ROCK1 and ROCK2 Are Required for Non-Small Cell Lung ... · ROCK1 and ROCK2 Are Required for Non-Small Cell Lung Cancer Anchorage-Independent Growth and Invasion Dominico Vigil1,

Therapeutics, Targets, and Chemical Biology

ROCK1 and ROCK2 Are Required for Non-Small Cell LungCancer Anchorage-Independent Growth and Invasion

Dominico Vigil1, Tai Young Kim1, Ana Plachco1, Andrew J. Garton4, Linda Castaldo4, Jonathan A. Pachter4,Hanqing Dong4, Xin Chen4, Brianna Tokar4, Sharon L. Campbell1,2, and Channing J. Der1,3

AbstractEvidence is emerging that the closely related ROCK1 and ROCK2 serine/threonine kinases support the invasive

andmetastatic growth of a spectrum of human cancer types. Therefore, inhibitors of ROCK are under preclinicaldevelopment. However, a key step in their development involves the identification of genetic biomarkers that willpredict ROCK inhibitor antitumor activity. One identified mechanism for ROCK activation in cancer involves theloss of function of the DLC1 tumor suppressor gene, which encodes a GTPase activating protein (RhoGAP) for theRhoA and RhoC small GTPases. DLC-1 loss may lead to hyperactivation of RhoA/C and its downstream effectors,the ROCK kinases. We therefore determined whether loss of DLC-1 protein expression identifies non-small celllung carcinoma (NSCLC) cell lines whose growth and invasion phenotypes are sensitive to ROCK inhibition. Weidentified and characterized a novel small molecule pharmacologic inhibitor of ROCK and additionally appliedgenetic approaches to impair ROCK1 and/or ROCK2 activity, and we determined that although NSCLCanchorage-dependent growth was ROCK-independent, both anchorage-independent growth and Matrigelinvasion were ROCK-dependent. However, loss of DLC-1 expression did not correlate with ROCK activationor with OXA-06 sensitivity. Unexpectedly, suppression of ROCK1 or ROCK2 expression alone was sufficient toimpair anchorage-independent growth, supporting their nonoverlapping roles in oncogenesis. Mechanistically,the block in anchorage-independent growth was associated with accumulation of cells in the G0–G1 phase of thecell cycle, but not increased anoikis.We conclude that ROCKmay be a useful therapeutic target for NSCLC.CancerRes; 72(20); 5338–47. �2012 AACR.

IntroductionThere is considerable and growing evidence for the impor-

tance of the ROCK serine/threonine kinases (ROCK1/ROKband ROCK2/ROKa) kinases in oncogenesis (1, 2). The highlyrelatedROCK1andROCK2kinases functionaskeydownstreameffectors of the RhoA small GTPase (3). ROCK regulates diversecellular processes such as actomyosin contractility, focaladhesion assembly, cytokinesis, and cell proliferation. ROCKhas also been implicated in colorectal, breast, gastric, andglioblastoma proliferation or anchorage-independent growth(4–7) and in prostate, bladder, fibrosarcoma, melanoma, and

hepatocellular cancer metastatic growth (8–13). An evaluationof ROCK as a therapeutic target for lung cancer has notbeen done.

Although preclinical development of ROCK inhibitors isongoing, a number of issues need to be resolved to facilitatetheir clinical development. First, genetic or biochemical deter-minants that identify cancers responsive to ROCK inhibitorsneed to be identified. Second, biomarkers that correlate withinhibitor antitumor response are needed for effective clinicalevaluation. Although the phosphorylation status of key sub-strates of ROCK is widely used, their value as biomarkers forROCK inhibition remains unresolved. Third, the majority ofstudies implicating ROCK in cancer growth used the Y-27632ROCK inhibitor (1, 2). Because Y-27632 can inhibit otherprotein kinases in vitro, whether the antitumor activitiesascribed to this inhibitor are target-based is unresolved.

One candidate molecular determinant for ROCK inhibitorsensitivity is the loss of expression of the DLC1 tumor sup-pressor (14). DLC1 mRNA expression was lost in 95% of non-small cell lung carcinoma (NSCLC) patient tumors and 58% ofNSCLC cell lines (15, 16). Due at least in part through itsfunction as a Rho GTPase activating protein and thus negativeregulator of RhoA and the related RhoB and RhoC, restorationof DLC-1 expression in DLC1 deficient NSCLC lines resultedin reduction of cell migration, proliferation, anchorage-inde-pendent growth, in vitro invasion, and tumorigenicity in nude

Authors' Affiliations: 1Lineberger Comprehensive Cancer Center;2Departments of Biochemistry and Biophysics, 3Pharmacology, Universityof North Carolina at Chapel Hill, Chapel Hill, North Carolina; and 4OSIPharmaceuticals, Farmingdale, New York

Current addresses: J. Pachter, Verastem, Inc., Cambridge, Massachu-setts. D. Vigil, Department of Biochemistry, Vanderbilt University School ofMedicine, Nashville, Tennessee.

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

Corresponding Author: Channing J. Der, University of North Carolina atChapelHill, LinebergerComprehensiveCancerCenter,ChapelHill,NC27599-7295. Phone: 919-966-5634; Fax: 919-966-9673; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2373

�2012 American Association for Cancer Research.

