Peloruside- and Laulimalide-Resistant Human Ovarian ... · laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the bI-tubulin isotype,

Preclinical Development

Peloruside- and Laulimalide-Resistant Human OvarianCarcinoma Cells Have bI-Tubulin Mutations and AlteredExpression of bII- and bIII-Tubulin Isotypes

Arun Kanakkanthara1, Anja Wilmes1, Aurora O'Brate4, Daniel Escuin4, Ariane Chan1, Ada Gjyrezi4,Janet Crawford1, Pisana Rawson1, Bronwyn Kivell1, Peter T. Northcote2, Ernest Hamel3,Paraskevi Giannakakou1,4, and John H. Miller1

AbstractPeloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of

action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin

remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed

peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were

able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold

resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A

resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide.

Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on b-tubulin nor was

there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/

laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in

the bI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to

support a b-tubulin–binding site for peloruside A and laulimalide. To determine whether the different

resistance phenotypes of the cells were attributable to any other tubulin alterations, the b-tubulin isotype

composition of the cells was examined. Increased expression of bII- and bIII-tubulin was observed in L4 cells

only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two

drugs, pelorusideA and laulimalide, that have a similarmode of action.Mol Cancer Ther; 10(8); 1419–29.�2011

AACR.

Introduction

Microtubules are major cytoskeletal polymers com-posed of heterodimers of a- and b-tubulin subunits (1).They are vital for several cellular functions including

mitosis, intracellular trafficking, and maintenance ofcell shape. The importance of microtubules in mitoticspindle formation and chromosome movement duringcell division makes them a major target for chemother-apeutic drugs to halt the uncontrolled division of cancercells (2).

Peloruside A and laulimalide (see SupplementaryFig. S1 for structures) are 2 microtubule-targetingagents isolated from different marine sponges Mycalehentscheli and Cacospongia mycofijiensis, respectively (3,4), although other sources of laulimalide have also beendescribed. The compounds have a similar mechanism ofaction to paclitaxel and are cytotoxic to a number ofmammalian cancer cell lines (5-7). Peloruside A andlaulimalide bind to and stabilize the polymerized formof microtubules, inhibiting microtubule dynamics. Thisinterferes with the function of the mitotic spindleand promotes mitotic arrest and apoptosis. Althoughhaving a similar mechanism of action to paclitaxel,peloruside A and laulimalide have a number of uniquefeatures that make them potentially useful second-gen-eration microtubule-stabilizing drugs for anticancertherapeutics.

Authors' Affiliations: Schools of 1Biological Sciences and 2Chemical andPhysical Sciences, Victoria University of Wellington, Wellington, NewZealand; 3Screening Technologies Branch, Developmental TherapeuticsProgram, Division of Cancer Treatment and Diagnosis, National CancerInstitute at Frederick, NIH, Frederick, Maryland; and 4Department ofMedicine, Division of Hematology/Clinical Oncology, Weill Cornell MedicalCollege, New York

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Current address for D. Escuin: Department of Medical Oncology, Institutde Recerca, Hospital de la Santa Creu i Sant Pau, Sant Antoni M. Claret,167, 08025 Barcelona, Spain.

Corresponding Author: John H. Miller, School of Biological Sciences,Victoria University of Wellington, PO Box 600, Wellington 6140,New Zealand. Phone: 644-463-6082; Fax: 644-463-5331; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-10-1057

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on November 5, 2020. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 8, 2011; DOI: 10.1158/1535-7163.MCT-10-1057

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Peloruside A and laulimalide share a similar or over-lapping binding site on the tubulin dimer that is distinctfrom the taxoid site (6-9); thus, peloruside A and lauli-malide retain their efficacy in paclitaxel- and epothilone-resistant cell lines that aremutated in the taxoid site (6, 7).Another possible advantage of the compounds is that,based on their chemical structure, they may have agreater polarity than paclitaxel. This greater polaritymay reduce the need to use a vehicle like CremophorEL for clinical delivery, as required for paclitaxel (10).Although these features may enhance the potential ofusing drugs like peloruside A or laulimalide for cancerchemotherapy, as with most of the anticancer drugs inclinical use, the possibility of tumors developing resis-tance to the drugs cannot be ignored.

Resistance by tumor cells to antimicrotubule agentsstems from, but is not limited to, overexpression of theP-glycoprotein (Pgp) drug efflux pump (11), elevatedlevels of microtubule-destabilizing factors (12), andchanges in the target molecule of drugs, tubulin (13-15). Tubulin alterations include tubulin mutations (14),altered expression of b-tubulin isotypes (13, 15), andincreased microtubule dynamics (16). Because peloru-side A and laulimalide are poor substrates for the Pgpdrug efflux pump (6, 7), resistance to these compoundsis unlikely to be dependent on development of an MDRphenotype.

