical response of the cell have a rolein regulating fundamental processes – including cell proliferation.

In this Commentary, we will cover recent advances in ourunderstanding of the regulation of cell proliferation by mechanicalsignaling. The emphasis is on the role of mechanical signaltransduction in the context of 3D microenvironments, whichinclude cell culture models of defined ECM composition andconcentration, such as collagen matrices that contain individualcells or cell communities, models of complex ECM mixturesincluding recombinant basement membrane (Matrigel) andmicroenvironments that are found within tissues in vivo. We payparticular attention to exciting new data describing the role ofmechanical signaling in cancer progression. In this area, the largestvolume of work focuses on breast cancer, which can be used as abeacon for understanding other cancers because many of thesignaling mechanisms might be similar.

For years, there was the suspicion that cell shape and spreadingregulates cell proliferation, but only recently have forces fromwithin and upon the cell been directly linked to these processes.Thus, we will discuss mechanisms by which stiffness of the ECMinfluences the cell–matrix interaction and focal adhesions, as wellas Rho GTPase signaling to transmit mechanical forces throughthe cytoskeleton, that lead to changes in cell proliferation. Althoughherein the emphasis is on cancer cell proliferation, the topic is ofbroad relevance because the mechanisms involved are likely to beat play in multiple cell types during developmental processes (e.g.stem cell differentiation, cellular morphogenesis and generation oftissue architecture) as well as in several proliferative pathologies(e.g. vascular disease, wound repair, lung and liver fibrosis, etc.)that are accompanied by changes in the ECM.

Linking the actin cytoskeleton to themicroenvironment – focal adhesions asregulators of mechanical signaling and cellproliferationCells form a functional force linkage between intracellular,contractile, force-generating motor proteins, the cytoskeleton andtranscellular adhesions (e.g. integrins) to transmit force to theECM. In addition, cell contacts between neighboring cells viacadherins are linked to the actin cytoskeleton and are also subjectedto, and respond to, forces between cells. The subject of cell–cellmechanical signaling is not covered in detail here; forcetransmission between cells has been reviewed elsewhere (Chen etal., 2004; Ingber, 2006; Janmey and McCulloch, 2007; Wozniakand Chen, 2009). The most-prominent cell adhesion structure isthe focal adhesion (FA), which has been defined for cells on 2Dsurfaces coated with ECM (Burridge et al., 1988). In 3Denvironments, specialized 3D-matrix adhesions are observed(Cukierman et al., 2001). The differences between 3D-matrixadhesions and those found in 2D systems might account for manyof the differences in cell signaling that are observed in 3D culture

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Mechanical propertiesMatrix stiffness,

external force

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alignment, etc.

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Fig. 1. The mechanically active 3D microenvironment. (A)Theprimary factors of the 3D microenvironment that influence cellbehavior. A dynamic dependence exists between all three factors,but here the focus is on mechanical signals to and from the ECMand the role these stimuli have in cell proliferation. (B)Outside–in(left) and inside–out (right) mechanical signals. During normalphysiological function, cells and tissues in the body experiencemulti-axial loading that result from a complex superposition ofexternal forces to produce stress in the cell. For example, tensilestress (T), compressive stress (C) and shear stress (; depicted asthe result of fluid flow over the cell) are commonly applied tocells during normal physiological tissue function. Of course, foreach of these stimuli there is an equal force that exists in the cell(not shown). Furthermore, during inside–out signaling, chemicalenergy is converted to mechanical energy in order to generatecontractile forces within the cell and to impart stress on the ECM(not shown), which results in an elevated force balance at the FAthat influences signal transduction within the cell.

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(referenced throughout this article). FAs are sites of integrinclustering that form a physical link between the actin cytoskeletonand the ECM (Burridge et al., 1988) to transduce force betweenthe cell and its microenvironment (Geiger et al., 2001). Consistentwith the concept that FA signaling increases when a cell facesresistance to intracellular contraction owing to the stiffness of theECM, force transmitted through the actin cytoskeleton results inincreased force at the cell-matrix interface, which further promotesFA assembly (Chrzanowska-Wodnicka and Burridge, 1996; Ridleyand Hall, 1992). Furthermore, external force applied to integrins,or exposure to a stiff 2D substrate promotes increased FA size andstrength (Choquet et al., 1997; Galbraith et al., 2002; Pelham andWang, 1997; Sniadecki et al., 2007). For 3D-matrix adhesions, wehave shown that disruption of either mechanisms of myosin-basedcontractility or the actin cytoskeleton itself – which releasesintracellular tension – diminishes the number of 3D-matrixadhesions that are formed in response to stiff collagen matrices(Provenzano et al., 2009). Moreover, 3D-matrix adhesions do notreadily form in compliant matrices but, rather, in stiff matrices thatoffer resistance to intracellular contractility (Provenzano et al.,2009; Wozniak et al., 2003). However, it should be noted that thepresence of FAs or punctate 3D-matrix adhesions in cells within3D microenvironments is controversial. For example, a recentstudy has demonstrated that FA proteins are diffuse in HT-1080fibrosarcoma cells that are cultured in 3D (Fraley et al., 2010),whereas another study using the same cell line has demonstratedrobust 1-integrin-positive 3D-matrix adhesions in 3D (Wolf et al.,2003). As more data emerge, evidence that 3D-matrix adhesionsare dependent on ECM stiffness might help in addressing thesediscrepancies.