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mice (16–18), supporting its role as a tumor suppressor (19).It is well established that aberrant RhoA and RhoC activationcan promote tumorigenic, invasive and metastatic growth(20–22). Thus, by analogy to the loss of the neurofibrominRasGAP or the tuberous sclerosis RhebGAP in cancer (23, 24),loss of DLC-1 results in hyperactivation and persistent RhoA/Ceffector signaling (15, 25). However, such as Ras, Rho GTPasesare not tractable molecules for drug discovery (14). Instead,in further analogy to Ras, where inhibitors of the Raf-MEK-ERKeffector protein kinase pathway are being considered for anti-Ras drug development (26), inhibitors of RhoA/C downstreameffector protein kinases, in particular ROCK, may also beattractive therapeutic targets for DLC1-deficient NSCLC. Insupport of this possibility, ectopic expression of DLC-1 sup-pressed ROCK activation and ROCK-dependent motility inDLC-1 deficient hepatocellular carcinoma cell lines (27), andsuppression of DLC-1 expression sensitized liver cancer cellsto reduced colony formation by pharmacologic inhibition ofROCK (19).In light of the frequent loss ofDLC1 expression in NSCLC, we

speculated thatDLC1 loss-induced activation of RhoA/Cwill inturn cause ROCK activation-driven NSCLC tumorigenic andmalignant growth. However, previous studies implicatingROCK in cancer growth have relied primarily on the use ofY-27632 ATP competitive ROCK1/2 kinase inhibitor, which hasadditional off-target inhibitory activity for other kinases suchas PKN and MSK1 (28). To offset this concern, we used astructurally distinct and more potent and selective smallmolecule ROCK1/2 inhibitor together with RNA interferencedepletion of ROCK1/2 expression to validate a role for ROCK inDLC1-deficient NSCLC growth. We determined that NSCLCanchorage-dependent growth was ROCK-independent, butanchorage-independent growth and Matrigel invasion wereROCK-dependent. However, loss of DLC-1 expression did notcorrelate with ROCK dependence. ROCK inhibition of growthwas associated with inhibition of cell-cycle progression ratherthan enhancement of cell death. Our studies provide validationof ROCK for NSCLC therapy.

Materials and MethodsIdentification and characterization of the OXA-06 ROCKinhibitorROCK1/2 cDNA sequences were subcloned into a bacu-

lovirus expression vector for protein expression as a C-terminal fusion protein with His6 in insect cells. Theexpressed protein (comprising residues 2–238 of ROCK1fused to residues 255–548 of ROCK2) was purified and usedin a fluorescence polarization assay-based high-throughputscreening campaign to identify ROCK kinase inhibitorswithin OSI Pharmaceuticals compound library. The screen-ing assay buffer consisted of 10 mmol/L Tris HCl (pH 7.2), 10mmol/L MgCl2, 0.1% bovine serum albumin, 1 mmol/Ldithiothreitol (DTT) and contained 100 nmol/L substratepeptide (50-FAM-AKRRRLSSLRA-COOH), 1 mmol/L ATP, and12.5 nmol/L ROCK kinase domain. A series of azaindole-based compounds was selected for medicinal chemistryoptimization, and synthetic routes for this series are

described within patent application WO2007084667. Todetermine the IC50 values of OXA-06 and Y-27632 againstROCK, we used the above fluorescence polarization assaywith 12.5 nmol/L ROCK fusion protein and 1.4 mmol/L ATP(roughly the measured Km value) as well as at 100 mmol/LATP. The IC50 for OXA-06 was measured 18 times, with theaverage and SD reported, and Y-27632 was measured 3 times,with the average and SD reported.

Protein kinase selectivity assaysKinase selectivity assays were conducted using ProfilerPro

Kinase Selectivity Kits (Caliper Life Sciences, Inc.) according tothe manufacturer's instructions. The extent of inhibition byeach compound ismeasured directly by quantifying the level ofboth unphosphorylated and phosphorylated peptide substrateafter electrophoretic separation. Assays were conducted atroom temperature following a 1-hour preincubation of com-pounds with the enzymes. Each assay was conducted on 2separate occasions.

Quantitative assays of MYPT1 phosphorylation and cellmigration in PANC-1 cells

Analysis of MYPT1 phosphorylation in PANC-1 cells byquantitative ELISA was conducted as described previously(29). PANC-1 cell migration was quantitated using 96-wellmodified Boyden chamber migration assay plates (Chemi-con). Cells were applied to the upper chamber in growthmedium supplemented with 0.5% fetal calf serum (FCS),either in the presence or absence of inhibitor, and the cellswere allowed to migrate toward growth medium supple-mented with 10% FCS for 16 hours. The number of migratedcells was then quantitated by labeling with Cyquant GR(Invitrogen).

Cell cultureNSCLC lines were obtained directly from the American Type

Culture Collection and grown in RPMI-1640 supplementedwith 10% FBS.

Soft agar colony formation, Matrigel invasion, andanoikis assays

Anchorage-independent growth soft agar assays were doneas previously described (30). For inhibitor treatments, vehicle(DMSO, dimethyl sulfoxide) or OXA-06 was also added to themedium at the final indicated concentrations. For siRNAtreatments, cells were suspended in soft agar 48 hours post-transfection and maintained at 37�C for 14 to 30 days, whenviable colonies were stained with the MTT viability stain. Thetotal number per plate of viable colonies of more than 10 cellswere quantified by counting the number of colonies in 5representative fields of view within each plate. Results areexpressed as mean � SD.

