blockade of the ras pathway by manumycin, a farnesyltransferase inhibitor, overcomes the resistance...

Abstract Ras activation (by point mutation or binding ofIL-6) is frequently observed in multiple myeloma (MM).As farnesylation of Ras protein by farnesyltransferase is acritical step for Ras functional activity, farnesyltransferaseinhibitors (FTI) have emerged as potential anti-canceragents. Manumycin, a natural FTI, prevents proliferationand induces apoptosis of myeloma cells refractory to Fas-and drug-induced cell death. Fas pathway analysis showedthat Fas-resistant apoptosis of Fas-positive myeloma cellsparallels FLIP (FLICE/caspase-8-inhibitory protein)

expression. Treatment of fresh purified myeloma cells,myeloma cell clone-2 and U266 cell line with manumycininduced down-regulation of FLIP expression with con-comitant expression of Apo 2.7 antigen, the marker ofearly apoptosis. Down-regulation of FLIP mRNA levels indrug-treated cells was associated to suppression of thetranscription factor NF-κB that plays a central role inchemoresistance, survival and proliferation of myelomacells. Further analysis showed that manumycin-inducedapoptosis involved caspases activation and was preventedby the addition of caspases specific inhibitors. Finally, pre-treatment of Fas-resistant/FLIP-positive cells withmanumycin sensitised them to Fas-triggered apoptosis.Overall results indicate that manumycin-induced apoptosisinvolves Fas pathway. FTIs may thus be proposed as apromising class of anti-cancer agents which can boost thecytotoxic effect of conventional drugs by overcoming NF-κB activation and Fas-resistant apoptosis.

Key words Farnesyltranferase inhibitors • Fas • FLIP •

Multiple myeloma • NF-κB

Introduction

Multiple myeloma (MM) is an incurable haematologicalmalignancy characterised by clonal expansion of malig-nant plasma cells secreting monoclonal immunoglobulins.Myeloma cells show a low proliferative index and anextended life span, suggesting that deregulation of apopto-sis is involved in the pathogenesis of MM [1]. Increasingevidence demonstrates that deregulation and/or overex-pression of anti-apoptotic signals allow tumour cells toescape apoptosis and develop resistance to cytotoxic drugs[1–4]. Gene expression analysis reveals that drug-resistantapoptosis involves alteration of genes associated withapoptosis, cell cycle and signal transduction [5, 6].

Clin Exp Med (2005) 4:174–182DOI 10.1007/s10238-004-0053-0

M.A. Frassanito • L. Mastromauro • A. Cusmai • F. Dammacco

Blockade of the Ras pathway by manumycin, a farnesyltransferaseinhibitor, overcomes the resistance of myeloma plasma cellsto Fas-induced apoptosis

Received: 5 December 2004 / Accepted 18 January 2005

O R I G I N A L

M.A. Frassanito • L. Mastromauro • A. Cusmai • F. Dammacco (�)Department of Internal Medicine and Oncology (DIMO),University of Bari Medical School,Piazza G. Cesare 11, I-70124 Bari, Italye-mail: [email protected].: +39-080-5478862Fax: +39-080-5478820

M.A. Frassanito et al.: Manumycin overcomes resistance of myeloma cells to apoptosis 175

Apoptosis can be induced by triggering of ligand-dependent receptors on the cell surface (extrinsic pathway)or by stimulation of intracellular receptor proteins (intrin-sic pathway). Among the death receptors (TNF-R, DR-3/TRAMP, TRAIL-R1 and TRAIL-R2), Fas is the mostpotent inducer of apoptosis [7]. Interaction of Fas with itscognate ligand (Fas-L) induces receptor trimerisation andhence recruitment of the adapter protein FADD (Fas-asso-ciated death domain), which interacts with a related motifin the prodomain of caspase-8 to form the death-inducingsignalling complex (DISC). Subsequent activation ofdownstream caspases leads to apoptosis. Apoptosis istightly regulated at different levels. FLIP (FLICE/caspase-8 inhibitory protein) powerfully inhibits Fas-mediatedapoptosis by blocking the signalling pathway furtherupstream before caspase-8 activation. It is recruited viaFADD to the Fas signalling complex and prevents caspase-8 activation and release [8]. Although the exact physiolog-ic role of FLIP is still unclear, accumulating evidence indi-cates that it is a tumour progression factor [9–11]. FLIPexpression correlates with resistance to Fas-induced apop-tosis in vitro and with tumour escape from T-cell immuni-ty and enhanced tumour progression in vivo.

