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Therapeutics, Targets, and Chemical Biology

RKI-1447 Is a Potent Inhibitor of the Rho-Associated ROCKKinases with Anti-Invasive and Antitumor Activities in BreastCancer

Ronil A. Patel1, Kara D. Forinash1, Roberta Pireddu1, Ying Sun1, Nan Sun1, Mathew P. Martin1,Ernst Sch€onbrunn1,2, Nicholas J. Lawrence1,2, and Saïd M. Sebti1,2

AbstractThe Rho-associated kinases ROCK1 and ROCK2 are critical for cancer cell migration and invasion, suggesting

they may be useful therapeutic targets. In this study, we describe the discovery and development of RKI-1447, apotent small molecule inhibitor of ROCK1 and ROCK2. Crystal structures of the RKI-1447/ROCK1 complexrevealed that RKI-1447 is a Type I kinase inhibitor that binds the ATP binding site through interactions with thehinge region and theDFGmotif. RKI-1447 suppressed phosphorylation of the ROCK substratesMLC-2 andMYPT-1 in human cancer cells, but had no effect on the phosphorylation levels of the AKT, MEK, and S6 kinase atconcentrations as high as 10 mmol/L. RKI-1447 was also highly selective at inhibiting ROCK-mediatedcytoskeleton re-organization (actin stress fiber formation) following LPA stimulation, but does not affectPAK-meditated lamellipodia and filopodia formation following PDGF and Bradykinin stimulation, respectively.RKI-1447 inhibited migration, invasion and anchorage-independent tumor growth of breast cancer cells. Incontrast, RKI-1313, a much weaker analog in vitro, had little effect on the phosphorylation levels of ROCKsubstrates, migration, invasion or anchorage-independent growth. Finally, RKI-1447 was highly effective atinhibiting the outgrowth of mammary tumors in a transgenic mouse model. In summary, our findings establishRKI-1447 as a potent and selective ROCK inhibitorwith significant anti-invasive and antitumor activities and offera preclinical proof-of-concept that justify further examination of RKI-1447 suitability as a potential clinicalcandidate. Cancer Res; 72(19); 5025–34. �2012 AACR.

IntroductionThe Rho family of small GTPases transduces biological

signals from cell surface receptors such as receptor tyrosinekinases to the nucleus. They act as molecular switches thatbind GTP (active) and GDP (inactive) to regulate cell prolif-eration, survival and cytoskeleton organization, thus, affectingcell shape/morphology, adhesion and movement (1–3). Someof these GTPases, particularly RhoA and RhoC, have attractedmuch attention because of their well-documented role inmalignant transformation hallmarks including migration,invasion, and metastasis. For example, RhoA and RhoC arerequired for Ras-driven malignant transformation (4), andtheir overexpression induces migration, invasion, and metas-tasis whereas their dominant negative forms block these hall-

marks of cancer (5, 6). Furthermore, several Rho GTPases areover expressed in a large number of human cancers (7–10), andthe expression of RhoA and RhoC has been shown to be muchhigher in metastatic tumors (11, 12). Several studies havesuggested RhoC as a prognostic biomarker for metastasis inmelanoma, breast and pancreatic cancer patients (5, 11, 12).The mechanism by which RhoA and RhoC contribute tometastasis is believed to involve the binding of RhoA-GTP andRhoC-GTP to their major effectors, the Rho-associated kinases1 and 2, together referred to here as ROCKs (13–19). ROCKscontrol cell shape, adhesion andmigration by regulating actin–myosin contractibility. This is accomplished by ROCKs phos-phorylating several major substrates such as myosin lightchain (MLC), the MLC phosphatase PP1 regulatory subunitMYPT-1 and LIM kinases 1 and 2. Phosphorylation of MLCactivates it to drive migration and invasion (14, 20). Phosphor-ylation of LIMK 1 and 2 activates it to phosphorylate andinactivate cofilin from inhibiting migration/invasion (21).Phosphorylation of MYPT-1 blocks dephosphorylation andinactivation of MLC (16).

The role of ROCKs inmigration, invasion andmetastasis hasbeenwell documented. For example, over expression of ROCKsinduces migration and invasion in several tumor types includ-ing breast cancer (22–24) whereas dominant negative ROCKsand ROCKs inhibitors block invasion and metastasis in vitroand in vivo (23, 25–30). Furthermore, the expression levels of

Authors' Affiliations: 1Drug Discovery Department, H. Lee Moffitt CancerCenter and Research Institute; and 2Departments of Oncologic Sciencesand Molecular Medicine, University of South Florida, Tampa, Florida

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthor:SaïdM.Sebti, DrugDiscoveryDepartment, H. LeeMoffitt Cancer Center and Research Institute, 12902 Magnolia Drive,SRB3-DRDIS, Tampa, FL 33612. Phone: 813-745-6734; Fax: 813-745-6748; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-0954

�2012 American Association for Cancer Research.

