G Proteins, Phosphoinositides, and Actin-Cytoskeletonin the Control of Cancer GrowthHIROSHI MARUTA,* HONG HE, ANJALI TIKOO, THAO VUONG, AND M.S.A. NUR-E-KAMALLudwig Institute for Cancer Research, Melbourne, Australia 3050

KEY WORDS Ras; Rho family GTPases; PIP2; tumor suppressors

ABSTRACT Almost three decades have passed since actin-cytoskeleton (acto-myosin complex)was first discovered in non-muscle cells. A combination of cell biology, biochemistry, and molecularbiology has revealed the structure and function of many actin-binding proteins and theirphysiological role in the regulation of cell motility, shape, growth, and malignant transformation. Asmolecular oncologists, we would like to review how the function of actin-cytoskeleton is regulatedthrough Ras/Rho family GTPases- or phosphoinosites-mediated signaling pathways, and howmalignant transformation is controlled by actin/phosphoinositides-binding proteins or drugs thatblock Rho/Rac/CDC42 GTPases-mediated signaling pathways. Microsc. Res. Tech. 47:61–66,1999. r 1999 Wiley-Liss, Inc..

REGULATION OF ACTIN-CYTOSKELETON BYRAS/RHO FAMILY GTPASES AND PIP2

Role of RasThe involvement of actin-cytoskeleton in malignant

transformation was first revealed when normal 3T3fibroblasts were transformed by a DNA tumor viruscalled SV40. In 1974, at the Cold Spring HarborSymposium on DNA Tumor Viruses, Weber and hiscolleagues demonstrated for the first time, using anti-bodies against actin or myosin II (double-headed non-muscle myosin), that (1) actin stress fibers or cables innormal cells consist of both actin filaments (F-actin)and myosin II filaments, and (2) upon malignant trans-formation of normal cells by SV40, actin stress fibersdisassemble (Weber et al., 1974). Furthermore, actinstress fibers can be reassembled when the transformedcells revert to normal cells by a spontaneous mutation(Weber et al., 1974). However, how SV40 causes thedisassembly of actin stress fibers still remains to beclarified.

Several years after oncogenic mutants of normal Rasgenes such as Ki-Ras and Ha-Ras in retroviruses calledKirstin and Harvey were found to be responsible formalignant transformation of normal 3T3 cells (Bar-bacid, 1987), it was shown by micro-injection that theseoncogenic Ras mutants also cause a rapid disassemblyof actin stress fibers (Bar-Sagi and Feramisco, 1986).Since then, in an attempt to understand the molecularmechanism of Ras-induced disruption of actin stressfibers and non-muscle cell motility in general, morethan 100 distinct actin-binding proteins were isolatedor cloned and biochemically characterized. Almost adecade ago, Vandekerckhove and his colleagues foundthat the levels of at least three distinct actin-bindingproteins that are associated with actin stress fibers,namely, vinculin, alpha-actinin, and gelsolin were foundto be markedly reduced upon malignant transforma-tion by SV40 or Ras (Vandekerckhove et al., 1990).Thus, it was quite reasonable to test whether over-expression of these proteins affects malignant transfor-

mation. Ben-Ze’ev and his colleagues found that over-expression of either vinculin or alpha-actinin suppressesSV-40-induced malignant transformation (Fernandezet al., 1992; Glueck et al., 1993).

Similarly, Kuzumaki and his colleagues found thatover-expression of a gelsolin mutant suppresses Ras-induced malignant transformation (Muellauer et al.,1993). Furthermore, we found that over-expression ofthe tumor sup-pressor NF2/merlin, which also bindsactin filaments, suppresses Ras transformation (Tikooet al., 1994). However, NF2 levels are not affected byRas transformation at all (Tikooet al., 1994). Recently,using an ectopic expression of HS1, an F-actin bun-dling/ PIP2-binding protein, we found that (1) Rascauses a dissociation of cortactin/EMS1 from the acto-myosin II (actin-myosin II complex) through PIP2, and(2) HS1 expression suppresses Ras transformation byrestoring the EMS1-actomysoin II complex (He et al.,1998).