Variations in the susceptibility of primary myelomacells and myeloma cell lines to Fas-triggered apoptosishave been described and possibly reflect different degreesof malignancy [12, 13]. Fas-resistance of myeloma cellshas been related to several factors, including the expres-sion of Fas splicing variants or complete absence of Fasreceptor [13], the anti-apoptotic effect of theparacrine/autocrine IL-6 [14, 15] and overexpression ofFLIP [16] and Bcl-2 family proteins [3] that interfere withthe Fas death signalling cascade. In the pathogenesis ofMM [17], IL-6 supports myeloma cell proliferation (byactivating the RAS-MAPK pathway) and survival (by acti-vating the Stat-3 and PI-3K-AKT pathways) [18, 19].

IL-6-positive myeloma cells show an IL-6-independentproliferative capacity and are refractory to drug-inducedapoptosis [15]. Moreover, transfection of the IL-6-depen-dent ANBL6 myeloma cell line with a constitutively activeRas mutant induces IL-6-independent cell growth andresistance to drug-induced apoptosis, suggesting that Rasactivation is involved in cell proliferation and response totherapy [20]. A suggestive hypothesis is that IL-6 activatesRas pathway in early MM, whereas during progressionactivating mutations of N- or K-Ras occur and allow anIL-6-independent tumour expansion [1]. Ras proteins thusplay an important role in immune cell signalling, in thatthey regulate cell proliferation as well as apoptosis [21].Transient expression of oncogenic Ras prevents Fas-medi-ated apoptosis by inhibiting Fas-triggered activation ofcaspases [22] or by down-regulating Fas expression onfibroblastic and epithelioid cells [23, 24]. An emergingconcept is that Ras acts as a branch-point in signal trans-duction pathways, in that it orchestrates the activity of

multiple cellular signals and thus may be a promising mol-ecular target for new therapeutic approaches [25–28].

We have previously shown that manumycin, a naturalfarnesyltransferase inhibitor (FTI), prevents cell prolifera-tion and induces apoptosis of fresh purified myeloma cellsand myeloma cell lines refractory to Fas- and drug-induced apoptosis [26]. The effect was related to inhibitionof post-translational Ras processing and activation of cas-pase-3, and was insensitive to the presence of exogenousIL-6. In the present study, we have explored the effect ofmanumycin on the Fas pathway. We show that treatment ofmyeloma cells with manumycin suppresses NF-κB activa-tion, reduces FLIP expression, activates caspase-8 andovercomes Fas-resistant apoptosis.

Materials and methods

Chemical

Manumycin was purchased from Sigma (St Louis, MO). It was dis-solved in dimethylsulphoxide (DMSO) and diluted with culture medi-um when used. Its final concentration (0.1%) was non-cytotoxic.

Myeloma cell purification and cell cultures

Fresh myeloma cells from bone marrow aspirates of 12 MMpatients (5 with a recent diagnosis and 7 with relapsing disease)were separated from bone marrow mononuclear cells with MACSCD138-MicroBeads (Miltenyi Biotec, Auburn, CA) according tothe manufacturer’s protocol. Cell preparations were investigated byimmunofluorescence analysis. Cultures containing 90% or more ofCD138+ myeloma cells were considered suitable for the study.

Myeloma cell clone (MCC)-2 was obtained from the bonemarrow of a myeloma patient, as previously described [13].Cryopreserved samples were thawed and cultured in culturemedium (RPMI 1640 containing 10% foetal calf serum, 2 mMglutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, allfrom Biochrom, Berlin, Germany). The human myeloma cellline, U-266, was obtained from American Type CultureCollection (ATCC) (Rockville, MD).