In addition to acting as a physical scaffold, FAs function as anode on which to assemble and regulate a complex signalingnetwork that influences fundamental cell processes including cellproliferation (Mitra et al., 2005; Playford and Schaller, 2004). Forinstance, FA kinase (FAK), a primary regulator of FA signaling,activates pathways known to promote cell proliferation (Fig. 3).Integrin-stimulated phosphorylation of FAK at tyrosine residue397 creates a high-affinity site that is recognized by several Srchomology 2 (SH2) domain-containing proteins, including Src andShc (Schaller et al., 1994; Schlaepfer et al., 1998; Xing et al.,1994). Moreover, FAK phosphorylation at tyrosine 925 by Srclinks FAK to the Ras pathway via growth factor receptor-boundprotein 2 (Grb2) (Schlaepfer et al., 1994; Schlaepfer and Hunter,1996; Schlaepfer et al., 1998). Interestingly, FAK activation hasbeen shown to occur with both outside–in and inside–outmechanical signaling. For example, several studies demonstrate arole for FAK in mechanotransduction by showing that cell ortissue loading affects FAK expression or activation in a numberof cell types, such as fibroblasts (Moalli et al., 2001; Molina etal., 2001; Wang, J. G. et al., 2001), osteocytes (Wozniak et al.,2000), chondrocytes (Lee et al., 2000), vascular smooth musclecells (Li et al., 2009; Nakayama et al., 2003), skeletal muscle cells(Fluck et al., 1999; Gordon et al., 2001) and epithelial cells(Basson et al., 2000; Provenzano et al., 2009; Zhang et al., 2003).FAK is also necessary for mechanically induced osteogenesis(Leucht et al., 2007) and has been implicated in mechanosensingin migrating fibroblasts (Wang, H. B. et al., 2001). FAK isphosphorylated at tyrosine residues 397 and 925 during fibroblastdeformation, which promotes phosphorylation of extracellularsignal-regulated kinases 1 and 2 (ERK1/2) (Wang, J. G. et al.,2001) and, subsequently, cell proliferation (Wang et al., 2005).Furthermore, mammary epithelial cells in stiff 3D matrices canupregulate FAK phosphorylation (Paszek et al., 2005; Provenzanoet al., 2009; Wozniak et al., 2003) and we have shown that anincrease in Rho-mediated cell contraction in response to a stiffmicroenvironment drives the localization of active FAK into 3D-matrix adhesions (Provenzano et al., 2009). It is worth noting thatthe stiff ECM microenvironment also increases the formation ofcomplexes between FAK and Src and Shc and the mitogen-activated protein kinase (MAPK) pathway member Grb2 – bothof which go on to enhance FAK-dependent activation of ERK1/2(Provenzano et al., 2009) (Fig. 3). Hence, the force balance at thecell-matrix interface, which is established by the contractileresponse to ECM stiffness, directly regulates classical pathwaysof proliferation, such as the Ras-MAPK pathway.

Regulation of cell proliferation by FA signaling is, however,more complex than providing merely a direct linkage to the Raspathway. In addition to this direct activation of pathways that areknown to regulate cell proliferation, FA components, such as FAKand Src, also display considerable crosstalk with receptor tyrosinekinases (RTKs) and affect the regulation of their activity (Aplinand Juliano, 1999; Baron et al., 1998; Benlimame et al., 2005;Casamassima and Rozengurt, 1998; Sieg et al., 2000). Examplesare the epidermal growth factor (EGF) and insulin-like growthfactor (IGF) family of receptors, which are also known to promotecell proliferation (Baselga and Swain, 2009; Pollak, 2008).Although a requirement for autocrine EGF signaling to inducephosphorylation of ERKs in compressed cells has beendemonstrated (Tschumperlin et al., 2004), little is currently knownregarding the role mechanical signals have in mediating crosstalkbetween FAs and RTKs.

1197Mechanical control of cell proliferation

FA

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Fig. 2. Cell contraction force as a function of ECM stiffness.(A)Contractile force is transmitted within the actin cytoskeleton through theFA to the ECM (kCS and kECM represent the stiffness of the actin cytoskeletonand the ECM, respectively). (B)The magnitude of cell-generated contractileforce is dependent upon the stiffness of the ECM (kECM). As the stiffness of themicroenvironment increases, the magnitude of contractile force also increasesin order to maintain tensional homeostasis. Of course, the cellular response tothe mechanical properties of the microenvironment is further complicatedbecause of the viscoelastic behavior of the ECM and within the cell itself (notshown; see Box 1). Although the time scale over which cells deform the matrixsuggests that the process can be well described by understanding elasticbehavior; the physiological implication of viscoelastic phenomena and thedegree to which mechanical behavior at cellular scale is dominated by eitherelastic or viscous effects in a certain situation is not well understood – but islikely to provide additional insight into mechanotransduction.