Role of Rho Family GTPasesSeveral years ago, Hall and his colleagues found that

Rho family GTPases, including Rho, Rac, and CDC42,are responsible for controlling the organization of avariety of actin-based cytoplasmic structures (Hall,1998; Ridley, 1994, 1998). Many isoforms of Rho GT-Pases in general (except for RhoB and RhoE) areessential for the formation of actin stress fibers, whileRac is responsible for membrane ruffling, and CDC42for the formation of microspikes (Hall, 1998; Ridley,1998). Interestingly, RhoB and RhoE appear to beinvolved in the disassembly (instead of assembly) ofactin stress fibers (Lebowitz et al., 1995; Ridley, 1998).Since Ras also causes disassembly of actin stress fibers,induction of both membrane ruffling and microspikeformation (Bar-Sagi and Feramisco, 1986), it has been

*Correspondence to: Hiroshi Maruta, Ludwig Institute for Cancer Research,Melbourne, Australia 3050. E-mail: hiroshi.maruta@ludwig.edu.au

Received 17 March 1999; accepted in revised form 6 May 1999.

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r 1999 WILEY-LISS, INC.

suspected that these Rho family GTPases, in particularRhoB/RhoE, Rac, and CDC42, might act downstream ofRas in controlling these actin cytoskeletons. Recentstudies on Ras signaling pathways have revealed thatRac is a component acting downstream of Ras (Down-ward, 1998). Ras directly activates PI-3 kinase, and anend-product of PI-3 kinase activates an activator of Raccalled Vav, which stimulates the GDP-dissociation ofRac (Downward, 1998). RhoB is also required for Rastransformation (Lebowitz et al., 1995). Furthermore, asdiscussed in detail later, we have recently obtaineddirect evidence indicating that Ras induces the activa-tion of CDC42 in NIH 3T3 fibroblasts and PC12 cells atleast (Nur-E-Kamal et al., 1999). However, it stillremains to be clarified how Ras activates CDC42, andwhether Ras induces the activation of RhoB/RhoE.

Rac activates several effectors including PAKs (Ser/Thr kinase family), WASP, n-Chimaerin, and Por1(Ahmed et al., 1998). It is of interest to note that PAKsinactivate myosin II light chain (MII LC) kinase, therebyreducing the phosphorylation of MII LC (Sanders et al.,1999), which is essential for the actin-activation ofmyosin II Mg21 ATPase activity. In contrast, a Rho-activated kinase called Rock phosphorylates MII LC(and also inactivates MII LC phosphatase), therebyincreasing the phosphory-lation of MII LC (Narumiyaet al., 1998). These opposite effects of PAKs and Rock onMII LC are compatible with the previous observationthat Rho and Rock induce stress fiber formation,whereas Rac and PAKs are involved in the disassemblyof actin stress fibers. Also, it is known that Rac some-how activates a few enzymes such as PI-4/PI-5 kinasesthat produce PIP2 (Hartwig et al., 1995), LIM-kinasesthat phosphorylate Ser3 of cofilin and blocks its F-actinsevering activity (Arber et al., 1998; Yang et al., 1998),and WAVE, a WASP-related protein of 80 kDa, that isresponsible for membrane ruffling (Miki et al., 1998).Interestingly, the anti-cancer drug SCH51344 thatblocks Rac-induced membrane ruffling, appears to blockthe Rac-induced activation of LIM-kinases (Kumar,1999; Walsh et al., 1997). The same drug also canrestore the EMS1-actomyosin II complex in Ras trans-formants and suppress Ras trans-formation (He et al.,1998; Kumar et al., 1995). However, this drug has noeffect on the parental normal fibroblasts or NGF-induced neurite outgrowth of PC12 cells (Kumar et al.,1995).