Invasion assays were conducted with Growth FactorReduced Matrigel Invasion Chambers (BD Biosciences)according to the manufacturer's protocol. For inhibitor treat-ments, vehicle or OXA-06 were also added to the upperchamber. RPMI-1640 containing 3% FBS as a chemoattractantwas added to the bottom well. Cells were allowed to invade for

ROCK as a Potential Therapeutic Target in NSCLC

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22 hours at 37�Cand thennoninvaderswere removed. Invadingcells were fixed and stained with the Diff-Quik Stain Set (DadeBehring Inc.). Five fields were counted for each chamber, andthe total number of cells counted per chamber was used forcalculating the average number of invading cells. Results areexpressed as mean � SD.

For the MTT anchorage-dependent growth viability assay, 2� 103 cells per well were seeded in 96-well plates in octuplet.For ROCK inhibitor studies, growth medium supplementedwith vehicle or OXA-06 was also added to the wells. Cells wereincubated for 2 days for the siRNA and 4 days for inhibitoranalyses. Optical density at 560 nm was recorded and normal-ized to either vehicle control nonspecific siRNA control. Valuesshown are the mean � SD.

For the anoikis assays, cells were plated on Ultra-LowAttachment plates (Corning) with RPMI-1640 plus 10% FCS,and either vehicle, 10 mmol/L OXA-06, or 10 mmol/L stauros-porine (positive control for induction of apoptosis). Cells withstaurosporine were harvested and lysed after 6 hours. All othercells were harvested and lysed after 48 hours, and then immu-noblotted for PARP and caspase-3 as described below in theWestern blot analyses section.

Flow cytometry cell-cycle analysisNSCLC cells were plated on Ultra-Low Attachment Plates

(Corning) with RPMI-1640 supplemented with 10% FCS, andeither vehicle or 10 mmol/L OXA-06 for 48 hours. After treat-ment, cells were collected, washed twice with PBS and fixed in70% ethanol overnight. After centrifugation, cells were stainedwith a buffer containing 20 mg/mL propidium iodide (Roche)and 200 mg/mL RNAse A (Qiagen) in PBS. Analyses wereconducted on a CyAn flow cytometer (Beckman-Coulter).Cell-cycle distribution was determined using FlowJo (TreeStar,Inc.). Experiments were carried out 2 independent times andvalues are shown as means � SD.

Statistical analysisData were analyzed using an unpaired t test. Values are

shown as means � SD. A P value <0.01 was consideredsignificant.

Western blot analysesFor OXA-06 treatment immunoblots, cells were treated with

1 hour with vehicle or the indicated OXA-06 concentration asthis treatment time was previously shown to be sufficient formaximal ROCK inhibition effect (29). The following antibodieswere then used for immunoblot analysis: pMYPT1(T853) (USBiologicals), MYPT1 (Covance), pCofilin (Ser3) and cofilin (CellSignaling Technology), and b-actin (Sigma-Aldrich). Pull-downanalyses to determine steady-state levels of RhoA-GTP weredone as we have described previously using a glutathione S-transferase fusion protein containing the RhoA-GTP bindingdomain of Rhotekin and anti-RhoA antibody (Cell SignalingTechnology; 31). For anoikis immunoblot analyses, suspensioncell cultures were treated with staurosporine (Calbiochem) for6 hours, or with vehicle or OXA-06 for 48 hours. The followingantibodies were then used to detect the indicated proteins:PARP (Cell Signaling Technology), caspase-3 (Cell Signaling

Technology), and vinculin (Sigma-Aldrich). For cell-cycleimmunoblot analyses, suspension cells were treated withvehicle or 2 or 10 mmol/L OXA-06 for 48 hours. The followingantibodies were used to detect: phosphorylated (Ser608) Rb(Cell Signaling Technology), total Rb (Santa Cruz Biotechno-logy), and b-actin. For the siRNA immunoblot analyses, immu-noblot analysis of ROCK1, ROCK2, phospho-MYPT1(T853),MYPT1, phospho-cofilin (Ser3), cofilin, and b-actin was done.

siRNA suppression of ROCK1 and ROCK2 expressionsiRNA oligonucleotides (Stealth siRNA, as we described

previously; ref. 29) were diluted to 20 mmol/L in nuclease-freewater and the transfection was conducted with Lipofecta-mine2000 (Invitrogen) according to manufacturer's protocolsand as described previously (29).

Forty-eight hours after transfection, transfected cellswere harvested for either analyses of anchorage-independentgrowth or for Western blot analyses. Data were analyzedusing a paired t test. Values are shown as means � SD. AP value <0.01 was considered highly significant.

ResultsOXA-06 is a novel and potent ROCK inhibitor

In light of the off-target activities of the Y-27632 ROCKinhibitor, we initiated a high-throughput screening of the OSIPharmaceuticals compound library using recombinant chime-ric ROCK1/2. A series of compounds was identified for medic-inal chemistry optimization, resulting in the identification of apotent ATP-competitive ROCK inhibitor, designated OXA-06,that is structurally distinct from the widely used Y-27632ROCK1/2 inhibitor (Fig. 1). We first compared the in vitropotency of OXA-06 with Y-27632 for ROCK in vitro. OXA-06exhibited an IC50 value of 0.01 � 0.005 mmol/L, althoughY-27632 measured 0.24 � 0.09 mmol/L when measured withan ATP concentration of 1.4 mmol/L, making OXA-06 roughly25-fold more potent in vitro. When conducted at 100 mmol/LATP concentrations, IC50 values of OXA-06 were similarlyabout 25-fold more potent than Y-27632. To evaluate selectiv-ity, we chose 200 nmol/L for OXA-06 and 10 mmol/L forY-27632, concentrations at which roughly 95% of ROCK1 andROCK2 are inhibited by each respective compound. We

Figure 1. OXA-06 is structurally distinct from Y-27632.