Several cellular studies have indicated a role for bI-tubulin mutations in the development of resistance topaclitaxel and other taxoid site microtubule-stabilizingagents (13, 14, 17-19). Mutations at the taxoid-binding siteon bI-tubulin impair drug–tubulin interactions (17); how-ever, the clinical occurrence of such b-tubulin mutationsin drug-resistant cancer patients seems to be rare,although it has yet to be thoroughly investigated inpatients with acquired resistance (20, 21). Differentialexpression of specific b-tubulin isotypes has also beenassociated with resistance to microtubule-targetingagents in cell lines and in patients (13, 15). The aim ofthis studywas, therefore, to generate cell lines resistant topeloruside A and laulimalide and to determine themechanisms of resistance by testing for tubulinmutationsand changes in tubulin isotype expression.

Materials and Methods

DrugsNatural peloruside A and laulimalide were isolated

and purified from the marine spongesM. hentscheli (NewZealand) and C. mycofijiensis (Tonga), respectively andstored as 1 mmol/L stock solutions in absolute ethanol at�80�C. Synthetic laulimalide was a gift from ArunGhosh, Purdue University, West Lafayette, IN, andsynthetic discodermolide was a gift from Ian Paterson,University of Cambridge, Cambridge, United Kingdom.Paclitaxel, epothilone A, vinblastine, vincristine, and col-chicine were purchased from Sigma Chemical Co.Epothilone B was from Novartis Pharmaceuticals Corp.

and 2-methoxy-estradiol was from Sigma or EntreMed,Inc.

Cell cultureThe human ovarian carcinoma cell line 1A9, a deriva-

tive of the A2780 cell line, was obtained from the NIH(17). Cells were cultured in RPMI 1640 medium (Invitro-gen) supplemented with 10% fetal calf serum (Invitro-gen), 0.25 units/mL insulin (Sigma), 100 units/mLpenicillin, and 100 units/mL streptomycin (Invitrogen).The cells were maintained at 37�C in a humidified airatmosphere containing 5% CO2.

Generation of peloruside A- andlaulimalide-resistant cell lines

A laulimalide-resistant cell line 1A9-L4 (L4) wasobtained by exposing 1A9 cells to increasing concentra-tions of laulimalide (10–150 nmol/L) over a period of 18months. The L4 cells that grew in the presence of 150nmol/L laulimalide exhibited the greatest resistance andwere selected for further analysis. The stability of theresistant phenotype in L4 cells was assessed by incubat-ing the cells in the presence of drug-free medium formore than 6 months with no reduction in the relativeresistance to laulimalide.

A peloruside A-resistant cell line 1A9-R1 (R1) wasderived from 1A9 human ovarian cancer cells grown fora number of months in medium with peloruside A at aconcentration just above the IC50 value (25 nmol/L).Verapamil (10 mmol/L) was included throughout toprevent selection of cells with the MDR phenotype, aspeloruside A has been shown to be a substrate, althougha poor one, for the Pgp pump (6). Over a period of 6months, the concentration of peloruside A was gradu-ally increased in steps to 30, 40, 50, 60, 80, 100, and 150nmol/L. The cells were typically exposed to each doseof peloruside A for 3 to 5 days and then allowed torecover in drug-free medium. Once confluent, cellswere treated with the next highest concentration ofpeloruside A until cells were able to grow at the higherpeloruside A concentrations. The cells were then clonedto ensure that resistant cell stocks were homogeneous.The resistant phenotype in R1 cells was stable in drug-free medium with no reduction in the relative resistanceto peloruside A observed over many months andpassages.

Cell proliferation assayTo determine the IC50 value for growth inhibition, we

used either a sulforhodamine B protein staining assay oran MTT cell proliferation assay as previously described(5, 17). IC50 values of the resistant cell lines were alwayscompared with parental 1A9 cells in the same (paired)experiment. Cells were tested for resistance against lau-limalide, peloruside A, 3 taxoid-binding site drugs (pacli-taxel, epothilone A, and discodermolide), and 3microtubule-destabilizing agents (VLB, colchicine, and2-methoxy-estradiol).

Kanakkanthara et al.

Mol Cancer Ther; 10(8) August 2011 Molecular Cancer Therapeutics1420




Analysis of drug effects on tubulin polymerizationand functionIntracellular polymerization of tubulin. Drug-

induced tubulin polymerization in cells was measuredby SDS-PAGE electrophoresis of soluble and pelletedfractions from centrifuged cell lysates (14,400 � g for10 minutes) and immunoblotting for tubulin. Cells weretreated with different concentrations of peloruside A orlaulimalide for 16 hours and then processed as previouslydescribed (17). For L4 cells, mouse monoclonal a-tubulinprimary antibody (1:1,000; Sigma) was used in conjunc-tion with an anti-mouse Alexa Fluor 680 secondary anti-body (Invitrogen). Immunoreactivity was detected in aLI-COR Odyssey infrared imaging system (LI-COR Bios-ciences) and quantified by densitometry with ImageJ(NIH). For Western blotting of R1 cells, a rabbit poly-clonal primary antibody to a-tubulin (1:1,000; Abcam)was used with a Cy5-conjugated goat anti-rabbit second-ary antibody (1:2,500; Amersham). The electrophoresedproteins were transferred to an Immobilon FL membrane(Millipore Corp), and fluorescence was measured using aFujifilm FLA-5100 imaging system (Fuji Photo Film Co.).The percentage of polymerized tubulin was calculatedfrom the band densities of soluble and pelleted tubulin.Immunocytochemistry and confocal microscopy.