CDC42 also activates several distinct effectors suchasACKs (Tyr-kinases), PAKs, MRCKs (myotonic dystro-phy kinase-related CDC42-binding kinases), N-WASP,and n-Chimaerin (Ahmed et al., 1998). At presentN-WASP, n-Chimaerin, PAKs, and MRCKs are knownto be responsible for CDC42-induced microspike forma-tion (Ahmed et al, 1998; Kozma et al., 1996; Miki et al.,1998). Recently a new pathway was found where CDC42induces activation of Rac. CDC42 activates PAKs, andPAKs then bind another protein called PIX or Coolthrough the interaction between the Pro-rich motif ofPAKs and the SH3 domain of PIX. PIX contains a GDSdomain that activates Rac (Manser et al., 1998). Inhibi-tion of the PAK-PIX interaction by the Pro-rich PAKdomain of 18 amino acids (PAK18) blocks Rac-inducedmembrane ruffling of PC12 cells and Ras-transformedfibroblasts (Maruta et al, 1999a; Obermeier et al.,1998). CDC42 is also essential for the formation of

cleavage furrow, an actomyosin II complex, which playsthe major role in cytokinesis (Drechsel et al., 1996), inaddition to the microspike formation.

The existence of several distinct isoforms of RhoGTPases (A, B, C, D, E, etc.), some of which haveapparently opposite effects on actin cytoskeleton, makesour straightforward understanding of their functionrather difficult. At least RhoB and RhoE disassembleactin stress fibers, while some of the remaining Rhoisoforms of Rho are essential for the stress fiber forma-tion. Using a bacterial exotoxin called C3 that inacti-vates selectively Rho GTPases by ADP-ribosylation attheir effector domain, it was also shown that some ofRho isoforms are required for the formation of cleavagefurrow during cytokinesis (Kishi et al., 1993; Mabuchiet al., 1995), as is CDC42. There are several distinctRho-activated kinases such as Rock (Narumiya et al.,1998). As we have briefly mentioned, the Ser/Thr-kinase Rock increases the phosphorylation of the myo-sin II light chain (MII LC). Since the phosphorylation ofMII LC is essential for the actin-activation of itsATPase activity and its bipolar filament formation,Rock favours the activation of actomyosin II ATPasesand myosin filament formation. In smooth muscle, Rockis involved in Ca21-sensitization of muscle fiber contrac-tion, while it is involved in the formation of actin stressfibers in non-muscle fibroblasts (Narumiya et al., 1998).Recently, the drug Y-27632 was found to selectivelyinhibit Rock activity and block both actin stress fiberformation in fibroblasts and Ca21-sensitization ofsmooth muscle contraction (Tominaga et al., 1998;Uehata et al., 1997). Interestingly, like C3, this drugsuppresses Ras transformation, but has no effect on thegrowth of normal cells (Maruta et al., 1999a; Sahai etal., 1999), indicating that some Rho effectors, includingRock, are essential for Ras transformation.

Role of PIP2 (Phosphatidylinositol 4,5Bisphosphate)

As we have briefly mentioned, Rac stimulates PIP2production by activating PI-4/PI-5 kinases. However,the exact molecular mechanism underlying Rac-in-duced PIP2 production still remains to be clarified.PIP2 then binds several distinct proteins includingF-actin capping proteins such as tensin, gelsolin, pro-filin, and Cap39 to inactivate their capping activity.Since these proteins bind selectively the plus-end ofactin filaments and block a rapid actin polymerizationat this end, PIP2 uncaps the plus-end of actin filamentsand induces actin polymerization at the same end.Interestingly, a large family of antibiotics called cytocha-lasins are known to cap the plus-end of actin filaments,and block membrane ruffling (Yahara et al., 1982).These observations clearly indicate that Ras/Rac-induced PIP2 production is involved in both uncappingand membrane ruffling. Is there any other pathway(s)in which PIP2 affects the organization of actin-cytoskeleton? The answer appears to be ‘‘yes.’’ Mostinterestingly, PIP2 binds all actin-binding proteins thatcan suppress malignant transformation caused by ei-ther SV40 or Ras (Maruta, 1998a). They include F-actincross-linking proteins such as vinculin, alpha-actinin,HS1, and NF2, as well as F-actin severing proteins suchas gelsolin and cofilin, in addition to F-actin cappingproteins such as tensin. It is worth noting that (1) PIP2