CancerResearch

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ROCKs is 10-fold higher in breast tumor biopsies as comparedto their corresponding normalmammary tissue, and these highexpression levels correlate with metastasis, poor clinical out-come and shorter survival time of breast cancer patients(26, 31). The overwhelming evidence implicating the RhoGTPases/ROCKpathway inmigration, invasion andmetastasisprompted the development of inhibitors to interfere with thispathway with the ultimate goal of discovering novel antitumordrugs (14, 26, 27, 29). To this end several approaches have beentaken. One of these is to develop geranylgeranyltransferase-1(GGT-1) inhibitors (GGTI), because GTPases such as RhoA andRhoC require the lipid posttranslation modification geranyl-geranylation for their ability to induce malignant transforma-tions (32). Although this approach has shown great promisepreclinically and one compoundGGTI-2418 has reached PhaseI clinical trials (33), a drawback is that GGTIs inhibit GGT-1, anenzyme that has dozens of substrates (32). A more selectiveapproach to interferingwith theRhoGTPase/ROCKpathway isto develop ROCK inhibitors as potential antimetastatic agents.In thismanuscript, we describe the discovery and developmentof RKI-1447 a highly potent ROCK inhibitor. First, using X-raycrystallography we solved the structure of the RKI-1447-ROCK1 complex and determined the mode of action of RKI-1447. Furthermore, we showed that RKI-1447 suppressesROCK-dependent signaling, cell morphology, anchorage-inde-pendent cell growth, migration invasion, and in vivo growth ofmammary tumors.

Materials and MethodsCell lines

Human breast (MDA-MB-231 and MDA-MB-468) and lung(H-1299) cell lines were purchased from American Type Cul-ture Collection. These cells have not been authenticated.

ROCK1 and ROCK2 kinase assaysThe Invitrogen Z-Lyte FRET kinase assay with KKRPQR-

RYSNVF peptide as substrate (from MLC sequence) was used(Invitrogen, cat. PV3793) exactly as described by us (34).

Enzyme purification and protein crystallographyEnzyme purification and protein crystallography were car-

ried out exactly as described by us previously (34). Data werereduced with XDS (35). The structure was solved by molecularreplacement using the MolRep program from CCP4 (36) withthe monomer of pdb 3TV7 as a starting model. PHENIX (37)was used for refinement, and model building was done usingCoot (38). Figures were prepared using PYMOL (39).

Evaluation of the phosphorylation levels of MLC-2 andMYPT-1 by Western blot analysis

Cells were plated and treated the next day with vehicle,RKI-1447 or RKI-1313 for 1 hour. [The chemical synthesis ofRKI-1447 and RKI-1313 was described by us (34). The mesylatesalts of the compounds were used to enhance solubility.]The cells were then harvested, lysed and processed forWestern blotting as described by us previously (40). Theantibodies that were used for Western blotting were MYPT1

(Cat#612164; BD Transduction Laboratories), Phospho-T696-MYPT1 (Cat#ABS45; Millipore), MLC2 (Cat#3672S),Phospho-S19-MLC2 (Cat#3671S), Akt (Cat#9272), Phospho-S473-Akt (Cat#9271), Phospho-S298-MEK(Cat#9128S), MEK(Cat#9122S), Phospho-S240-S244-S6 (Cat#2215S), S6 (Cat#2217),Cleaved PARP (Cat#5625S; Cell Signaling Technology) andTubulin (Cat#T5168; Sigma).

Determination of anchorage-independent growth usingsoft agar assays

The soft agar assays were carried out exactly as described byus (41).

Migration assayMDA-MB-231 cells were seeded at 4� 105 cells per well in a

6-well plate and allowed to grow overnight. The cells were thenstarved for 24 hours. The media was then aspirated and thecells were scratched with a pipette tip, and treated either withvehicle, RKI-1447 or RKI-1313 at the indicated concentrationsfor 24 hours. Pictures were taken with a Leica Microscopebefore (time 0) and 24 hours after drug treatments.

Invasion assayInvasion assay was carried out in Corning Transwell inserts

coated with Matrigel. MDA-MB-231 cells were seeded at3.5 � 105 and allowed to grow overnight in a 6-well plate. Thecells were then treated with either vehicle, RKI-1447 or RKI-1313 for 21 hours and trypsinized, resuspended in DMEMcontaining 0.1% BSA and plated at 20,000 cells/insert in thetop chamber over the Matrigel. The bottom chamber con-tained 20% FBS as the "chemoattractant". The cells in the topchamber were carefully removed after 48 hours and the filtermembranes containing the invaded cells were fixed withmethanol, stained with crystal violet and photographed. Themembranes were then dis-solved in 10% acetic acid and theabsorbance was read at 540 nm.