Immunocytochemistry and confocal microscopy of the1A9, L4, and R1 cells was carried out as previouslydescribed (22, 23).Cell-cycle analysis. Cell-cycle analysis was con-

ducted by flow cytometry following staining of theDNA with propidium iodide as previously described(5).

Analysis of tubulin structural alterations andisotype expression patternsSequence analysis of human a- and b-tubulin genes.

Total RNA was isolated from the cells using a QiagenRNeasy kit, and reverse transcriptase-PCR was carriedout using a Protoscript First Strand cDNA SynthesisKit (New England Biolabs). PCR amplification of cDNAof bI-tubulin (HM40/TUBB gene; RNA accessionNM_178014) was carried out using the following primerspurchased from Invitrogen: 50-CTTGCCCCATACA-TACCTT-30 and 50-GTAAGACGGCTAAGGGAACTG-30.PCR products were then direct sequenced using the

following 7 primers:50-TCTGGGGCAGGTAACAACT-30; 50-AGTTGTTAC

CTGCCCCAGA-30; 50-CTCCGCAAGTTGGCAGTCAAC-30; 50-TGGCCTCCAGATGGCAGTC-30; 50-GGGG ATCCATTCCACAAAGTA-30; 50-GGACCATGTTGACT GCCAAC-30; and 50-GACTGCCATCTTGAGGCCAC-30.Subcloning of the cDNA PCR-amplified products

was conducted using the TOPO TA cloning system(Invitrogen), followed by direct sequencing with theprimers listed above. A minimum of 20 clones wereanalyzed for each sample. All PCR reactions werecarried out in 50 mL reaction mixtures, as previouslydescribed (24). Multiple sequence alignment of b-tubu-

lin isotypes was conducted using the Clustal Wmethod and the MegAlign program (Lasergene,DNASTAR). All a-tubulin primer sequences for thepredominant a-tubulin isotype (Ka1) were obtainedfrom Poruchynsky and colleagues (25). Primers werepurchased from Invitrogen.

Quantitative real-time PCR. mRNA expressionlevels of the 7 b-tubulin isotypes and a house-keepinggene were determined using a quantitative SYBR greenPCRmethod. Several common house-keeping genesweretested, but only 18S rRNA showed similar expression inthe parental and the resistant cells. Total RNA wasextracted from frozen cells using an RNeasy Protect CellMini kit (Qiagen) following the manufacturer’s instruc-tions. Extracted total RNA (1 mg) was treated with DNaseI (amplification grade; Invitrogen) to remove any geno-mic DNA, and cDNA was synthesized using SuperScriptIII First-Strand Synthesis Supermix (Invitrogen) in accor-dance with the manufacturer’s instructions. Excluding apreviously described primer set for the bIII-tubulin gene(26), all primers were designed using Beacon Designer(Premier Biosoft International) and manufactured byInvitrogen and are listed in the SupplementaryTable S1. For quantification of expression levels of eachof the 7 b-tubulin isotypes, and 18S rRNA, singleplexreaction mixes were prepared containing a single set ofisotype-specific or 18S rRNA primers at validated con-centrations (200 nmol/L) and reagents supplied in theSYBR GreenER qPCR SuperMix Universal Kit (Invitro-gen) according to the manufacturer’s instructions. Sam-ples were prepared in duplicate by adding the cDNAsample (200 ng) into the prepared reaction mix (totalvolume of 52 mL), and then transferring two 25-mL ali-quots containing 100 ng of cDNA/aliquot into 0.2 mLoptical PCR tubes. The amplification reaction was run onan iCycler real-time PCR detection system (Bio-Rad)under the following conditions: 1 cycle of 50�C for 2minutes, 1 cycle of 95�C for 10 minutes, 40 cycles of95�C for 15 seconds, and 60�C for 60 seconds. Controlsincluded reaction mixtures that did not include templateand samples that underwent reverse transcription PCRwith the exclusion of SuperIII/RNaseOUT Enzyme Mixto check the effectiveness of the DNase treatment. ThePCR amplification efficiency for each set of primers,evaluated using the slope of a serially diluted (1:1–1:64) cDNA sample, was above 96% (SupplementaryTable S2). The primer specificity was confirmed bysequencing the PCR product. The 1A9 parental cell linewas used as a control for the resistant cell lines andquantification of samples was determined by the

2ð�DDCt Þ method (27).One-dimensional gel electrophoresis and Western

blotting. Cell lysates were prepared using a cell lysisbuffer containing 30 mmol/L Tris-HCl, 7 mol/L urea, 2mol/L thiourea, and 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), pH8.5. Total protein was quantified with a protein assaydye reagent kit (Bio-Rad Laboratories). Lysate containing

Peloruside- and Laulimalide-Resistant Cells

www.aacrjournals.org Mol Cancer Ther; 10(8) August 2011 1421




25 mg of proteinwas electrophoresed on a 10% SDS-PAGEgel at 120 V for 1 hour. Western blot transfer to anImmobilon FL membrane was carried out for 17 hoursat 20 V in a wet transfer unit. Immunoblotting wascarried out with mouse monoclonal bII-tubulin(1:1,000; MMS-422P; Covance), bIII-tubulin (3:1,000;T8660; Sigma), total b-tubulin (3:1,000; T4026; Sigma),b-actin (1:3,000; A2228; Sigma), and rabbit polyclonala-tubulin (1:1,000; ab18251; Abcam). Primary antibodieswere incubated at room temperature with the membranefor 2 hours with rocking. A Cy5-conjugated anti-mouse(1:2,500; PA45010V; Amersham) or anti-rabbit (1:2,500;PA45011V; Amersham) secondary antibody was used,and bands were detected with a Fujifilm FLA-5100 ima-ging system. Immunoblots were analyzed with ImageJsoftware.