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An additional level of complexity arises from reports that ERKis localized at FAs in complexes that also contain the FA proteinspaxillin, Src and FAK [together with indications that paxillinassociates with Raf and MAPK kinase (MEK)] to regulate epithelialcell migration and morphogenesis (Ishibe et al., 2004; Ishibe et al.,2003; Liu et al., 2002; Webb et al., 2004). These findings haveinteresting implications for a possibly direct mechanical regulationof the MAPK pathway to cause cell proliferation, because FAK,Src and paxillin are regulated in response to mechanical signals.

FAK can also directly bind to, and promote Src-mediatedphosphorylation of, p130Cas, an adaptor protein that mediatesseveral cellular events associated with cell adhesion and mitogensignaling, and that undergoes stretch-dependent activation thatresults in its increased phosphorylation by Src family kinases(Sawada et al., 2006). Activation of p130Cas, in turn, scaffolds acomplex between Crk, Dock180 and the engulfment and cellmotility (ELMO) protein, which serves as a guanine exchangefactor (GEF) for Rac. Moreover, p130Cas provides a scaffold forthe non-catalytic region of tyrosine kinase adapator protein (Nck),which then activates the Ras–ERK pathway, suggesting that

mechanical signals regulate cell proliferation in part throughdifferent signaling routes that all lead to activation of ERKs (Fig.3). Hence, although it remains to be fully elucidated whetherMAPK components that are localized to FAs have a role inregulating proliferation, current data suggest that additional linksexist between mechanical signals transmitted through the actincytoskeleton to FAs and the signaling cascades that regulate cellproliferation.

Finally, how mechanical signals are transmitted through thestructural components of the cytoskeletal to FAs is likely to havea role in signaling events that regulate cell proliferation. Foremost,mechanical activation of signaling pathways at FAs requires anintact and well-regulated cytoskeleton to effectively transmitforce from the contractile machinery to the FA proteins, suggestingthat changes in the cytoskeleton structure can differentiallyinfluence the mechanical stimuli presented to FAs. It is knownthat the actin cytoskeleton displays complex elastic andviscoelastic behavior (e.g. Gardel et al., 2006; Liu et al., 2006;Luan et al., 2008; Tseng et al., 2004; Xu et al., 2000). As such,it is possible that an increased understanding of how mechanical

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Box 1. Glossary of mechanics terminology

MechanicsThe sub-discipline of physics and engineering devoted to the action of forces and displacements on physical bodies.

Force and stressForce is a vector (it has magnitude and direction) that produces acceleration of a body in the direction of the applied force. When the resultantforce that acts on a body is zero the body is in equilibrium. Stress () is the force per unit area and is a measure of the forces acting betweenparticles in a deformable body.

Deformation and strainIn solid continuum mechanics (i.e. the study of mechanical behavior of materials modeled as a continuous mass rather discrete particles),deformation is an alteration in the shape or size of a body as the result of an applied force. Strain () is a unitless normalization of deformationand for small strains can be described as (L–L0)/L0, where L is the final length of the body and L0 is the initial length (known as‘engineering’ or ‘Cauchy’ strain). For larger strains (>5–10% strain) as commonly seen in biologic materials, the use of ‘Green’ strain isappropriate: 1/2*[(L/L0)2–1].

Stiffness and elastic modulusStiffness of an elastic material is the internal resistance to deformation produced by the application of force. It is a structural property (alsoknown as an extensive property) because it depends on the size, organization and shape of the material. By contrast, the elastic modulus is amaterial property (also known as an intensive property) as it is a normalized metric that is independent of geometric considerations. Theelastic modulus (E) describes the tendency of a material to undergo elastic strain when experiencing stress; e.g. the tensile (or Young’s)modulus is defined as E/ (stress/strain) and can be determined by the slope of the stress–strain curve. Compliance, a term commonlyused in cell biology, is the inverse of stiffness and is a measure of the softness as opposed to the stiffness of a material.

Contractile and traction forceA cell converts chemical energy to mechanical energy during the process of contraction. Contractile force refers to the force generated by acell in a 3D environment and is resisted by the stiffness of microenvironment. Because cell-generated contractile force pulls on the ECM, cellsstretch the ECM. Therefore, tensile structural and material properties are the most appropriate measures to understand the mechanicalinteraction between cells and the microenvironment. Traction force is commonly used to describe force generated by a contracting cell on a2D substrate. Because the cell sits on top of the substrate, the primary force is in-plane with the area experiencing the force, i.e. shear force.Resistance to traction force by the substrate is, therefore, described by the materials resistance to shear stress, known as the shear modulusor modulus of rigidity.