62 H. MARAUTA ET AL.

strongly binds HS1, (2) PIP2 strongly inhibits theF-actin cross-linking activity of the HS1-related proteinEMS1, and (3) HS1 abolishes the PIP2-mediated inhibi-tion of EMS1’s F-actin cross-linking, presumably bysequestering PIP2, and restores the EMS1-acto-myosinII complex in Ras transformants (He et al., 1998). Thesefinding suggest the possibility that (1) F-actin cross-linkers are also potentially tumor suppressors, and (2)PIP2-binding compounds are also potentially tumorsuppressors, as is HS1, which is an F-actin cross-linkerand PIP2-binder. The latter notion is supported by aprevious finding that micro-injection of a PIP2-specificantibody blocks the growth of Ras transformants atleast transiently (Fukami et al., 1988).

SUPPRESSION OF MALIGNANCY BYACTIN/PIP2-BINDING MOLECULES ANDINHIBITORS OF RHO FAMILY GTPASESF-Actin Cross-Linkers/Cappers as Tumor

SuppressorsWe have shown previously that over-expression of

tensin, an F-actin capper of 1,744 amino acids, sup-presses almost completely Ras transformation (Ma-ruta, 1998a). However, since tensin has both F-actincapping domain (III) and F-actin cross-linking domains(I and II), it is not clear whether the tumor suppressionis solely due to the capping of F-actin plus-ends, or alsodue to F-actin cross-linking.

To clarify this point more directly, we have tested theanti-oncogenic effect of a unique cytochalasin calledchaetoglobosin K or CK (for the chemical structure, seeFig. 1).We found that 2 µM of CK, which caps theF-actin plus-end and blocks membrane ruffling (Yaharaet al., 1982), almost completely suppresses Ras transfor-mation (Maruta, 1998a). This finding clearly indicates

that Ras/Rac/PIP2-induced uncapping of the F-actinplus-end is essential for Ras transformation. In otherwords, the F-actin cross-linking activity of tensin is notabsolutely required for its tumor suppressor activity.

Our next question was whether F-actin cross-linkingis also sufficient for suppressing Ras transformation.The answer also appears to be ‘‘yes.’’ During our recentinvestigation of the molecular mechanism underlyingthe anti-cancer action of the rhodacyanine dye MKT-077 (for the chemical structure, see Fig. 2), we foundthat (1) this dye, a photo-sensitizer, selectively blocksthe growth of Ras transformants, without any signifi-cant effect on the parental normal cells, (2) the dyebinds selectively actin, and bundles actin filaments bycross-linking, and (3) its binding to actin inhibits theactin-myosin II interaction (Maruta et al., 1999b).These findings strongly suggest that either F-actincapping or cross-linking drugs are potentially useful forthe chemotherapy of Ras-associated cancers, that repre-sent more than 30% of all human cancers, notably morethan 90% of pancreatic cancers and 50% of coloncancers.

PIP2-Binding Molecules as Tumor SuppressorsOur previous finding that PIP2 binds all tumor-

suppressive F-actin binding proteins such as HS1,gelsolin, tensin, and NF2 hints at the possibility thathigh-affinity PIP2-binding is sufficient to suppress Rastransformation by sequestering PIP2 that either un-caps the plus-end of actin filament or blocks cross-linking of actin filaments (or disassembles actin stressfibers). In an attempt to test this possibility, we haveexamined the anti-oncogenic effect of a unique cofilinmutant called 112Q/114Q. Cofilin is a proton-depen-dent F-actin severing protein, and binds PIP2. The

Fig. 1. Chemical structure of Chaetoglobosin K (Cytochalasin K), an F-actin capper.