Two-dimensional gel immunoblot. The cell lysatepreparation and protein quantification were carried outas described in theWestern blotting procedure. A samplecontaining 80 mg of protein was mixed with rehydrationbuffer [2 mol/L thiourea, 7 mol/L urea, 2% IPG buffer,2% dithiothreitol (DTT), 4% CHAPS], followed by over-night incubation with immobilized pH gradient strips(pH ¼ 3–5.6). Isoelectric focusing was conducted in anEttan IPGphor Isoelectric Focusing Unit (GE Healthcare)with the following settings: (i) step and hold 300 V for 30minutes; (ii) gradient 1,000 V for 30 minutes; (iii) gradient5,000 V for 90 minutes; and (iv) step and hold 5,000 V for25 minutes. After the isoelectric focusing, the strips wereequilibrated in equilibration buffer (50 mmol/L Tris, 6mol/L urea, 30% glycerol, and 2% SDS) containing 1%

DTT for 10 minutes, then in equilibration buffer contain-ing 2.5% iodoacetamide for 10 minutes. The seconddimension was conducted on a 4% to 12% gradientNuPage Bis-Tris gel (Invitrogen) for 55 minutes at200 V. The separated proteins were transferred to anImmobilon FL membrane and probed with mouse mono-clonal primary antibodies to bII-tubulin (1:1,000; MMS-422P; Covance), bIII-tubulin (3:1,000; T8660; Sigma), andb-actin (1:3,000; A2228; Sigma). The protein bands werevisualized with a Cy5-conjugated anti-mouse secondaryantibody (1:2,500; PA45010V; Amersham). The blots werescanned with the Fujifilm FLA-5100 imaging system, andthe densities of the protein spots were quantified withImageJ software and normalized to the b-actin banddensity.

Data analysisTests for significant differences used ANOVA or

Student’s t tests. P < 0.05 was taken as significant, andall data are presented as mean � SEM.

Results

Resistance profile of L4 and R1 cellsTo gain a better understanding of the specific effects

and interactions of peloruside A and laulimalide withthe microtubule cytoskeleton, we selected 1A9 ovariancarcinoma cells with increasing concentrations ofeither peloruside A or laulimalide and generated 2stable resistant cell lines named R1 and L4, respec-tively. The ability of drugs to inhibit growth of these

Table 1.Resistance of cells to various microtubule-targeting agents in parental 1A9 and mutant L4 and R1cells

Giannakakou laboratory Miller laboratory

1A9 L4 Fold change 1A9 R1 Fold change

LAU 3.6 � 0.2 141 � 6a 39.2 14.9 � 2.9 20.1 � 3.4 1.3PLA 10.1 � 0.8 396 � 23a 39.2 12.9 � 1.5 72.4 � 3.7a 5.6PTX 1.3 � 0.1 1.8 � 0.1b 1.4 14.4 � 5.1 12.1 � 4.8 0.8EPOA 1.3 � 0.1 1.6 � 0.1b 1.2 8.1 � 0.2 9.0 � 0.8 1.1EPOB 0.17 � 0.07 0.47 � 0.12 2.8DISC 48.1 � 10.8 31.6 � 7.01 0.72ME 317 � 60 233 � 33 0.7VBL 0.40 � 0.06 0.33 � 0.09 0.8 0.84 � 0.11 1.00 � 0.13 1.2VCR 6.2 � 0.5 6.7 � 0.2 1.1COL 5.4 � 1.1 4.7 � 0.7 0.9

NOTE: Growth inhibition by various microtubule-targeting agents was measured in 1A9, L4, and R1 cells. Data are the average IC50

values for growth inhibition in nmol/L. Data are presented as mean � SEM.Abbreviations: LAU, laulimalide; PLA, peloruside A; PTX, paclitaxel; EPOA, epothilone A; EPOB, epothilone B; DISC, discodermolide;2ME, 2-methoxy-estradiol; VBL, vinblastine; VCR, vincristine; COL, colchicine.aP < 0.0001 (unpaired Student's t test; n¼ 3–7 biological replicates). Data on left of table (1A9 and L4) are from PG lab, except for PLA.bP < 0.05.






resistant cells is shown in Table 1. The L4 cellline exhibited 39-fold resistance to laulimalide com-pared with the parental 1A9 cells but showed nosignificant cross-resistance to microtubule-targetingagents known to bind at the taxoid site (paclitaxel,epothilone A, and epothilone B), the vinca site (VLBand vincristine), or the colchicine site (colchicine and2-methoxy-estradiol). L4 cells showed a slightlyincreased resistance to paclitaxel and epothilone A,but the magnitudes of the effects were very small andunlikely to be biologically significant. Interestingly,the L4 cells were highly cross-resistant to pelorusideA, confirming earlier evidence that the 2 drugs havean identical or overlapping binding site (6). The R1cells were 5.6-fold resistant to peloruside A butremained sensitive to the other microtubule-targetingdrugs tested (Table 1). Interestingly, unlike the case ofL4 cells that were resistant to both peloruside A andlaulimalide, there was no significant resistance of R1cells to laulimalide. This suggests that the bindingsites for peloruside A and laulimalide on tubulinmight not be identical, but overlapping.