ViscoelasticityBiologic materials display both elastic and viscoelastic behavior. Viscoelastic materials display both elastic (solid) and viscous (fluid)characteristics. Whereas elastic materials store energy under deformation, viscoelastic materials dissipate energy (e.g. the gel cushion inrunning shoes). In addition, a main feature of viscoelastic materials is the presence of a time-dependent relationship between stress andstrain. This time dependence is commonly described by two main characteristics of viscoelastic materials: creep (the time-dependent changein strain under constant stress) and stress relaxation (the decrease in stress over time when material strain is constant). Interestingly, in mostbiologic materials the most significant creep or stress relaxation occurs very rapidly (often on the order of seconds) and then the strain orstress, respectively, approaches steady-state levels. As such, owing to the dynamic nature of tensional homeostasis over a long period of time(relative to the time scale for which many viscoelastic materials approach steady-state value for creep and relaxation), the influence ofviscoelastic behavior in the microenvironment is currently not well-understood and an increased understanding of viscoelastic behavior in themicroenvironment is thus likely to provide additional insight into mechanotransduction.

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signals regulate cytoskeleton remodeling through actin modifyingproteins and how temporal behavior of cytoskeleton mechanicsmodulate differential signal transduction, will improve ourunderstanding of mechanotransduction. For instance, suchinformation might provide insight into whether there are directmechanisms of force transmission through the cell to the nucleusthat influence gene expression (Wang, N. et al., 2009), which inturn could regulate the expression of cell proliferation genes.Thus, although our understanding of the link between cytoskeletalmechanics and mechanotransduction remains incomplete atpresent, it is clear that force transmission through the cytoskeletonsignificantly influences FA signaling, and that a complex feedbackloop exists between cell proliferation and pathways that regulateintracellular contraction to produce force through the cytoskeletonto regulate FA signaling.

Rho GTPases and the proliferative response tomechanical cuesCells sense the stiffness of their environment by generating forcesfrom within the cell that pull against the extracellular matrix.These contractile forces are largely based on actin–myosininteractions, because inhibition of actin or myosin activity abolishesthe ability of epithelial cells to contract 3D matrices and to undergotubulogenesis (Wozniak et al., 2003). The small GTPase Rho is akey regulator of intracellular contractility and, thus, allows cells tosense matrix stiffness and respond to mechanical cues. This functionis largely exerted through the Rho effector protein Rho-associatedprotein kinase (ROCK) because epithelial cells with modifiedROCK or Rho activity – i.e. with levels outside of the physiologicalrange required for appropriate morphogenesis – no longer respondeffectively to matrix stiffness (Paszek et al., 2005; Provenzano et

1199Mechanical control of cell proliferation

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Fig. 3. Inside–out contractile force as a regulator of cell signaling. Stiffness of the extracellular matrix influences the magnitude of the contractile force of thecell that is transmitted to the ECM through integrins. (A)Increased ECM deposition by carcinoma-associated fibroblasts (CAFs) produces a stiff microenvironmentfor carcinoma cells. The dense ECM contains CAFs that signal to carcinoma cells, provides micro- and/or nano-structures that influence cell behavior, and has astiffness to which the cells respond. (B)Elevated stiffness of the extracellular matrix results in elevated forces (F) at the cell–matrix interface, and promotes 3D-matrix adhesion formation and maturation that leads to the activation of highly dynamic signaling networks that regulate fundamental cell processes such asproliferation.

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al., 2009; Wozniak et al., 2003). As a result of Rho- or ROCK-mediated contractility, cells in compliant matrices are able toreorganize the matrix and assume a more-rounded morphology.Interestingly, cell shape has long been linked to regulatedproliferation (Chen et al., 1997; Folkman and Moscona, 1978;Huang et al., 1998), although the underlying mechanism is notwell-understood. Data suggest that cell shape has a dominant rolein regulating cell proliferation: when cells are placed on defined2D surfaces in which their shape is confined, they have restrictedproliferation (Chen et al., 1997). Thus, an important mechanism bywhich matrix compliance regulates proliferation is likely to be theeffect it has on cell shape.

Several cell types cultured in a compliant environment showdiminished proliferation and increased differentiation comparedwith cells in stiff environments (Fringer and Grinnell, 2003;Grinnell et al., 2003; Helm et al., 2007; Koyama et al., 1996; Nget al., 2005; Wang et al., 2000; Wozniak et al., 2003). Stiff matrices,in turn, lead to increased Rho activation (Bhadriraju et al., 2007;Provenzano et al., 2009; Wozniak et al., 2003). The exactmechanism by which this occurs is still being elucidated. SeveralRho GEFs are regulated by phosphorylation and, therefore, mightbe targets of increased integrin-mediated signaling through FAKand ERK1/2 that, because of increased mechanical tension, occursin stiff environments (Provenzano et al., 2009; Rosenfeldt andGrinnell, 2000). Some of these pathways are discussed below.