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conducted kinase selectivity assays using the ProfilerProKinase Selectivity Kits composed of 216 pharmacologicallyrelevant wild type or mutant protein kinases. Consistent datawere generated for 183 kinases within this analysis (assays thatfailed in one or more assay, or that did not generate datawith coefficient of variation (CV) values below 25% are notincluded; Supplementary Table S1). Clear selectivity differ-ences were noted between the 2 compounds. OXA-06 showedmore than 50% inhibition of 9 out of 183 (5.4%) whereasY-27632 inhibited 17 out of 183 (10.2%; Fig. 1B). Both sharedactivity for 5 additional protein kinases, whereas OXA-06 wasactive on 2 other kinases and Y-27632 was active on 10additional kinases. These results show the greater selectivityof OXA-06 rather than Y-27632. In addition, this degree ofselectivity is comparable or better than highly selective proteinkinase inhibitors (e.g., imatinib, gefitinib) currently in clinicaluse (32, 33). We conclude that the different and improvedkinase inhibition selectivity profile, together with being struc-turally distinct fromY-27632, support the usefulness of OXA-06to investigate the role of ROCK kinase activity in cellularprocesses.We next determined if OXA-06 could block ROCK activity

in intact cells. We determined previously that siRNA suppres-sion of ROCK1 and ROCK2 expression, and additionally treat-ment with 3 different ROCK1/2 inhibitors, reduced the phos-phorylation of MYPT1 at T853 indicating that this site wasROCK-dependent in PANC-1 pancreatic tumor cells (29).PANC-1 migration was also shown previously to be ROCK-dependent (34). We determined that OXA-06 treatment ofPANC-1 cells showed dose-dependent inhibition of MYPT1phosphorylation (IC50 300 nmol/L) and migration in vitro(Supplementary Fig. S1). In comparison Y-23732 showed areduced potency to inhibit MYPT1 phosphorylation (IC50

1.4 mmol/L) and migration.

OXA-06 blocks anchorage-independent growth andinvasion of NSCLC linesWe next used OXA-06 to validate the importance of

ROCK1/2 as therapeutic targets. Western blot analyses of16 NSCLC cell lines determined that all lines express ROCK1and ROCK2 protein (Fig. 2A). Based on previous studies withother cancer types (19, 27), we hypothesized that theabsence of DLC-1 expression might be a predictor of sen-sitivity to inhibition of ROCK1 and/or ROCK2. For theseanalyses, we evaluated the A549, H23, and H358 cell lines,which lack DLC-1 protein expression, and the H1299 andH1703 cell lines that express high levels of DLC-1 (18).Surprisingly, none of the cell lines was inhibited in an-

chorage-dependent proliferation by treatment with 2 or 10mmol/L OXA-06 (Fig. 2B). However, at the highest concen-tration of OXA-06 used, 10 mmol/L, greater than 90% anchor-age-independent growth inhibition was achieved in all thelines tested (Fig. 2C). Although 400 nmol/L was sufficient tocause approximately 90% reduction in the colony formationactivity of the DLC-1 positive H1299 cell line, this concen-tration did not cause statistically significant inhibition of theDLC-1 negative A549 and H358 or the DLC-1 positive H1703cell lines.

Because OXA-06 also inhibited 7 other kinases in vitro(e.g., PKA), we also assessed the ability of Y-27632 to inhibitanchorage-independent growth of H1299 and A549 cells. Inboth lines, Y-27632 also caused a decrease in anchorage-independent growth, suggesting a ROCK-dependent foranchorage-independent growth, although Y-27632 had muchweaker potency than OXA-06 (Fig. 3). This increased potencyof OXA-06 compared with Y-27632 in blocking anchorage-independent growth is consistent with its increased potencyin vitro and in ROCK-dependent cell-based assays (Fig. 1B,Supplementary Table S1 and Supplementary Fig. S1). Similarto their sensitivity to inhibition of anchorage-independentgrowth by OXA-06, both DLC-1 negative and positive NSCLCcell lines showed comparable dose-dependent sensitivitiesto OXA-06 suppression of Matrigel invasion, with approxi-mately 70% inhibition seen at 2 mmol/L (Fig. 2D). Theobservation that Matrigel invasion and anchorage-indepen-dent growth were inhibited at similar concentrations ofOXA-06 is consistent with the inhibitor blocking both tumorphenotypes through inhibition of the same target(s). Weconclude that loss of DLC-1 did not correlate with increasedsensitivity to OXA-06.

One possible basis for the lack of correlation between DLC-1loss of expression and increased sensitivity to OXA-06 is thatDLC-1 expression alone does not determine the steady-statelevel of RhoA activation. The activities of RhoGEFs as well asRhoGAPs will also influence RhoA steady-state levels. Consis-tent with this possibility, we carried out pull down analyses toquantitate the steady-state levels of active RhoA-GTP in a panelof NSCLC cell lines. We found no direct correlation betweenelevatedRhoA-GTP levels the absence ofDLC-1 protein expres-sion (Fig. 2E).

OXA-06 antitumoractivity correlateswith suppressionofcofilin phosphorylation

To investigate the mechanism of OXA-06-mediated inhibi-tion of growth and invasion, we determined if the antitumoractivity of OXA-06 correlated with inhibition of ROCK activity.Two well-characterized ROCK-dependent phosphorylationactivities are direct ROCK phosphorylation of the MYPT1 andthe indirect phosphorylation of cofilin through ROCK phos-phorylation and activation of LIMK1/2. However, as bothMPYT1 and cofilin can be phosphorylated by ROCK-indepen-dent mechanisms, their phosphorylation state may or may notaccurately monitor ROCK activity.