Impaired drug-induced tubulin polymerizationTo determine whether the drug-resistant cells dis-

played altered drug–tubulin interactions, we testedthe ability of the drugs to induce tubulin polymerizationusing a cell-based assay. Treatment of 1A9 cells with aslittle as 10 nmol/L laulimalide resulted in near-max-imum polymerization (90% P) compared with untreatedcells in which no polymerized tubulin was detected(Fig. 1A). In contrast, laulimalide treatment of the L4cells had only a minimal effect at concentrations ashigh as 100 nmol/L (24% P), consistent with thedrug-resistant phenotype of these cells. Paclitaxel-induced polymerization in both cell lines, however,

was not compromised. With peloruside A (Fig. 1B),100 nmol/L peloruside A induced 61% tubulin poly-merization in 1A9 cells; whereas, in L4 and R1 cells,100 nmol/L peloruside A only induced 13% and 22%polymerization, respectively. These results for peloru-side A and laulimalide mirror the antiproliferativeassays summarized in Table 1.

Using confocal microscopy, we showed that themicrotubule-stabilizing effects of peloruside A andlaulimalide were significantly impaired in the drug-resistant cell lines (Fig. 2). Whereas treatment of 1A9cells with as little as 10 nmol/L laulimalide inducedmicrotubule stabilization (bundling; Fig. 2A), thiseffect was only seen at much higher drug concentra-tions in L4 and R1 cells [�300 nmol/L laulimalide forL4 (data not shown) and �50 nmol/L for R1]. Withpeloruside A, microtubule bundling in interphase cellsand multiple asters in mitotic cells were observed at apeloruside A concentration of 40 nmol/L in 1A9 cells,but only at much higher concentrations in L4 and R1cells (�500 nmol/L or �200 nmol/L peloruside A,respectively; Fig. 2B). Paclitaxel induced microtubuleaberrations at low concentrations in all 3 cell lines.Quantitative counts of cells with aberrant microtubulemorphologies after treatment with microtubule-stabi-lizing agents are presented in the SupplementaryMaterial (Supplementary Table S3). Taken together,these results corroborate and extend our cytotoxicityand cell-based tubulin polymerization assays and sug-gest that impaired drug–tubulin interaction mediatesthe resistance phenotype.

We also evaluated the ability of peloruside A andlaulimalide to induce cell cycle arrest in the resistantcells (Fig. 3). Again, we found that the resistant cellsrequired higher concentrations of peloruside A andlaulimalide, but not paclitaxel, to obtain a given levelof G2–M block.

Figure 1. Impaired ability ofpeloruside A and laulimalide toinduce tubulin polymerization inL4 and R1 cells. An in situ cellularassay was used to quantifydrug-induced tubulinpolymerization by SDS-PAGEelectrophoresis of soluble (S) andpelleted (P) fractions ofcentrifuged cell lysates, asdescribed in Materials andMethods. Cells were treated withlaulimalide and paclitaxel (A) orpeloruside A (B) for 16 hours. Thepercentage soluble or pelletedtubulin is given below each lane.LAU, laulimalide; PTX, paclitaxel;PLA, peloruside A.

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Distinct tubulin mutations are identified inpeloruside A- and laulimalide-resistant cell lines

The higher IC50 values of peloruside A and laulimalidein the R1 and L4 cells prompted us to investigate whether

a sequence alteration in the tubulin gene itself (13, 14)might account for the resistance of the cells. In addition,we reasoned that Pgp overexpression was an unlikelymechanism of resistance, as we did not observe any

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Control LAU 10 nmol/L LAU 50 nmol/L PTX 5 nmol/L PTX 10 nmol/L

Figure 2. Microtubule aberrations in 1A9, L4, and R1 cells treated with laulimalide, peloruside A, or paclitaxel. A, immunofluorescent staining of tubulinand DNA is presented in fixed cells following overnight treatment with laulimalide or paclitaxel. Arrows point to microtubule bundles. B, 1A9, L4, and R1cells were treated with peloruside A and paclitaxel for 12 hours. The cells were fixed and stained with anti-a-tubulin antibody and 40,6-diamidino-2-phenylindole nuclear stain. Staining was visualized by confocal microscopy. Arrows point to microtubule bundles. Both interphase and mitotic cells illustratemicrotubule bundles and multiple asters, respectively. The images are representative of 4 independent experiments at each drug concentration.