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wild-type cofilin contains two critical Lys residues atpositions 112 and 114. When these Lys residues arereplaced by Gln residues, it no longer binds actin, but isstill able to bind PIP2. We found that over-expression ofthis double-mutant strongly suppresses Ras transforma-tion, indicating that PIP2-binding is sufficient for thetumor suppression (Maruta, 1998a). This finding sug-gests the possibility that a specific compound(s) withhigh-affinity for PIP2 would also be potentially usefulfor the chemotherapy of Ras-associated cancers.

Antagonists of Rho Family GTPases as TumorSuppressors

As we have mentioned briefly, blocking the Rac/Rho-mediated pathways by either SCH51344 or C3 is suffi-cient for suppressing Ras transformation, establishingthat both Rac and Rho are required for Ras transforma-tion. However, until very recently, it remained unclearwhether CDC42 is also required for Ras transforma-tion. Qiu and his colleagues suggested, using the N17mutant of CDC42, that Ras transformation mightrequire CDC42 (Qiu et al., 1997). However, this mutantis not a specific inhibitor of CDC42, simply because itcould sequester not only CDC42-specific GDSs such asFGD1, but also many other GDSs such as DBL, OST,and Tiam-1 that activate Rho or Rac, in addition toCDC42 (Maruta, 1998b). Thus, it is possible that theobserved effect of this CDC42 mutant might be due toblocking Rac/Rho pathways, instead of CDC42 pathways.

In an attempt to clarify this point, we have createdthe first CDC42-specific inhibitor called ACK42 (for thesequence, see Fig. 3). It is a 42-amino acid fragment ofthe Tyr-kinase ACK-1 that binds only CDC42 in theGTP-bound form. Thus, ACK42 would selectively blockthe inter-action of CDC42 with any of its effectors. Wefound that (1) over-expression of ACK42 suppressesRas transformation, and that (2) a cell-permeable de-rivative of ACK42 called WR-ACK42 also selectivelyblocks the growth of Ras transformants without anysignificant effect on the parental normal cells (Marutaet al., 1999a). These observations unambiguously haveproved that CDC42 is indeed essential for Ras transfor-mation, and suggested that ACK42 peptidomimeticswould be potentially useful for the chemotherapy ofRas-associated cancers.

Why is CDC42 essential for Ras transformation?Does CDC42 act downstream of Ras? Until recently,nobody has demonstrated that Ras induces the activa-tion of CDC42. However, using the ACK42-GST fusionprotein as a specific probe for the active GTP-boundform of CDC42, we have provided direct evidenceindicating that oncogenic Ras mutants such as v-Ha-Ras indeed induces the conversion of CDC42 from theGDP-bound to GTP-bound forms in NIH 3T3 fibro-blasts, and NGF, which activates Ras, also induces theactivation of CDC42 in PC12 cells (Maruta et al., 1999a;Nur-E-Kamal et al., 1999). Is which CDC42 is GDS (s)activated by Ras/NGF? Since Ras up-regulates bradyki-nin B2 receptor (BB2R) gene expression (Downward etal., 1988), and BB2R directly activates DBL that acti-vates CDC42 (Kozma et al., 1995), it is conceivable thatthis BB2R pathway might be involved in Ras-inducedactivation of CDC42.

To identify the specific residues of ACK42 that areessential for the binding to CDC42/ GTP complex, wehave created a series of ACK42 mutants. Replacementof His17 by any other amino acids including Arg andLys abolishes its binding to CDC42. Replacement ofArg34 by Leu, but not by Lys, also abolishes its bindingto CDC42 (Vuong and Maruta, unpublished data). Thisfinding indicates that both His17 and Arg34 are essen-tial for the binding to CDC42.