Flow cytometry

Phenotypic analysis of myeloma cells included both single- anddouble-fluorescence staining using specific monoclonal antibod-ies (mAb), namely the phycoerythrin (PE)-conjugated anti-CD38mAb (Becton Dickinson, Mountain View, CA), the FITC-conju-gated anti-CD138 mAb (Biosource, Camarillo, CA) and theFITC-conjugated anti-Fas, clone UB2 (Immunotech, Marseille,France). The intracellular expression of FLIP was determinedwith the specific anti-FLIP polyclonal antibody (Santa CruzBiotechnology, Santa Cruz, CA), according to the procedure sug-gested by the manufacturer. This included cell permeabilisation.

176 M.A. Frassanito et al.: Manumycin overcomes resistance of myeloma cells to apoptosis

The negative controls were isotype-matched irrelevant antibod-ies. Cytometry was carried out with the CellQuest programme ina FACScan (Becton Dickinson).

Caspase activation and inhibition

Activation of caspase-8 by manumycin was evaluated by flowcytometry with a CaspTag commercial kit (Intergen Company,Purchase, NY). The method is based on carboxyfluorescein (FAM)-labelled fluoromethyl ketone-peptide inhibitor (LETD-fmk) of cas-pase-8, which is cell permeable and non-cytotoxic. Once inside thecell, FAM-LETD-fmk irreversibly binds to the activated caspase-8.

Irreversible inhibition of caspases was achieved with syn-thetic peptides. Cell suspensions (1x106/ml) were incubated for 2h with a specific caspase-8 inhibitor, IETD-fmk (2 mM) and apan-caspase inhibitor, VAD-fmk (2 mM), purchased fromMedical and Biological Laboratories (Naka-ku Nagoya, Japan).

MTT colorimetric assay

The cytocidal effect of manumycin and anti-Fas mAb on myelo-ma cells was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Briefly,myeloma cells were cultured in 96-well plates at a density of1x106 cells/ml for 24 h at 37°C in culture medium or in the pres-ence of 5 or 10 mM manumycin and/or 100 ng/ml anti-Fas mAb,clone CH11 (Immunotech). At the end of each treatment, cellswere incubated with 1 mg/ml MTT for 4 h at 37°C. A mixture ofisopropanol and 1N HCl (23:2, v/v) was then added under vigor-ous pipetting to dissolve the formazan crystal. Dye absorbance inviable cells was measured at 570 nm, with 630 nm as a referencewavelength. Cell survival was estimated as a percentage of thevalue of untreated controls. Percentage of cell death was calcu-lated as 100% minus percentage survival. All experiments wererepeated at least three times, and each experimental conditionwas repeated in quadruplicate wells in each experiment.

Apoptosis

The susceptibility of myeloma cells to manumycin- and Fas-induced apoptosis was evaluated by flow cytometry afterAnnexin V-FITC (Molecular probes, Eugene, Oregon)/propidiumiodide staining and determination of the expression of Apo 2.7antigen (Ag). Briefly, myeloma cells (1x106) were incubated with5 or 10 mM manumycin and/or 100 ng/ml anti-Fas mAb for 6-24h and in their absence (controls). Cells were suspended in bind-ing buffer and incubated with Annexin V-FITC for 10 min.Finally, cells were resuspended in binding buffer containing pro-pidium iodide (20 µg/ml) and analysed by flow cytometry.

Expression of Apo 2.7 Ag on the mitochondrion membrane isan early identifying event in apoptosis. Treated myeloma cells andcontrols were permeabilised with 50 mg/ml digitonin (SigmaChemical CO, St Louis, MO) for 20 min at room temperature,incubated with PE-conjugated anti-Apo 2.7 mAb (Immunotech)and analysed by flow cytometry.