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Figure 3. G2–M block of 1A9, L4, and R1 cells by microtubule-stabilizing agents. Cells were treated with peloruside A, laulimalide, or paclitaxel for 16 hoursat 37�C. Cell-cycle analysis was carried out by flow cytometry. Values represent the mean� SEM of 3 to 6 independent preparations. PLA, peloruside A; LAU,laulimalide; PTX, paclitaxel.






significant cross-resistance to paclitaxel, which is anexcellent Pgp substrate (Table 1).Given that the exact binding site for peloruside A or

laulimalide on tubulin remains unknown and in light ofmodeling studies that suggest that peloruside A andlaulimalide bind to a-tubulin (28-30), we first sequencedthe predominant a-tubulin isotype in these cells, Ka1. Nomutations in this gene were identified in either resistantcell line. Sequencing the predominant b-tubulin isotype,bI-tubulin, revealed distinct acquired mutations in bothcell lines. L4 cells had acquired 2 single-point mutations,both of them at amino acid 306 that changed the wild-type R306 to either H306 or C306 (SupplementaryFig. S2A and B). Residues are numbered according totheir positioning in the sequence of the human b2atubulin isoform (UniProt accession number Q13885).RNA was extracted from the L4 cells and subcloned intobacteria. Individual bacterial cloneswere then picked andsequenced. This revealed that approximately 67% of theclones expressed histidine at position 306, whereas 33%expressed cysteine at 306. No trace of wild-type 306 wasfound. The R1 cell line harbored a single-point mutation,A296T, relative to the 1A9 parental cells (SupplementaryFig. S3). As with L4 cells, the R1 cells showed no expres-

sion of the wild-type b-tubulin allele at position 296, norwas there any evidence of a mutation at position 306 inthe bI-tubulin of R1 cells.

Alterations in b-tubulin isotype compositionb-Tubulin isotype mRNA levels in 1A9, L4, and R1

cells. Next, we sought to determine the relative mRNAexpression of b-tubulin isotypes in parental and resistantcells. The tubulin isotype levels were normalized to 18SrRNA, and their expression relative to parental cells wasdetermined. In L4 cells, mRNA expression of bII (class II)-and bIII (class III)-tubulin isotypes increased 16.8 � 1.6-fold and 5.7 � 0.8-fold, respectively (Fig. 4A and Supple-mentary Table S4). In contrast, the expression levels ofthese genes in R1 cells were similar to that in parentalcells. The mRNA expression levels of the other isotypes,bI (class I), bV (class V), and bVI (class VI), were similar inthe parental and the resistant cells. We were unable toamplify bIVa- and bIVb-tubulin despite trying variousprimer sets and different amplification conditions for thePCR. There are 2 likely reasons for this problem, lowmRNA abundance or nonspecific primers.

Tubulin protein levels in 1A9, L4, and R1 cells. Theprotein expression levels of the 2 b-tubulin isotypes, bII

βI βII βIII βV βVI0

5

10

15

20R1L4

1A9

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*

β -Tubulin isotypes

mR

NA

(rela

tive f

old

exp

ressio

n level)

βII

β-Tubulin

α-Tubulin

1A9 R1 L4

β-Actin

βIII

βII β-Tubulin α-TubulinβIII

β-Actin

β-Actin

β-Actin

A

B

0

2

4

6

8

10R1L4**

*

1A9

Pro

tein

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Figure 4. b-Tubulin isotype mRNA and protein expression in parental and resistant cells. A, mRNA expression of b-tubulin isotypes. The mRNA expressionprofile of b-tubulin isotypes in peloruside A and laulimalide resistant cells was analyzed using qRT-PCR. The relative fold expression of mRNA for eachisotype is compared with the parental 1A9 cells (dashed line). Data are the mean � SEM of 5 to 7 experiments. *, P < 0.05; **, P < 0.001, Student's t test. B,protein abundance in parental and resistant cells by immunoblotting. Total cellular protein (25 mg) was resolved on 10% SDS-PAGE and analyzed by Westernblotting with bII-, bIII-, total b- and a-tubulin antibodies. b-Actin was used as a loading control. The image is a representative blot from 5 independentexperiments. C, summary of the protein abundance data. Quantitation of immunoblot density in Western blots was determined relative to the actin loadingcontrol and presented as the fold expression relative to 1A9 cells. *, P < 0.05; **, P < 0.01, Student's t test. Data are the mean � SEM (n ¼ 5 experiments).






and bIII, and the 2 microtubule subunits, a- and b-tubu-lin, in the parental and the resistant cells were investi-gated by Western blotting (Fig. 4B and C). The total a-and b-tubulin protein levels were similar in all 3 cell lines,but bII- and bIII-tubulin isotype protein expression wasincreased by 7.4 � 1.2-fold and 5.6 � 0.4-fold, respec-tively, in the resistant L4 cells (Fig. 4C). Conversely, bII-and bIII-tubulin isotype protein levels in R1 cells weresimilar to that in parental cells. The increased expressionof bII and bIII isotypes in L4 cells was confirmed byimmunocytochemistry (Supplementary Fig. S4).