Effects of RKI-1447 and RKI-1313 on cell morphologyNIH 3T3 cells were plated at 8000 cells/well in 8-chamber

slides in serum free media for 24 hours, and treated withvehicle, 1 mmol/L RKI-1447 or 1 mmol/L RKI-1313 for 1 hour.The cells were then stimulated with complete media,10 mmol/L LPA, 200 ng/mL bradykinin, or 30 ng/mL PDGFfor 30 minutes. After stimulation, the cells were fixed in 4%para-formaldehyde and stained with Texas-Red Phalloidin.Mounting medium containing DAPI (Cat#H-1200; VectorLaboratories) was then added and at least 100 cells per wellwere observed. The cells were imaged using Zeiss Uprightfluorescence microscope.

RKI-1447 antitumor efficacy studiesMMTV/neu transgenic mice [FVB/N-Tg (MMTVneu) 202

Mul/J] were purchased from the Jackson Laboratory and bredto produce multiple litters to maintain the colony. The anti-tumor efficacy studies were carried out exactly as describedby us previously (41). In brief, the mice were treated i.p. dailyfor 14 days with either vehicle [20%–2-hydroxypropyl-beta-cyclodextrin (HPCD)] or 200mpk RKI-1447 dissolved in freshly

Patel et al.

Cancer Res; 72(19) October 1, 2012 Cancer Research5026




prepared HPCD. The percentage change in volume was cal-culated on the basis of the tumor volume on the last day oftreatment (Vf) relative to that on the day of initiation oftreatment (V0). The average percentage change in tumorvolume was then calculated for each treatment group.

ResultsDiscovery of RKI-1447, a highly potent ROCK1 andROCK2 inhibitorOur chemistry efforts resulted recently in the identification

of a family of pyridylthiazole ROCK inhibitors (34). Structureactivity relationship studies of this family of ROCK inhibitorsled to the discovery of a pair of closely related analogues; RKI-1447 (potent ROCK inhibitor) and RKI-1313 (weak ROCK

inhibitor). Figure 1A and B show that RKI-1447 inhibitspotently ROCK1 and ROCK2 in a dose-dependentmanner withIC50 values of 14.5 and 6.2 nmol/L, respectively. In contrast,RKI-1313 where the meta-hydroxyl on the phenyl ring of RKI-1447 was replaced with a para-methoxy group was much lessactive and had IC50 values 34 mmol/L for ROCK 1 and 8 mmol/Lfor ROCK2 (Fig. 1A and B).

Crystal structure of the RKI-1447-ROCK1 complexreveals that RKI-1447 is a Type 1 inhibitor that bindsboth thehinge region and theDFGmotif of theROCKATPbinding site

To determine the mode of binding of RKI-1447 to ROCK, wedetermined the crystal structure of RKI-1447 in complex with

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Figure 1. RKI-1447 and RKI-1313 chemical structures; in vitro ROCK inhibitory activity and RKI-1447-ROCK1 co-crystal structure. A, chemical structures ofRKI-1447 and RKI-1313. B, RKI-1447 is much more potent than RKI-1313 at inhibiting both ROCK1 and ROCK2. C, structure of the RKI-1447-ROCK1complex. a, surface presentation of 1 of the 2 ROCK1 dimers in complex with RKI-1447 determined by X-ray crystallography at 2.9 Å resolution anddetailed binding interactions in the ATP site. The hinge region is indicated in orange, the DFGmotif in cyan, and RKI-1447 in yellow. Displayed in blue is the 2Fo-Fc electron density, contoured at 1s around the RKI-1447 inhibitor. The Fo-Fc electron density map with the RKI-1447 inhibitor omitted duringrefinement is shown in the supplementary data (Supplementary Fig. S4). The hydrogen bonding and van der Waal interactions are shown as black and greendotted lines, respectively. b, schematic presentation of the binding interactions between RKI-1447 and the ATP site. c, model of RKI-1313 bound to thebinding site of RKI-1447.