Rho has been linked to increased cell proliferation in manycancers, in which it is increased in both amount and activationstate (Faried et al., 2007; Sterpetti et al., 2006; Wang, H. B. et al.,2009; Zhang et al., 2009). Mammary carcinoma cells proliferate inresponse to stiff matrices, and this was shown to be mediated inpart by Rho (Paszek et al., 2005; Provenzano et al., 2009; Wozniaket al., 2003). In addition to cancer, a link between Rho activation,mechanical signaling and proliferation has also been demonstratedin other cell types. For example, vascular smooth muscle cells inblood vessels proliferate in a Rho-dependent manner in responseto deformation (Halka et al., 2008; Qi et al., 2010). Similarly,skeletal muscle cells proliferate following cyclic mechanical strain,and this involves regulation of FAK, NFB and the Rho GTPaseRac1 (Kumar et al., 2004). The interplay between Rho and FAK iscomplex, as non-phosphorylated FAK inhibits Rho-mediatedcellular proliferation in endothelial cells (Pirone et al., 2006). OnceRho is activated in response to stiff matrices or by mechanicalstrain, there are several pathways by which it may signal in orderto stimulate proliferation.

The Rho family of GTPases is involved in cell-cycle progressionat several points. Activation of the family members Rho, Rac andCdc42 can lead to expression of cyclins D and E (for reviews, seeAssoian and Klein, 2008; Pruitt and Der, 2001). They are alsoinvolved in timing the activity of cyclin D1, because Rho activityswitches off the early activation of cyclin D1 by Rac and Cdc42and, instead, sustains ERK1/2 activation, which ensures subsequentcyclin D1 expression in the mid-G1 phase of the cell cycle (Welshet al., 2001). Rho GTPases also activate p38 mitogen-activatedprotein kinase and Jun N-terminal kinase (JNK) through the MAPKkinase – mixed-lineage kinase (MKK–MLK) pathway, whichresults in the transcriptional regulation of genes associated withcell proliferation (Minden et al., 1995; Neisch et al., 2010; Philipset al., 2000; Whitehead et al., 1999). In addition, Rho familyGTPases activate serum response factor (SRF), which can enhanceproliferation through increased transcription of cyclins (Hill et al.,1995). Transcription mediated by SRF is regulated in a competitive

manner by either the ternary complex factor (TCF) or themyocardin-related transcription factor (MRTF), depending on thesignaling inputs (Gineitis and Treisman, 2001; Wang et al., 2004).MRTF is particularly relevant to mechanical regulation oftranscription and cellular proliferation because it is inhibited by G-actin; thus, the effect of Rho GTPases on stimulating actinpolymerization allows MRTF to enter the nucleus and regulateSRF (reviewed in Olson and Nordheim, 2010). In addition toproliferation, MRTF and SRF contribute to metastasis of bothbreast carcinoma and melanoma cells (Medjkane et al., 2009).Here, a role of cellular contractility depending on actin and myosinis suggested by the finding that one of the targets of MRTF andSRF, myosin 9, is also required for metastasis (Medjkane et al.,2009). A further level of regulation occurs through suppressor ofcancer cell invasion 1 (SCA1), which can form a ternary complexwith SRF to inhibit its target proteins. In this case, a key target is1-integrin, which is upregulated upon loss of SCA1 and inducesinvasion of breast carcinoma cells (Brandt et al., 2009). A numberof MRTF and SRF targets are also found in skeletal and smoothmuscle cells, and here Rho signaling to SRF induces bothproliferation and expression of differentiation-specific genes thatare involved in myogenesis (Gopinath et al., 2007; Kuwahara etal., 2005; Lockman et al., 2004).

Rho GTPases also signal to phosphoinositide 3-kinase (PI 3-kinase), which activates proliferation through the Akt pathway. Forexample, both RhoA and RhoC are involved in promotingproliferation in gastric carcinoma that depends on PI 3-kinase andAkt (Sun et al., 2007). Of relevance to mechanical regulation ofcell proliferation is the observation that cells cannot progressthrough G1 into S phase in the absence of integrin-mediatedadhesion, which appears to rely on Rho GTPases. Rho activationis necessary to remove the inhibition of cell-cycle progression thatis imposed by p21 and p27 (Zhang et al., 2009). Thus, enhancedintegrin-mediated signaling that occurs when cells are in stiffmatrices will activate Rho GTPases and promote progressionthrough G1 by induction of cell-cycle stimulators and loss of cell-cycle inhibitors.