Evidence for posttranslational modifications oftubulin isotypes. Posttranslational modifications ofthe bII- and bIII-tubulin isotypes in the resistant cellsare illustrated in Fig. 5. In L4 cells, 1 additional proteinspot of bII-tubulin (black arrow) and 2 additional proteinspots for bIII-tubulin (white arrows), which were notpresent in the parental 1A9 cells, were detected. One faintadditional protein spot for each of the bII- and bIII-tubulinisotypes was found in R1 cells, compared with 1A9 cells.These additional spots indicate posttranslational modifi-cations of the tubulin isotypes in the resistant cells.

Discussion

bI-Tubulin structural alterationsTo verify that the resistance of the L4 and R1 cells was

due to altered interactions of peloruside A or laulimalide

with tubulin, we examined the ability of pelorusideA andlaulimalide to induce tubulin polymerization in the resis-tant cells, formmicrotubule bundles or multiple asters, orblock cells in G2–M of the cell cycle. We then showed thatL4 and R1 cells have a single-nucleotide mutation atamino acid positions 306 and 296, respectively, in thebI-tubulin gene. Nomutations in the a-tubulin gene werefound. Recent studies have shown that peloruside A andlaulimalide bind to an exterior site on b-tubulin (8, 31, 32)and not on a-tubulin as originally proposed from com-puter modeling studies (28-30). For peloruside A-bindingsite modeling, Huzil and colleagues (8) used hydrogen–deuterium exchange mass spectrometry (HDX-MS) andfound that there was a reduction in labeling on peptidesb294-301 (H9–H90 loop), b302-314 (H90–S8), and b332-340(H10 loop), suggesting that these regions of b-tubulin areinvolved in peloruside A binding. Importantly, the pep-tide b302-314 (H90–S8) had a large reduction in deutera-tion and thus represents a strong candidate for residing inthe pelorusideA-binding site. Thiswas further confirmedby data-directed molecular docking simulations. Model-ing laulimalide binding, Bennett and colleagues (31) usedmass shift perturbation analysis and data-directed dock-ing, proposing that laulimalide binding promotes a reor-ganization of the R306 residue. This reorganizationcauses stabilization of the loops in this region and gen-erates a polar contact with the oxygen of the dihydro-pyran side chain of laulimalide. Recently, Nguyen and

HighLow

pH

L4R11A9

L4R11A9

L4R11A9

βII

βIII

βII + βIII ActinActinActin

Actin Actin Actin

ActinActinActin

Figure 5. Two-dimensional gel immunoblot analysis of b-tubulin isotypes. Protein lysates (80 mg) from 1A9, L4, and R1 cells were isoelectrofocused on a pHgradient strip (pH 3–5.6) and separated in the second dimension on a 4% to 12% PAGE. The proteins were transferred and probed with bII- and bIII-tubulinisotype–specific antibodies. The bII- and bIII-tubulin region (denoted by a rectangle on the gel image near the middle) is enlarged in the top right inset.Posttranslational modification of bII-tubulin (black arrows) and bIII-tubulin (white arrows) is indicated. Membranes were reprobed with b-actin to confirm equalprotein loading. The image is a representative example from 3 experiments.






colleagues (32) modeled both peloruside A and lauli-malide into the b-tubulin site identified by Huzil andcolleagues (8). They showed that the peptide containingamino acid b296 provided a favorable binding surfacefor the compounds. These results (8, 31, 32) show thatthe above amino acid residues are necessary for thestable binding of peloruside A and laulimalide tob-tubulin, but that they have no effect on taxoid sitedrug binding. Therefore, a mutation in 306 (as seen in L4cells) and 296 (as seen in R1 cells) could cause impaireddrug-induced tubulin polymerization and, therefore,confer resistance to the drugs. Notably, amino acidR306 seems to be of major importance for pelorusideA and laulimalide binding. This might explain why theL4 cell line shows a very rare mutation event in which 2point mutations have occurred at the same location inboth alleles. Alternatively, however, it is possible thatL4 may comprise 2 cell populations, 1 with the H306 and1 with the C306 mutation. In any case, the fact that therewas no wild-type 306 left suggests that indeed thisresidue is important for laulimalide binding and thatthe cells had to mutate it to adapt to the drug selectionpressure. The fact that L4 cells are also resistant topeloruside A provides strong support that pelorusideA and laulimalide share the same binding site. Mutationof amino acid A296 has a selective effect on pelorusideA activity with no impact on laulimalide activity, butthe drug–amino acid interactions have not been speci-fically modeled at this stage, although alanine 296, likearginine 306, is located in the proposed laulimalide/peloruside A site.These results are the first to provide cell-based support

for a b-tubulin–binding site of peloruside A and lauli-malide. All other evidence is based on computer-dockingsimulations and HDX-MS (8, 31, 32). The clinical rele-vance of bI-tubulin mutations in drug resistance is notclear, as such mutations are rarely seen in tumors (20, 21).This led us to search for other tubulin-related mechan-isms that contribute to a cell’s distinct resistance profilesto peloruside A and laulimalide.

b-Tubulin isotype alterationsb-Tubulin in humans exists as 7 different isotypes: bI