RKI-1447, a Potent ROCK Inhibitor with Antitumor Activity

www.aacrjournals.org Cancer Res; 72(19) October 1, 2012 5027




the kinase domain of human ROCK1 (residues 6-415). The RKI-1447-ROCK1 complex was crystallized in space group C2221with 2 ROCK1 dimers in the asymmetric unit. The structurewas refined to 2.9 Å with Rcryst and Rfree values of 22.8% and29.0%, respectively. Figure 1C shows the overall structure of thedimer and the interactions that RKI-1447 establishes in theATP binding site. Specifically, RKI-1447 is a Type I inhibitorthat binds to the hinge region through a hydrogen bond formedbetween the pyridine ring nitrogen and the main chain amideNH of Met156. The inhibitor extends from the hinge regionalong Asp216 of the DFGmotif towards the b-turn comprisingresidues Gly85–Gly88. The urea moiety interacts with thee-amino group of Lys105 and the carboxyl group of Asp216.The phenol hydroxyl is in hydrogen bonding distance with themain chain carbonyl oxygen of Gly85. The pyridine, thiazole,and phenol moieties of the inhibitor form multiple van derWaals (hydrophobic) interactions with surrounding residues(3.4 Å < d < 4 Å). The network of noncovalent bindinginteractions between RKI-1447 and ROCK1 together with theextended, low energy conformation of the inhibitor, explain thehigh inhibitory potency of RKI-1447 towards ROCKs. Thisbinding mode also explains the weak inhibitory activity of theanalogue RKI-1313. Introduction of the methoxy group in paraposition of the phenyl ring is likely to cause substantial sterichindrance with Gly88 and Phe87 [Fig. 1C (panel (c)]. Inaddition, elimination of the hydroxyl group from the metaposition of the phenyl ring will result in the loss of hydrogenbonding potential with the main chain carbonyl of Gly85.

RKI-1447 is much more potent than RKI-1313 atinhibiting the phosphorylation of the ROCK substratesMLC-2 and MYPT-1 in human cancer cells

We next determined the ability of RKI-1447 and RKI-1313 toinhibit ROCKs in intact human cancer cells. To this end, wedetermined the effects of RKI-1447 and RKI-1313 on thephosphorylation levels of 2 ROCK substrates: MLC-2 andMYPT-1. This was carried out by treating cells with variousconcentrations of the compounds and processing the cells forWestern immunoblotting to determine their effects on thelevels of P-MLC-2, P-MYPT-1, totalMLC-2, and totalMYPT-1 asdescribed under Materials and Methods section. Figure 2Ashows that RKI-1447 treatment of MDA-MB-231 human breastcancer cells decreased the levels of P-MLC-2, but not totalMLC-2, in a concentration-dependent manner with significanteffects starting at 100 nmol/L. RKI-1313 did not decrease P-MLC-2 at 10mmol/L consistentwith itsweak inhibitory activityagainst ROCK1 and ROCK2 in vitro (Fig. 1B). RKI-1447 alsodecreased the levels of P-MYPT-1 in MDA-MB-231 cells in adose-dependent manner (Supplementary Fig. S1). Similarresults were obtained with another human breast cancer cellline, MDA-MB-468, where RKI-1447 decreased the levels ofboth P-MLC-2 and P-MYPT-1 in a dose-dependent manner(Supplementary Fig. S1). Furthermore, RK1-1447 but not RKI-1313 inhibited the levels of P-MYPT-1 in a concentration-dependent manner in H1299 human lung cancer cells (Fig.2B). Figure 2A also shows that RKI-1447 had no effects on thephosphorylation levels of Akt,Mek, and S6 suggesting that RKI-

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Figure 2. RKI-1447 but not RKI-1313 inhibits selectively thephosphorylation of MLC-2 andMYPT-1. MDA-MB-231 (A) andH1299 (B) cells were treated withRKI-1447 or RKI-1313 andprocessed for Western blottingas described in Materials andMethods. GSK-429286 andY-27632 were used as controls.Data are representative of 2independent experiments.

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1447 is selective for ROCK over kinases that phosphorylate Akt(i.e., mTORC2), Mek (i.e., PAK), and S6 (i.e., S6K).

RKI-1447 inhibits LPA-induced actin stress fiberformation but not PDGF-induced lamellipodiaformation or bradykinin-induced filopodia formation.The ability of LPA to induce actin stress fiber formation is

known to be mediated by activation of the RhoA/ROCKpathway whereas the ability of PDGF and bradykinin to inducelamellipodia and filopodia is known to be mediated by theRAC1/PAK and the CDC42/PAK pathways, respectively. Wereasoned that if RKI-1447 is selective for ROCKs, then it shouldonly inhibit LPA-induced actin stress fiber formation but notlamellipodia and filopodia formation by PDGF and Bradykinin.To this end, we starved NIH3T3 cells and treated them witheither vehicle or RKI-1447 before stimulation with either LPA,PDGF or Bradykinin, stained the cells with phalloidin toevaluate their morphological changes as described underMaterials and Methods section. Figure 3A shows that starvedcells contain no actin stress fibers, filopodia or lamellipodia.Stimulation with LPA resulted in actin stress fiber formationand this was blocked by RKI-1447 but not RKI-1313 treatment.Stimulation with PDGF resulted in the formation of lamelli-podia and this was not affected by RKI-1447 treatment (Fig.3B). Similar results were obtained with Bradykinin where RKI-1447 had no effects on bradykinin-induced filopodia formation

(Fig. 3B). These results further support the selectivity of RKI-1447 as a ROCK inhibitor.