We found that OXA-06 treatment reduced pMYPT1 andpCofilin levels at concentrations that coincided with thatrequired to block anchorage-independent growth. Thesedata are consistent with ROCK inactivation as a key mech-anism for OXA-06 antitumor activity. However, the extent ofphosphorylation reduction and the inhibitor concentrationrequired for this effect varied between different cell lines(Fig. 4). Generally, we found that pCofilin provided themost accurate marker for OXA-06 growth inhibition activityfor all 5 NSCLC cell lines evaluated. For example, H1299 cellswere the most sensitive to OXA-06 colony suppression(Fig. 2C) and pCofilin reduction was seen at the lowestconcentrated used, 80 nmol/L. In contrast, H358 cells were






the most resistant to OXA-06 colony suppression and evenat the highest concentration studied (10 mmol/L), incom-plete reduction in pCofilin was seen. For the remaining 3NSCLC cell lines, 10 mmol/L was required for near-complete

suppression of pCofilin as well as soft agar growth. Incontrast, pMYPT1 levels were less informative, largelybecause 2 cell lines lacked detectable phosphorylation inuntreated cells. For A549, but not H358 cells, this was most

Figure 2. OXA-06 treatment blocks anchorage-independent growth and invasion in NSCLC cell lines, independent of DLC1 status. A, ROCK1 and ROCK2proteins are expressed all NSCLC cell lines. The indicated NSCLC cell lines were lysed and resolved by SDS-PAGE, and then blotted with ROCK isoform-specific antibodies. B, cell viability was measured using the MTT assay after treatment of the indicated cell lines with vehicle (DMSO) or the indicatedconcentrations of OXA-06 for 4 days. Data shown are the optical density (OD) values relative to vehicle and represent themean�SD of octuplet wells and arerepresentative of 2 independent experiments. C, colony formation ofNSCLCcell lines in soft agar in growthmedium supplementedwith vehicle (DMSO) or theindicated concentrations of OXA-06. The number of viable proliferating colonies were stained byMTT and counted after 30 days, except for A549 and H1299cells, which were counted after 14 days. Data shown are the percentage colonies relative to vehicle, are the average � SD of triplicate wells, and arerepresentative of at least 2 independent experiments. D, invasion of the indicated NSCLC cell lines was assayed by Matrigel transwell assays with vehicle(DMSO) or the indicated concentrations of OXA-06 after 22 hours. Data shown are the percentage invaded cells relative to vehicle, are the average � SD oftriplicate chambers and are representative of at least 2 independent experiments. For panels B–D, DLC-1 protein expression as determined by Western blotanalyses is indicated as detectable (DLC-1 positive) or undetectable (DLC-1 negative, as we determined previously). E, elevated steady-state RhoAactivity levels do not correlate with loss of DLC-1 expression. Pull-down analyses, followed by blot analysis for RhoA, was done to measure the level ofactivated RhoA-GTP and total RhoA expression. b-Actin blot analysis was done to verify equivalent total protein loading.

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likely due to the barely detectable levels of MYPT1 ex-pression. Where it could be detected, pMYPT1 did showdose-dependent reduction that coincided with OXA-06 con-centrations needed for growth inhibition. Thus, cofilin phos-phorylation may provide a useful biomarker for ROCKinhibition in NSCLC.

Genetic suppression of ROCK1 and/or ROCK2expression phenocopies the biochemical and biologicactivities of OXA-06Although ROCK1/2 were the protein kinases most potently

inhibited by OXA-06 in vitro (Table 1), it is possible that theeffects of OXA-06 on NSCLC anchorage-independent growthand invasion resulted from inhibition of other kinases. Toconfirm that ROCK kinase inhibition contributes to the anti-tumor activity of OXA-06, we specifically reduced ROCK1 andROCK2 expression with siRNA validated previously to beselective inhibitors of ROCK1 and ROCK2 (29). We used 2independent siRNA sequences each for ROCK1 and ROCK2 in

H1299 cells that caused near complete knockdown of ROCK1and/or ROCK2 protein expression (Fig. 5A). Interestingly,neither ROCK1 nor ROCK2 depletion individually was able tostrongly reduce pMYPT1 or pCofilin, and instead, concurrentdepletion of both was required to reduce pMYPT1 and to alesser extent pCofilin (Fig. 5A). This result shows that bothROCK isoforms are functionally redundant for these signalingactivities.

In contrast, we found that depletion of either ROCK1 orROCK2 alone, using 2 independent siRNA sequences each,was sufficient to potently (�90%) inhibit the anchorage-independent growth of H1299 cells. Concurrent suppressionof both ROCK1 and ROCK2 more effectively suppressedgrowth with a near complete suppression of colony forma-tion (Fig. 5B). Thus, ROCK1 and ROCK2 each contributenonredundant activities to support anchorage-independentgrowth. Finally, we determined that, similar to OXA-06 treat-ment, concurrent depletion of ROCK1 and ROCK2 did notsignificantly impair anchorage-dependent proliferation (Fig.5C). That siRNA for ROCK1 and ROCK2 phenocopied thebiologic activities of OXA-06 suggests that the antitumoractivities seen with OXA-06, despite having other proteinkinase targets, are likely due primarily to inhibition of ROCK1and ROCK2.