Real-time PCR

Total RNA was isolated from myeloma cells incubated in theabsence or presence of manumycin (10 µM) for 6 h by using theTrizol reagent (InVitrogen, Life Technologies, Carlsbad, CA)according to the manufacturer’s instructions. For cDNA synthesis,5 µg of total RNA was added in a single 100-µl reaction using thehigh-capacity cDNA archive kit (Applied Biosystem, Branchburg,NJ). FLIP and β-actine cDNA were amplified using primers andprobes from TaqMan® Assays-on-Demand™ Gene ExpressionProducts (Applied Biosystem). The probes were labelled at the 5´end with the reporter dye molecule (FAM and VIC for FLIP andβ-actine, respectively) and at the 3´ end with the quencher dyemolecule (MGB). The PCR reaction (25 µl total volume) washeated to 95°C for 10 min and then amplified for 40 cycles at95°C for 15 s and at 60°C for 60 s by using the ABI Prism 7000sequence detection system (Applied Biosystem). Relative quanti-tation of gene expression was performed as described in the man-ual using the comparative Ct (threshold cycle) method. RelativeFLIP mRNA levels were normalised to β-actine mRNA levels(∆Ct), and finally expressed as ∆∆Ct and as [2-(∆∆Ct)] values.

Evaluation of NF-κB activity

NF-κB activation in myeloma cells was evaluated by ELISAusing the TransAM NF-κB 65 transcription factor assay kit(Active Motif Europe, Rixensart, Belgium), according to themanufacturer’s instructions. Briefly, nuclear extracts of myelomacells cultured with or without manumycin (10 µM) for 6 h wereprepared by using the TransAM nuclear extract kit and incubatedin a 96-well plate coated with immobilised oligonucleotide con-taining the NF-κB consensus site (5´-GGGACTTTCC-3´). Theactive form of NF-κB specifically bound to this oligonucleotidewas detected by incubation with a primary antibody which recog-nised an epitope on the p65 subunit of activated NF-κB, visu-alised by an anti-IgG horseradish peroxidase conjugate anddeveloping solution, and quantified by spectrophotometry at 450nm with a reference wavelength of 655 nm.

Statistical analysis

The Student’s t-test and in several instances the Wilcoxon non-parametric method were used to compare the mean values of spe-cific phenotype expression and in vitro variables. P-values lessthan 0.05 were considered significant.

Results

Resistance of myeloma cells to Fas-induced apoptosis par-allels FLIP expression

Phenotypic analysis of freshly purified myeloma cells,MCC-2 and U266 myeloma cell line revealed a wide range


(5%–90%) of Fas and FLIP distribution (Table 1).Analysis of the in vitro susceptibility to Fas stimulation byflow cytometry showed that FLIP expression was associat-ed with Fas-resistant apoptosis. Addition of CH11 agonistmAb (0.1 mg/ml) induced apoptosis (Annexin-V+

cells>20%) of FLIP-/Fas+ cells (patients 2, 5, 7, 9, 12),whereas FLIP+/Fas+ myeloma cells were insensitive toFas-triggered apoptosis (Annexin V+ cells≤20%) (Table1). By contrast, manumycin-induced apoptosis was inde-pendent of FLIP and Fas expression.

Manumycin reduces FLIP expression

Our previous study [26] showed that blockade of the Raspathway by manumycin induced in vitro apoptosis ofdrug- and Fas-resistant IL-6-producing myeloma cells.Involvement of the Fas pathway in manumycin-inducedapoptosis was further investigated by evaluation of theeffect of drug on FLIP expression. Myeloma cells wereincubated for 6 h in the absence and presence ofmanumycin (10 mM). Their susceptibility to apoptosiswas evaluated in terms of Apo 2.7 Ag expression, whichidentified the early apoptotic event. A significant reductionof FLIP expression with a concomitant expression of Apo2.7 Ag was observed (Fig. 1a) in drug-treated cells. Datafor freshly purified myeloma cells from 2 patients and theU266 myeloma cell line are shown in Figure 1b.

The effect of manumycin on FLIP expression was alsoevaluated by real time quantitative RT-PCR. To this pur-

pose, FLIP mRNA levels normalised to β-actine mRNAlevels of manumycin-treated cells were compared withthose observed in untreated cells. As shown in Figure 2a,FLIP mRNA levels were significantly reduced in drug-treated cells that showed a higher Ct value. Analysis ofdata normalised to β-actine mRNA levels as ∆∆Ct andthen expressed as [2-(∆∆Ct)] clearly demonstrated thatmanumycin induced a down-expression of FLIP mRNAlevels (values<1) (Fig. 2b).