(class I), bII (class II), bIII (class III), bIVa (class IVa),bIVb (class IVb), bV (class V), and bVI (class VI; ref. 33).Some of these isotypes exhibit tissue-specific expres-sion; whereas, other isotypes are constitutivelyexpressed in all tissues (34, 35). The present studyinvestigated mRNA expression of 5 of these isotypesin the resistant L4 and R1 cells. bII- and bIII-tubulinmRNAs were found to be increased in L4 cells but notR1 cells. The protein abundance of the isotypes in thecells mirrored the mRNA results, but there were nosignificant alterations in the total b-tubulin and totala-tubulin protein levels. Based on the qRT-PCR results,in 1A9 parental cells, bII and bIII mRNA levels wereeach 7% of the total mRNA (excluding bIVa/b) and inthe resistant L4 cells, bII and bIII mRNA levels were

increased to 55% and 19% of the total mRNA, respec-tively. Comparative protein levels in the cells could notbe determined from the Western blots, as differentantibodies were used for each isotype; however, it isexpected that the bI isotype will still be the predominantb-isotype in the L4 cells. Overexpression of bII- and bIII-tubulin has been reported to be a predictive marker fordrug resistance in cancer patients (13, 15). bIII-Tubulinis a multifunctional protein, and its upregulation isassociated with tumor progression and resistance totubulin-binding and DNA-damaging agents (36, 37).There is now a clear indication that bIII-tubulin playsa vital role in microtubule dynamic instability and inopposing the ability of tubulin-binding agents to sup-press spindle dynamics. A recent study by Gan andcolleagues (38) showed that bIII-tubulin knockdown innon–small cell lung cancer cells enhanced the suppres-sion of microtubule dynamics at low concentrations ofpaclitaxel and vincristine. Given that microtubule poly-merization and stabilization are the major modes ofaction of peloruside A and laulimalide, increasedexpression of bIII-tubulin presumably counteracts theaction of these 2 drugs. The functional significance ofbII-tubulin expression with regard to drug sensitivitydiffers from bIII-tubulin (13). It is not clear at this pointwhat the role in resistance of bII-tubulin isotype over-expression is in L4 cells. Because the cells also have amutation in the bI-tubulin gene, the resistance is likelyto be due to a combination of decreased binding ofpeloruside A and laulimalide as a result of the bI-tubulin structural mutation and the effects of bII-and/or bIII-tubulin isotype overexpression.

Two-dimensional gel immunoblots revealed post-translational modification of bII- and bIII-tubulin inL4 cells. Increased levels of tyrosinated a-, bIII-, andbIV-tubulin have been associated with paclitaxel resis-tance in MCF-7 breast cancer cells (39). The additionalprotein spots observed in the L4 and R1 cells, not seen inthe 1A9 cells (Fig. 5), were in the low pH region of theisoelectric focusing but were at a similar molecularweight (50 kDa) as the main bII- and bIII-tubulin iso-types, indicating that the additional spots were likely tobe due to posttranslational modifications such as phos-phorylation or glutamylation. A study by Khan andLuduena (40) has shown that the phosphorylation ofbIII-tubulin can regulate microtubule assembly in vivo.Phosphorylation of bIII-tubulin occurs at serine or tyr-osine residues near the C-terminus (40). Tubulin mod-ification at the C-terminus can affect the conformationof the tubulin protein, preventing the binding of a drugto tubulin (41). It would be of great interest to determinethe precise posttranslational modifications of bII- andbIII-tubulin in L4 cells and their contribution to theresistance phenotype.

Overall, our results provide the first cell-based evi-dence in support of a b-tubulin–binding site for peloru-side A and laulimalide that involves R306 for bothcompounds and A296 for peloruside A. We also showed






that resistance to peloruside A and laulimalide mayarise from altered drug–tubulin interactions as a resultof not only bI-tubulin structural mutations but alsoincreased bII- and bIII-tubulin isotype expression andposttranslational modification. Future directions of studywill be to elucidate the functional significance of theseb-tubulin mutations and isotype alterations in the resis-tant cells. Understanding the role of multiple b-tubulinalterations will help improve targeting of anticancerdrugs whose mechanisms of action involve interactionswith microtubules.

Disclosure of Potential Conflicts of Interest

P.T. Northcote and J.H. Miller hold a patent on peloruside in the USAonly.

Acknowledgments

The authors thank Dr. Thomas Gaitanos for early development of thepeloruside A-resistant cells.

Grant Support

This work was supported in part by grants to J.H. Miller from the NewZealand Foundation of Research, Science, and Technology, the CancerSociety of NZ, the Wellington Medical Research Foundation, and VictoriaUniversity of Wellington. This work was also supported in part by NIHRO1 grants CA100202 and RO1 CA114335 to P. Giannakakou.

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 indicatethis fact.

Received November 22, 2010; revised May 10, 2011; accepted May 27,2011; published OnlineFirst June 8, 2011.

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2011;10:1419-1429. Published OnlineFirst June 8, 2011.Mol Cancer Ther Arun Kanakkanthara, Anja Wilmes, Aurora O'Brate, et al. III-Tubulin Isotypesβ

II- and βI-Tubulin Mutations and Altered Expression of βCells Have Peloruside- and Laulimalide-Resistant Human Ovarian Carcinoma

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Peloruside- and Laulimalide-Resistant Human Ovarian ... · laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the bI-tubulin isotype,