RKI-1447 is much more potent than RKI-1313 atinhibiting anchorage-independent colony formation,migration and invasion of breast cancer cells

The results presented in Figs. 1–3, and Supplementary Fig.S1 clearly showed that RKI-1447 is a potent and selectiveROCK1 and ROCK2 inhibitor capable of reaching its targetand inhibiting selectively the phosphorylation of the ROCKsubstrates MLC-2 and MYPT-1. Because of the prominent rolethat ROCKs play in adhesion, migration and invasion wereasoned that inhibition of ROCKs should result in suppressionof these hallmarks of malignant transformation. To this end,MDA-MB-231 cells were treatedwith various concentrations ofRKI-1447 and its less active analogue RKI-1313 and processedfor anchorage-independent tumor colony formation in softagar, migration using wound healing assays and invasion usingBoyden Chamber assays as described under Materials andMethods section. Figure 4 shows that treatment of MDA-MB-231 cells with RKI-1447 hampered their ability to form soft agarcolonieswith an IC50 value of 709 nmol/L. In contrast, RKI-1313up to 30mmol/L had little effect. Furthermore, in the absence ofRKI-1447, MDA-MB-231 cells migrated after being scratchedand filled the wound created by the scratch (Fig. 5). Treatmentwith RKI-1447 inhibited this scratch-inducedmigration at both

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Figure 3. RKI-1447 suppresses LPA-induced actin stress fiber but not PDGF-induced lamellipodia or bradykinin-induced filopodia formation. A, starved NIH-3T3 cells were treated with either vehicle, RKI-1447, or RKI-1313 before treatment with LPA. B, starved NIH-3T3 cells were treated with either vehicleor RKI-1447 before treatment with LPA, bradykinin or PDGF, and then stained with phalloidin as described inMaterials andMethods. Data are representativeof 2 independent experiments.






1 and 10 mmol/L (Fig. 5); whereas RKI-1313 had minimal effecton migration consistent with its much less potent activityagainst ROCKs in vitro and in intact cells. Finally, treatment ofMDA-MB-231 cells with RKI-1447 inhibited their serum-induced invasion by 53% and 85% at 1 and 10 mmol/L, respec-tively (Fig. 6). Consistent with the soft agar and migrationassays, RKI-1313 had little effect on inhibiting MDA-MB-231tumor cell invasion (Fig. 6). It is important to point out thatRKI-1447 at these concentrations (1 and 10 mmol/L) was morethan 10-fold less active at inhibiting anchorage-dependentproliferation/survival of MDA-MB-231 breast cancer cellsas measured by MTT assay (see Supplementary Fig. S2),indicating that the ability of RKI-1447 to inhibit soft agar

growth, migration and invasion is most likely not due to itsability to inhibit anchorage-dependent proliferation/survival.Consistent with this, RKI-1447 concentrations as high as10 mmol/L did not induce apoptosis (PARP cleavage) and hadlittle effect on cell-cycle progression (Supplementary Fig. S3).

RKI-1447 inhibits mammary tumor growth in vivoThe results described in Figs. 1–6 show that RKI-1447 is a