OXA-06 stimulates accumulation in G0–G1 phase but notapoptosis in nonadherent NSCLC cells

Although we found that prolonged OXA-06 treatment didnot reduce NSCLC cell line viability on plastic (Fig. 3B), thisresult did not exclude the possibility that OXA-06 renderedNSCLC cells more sensitive to suspension-induced apoptosis(anoikis). We therefore investigated possible apoptotic effectsof OXA-06 during blockade of anchorage-independent growthof NSCLC cells. We saw no evidence for caspase-3 cleavage inA549 orH1299 cells, and only partial PARP cleavage in A549 butnot H1299 cells in suspension cells, even when treated with thehigh concentration of 10mmol/L OXA-06, although the positivecontrol of 10 mmol/L staurosporine showed clear PARP andcaspase-3 cleavage (Supplementary Fig. S2A). Thus increased

Figure 3. OXA-06 exhibits greater potency than Y-27632 for inhibition ofNSCLC anchorage-independent growth. Colony formation of A549 (A) orH1299 (B) NSCLC cell lines in soft agar in growth medium supplementedwith vehicle (DMSO) or the indicated concentrations of OXA-06 or Y-27632. The number of viable proliferating colonies were stained by MTTand counted after 14 days. Data shown are the percentage coloniesrelative to vehicle, are the average � SD of triplicate wells, and arerepresentative of at least 2 independent experiments.

Figure 4. OXA-06 treatment reduces steady-state levels of phospho-cofilin and phospho-MYPT1 in NSCLC lines. DLC-1 negative or positivecell lines were treated with vehicle (DMSO) or the indicatedconcentrations of OXA-06 for 1 hour. Western blot analysis usingphospho-specific antibodies for pMYPT1 (pT853) and pCofilin (pS3) andfor total MYPT1 and cofilin.






anoikis sensitivity is not a mechanism for the decreasedanchorage-independent growth seen with OXA-06.

We next determined if OXA-06 impaired soft agar colonyformation by disruption of cell-cycle progression underanchorage-independent conditions. We used propidiumiodide staining and flow cytometry to investigate possiblecell-cycle distribution effects of OXA-06 on 2 NSCLC cell linestreated with inhibitor in suspension. OXA-06 treatment at 1mmol/L in suspension (a concentration that is around thecellular IC50 value for reduced anchorage-independent growth,invasion, and downstream ROCK target phosphorylation)caused a statistically significant reduction in S-phase and anincrease in G0–G1 (Fig. 6A).

To confirm that ROCK inhibition is required for theseeffects of OXA-06, we used siRNA to reduce ROCK1 andROCK2 protein expression and we found that concurrentsuppression of ROCK1 and ROCK2 expression also caused avery similar cell-cycle phenotype (Fig. 6B). This result sug-gests that these activities of OXA-06 on the cell cycle are dueto inhibition of ROCK. Furthermore, we did not detect anysub-G0 peaks in the analysis of OXA-06 at 1 mmol/L (Fig. 6) orat 10 mmol/L (data not shown), or ROCK1/ROCK2 siRNA(Fig. 6), which when taken together with lack of full caspase-3 or PARP cleavage, excludes anoikis or necrosis as a

mechanism for the ROCK inhibition-dependent block inanchorage-independent growth.

Finally, we evaluated the effects of OXA-06 treatment on 2key regulators of G1 progression, the Rb tumor suppressor andcyclin D1. UsingWestern blot analysis on OXA-06 treated A549or H1299 cells in suspension at 2 or 10 mmol/L, we did notobserve increased Rb phosphorylation at S608 (a site phos-phorylated by cyclin/cyclin-dependent kinase complexes) nordecreased levels of cyclin D1 (Supplementary Fig. S2B).We alsodid analysis for 2 regulators of Rb phosphorylation, p21/CIP1and p27/KIP1, and we found no consistent and significantchanges in their levels of expression upon OXA-06 treatment(data not shown). These results indicate that OXA-06 blocksanchorage-independent growth by causing cell-cycle arrest inG0–G1 through a mechanism involving other regulators of G1

progression.

DiscussionIn light of their role as key effectors of the RhoA and RhoC

small GTPases, which have validated roles in oncogenesis (20,21), ROCK1/2 are being considered as therapeutic targets forcancer (1, 2). The first goal of our study was to rigorouslyvalidate ROCK as a useful therapeutic target for NSCLC. Themajority of previous studies with this goal used Y-27632 or itsclose structural relativesWf-536 or fasudil, which are known toalso potently block the activities of other protein kinases (28).To overcome this limitation with Y-27632, we used a secondnovel ROCK inhibitor. OXA-06 exhibited increased potencyto block ROCK-dependent signaling in cell-based assays, andadditionally exhibited less off-target protein kinase inhibitoryactivities in vitro. Therefore, when used in conjunction with astructurally distinct ROCK inhibitor with a distinct off-targetactivity profile, concurrently with 2 independent siRNAs eachtargeting ROCK1 and/or ROCK2, we feel that our analysesprovide an accurate assessment of ROCK function in lungcancer growth. Generally, RNAi depletion of a protein kinase,where there is a loss of entire protein expression, may notaccurately model pharmacologic inhibition of the catalyticactivity alone, where a kinase-inactive protein persists andmay retain nonkinase functions. However, our finding thatRNAi depletion and pharmacologic inhibition of ROCK exhib-ited essentially identical consequences on NSCLC growthsuggests that the anchorage-independent growth andMatrigelinvasion inhibition seen is because of loss of ROCK proteinkinase function. Furthermore, as discussed below, our findi-ngs that OXA-06 antitumor activity correlated well withOXA-06 inhibition of phosphorylation of known direct andindirect ROCK substrates and that siRNA for ROCK phenocop-ied the effects of OXA-06 on anchorage-dependent and -inde-pendent growth strongly support our conclusion that ROCKkinases represent functionally relevant targets of OXA-06 inNSCLC growth. Thus, although OXA-06 can inhibit otherprotein kinases, our siRNA results argue that the antitumoractivity observed in NSCLC cell lines may be due primarilyto ROCK inhibition. The U.S. Food and Drug Administra-tion (FDA) approval of the dasatinib and sunitinib proteininhibitors for cancer treatment, both of which show activity