Rho GTPases also regulate several of the steps involved inmitosis (for a review, see Schlessinger et al., 2009). Rho, inparticular, is involved in initial cell rounding that heralds the startof mitosis and, later, in the development of the cleavage furrowthat leads to cytokinesis (Glotzer, 2005). Two Rho-specific GEFs,epithelial cell transforming sequence 2 (Ect2) and GEF-H1, areinvolved in the precise regulation of Rho during cytokinesis.Ect2 is recruited to the central spindle through the action of Polo-like kinase 1 (Burkard et al., 2007; Petronczki et al., 2007),which also regulates the positioning of RhoA during cytokinesis(Burkard et al., 2007), whereas the Rho-specific GTPaseactivating protein (GAP) p190RhoGAP is degraded duringcleavage-furrow formation in order to sustain mitosis (Su et al.,2009). Thus, an intriguing possibility – although there arecurrently no data to support or refute this idea – is that increasedcell proliferation within stiff matrices is, in part, due to anupregulation of Rho pathways that are also necessary for mitosis.As such, it is important to note that GEF-H1 is activated bymechanical strain in vascular endothelial cells (Birukova et al.,2010), thus, suggesting it has a direct role in mediating cellproliferation in response to mechanical signals.

In polarized epithelial cells, GEF-H1 is kept inactive by beingsequestered to tight junctions (TJs). There, it binds to cingulin, aTJ protein whose function is not well understood and, thus, might

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contribute to contact inhibition of cell proliferation (Aijaz et al.,2005; Citi et al., 2009; Nie et al., 2009). Release of GEF-H1 fromtight junctions, for example during epithelial–mesenchymaltransition (EMT), activates Rho, which then – to promoteproliferation – collaborates with ZO-1 associated nucleic-acidbinding protein (ZONAB), a transcription factor that increasesexpression of cyclin D1 (Nie et al., 2009). In addition to GEFs,RhoGAPs are also a mechanism through which mechanicalsignaling can regulate cell proliferation. For instance, p190RhoGAPis mechanosensitive (Mammoto et al., 2009) and is regulatedduring cell adhesion (Arthur and Burridge, 2001; Arthur et al.,2000). p190RhoGAP is phosphorylated by Src (Arthur et al., 2000;Parsons and Parsons, 2004), which allows it to be regulateddownstream of both integrin and growth factor pathways (Parsonsand Parsons, 2004). Furthermore, both vascular endothelial growthfactor (VEGF) and matrix elasticity can control p190RhoGAP inorder to regulate two antagonistic transcriptional factors (TFII-Iand GATA2) that control angiogenic factors (Mammoto et al.,2009), suggesting that RhoGAPs and GEFs are potentialconvergence points, at which the microenvironment and solublefactors coordinate cell responses.

Rho GTPases also have a role in inhibiting apoptosis and,through this function, might also contribute to cancer progression.Rho–ROCK and Cdc42–Rac–PAK signaling networks are requiredto regulate Bcl2 family members and inhibit activation of caspase3 in gastric carcinoma cells (He et al., 2008). Furthermore, Rhoactivation in hepatocellular carcinoma results in upregulation ofthe anti-apoptotic Bcl2 and increased phosphorylation of the pro-apoptotic Bcl2-associated death promoter (BAD), thus reducingapoptosis and resulting in cells that are resistant to genotoxic stress(Sterpetti et al., 2006). Interestingly, mammary epithelial cellsdownregulate several pro-apoptotic genes when they are culturedin a stiff matrix (Provenzano et al., 2009), further supporting theidea that mechanical stimuli promote increased cell numbersthrough both increased proliferation and resistance to apoptosis.

Regulation of stem cell fate, as well as maintenance of the self-renewing population of stem cells, are nuances of cell proliferationthat are also regulated by Rho. Self-renewal is maintained in partthrough Wnt signaling. A growing body of work suggests that boththe canonical and non-canonical Wnt pathways are partly regulatedby Rho (reviewed in Schlessinger et al., 2009). The commitmentof cells that differentiate to a particular lineage is also regulated bycytoskeletal tension and Rho. In a manner that is also linked to cellshape, mesenchymal stem cells (MSCs) adopt an osteogenic fatewhen RhoA is activated and an adipocyte fate when RhoA isinhibited (McBeath et al., 2004). Furthermore, MSCs cultured onsubstrates of a different stiffness will commit to a lineage whosemicroenvironment corresponds to the substrate stiffness, i.e. toosteogenic lineages on stiffer substrates with an elastic modulusapproximating that of embryonic bone microenvironments, and toneurogenic lineages on softer substrates that have an elasticmodulus similar to that of neural tissue (Engler et al., 2006).