potent and selective ROCK inhibitor that suppresses ROCK-driven cytoskeleton re-organization, signaling,migration, inva-sion, and anchorage-independent tumor cell growth of breastcancer cells. Although these results are encouraging they wereall carried out in cultured cells. We next determined whetherRKI-1447 can inhibit tumor growth in an in vivo environment.To this end, we used the ErbB2 mammary cancer transgenicmouse model. In this model, the mice harbor the ErbB2 geneunder the control of the MMTV promoter, and spontaneouslydevelop tumors in their mammary tissues. Mammary tumorswere measured beginning at the time of tumor onset andtreatment (once a day for 14 days) with vehicle (20% HPCD) orRKI-1447 (200 mpk/day) began when tumor volumes reached75 to 2,300 mm3. A wide range of tumor volumes was used toensure that responses were not volume dependent. Supple-mentary Tables S1 and S2 show initial volume before treat-ment, final volume at the end of treatment and the percentagechange in tumor volume for 57 tumors from 28 mice treatedwith RKI-1447 and 14 tumors from 13 mice treated withvehicle. The percentage change was calculated from the tumorvolume on the last day of treatment relative to the volume onthe day of initiation of treatment. Figure 7A shows the per-centage change in tumor volume for each individual tumorwhereas Fig. 7B shows the average percent change for eachtreatment group. Tumors from mice treated with vehicleincreased in size with an average percent change in tumorvolume of 68.3% (Fig. 7B). In contrast, tumors from micetreated with the RKI-1447 increased in size with an averagepercent change in tumor volume of only 8.8% (P < 0.0001).Thus, RKI-1447 inhibited mammary tumor growth by 87%([1� (8.8/68.3)] � 100), and on average the mammary tumorsfrom RKI-1447 treated mice were 7.7-fold smaller comparedwith those tumors frommice treatedwith the vehicle control. Acloser look at the data from Supplementary Table 1 and Fig. 7A

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Figure 5. RKI-1447 but not RKI-1313 inhibits migration of breast cancercells. MDA-MB-231 cells were treated with RKI-1447 or RKI-1313 andprocessed for scratch-wound healing migration assays as described inMaterials and Methods. Data are representative of 3 independentexperiments.

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lead us to divide the RKI-1447 treated group into 3 subgroups:those tumors that regressed (tumorswhose volumes decreasedby 10% or greater—see representative growth curve in Fig. 7Ctop right panel), those tumors that continued to grow (tumorswhose volumes increased by 10% or more-see representativegrowth curve in Fig. 7C bottom right panel) and those tumorswhose growth was inhibited (tumors whose volume decreasedor increased by less than 10%—see representative growth curvein Fig. 7C bottom left panel). Figure 7B shows that RKI-1447treatment caused 20 tumors (35% of the total of 57 tumorstreated) to regress, 14 tumors (25%) to be growth inhibited andhad no effect on the growth of 23 tumors (40%). The 20 tumorsthat regressed did by an average of 35.1%. The 23 tumors thatwere not affected grew on average by 51.8% similar to thevehicle treated tumors (average of 68.3%; Fig. 7B). Finally, RKI-1447 treatment inhibited the growth of 14 tumors with achange in volume of only 1.04%. Therefore, as compared withthe vehicle treated groupwhere tumors grew on average 68.3%,the tumors in this group grew only by 1.04% indicating a 98%([1 � (1.04/68.3)] � 100) growth inhibition. Thus, in themajority (60%) of tumors, RKI-1447 treatment caused eitherregression (in 35% of tumors) or tumor growth inhibition (in25% of tumors). RKI-1447 or vehicle treatments did not resultin mouse weight loss (see Supplementary Tables S3 and S4).

DiscussionBreast cancer accounts for 450,000 of cancer deaths and is

the leading cause of cancer death in women worldwide (42). Aswith other cancers, metastasis is the major reason breastcancer patients succumb to this disease. The role of the Rhofamily of GTPases in malignant transformation, and particu-larly the role of RhoA and RhoC in migration, invasion andmetastasis has been well documented both in preclinicalmodels as well as in human patients (4–6, 8–12). The abilityof RhoA and RhoC to inducemigration and invasion is believedto be mediated primarily through binding and activation oftheir effectors ROCK1 andROCK2 (ROCKs; refs. 14–19, 32). TheRhoA/C-ROCKs pathway has been shown to be elevated in

metastatic tumors as compared with their nonmetastaticcounter parts, and this has been associated with poor prog-nosis, resistance to therapy and shorter survival time of breastcancer patients (26, 31). In this manuscript we describe thediscovery of RKI-1447 as a potential anti-invasive and antitu-mor agent in breast cancer. As control we have used RKI-1313 aclosely related analogue that is amuchweaker ROCK inhibitor.RKI-1447 inhibited potently ROCKs and blocked selectivelyROCK-dependent signaling and morphological changes. Fur-thermore, RKI-1447 was muchmore effective than RKI-1313 atinhibiting breast cancer anchorage-independent growth,migration and invasion. Finally, RKI-1447 significantly inhib-ited mammary tumor growth in animal models.