Manumycin down-regulates NF-κB activity in myeloma cells

As FLIP expression is regulated by the transcription factorNF-κB [29], which is constitutively activated in myelomacells [30], the effect of manumycin on NF-κB was furtherinvestigated. NF-κB DNA-binding activity was evaluated inthe nuclear extract of fresh myeloma cells, U266 and MCC2incubated in the absence (control) or presence of 10 mMmanumycin. Drug-treated cells showed a reduced NF-κBDNA-binding activity compared with untreated cells ascontrol (Fig. 3), suggesting that manumycin inhibits NF-κB.

Caspase-8 inhibitor prevents manumycin-induced apoptosis

Recruitment of FLIP via FADD to the Fas signalling com-plex inhibits Fas-induced apoptosis by preventing caspase-8activation [8]. Thus, in the next set of experiments, we inves-

Table 1 Fas-resistant apoptosis of myeloma cells parallels FLIP expression

Myeloma cells, % Apoptotic cells*, %

FLIP-positive Fas-positive Anti-Fas mAb Manumycin

Patient 1 32.6 77.8 13.8 73.3

Patient 2 12.0 63.5 58.5 80.0

Patient 3 63.3 31.6 12.4 47.3

Patient 4 75.0 46.4 15.5 55.2

Patient 5 11.4 60.8 47.1 68.6

Patient 6 47.4 26.2 9.7 83.3

Patient 7 20.8 52.1 31.5 90.1

Patient 8 64.2 78.9 15.0 73.8

Patient 9 19.3 30.3 45.9 67.1

Patient 10 63.4 85.1 8.9 81.3

Patient 11 68.1 71.0 10.5 79.0

Patient 12 17.2 56.4 43.8 63.9

MCC-2 13.2 5.4 3.6 98.5

U-266 89.0 90.9 5.7 95.9

*Myeloma cells were incubated with anti-Fas mAb (0.1 mg/ml) or manumycin (10 mM) for 24 h. Apoptosis was evaluated by flowcytometry after Annexin V-FITC/propidium iodide staining


Fig. 1a Manumycin treatment of myeloma cellsdown-regulates FLIP expression. Fresh myelo-ma cells purified from 12 patients, U266 cellline and MCC-2 were incubated for 6 h in theabsence (control) and presence of 10 mMmanumycin. Expression of FLIP, and Apo 2.7Ag was evaluated by flow cytometry. Data areexpressed as mean±SD. b Cytofluorimetric pat-tern of FLIP and Apo 2.7 Ag expression in U266cell line and in fresh myeloma cells purifiedfrom patient 1 and patient 4. Cells were treatedas described above. Numbers within histogramsindicate the percentage values of positive cellsin manumycin-treated cell population (M1) andcontrol (M2). Dotted lines represent the negativecontrol by using isotype-matched antibodies.

Fig. 2a Representative analysis by realtime quantitative RT-PCR of FLIPexpression in fresh myeloma cells puri-fied from patient 6, patient 8 and U266cell line. Myeloma cells were incubatedin the absence (control) or presence of10 µM manumycin for 6 h. Manumycin-treated cells show lower FLIP mRNAlevels, represented by a higher thresholdcycle (Ct), than control cells. b Analysisof data of real time quantitative RT-PCRas ∆∆Ct and expressed as [2-(∆∆Ct)] val-ues. [2-(∆∆Ct)] values <1 indicate a down-expression of FLIP mRNA levels. FLIPmRNA levels in control cells=1

Fig. 3 Manumycin suppresses NF-κB activation in myelomacells. NF-κB DNA-binding activity was evaluated in nuclearextracts of fresh purified myeloma cells, U266 cell line andMCC-2 incubated in the absence (control) or presence of 10µM manumycin for 6 h

a

b

a b


tigated the effect of manumycin on caspase-8. In particular,its activation was evaluated by flow cytometry by employinga fluorogenic peptide (FAM-LETD-fmk) inhibitor that irre-versibly and specifically binds to activated caspase-8.Treatment with manumycin induced a remarkable expres-sion of activated caspase-8 and Apo 2.7 Ag (Fig. 4).