Mechanical regulation of cell proliferation incancerThe increased density and altered composition of the fibroblast-derived ECM, and its influence on carcinoma cells during tumorprogression (Amatangelo et al., 2005; Cukierman and Bassi, 2010;Serebriiskii et al., 2008) suggests a role for the physical propertiesof the ECM in tumor progression. In addition, reports by Grinnelland co-workers demonstrated that reducing resistance to cell

contractile force in fibroblasts cultured in 3D collagen matricesinhibits cell proliferation by reducing phosphorylation of ERKs,downregulation of cyclin D1 and increasing levels of the cell-cycleinhibitor cyclin-dependent kinase inhibitor 1B (CDKN1B, alsoknown as Kip1) (Fringer and Grinnell, 2001; Rosenfeldt andGrinnell, 2000). Extending these studies in breast carcinoma cells,Wozniak and colleagues demonstrated a link between cellularcontractility and proliferation (Wozniak et al., 2003). Underconditions of high resistance to cell contraction by the 3D matrix,proliferation is high, whereas reduced resistance to cell contractiondecreases the proliferation of carcinoma cells (Wozniak et al.,2003). Consistent with these findings, normal and transformedmammary epithelial cells cultured in 3D matrices of increasingstiffness result in increasingly larger colonies (Paszek et al., 2005)and a more proliferative population of cells (Provenzano et al.,2009; Provenzano et al., 2008a). Indeed, culturing mammaryepithelial cells in matrices with high stiffness increases theexpression of genes that have been identified as the breastcarcinoma ‘proliferation signature’ (Whitfield et al., 2006), whichnegatively correlates with patient survival as shown by us andothers (Provenzano et al., 2009; Whitfield et al., 2006). Expressionof this proliferation signature is dependent on ERKs, and alsoserves to induce an invasive phenotype (Provenzano et al., 2009).Furthermore, in addition to transcripts associated with the G1phase of the cell cycle, there is an upregulation of transcripts thatregulate G2 and G2–M transition and that display a strong statisticalenrichment for computationally predicted transcription factorbinding sites for, and hence regulation by, the transcription factorp53. The tumor suppressor protein p53 is known to be deleted ormutated in numerous human cancers (Junttila and Evan, 2009) andfunctions as a regulator of G1–S and G2–M transitions (Taylor andStark, 2001). Intriguingly, FAK binds directly to p53, which resultsin reduced transcriptional activity of p53 (Golubovskaya et al.,2005) and its inactivation through murine double minute 2 (Mdm2)-dependent p53 ubiquitylation, whereas loss of FAK results inincreased amounts of active p53 that impair cell proliferation (Limet al., 2008). Taken together, these studies suggest that mechanicalsignals that active FA proteins including FAK emerge as directmediators of proteins known to be important in cancer – such asp53.

In addition to findings in epithelial carcinoma, recent work hasshown that cell lines derived from glioma also respond to substratestiffness (Ulrich et al., 2009). Here, increasing rigidity offibronectin-coated substrates substantially increase glioma cellproliferation, suggesting a role for abnormally stiffmicroenvironments in promoting brain tumor progression.Interestingly, the authors showed that inhibition of ROCK- andmyosin-based cellular contractility impede the stiffness-inducedactin architecture of the cytoskeleton and the associated spread-cell phenotype (Ulrich et al., 2009), suggesting a role for thecontractile force in regulating the cellular response to stiff matricessuch as elevated proliferation. Hence, although the exact molecularmechanisms have not yet been fully elucidated across a diverserange of cancers, emerging evidence advocates that mechanicshave a fundamental role in human cancer.

Furthermore, the links between Rho- and myosin-mediatedcontractility in regulating both cancer cell and stem cell behavior,and the potential role for adult stem or progenitor cells in humancancer raises additional questions regarding the interplay betweenmechanics and tumor initiation. As some human cancers, or asubset of cancers in a particular organ, have been proposed to

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result from aberrant adult tissue progenitor cells (Stingl and Caldas,2007; Visvader and Lindeman, 2008), it is reasonable to assumethat the mechanical properties of the microenvironment influencesprogenitor cell differentiation, as was shown for mesenchymalstem cells (Engler et al., 2006). Evidence that abnormal ECMstiffness disrupts differentiation and contributes to transformationis provided by the observation that altering stiffness and fibronectinlevels in the ECM can control both normal acinar differentiationas well as over-proliferation of breast cells (Williams et al., 2008).Loss of normal intestinal epithelial differentiation, and a switch toenhanced proliferation and tumor formation is also observed inmice that lack decorin (Bi et al., 2008), a proteoglycan that has arole in collagen fibrillogenesis and organization. It makes sensethat a substantial increase in the turnover rate of progenitor cellsdue to elevated proliferation induced by aberrant matrix stiffnessmakes these cells more susceptible to events that promotetransformation. Thus, although the exact mechanisms by whichadult progenitor cells are regulated are currently not known, themechanical properties of the microenvironment are likely to havea significant influence over their behavior and, thus, offer a unifyingprinciple in understanding cancers in which progenitor cells havea role.

Mechanical signals and breast cancer risk – acase for mechanotransduction in breast cancerrisk associated with high breast tissue densityHigh mammographic density of breast tissue has been correlatedwith a greater than fourfold increase in the relative risk fordeveloping breast cancer, making high density of breast tissue oneof the most significant independent risk factors for developing thedisease (Boyd et al., 1998; McCormack and dos Santos Silva,2006). In fact, high mammographic density throughout the breastmay account for up to 30% of breast cancers, whereas mutationsin BRCA1 or BRCA2 – although conferring a greater relative risk– account for only 5% of total breast cancers [see Boyd et al.(Boyd et al., 2005) and references therein]. Furthermore, a recentstudy revealed that ductal carcinoma in situ occurs overwhelminglyin dense regions of the breast (Ursin et al., 2005), suggesting thatlocal densities in breast tissue increase cancer risk.