The co-crystal structure of the RKI-1447-ROCK1 complexrevealed that RKI-1447 belongs to the Type I class of kinaseinhibitors. The high potency of RKI-1447 is reflected in anelaborate network of noncovalent interactions in the bindingsite that involve all inhibitormoieties. This provides a very tightfit of the entire inhibitor molecule and the ATP site over adistance of �18 Å. Consequently, a slight modification in thephenyl ring, which is opposite to the hinge-binding pyridine,disturbs the binding interactions between the inhibitor andROCK1 substantially and leads to a significant loss of potencyas seen with RKI-1313. Indeed, the hydrogen bond between thephenyl meta-hydroxyl in RKI-1447 is lacking in RKI-1313, andthis coupled with the steric hindrance caused by the para-methoxy in RKI-1313 offers a possible explanation for the largedifference in potency between RKI-1313 and RKI-1447.

Our studies also showed that RKI-1447 is selective andinhibited the phosphorylation levels of ROCK substrates suchas MLC-2 and MYPT-1 but not substrates for PAK (Mek), S6K(S6), and mTORC2 (Akt). This selectivity translated into highlyspecific morphological effects of RKI-1447. Indeed, RKI-1447inhibited LPA-induced, ROCK-dependent, actin stress fiberformation but not the PAK-dependent, PDGF- and bradyki-nin-induced lamellipodia and filopodia formation, respective-ly. This inhibition of stress fiber formation and the resultingdisruption of the cytoskeleton organization suggested that

Figure 6. RKI-1447 but not RKI-1313 inhibits invasion of breastcancer cells. MDA-MB-231 cellswere treated with RKI-1447 or RKI-1313 and processed for invasionassays as described in Materials andMethods. S, serum. Data showaverages of 3 independentexperiments.

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0.20

Serum +++++-

*

***

VehicleRKI-1447 RKI-1313

10 µmol/L1 µmol/L10 µmol/L1 µmol/L

* P = 0.0001

*** P = 0.444** P = 0.0009

**

****

**** P = 0.1

O.D

at

540 n

m






RKI-1447might have effects on cellmotility, invasion and othercancer cell characteristics required for metastasis. Our resultsclearly show that the ability of breast cancer cells to formcolonies in an anchorage-independent manner in soft agar ishampered by RKI-1447. Similarly, RKI-1447 was very potent atinhibiting breast cancer cell migration in a wound healingassay as well as breast cancer cell invasion through matrigel.In contrast, RKI-1313 was much less effective at inhibitinganchorage-independent growth, migration and invasion,consistent with it its very weak activity to inhibit ROCKsin vitro and in intact cells. The fact that RKI-1313 and RKI-1447 are very close analogues structurally coupled with thefact that their ability to inhibit ROCKs in vitro and in vivocorrelates with their biological effects suggests that theability of RKI-1447 to inhibit anchorage-independentgrowth, migration and invasion is mediated by its abilityto inhibit ROCKs. Furthermore, the ability of RKI-1447 toinhibit anchorage-independent growth, migration and inva-sion is most likely not due to inhibition of tumor prolifer-ation because the concentrations that inhibited the former

had little effects on the later. This is not surprising becauseseveral studies have shown that the involvement of ROCKsin oncogenesis is primarily by mediating invasion andmetastasis and not proliferation of tumors (22–30).

The receptor tyrosine kinase family plays a major role inbreast cancer oncogenesis. In particular, ErbB2 is a significantbiomarker that predicts poor prognosis, therapy resistance andpoor survival of breast cancer patients. We have used atransgenic mouse model where mammary tumor oncogenesisis driven by ErbB2 to evaluate the effectiveness of RKI-1447 invivo. This model has extensively and successfully been used toevaluate the effects of a variety of agents on mammary tumor-igenesis. For example, this model was used to show that thesmall molecule tyrosine kinase inhibitors lapatinib that targetsboth ErbB2 and EGFR (43) and gefitinib that targets EGFR (44)inhibited mammary tumorigenesis. Cytotoxic agents such astheHSP90 inhibitor 17-AAG (45) and carboplatin (46) as well asmonoclonal antibodies against ErbB2 (47) were also veryeffective in this model. In addition, this model was alsosuccessfully used by us to show that the combination of the

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Figure 7. Effects of RKI-1447 ontumor growth in ErbB2 mammarytumor transgenic mouse model.Mice bearing mammary tumorswere treated (once a day for 14days) with vehicle (20% HPCD) orRKI-1447 (200 mpk/day). A, showsthe percent change in tumorvolume for each individual tumor.B, shows the average percentchange for each treatment group.C, shows representative tumorgrowth curves from vehicle treatedmice (top left) as well as from RKI-1447 treated mice where tumorsregressed (top right), were growthinhibited (bottom left), or continuedto grow (bottom right). Statisticalsignificance was evaluated bydetermining the P value for thedifferences between vehicle andRKI-1447 treated groups:�, P ¼ 0.0001 (all tumors);��, P < 0.0001 (tumor regression);��, P < 0.0001 (tumor inhibition);and ��, P ¼ 0.2928 (tumorgrowth).