The involvement of caspases was further corroboratedby the use of synthetic peptide inhibitors that irreversiblyinhibit their activity: a specific caspase-8 inhibitor (IETD-fmk) and a positive pan-caspase inhibitor (VAD-fmk). Asshown in Fig. 5a, incubation of myeloma cells with theinhibitors protected them from the apoptogenic effect ofmanumycin: the percentage value of Apo 2.7+ cells wassignificantly reduced by the specific caspase-8 inhibitor.

The protective effect of caspase inhibitors was alsoevaluated by MTT colorimetric assay. Fig. 5b shows thatthe cytocidal properties of manumycin were blocked by

the three inhibitors. The apoptosis percentage was signifi-cantly reduced in their presence compared to that observedin manumycin-treated cells.

Manumycin overcomes Fas-resistance of Fas+/FLIP+

myeloma cells

Finally, we determined whether manumycin sensitisedmyeloma cells to Fas-mediated apoptosis by inducingdown-expression of FLIP. To this purpose, fresh myelomacells, U266 and MCC-2, were pretreated for 6 h with 5 µMmanumycin and then incubated in the absence or presenceof the CH11 agonist mAb (0.1 mg/ml). As shown in Fig. 6,pretreatment of myeloma cells with manumycin increasedtheir susceptibility to Fas-induced apoptosis. The percent-

Fig. 4 Manumycin treatment of myeloma cellsactivates caspase-8. Fresh myeloma cells puri-fied from 12 patients, U266 cell line and MCC-2 were incubated for 6 h in the absence (con-trol) and presence of 10 mM manumycin.Expression of active caspase-8 and Apo 2.7 Agwas evaluated by flow cytometry. Data areexpressed as mean±SD

Fig. 5a,b Caspase inhibitors preventmanumycin-induced apoptosis. a Ad-dition of synthetic peptide inhibitors(2 mM) that irreversibly inhibit cas-pase activity significantly reduced thepercentage value of Apo 2.7 Ag+ ma-numycin-treated cells. b Caspaseinhibitors protected myeloma cellsfrom death induced by manumycin(10 mM for 24 h) assessed by MTTcolorimetric assay. IETD-fmk, specificcaspase-8 inhibitor; VAD-fmk, pan-caspase inhibitor. *P<0.05

a b


age of apoptotic cells in the presence of both manumycinand anti-Fas mAb was significantly increased comparedwith the values observed in the presence of either the drugor the mAb. Moreover, the effect was particularly promi-nent in Fas+/FLIP+ cells, thus confirming that cell apopto-sis occurred through Fas.

Discussion

Several chemotherapeutic agents such as cisplatin, dox-orubicin, etoposide, methotrexate and cytarabine inducetumour cell death by apoptosis [31–33]. Consequently,apoptosis defects contribute to drug resistance. Recent evi-dence supports the view that selection for drug resistancein a haematopoietic cell line results in simultaneous resis-tance to Fas-mediated apoptosis [4, 5, 31]. Althoughmyeloma cells express Fas antigen on their surface, onlysome of them are sensitive to Fas-triggered apoptosis [12,13, 34]. Fas expression thus does not fully correlate withbiological response: the myeloma cell line U266 express-es high levels of Fas but its treatment with anti-Fas mAbdoes not induce apoptosis. A similar picture has beenobserved in freshly purified myeloma cells.