Importantly, regions of increased breast density havesubstantially increased deposition of fibrillar collagen and collagen-associated proteoglycans such as decorin (Alowami et al., 2003;Guo et al., 2001; Li et al., 2005). Until recently, the link betweenmammographically dense breast tissue and the composition ofbreast tissue had only been correlative, as there was no directevidence for a causative link. Therefore, we set out to test whetherincreased stromal collagen is sufficient to promote mammarycarcinoma. Using transgenic mouse models with increased stromalcollagen, we indeed found a causal link between breast tissuedensity and breast carcinoma, because increased collagen promotestumor formation, growth and progression (Provenzano et al.,2008a). It is interesting that one of the primary factors that promotesmammary epithelial cell proliferation and invasion in collagendense 3D microenvironments is the high stiffness of these collagenmatrices (Provenzano et al., 2009). The stiff ECM resists cellularcontraction, resulting in elevated Rho-dependent intracellularcontractile force, integrin clustering and FAK-dependentproliferation mediated by ERKs. These observations suggest thata chronically stiff microenvironment results in the increased (pre-tumor) epithelial content that is found in patients with high densityof breast tissue (Guo et al., 2001; Li et al., 2005). Moreover,

abnormally elevated cell growth and cell turnover that occurs in astiff microenvironment might be subjected to mutagenic damagearising from chronic inflammatory disease (Martin and Boyd,2008), leading to transformation. Furthermore, the emerging rolefor mammary progenitor cells that reside in the basal compartmentof the mammary gland in breast cancer (Stingl and Caldas, 2007)raises the interesting possibility that a collagen-dense (stiff) stromaleads to hyperproliferation of progenitor cells in women with highdensity of breast tissue, ultimately contributing to the transition tocarcinoma.

In addition to a direct effect on mammary epithelial cell growth,the stiff collagen-dense stroma in women with high density ofbreast tissue is likely to also promote an activated stromal cellpopulation (commonly referred to as ‘reactive’ stroma), i.e.upregulation of growth factor, cytokine and/or excessive ECMsecretion by the stromal cell population that is often associatedwith a pathological condition. Consistent with this hypothesis,fibroblast proliferation is amplified in 3D environments that resistthe contractile force of the cell (Fringer and Grinnell, 2001;Rosenfeldt and Grinnell, 2000), and higher numbers of fibroblastsare present in high-density breast tissue (Alowami et al., 2003;Guo et al., 2001). Mechanical signals may promote the secretionof mitogenic factors by activating the fibroblasts that contributeto abnormal epithelial behavior in collagen-dense tissue becauseit is known that stromal fibroblasts can regulate the epithelium,in part through secreting growth factors and cytokines (Allinenet al., 2004; Bavik et al., 2006; Orimo et al., 2005). Furthermore,chronic inflammation is known to promote tumor progression(Condeelis and Pollard, 2006) and fibroblast activation mighthelp recruit immune components that can promote activatedstroma. Hence, it is likely that the stiff environment associatedwith high density of breast tissue activates a series of relatedmechanotransduction cascades in both epithelial and stromal cellsthroughout the breast, which result in chemical changes in thebreast and in paracrine signaling that drives the epitheliumtowards transformation.

ConclusionsHere we have discussed the mechanisms by which mechanicalsignals, particularly those related to 3D matrix mechanicalproperties, regulate cellular responses such as cell proliferation.Cell proliferation is subject to many levels of control, but it isbecoming clear that mechanical signaling through the cytoskeletonlinkage between FAs and regulators of cellular contractilitycontribute to the regulation of cell proliferation. In addition,mechanical signals that are associated with cell and matrixchanges during cancer formation and progression have afundamental role in tumor growth and invasion. Moreover, a stiffextracellular matrix will contribute to the activation andproliferation of tumor-associated stromal cells. All of these factorsmight support carcinoma growth and progression in general, andin dense breast tissue in particular. Thus, although ourunderstanding of the physical mechanisms of force transmissionin cells and the underlying molecular mechanisms ofmechanotransduction remains incomplete, it is clear that theseprocesses are fundamental regulators of cell behavior. As such,carefully executed studies to determine the mechanical propertiesof the cell microenvironment in vivo and the use of 3D culturesystems that closely mimic these properties will shed substantiallight on the mechanisms by which mechanical signals regulatecell behavior.

1202 Journal of Cell Science 124 (8)

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We thank the members of the Keely and LOCI Laboratories forhelpful discussions and comments on this article. This work wassupported by grants from the National Institutes of Health (NIH):CA152249 to P.P.P. and CA142833 and CA114462 to P.J.K. Depositedin PMC for release after 12 months.

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