Patel et al.





Akt inhibitor TCN-P and the farnesyltransferase inhibitorZarnestra is more effective than single agent treatment (41).In this study, this mammary tumor model was used to showthat RKI-1447 treatment of 28 mice (57 tumors) resulted in anaverage tumor growth inhibition of 87%; on average tumorsfrom mice treated with RKI-1447 were 7.7-fold smaller thanthose frommice treated with vehicle. To our knowledge this isthe first demonstration that a ROCK inhibitor affects thegrowth of mammary tumors in an ErbB2-driven breast cancermodel. The draw back of this antitumor in vivo study is that itdid not investigate metastasis directly. However, the growth ofthese mammary tumors as a 3-dimensional mass depends ontheir ability to grow in an anchorage-independent mannerthat is ROCK-dependent and that was potently inhibitedby RKI-1447 in the soft agar studies.The effect of RKI-1447 in this in vivo model was heteroge-

neous with 60% of the 57 tumors treated being sensitive and40% resistant. Among the 34 sensitive tumors, 20 regressed,and 14 were growth inhibited. There are several potentialmechanisms that could account for these heterogeneousresponses. One possibility could be related to expression levelsof certain biomarkers known to influence resistant/sensitivityof beast tumors. For example, the expression levels of estrogenreceptors (ER) and progesterone receptors (PR) is often asso-ciated with sensitivity/resistance to chemotherapeutic agentsin both preclinical and clinical settings (48–50). However, thismechanism is unlikely to contribute to the response differ-ences that we have seen as it has been shown that themammary tumors in this MMTV-Her2 model all express verylow levels of ER and are PR negative (51). Another mechanismthat could account for the differences in response to RKI-1447could be related to differences in the epithelial-to-mesenchy-mal transition (EMT) status. In deed, EMT has been shown togenerate cells with properties of stem cells (52), and these canbecome resistant to treatment (53). Therefore, it would be ofgreat interest to evaluate the EMT status in responders com-pared with nonresponders to RKI-1447. Furthermore, thetumor stroma plays a pivotal role in breast tumor angiogenesisand metastasis (54), and differences in the relative tumorstroma/cancer cell mass could contribute to differences inresponses to RKI-1447. It is also possible that tolerance,pharmacokinetics and bioavailability influenced RKI-1447response, but this is unlikely because these mice are in-bred

and should behave similarly with regards to these parameters.Understanding themechanisms of resistance will pave the wayto designing combination therapies of RKIs with other anti-signaling agents including ErbB2 inhibitors and ErbB2 neu-tralizing antibodies.

In summary, we have designed a highly potent and selectiveROCK Type 1 inhibitor that binds to the ATP site of ROCK 1 atthe hinge region and the DFG motif. RKI-1447 but not itsclosely related analogue RKI-1313 was highly effective atinhibiting ROCK-dependent signaling, cytoskeletal changes,anchorage-independent colony formation, migration, andinvasion. Finally, RKI-1447 inhibited tumor growth and causedtumor regression in animal models with little side effects. Ourstudies warrant further advanced preclinical studies to deter-mine the suitability of RKI-1447 as a potential clinicalcandidate.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R.A. Patel, N.J. Lawrence, S.M. SebtiDevelopment of methodology: R.A. Patel, K.D. Forinash, N.J. Lawrence, S.M.SebtiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.A. Patel, Y. Sun, N. Sun, M.P. Martin, E. SchonbrunnAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.A. Patel, Y. Sun, N. Sun, M.P. Martin, E. Schon-brunn, N.J. Lawrence, S.M. SebtiWriting, review, and/or revision of themanuscript: R.A. Patel, M.P. Martin,N.J. Lawrence, S.M. SebtiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.A. Patel, K.D. Forinash, S.M. SebtiStudy supervision: N.J. Lawrence, S.M. SebtiDesign and synthesis of the inhibitors RKI1447 and RKI1313: R. Pireddu

AcknowledgmentsThe authors would like to thank the Moffitt Cancer Center Chemical Biology

Core, the Analytical Microscopy core and the animal facility.

Grant SupportThis work was supported by NCI grant U19 CA 067771.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 9, 2012; revised June 22, 2012; accepted July 10, 2012;published OnlineFirst July 30, 2012.

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2012;72:5025-5034. Published OnlineFirst July 30, 2012.Cancer Res Ronil A. Patel, Kara D. Forinash, Roberta Pireddu, et al. with Anti-Invasive and Antitumor Activities in Breast CancerRKI-1447 Is a Potent Inhibitor of the Rho-Associated ROCK Kinases

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