Table 1. Protein kinase inhibition profiles ofOXA-06 and Y-27632

Compound

Protein kinase OXA-06 Y27632

ROCK2 95 95ROCK1 94 94PKA 98 29PKG1b 73 68PKG 72 64STK10 58 87PRKX 56 78ARK5 56 1IKKbeta 56 82PKCdelta 36 55PKCeta 27 81PKCepsilon 26 84MST1 19 59RSK2 17 70MNK1 16 78PKCtheta 12 82RSK3 10 82RSK1 6 51PKCbeta1 �3 52

NOTE: Percentage inhibition of a panel of protein kinasesconducted using ProfilerPro Kinase Selectivity Kits with 10mmol/L Y-27632 or 200 nmol/L OXA-06. This table shows allkinases inhibited by >50% by either OXA-06 or Y-27632compiled from Supplementary Table S1. Data shown arerepresentative of 2 independent experiments.

Vigil et al.





(Kd < 100 nmol/L) for greater than 15% of the 290 proteinkinases tested (36), clearly show that multikinase inhibitorscan be developed successfully for therapeutic use. OXA-06 doesnot have sufficient pharmacokinetic/pharmacodynamic prop-erties for use in animal studies (unpublished observations), butit provides a more potent and selective inhibitor than thewidely used Y-27632 compound to study ROCK function in cellculture. More importantly, our results support the discoveryand development of potent and selective ROCK inhibitors inanimal models of NSCLC.

The second goal of our study was to determine if DLC-1 lossof expression is a reliable biomarker for NSCLC tumor cellROCK dependency. In contrast to 2 recent studies that foundROCK important for DLC-1 tumor suppressive effects inhepatocellular carcinoma (19, 27), we found that DLC-1 expres-sing and deficient cells were comparably sensitive to OXA-06and additionally showed comparable levels of phosphorylationof ROCK substrates. This disconnect is not entirely surprisingfor several reasons. First, there exist a multitude of guaninenucleotide exchange factors and GTPase-activating proteins(GAP) for RhoA/C (14). Thus, the steady-state activation ofRhoA-C and ROCKmay not be dictated solely by loss of DLC-1alone. Consistent with this possibility, we found no directcorrelation between the level of DLC-1 protein reduction andincreased cellular RhoA-GTP levels. Furthermore, in studies ofa DLC-1 mutant that failed to associate with focal adhesions,this disrupted subcellular localization was not associated witha loss in the ability to lower total cellular RhoA-GTP levels (37).Thus, it is not surprising that total RhoA-GTP levels are nottightly linked to the level of DLC-1 expression. Second, RhoA/Cmay use non-ROCK effectors to promote tumorigenesis (35).Finally, DLC-1 can also serve as a GAP for other Rho familyproteins and DLC-1 tumor suppressor function involves bothRho-dependent and Rho-independentmechanisms (18). Theseadditional complexities of DLC-1 function may prevent asimple and direct relationship between DLC-1 expression lossand ROCK-dependent growth. Perhaps the combined use ofDLC-1 loss together with hyperphosphorylation of ROCKsubstrates will provide amore reliablemarker to predict ROCKdependency.

The third goal of our study was to determine if the widelystudied substrates for ROCK activity provided accurate bio-chemical markers that correlated with OXA-06-mediatedgrowth inhibition. Although our studies found that cofilin,rather than MYPT1, provided a more accurate biomarker forpan-ROCK inhibitor treatment, our use of isoform-specificsiRNA to selectively silence ROCK1 or ROCK2 found thatinhibition of either isoform alone inhibited anchorage-inde-pendent growth, yet inhibition of both ROCK1 and ROCK2were required to suppress cofilin phosphorylation. Thus,ROCK1 and ROCK2may be functionally overlapping for cofilinphosphorylation but serve nonoverlapping roles in NSCLCgrowth, although whether they are truly functionally distinctremains to be rigorously evaluated. These results support thevalue of investigation of isoform-specific ROCK inhibitors,which may provide reduced off-target activities, and the needfor additional biomarkers for ROCK isoform-selective inhibi-tors. Nevertheless, our observation that combined inhibition ofROCK1 and ROCK2 has strong anticancer activity in NSCLClines supports the further development of ROCK dual-selectiveinhibitors for lung cancer.