Interestingly, the position of all CDC42/Rac-bindingproteins corresponding to His17 of ACK-1 is highlyconserved, Furthermore, ACK-1 and ACK-2 share thealmost identical CDC42-binding motif (ACK42), exceptfor position 34 where ACK-2 contains Lys, instead ofArg (Yang and Cerione, 1997), suggesting that position34 should be a basic residue for binding to CDC42.Furthermore, we found that Lys34 mutant of ACK42has 15 times higher affinity for CDC42 than the Arg34form of ACK42 (Vuong and Maruta, unpublished data).Since position 38 in the effector domain of Ras/Rhofamily GTPases is invariably an acidic residue (Asp/Glu), and their effectors or GAPs require a basic residueto interact with the position 38 of GTPases, we specu-late that either His17 or Lys/Arg34 of ACK42 is respon-sible for the interaction with Asp38 of CDC42. Cur-rently, we are screening for ACK42 mutants that showa further higher affinity for CDC42, thereby being more

Fig. 2. Chemical structure of MKT-077, an F-actin bundler.

64 H. MARAUTA ET AL.

potent tumor suppressors than the Lys34 mutant ofACK42.

As we have briefly mentioned, the PAK-PIX interac-tion is required for a CDC42/PAK/PIX/Rac-mediatedmembrane ruffling of PC12 cells. Since Rac-dependentmembrane ruffling is essential for Ras transformation,we have created a cell-permeable inhibitor of PAK-PIXinteraction that could suppress Ras transformation. Itis called WR-PAK18. PAK18 is an 18-amino acid frag-ment of PAK that binds the SH3 domain of PIX. WR is asimple derivative of the cell-permeable peptide vector of16 amino acids called penetratin which consists of onlyArg and Trp residues (Derossi et al., 1998). LikeWR-ACK42, WR-PAK18 enters Ras transformants andblocks their growth, but has little effect on the growth ofthe parental normal cells (Maruta et al., 1999a). Thisclearly confirmed that the PAK-PIX interaction is re-quired for Ras transformation, and suggested thatPAK18 peptidomimetics would be useful for the chemo-therapy of Ras-associated cancers.

Future PerspectivesOncogenic Ras mutants activate their effectors such

as Raf and PI-3 kinase. During the last few years it hasbeen established that at least a pathway downstream ofPI-3 kinase, which includes Rac, is involved in Ras-induced re-organization of actin-cytoskeleton, in particu-lar membrane ruffling, and is required for Ras transfor-mation. This finding has promoted our effort to developa series of anti-Ras cancer drugs such as SCH51344that selectively block one of the critical steps in thisoncogenic pathway. Now it is also clear that oncogenic

Ras mutants induce the activation of CDC42, and aspecific inhibitor has been developed to selectivelyblock the function of CDC42 that activates severaleffectors such as ACKs, PAKs, N-WASP, and n-Chimae-rin. Once the detailed pathway that leads to theCDC42/N-WASP-induced formation of microspikes, itwould allow us to develop a series of new anti-Rascancer drugs that selectively block one of the criticalsteps in this new oncogenic pathway, too. Some mem-bers of Rho GTPases such as RhoB are also essential forRas transformation, as the Rho inactivators such as C3and farnesyltransferase inhibitors as well as the Rockinhibitor Y-27632 suppress Ras transformation. How-ever, it still remains to be clarified whether Ras inducesthe activation of Rho or not.

Since these Rho family GTPases control actin-cytoskeleton, and in fact over-expression of many actin-binding proteins has been shown to suppress Ras/SV40-induced malignant transformation, it is most likelythat a series of new anti-Ras cancer drugs, other thanCK and MKT-077, would be developed from a fairlylarge stock of unique actin-binding compounds, in earlyyears of the coming century, to open a new era of‘‘rational’’ cancer chemotherapy.

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66 H. MARAUTA ET AL.

REGULATION OF ACTIN-CYTOSKELETON BY RAS/RHO FAMILY GTPASES AND PIP2SUPPRESSION OF MALIGNANCY BY ACTIN/PIP2-BINDING MOLECULES AND INHIBITORSFig. 1.Fig. 2.REFERENCESFig. 3.

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