One surprising observation was the failure of OXA-06 toimpair anchorage-dependent NSCLC tumor cell growth at aconcentration (10 mmol/L) that potently blocked soft agarcolony formation and Matrigel invasion. This activity beginsto shed some light on the role of ROCK in NSCLC tumorgrowth. When evaluated on cells in suspension, we found thatOXA-06 treatment did not increase apoptosis or necrosis and

Figure 5. Concurrent suppression of ROCK1 and ROCK2 expressiondoes not impair cell viability, but is required to block downstreamphosphorylation but not anchorage-independent growth. H1299NSCLC cells were transfected with nonspecific (NS) siRNA or2 different siRNA sequences targeting ROCK1 or ROCK2. A,Concurrent suppression of ROCK1 and ROCK2 expression blocksphospho-MYPT1 and to a lesser degree phospho-cofilin. After 48hours, lysates were harvested and Western blot analysis wasconducted using the indicated antibodies. B, suppression of ROCK1or ROCK2 expression alone is sufficient to impair anchorage-independent growth. Forty-eight hours posttransfection, cells weresuspended in soft agar. The number of viable proliferating colonieswere stained by MTT and counted after 14 days. Data shown are thepercentage colonies relative to vehicle, are the average � SD oftriplicate wells, and are representative of at least 2 independentexperiments. C, concurrent suppression of ROCK1 and ROCK2expression does not affect cell viability. Forty-eight hoursposttransfection, cells were split at 5 � 103 cells/well and cell viabilitywas measured MTT assay analysis. Data shown are the optical density(OD) values relative to nonspecific siRNA and represent the mean �SD of octuplet wells and are representative of 2 independentexperiments.






instead caused a G0–G1 arrest, although it is possible thatother less well understood types of cell death in combina-tion with the cell-cycle arrest seen are responsible for thestriking inhibition of anchorage-independent growth seenwithROCK inhibition and knockdown. Although our analysesdid not establish a mechanism for why ROCK dependencywas not evident for cells grown on plastic, previous studiessuggest that loss of integrin-dependent signaling events(36, 37) are a likely basis for ROCK dependency for cells insuspension. Finally, how tumor cells will respond to ROCKinhibition within the context of host stromal tissue will bean important future question to address using both pharma-cologic inhibitors and conditional ROCK knockouts in mousemodels of lung cancer.

In summary, our studies provide strong validation of ROCK1and/or ROCK2 as useful targets for NSCLC therapy. Further-more, despite the potentially significant off-target activities ofour newly identified OXA-06 ROCK inhibitor, our demonstra-tion that OXA-06 NSCLC antitumor activity correlates wellwith ROCK inhibition argues that, as with other multikinaseinhibitors FDA-approved for cancer treatment, a highly selec-

tive ROCK inhibitor may not be essential for clinical success ofROCK inhibitors. Finally, our failure to validate DLC-1 loss as agenetic marker for ROCK dependency or pCofillin as a bio-marker for ROCK inhibition defines these 2 areas where moreprogress will be needed if ROCK inhibitors will be developedsuccessfully.

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

Authors' ContributionsConception and design: D. Vigil, A. Plachco, A.J. Garton, J. Pachter, H. Dong, X.Chen, C. DerDevelopment of methodology: D. Vigil, A.J. Garton, L. Castaldo, H. DongAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Vigil, T.Y. Kim, A. Plachco, H. Dong, X. Chen, B.TokarAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.Vigil, A. Plachco, A.J. Garton, J. Pachter, H. Dong, X.Chen, C. DerWriting, review, and/or revision of themanuscript:D. Vigil, A. Plachco, A.J.Garton, C. DerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Castaldo, C. DerStudy supervision: A.J. Garton, S. Campbell, C. Der

Figure 6. OXA-06 and suppressionof ROCK1/2 expression causes ablock in G0–G1 in NSCLC lineswhen in suspension. The indicatedNSCLC cell lines were plated onUltra-Low Attachment plates andincubated with DMSO (vehicle) or1 mmol/L OXA-06 for 48 hours (A)or transfected with nonspecific(NS) siRNA or ROCK1 siRNA #1and ROCK2 siRNA #1 sequencestargeting ROCK1 or ROCK2 (B).Forty-eight hours posttransfection,the cells were plated on Ultra-LowAttachment plates (Corning). Thecell-cycle dependent DNA contentfor the indicated treatments isshown on the left (eachrepresentative of the 3experiments), and the means andSD of cell-cycle percentages forthe 3 independent experiments areshown on the right.

Vigil et al.





Providing compound featured in the study, and data on the compoundthat were included in the manuscript: J. PachterDesigning and synthesizing different series of compounds to generateSAR agaist ROCK: X. ChenDesigning and synthesizing analogs from high-through-put hits, leadingto the discovery of potent and selective compound OXA-06 shown in Fig.1: H. Dong

AcknowledgmentsThe authors thank the UNC FlowCytometry Facility for technical expertise and

data interpretation. The authors thank Adrienne Cox for helpful discussions andLanika DeGraffenreid and Jenni Sells for assistance in manuscript preparation.

Grant SupportThis work was supported by grants from the National Institutes of

Health (CA67771 and CA129610 to C. Der) and by postdoctoral fellowshipsfrom the American Cancer Society to D. Vigil and from Susan Komen toT.Y. Kim.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received July 14, 2011; revised July 25, 2012; accepted August 1, 2012;published OnlineFirst August 31, 2012.

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2012;72:5338-5347. Published OnlineFirst August 31, 2012.Cancer Res Dominico Vigil, Tai Young Kim, Ana Plachco, et al. Anchorage-Independent Growth and InvasionROCK1 and ROCK2 Are Required for Non-Small Cell Lung Cancer

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ROCK1 and ROCK2 Are Required for Non-Small Cell Lung ... · ROCK1 and ROCK2 Are Required for Non-Small Cell Lung Cancer Anchorage-Independent Growth and Invasion Dominico Vigil1,