In this study we demonstrate that Fas-resistant apopto-sis parallels expression of FLIP [16], which exerts its anti-apoptotic properties by directly binding to the proteasedomain of caspase-8 and thus preventing its activation [8,9, 35, 36]. FLIP-transfected tumour cells expressing highprotein levels were completely protected from TNF-,FasL- and TRAIL-induced apoptosis [31]. Their injectioninto mice consistently resulted in tumour development [9],whereas in vitro treatment with FLIP antisense oligodeo-xynucleotides sensitised myeloma cells to Apo2/TRAIL-induced apoptosis [35]. Recently, Anderson et al. [37], byusing microarray analysis in genetically identical twin

samples, have identified several genes highly expressed inmyeloma cells, including cell survival genes such as cas-pase-8 and FLIP.

Manumycin, a natural FTI, induces apoptosis of Fas-resistant/FLIP+ cells. Its effect is related to inhibition ofFLIP expression and activation of caspase-8. Treatment withthe cell-permeable IETD-fmk peptide, which irreversiblyinhibits the initiator caspase-8, protects tumour cells fromthe apoptotic effect of manumycin. In addition, Fas+/FLIP+

cells become susceptible to the apoptogenic effect of anti-Fas mAb after treatment with manumycin, suggesting thatmanumycin overcomes Fas-resistant apoptosis.

Cellular transformation by the Ras oncogene involvesactivation of several signal transduction pathways thatpromote deregulated cell cycle progression and enhancecell survival [38]. Transient expression of Ras mutant infibroblastic and epithelioid cells inhibits Fas-mediatedapoptosis by down-regulating Fas expression [23, 24, 39]or by inhibiting caspase activation [22]. In this study, wedemonstrate that treatment of myeloma cells with FTIrestores Fas susceptibility by down-regulating FLIPexpression.

Tumour progression is the result of a multistep trans-formation, leading to a deregulated balance between cellgrowth and apoptosis [1]. The evidence that tumour sup-pressor genes and oncogenes regulate apoptosis, and there-by promote neoplastic progression, strengthens the ideathat escape from apoptosis is an important step in tumourdevelopment. Activating mutations of N- and K-Ras seemto mark the MGUS to MM transition, although they canalso occur as a later progression event [1]. In fact, Rasmutations are rare or absent in MGUS, whereas they occurin 30-40% of early MM. On the other hand, IL-6, the mostimportant growth factor for MM, induces myeloma cellproliferation by activating the Ras pathway [40]. Ectopicexpression of mutated N-Ras or K-Ras in IL-6-dependentANBL6 myeloma cell lines induces cytokine-independent

Fig. 6 Manumycin sensitises myeloma cells toFas-induced apoptosis. Myeloma cells werepretreated with manumycin (5 µM) for 6 h,then incubated for an additional 24 h with anti-Fas mAb (0.1 µg/ml) (�). Myeloma cells incu-bated with manumycin (��) or anti-Fas mAb(�) without pretreatment were used as control.Pretreatment of cells with manumycin increa-sed their sensitivity to Fas-mediated apoptosis


growth and resistance to drug-induced apoptosis [20, 41].Mutated Ras-transfected myeloma cells showed constitu-tive upregulation of several intracellular signals, includingMEK/ERK, PI3K/AKT, mTOR and NF-κB [38].

Constitutive activation of NF-κB has been reported infresh myeloma cells and myeloma cell lines [30]. It paral-leled resistance of tumour cells to apoptosis induced bychemotherapy and radiotherapy. The transcription factorNF-κB plays a central role in chemoresistance, cell sur-vival and proliferation [42], in that it regulates the tran-scription of several target genes including cyclin D1, Bcl2,survivin, cellular inhibitor of apoptosis protein 1 (cIAP-1),cIAP-2 and FLIP. Thus, overexpression of NF-κB regulatesapoptosis induced by extrinsic as well as intrinsic pathwaysand has emerged as a new promising target for myelomatherapy [43, 44]. Treatment of myeloma cells with SN50, acell-permeable specific NF-κB inhibitor, induces myelomacell apoptosis and sensitises tumour cells to the apoptoticeffect of dexamethasone and doxorubicin [43].

Our study demonstrates that manumycin treatment sup-presses NF-κB activation in fresh myeloma cells andU266. Down-regulation of NF-κB activity is associatedwith decreased expression of FLIP, cyclin D1 and phos-phorylated Rb (data not shown), and with activation ofcaspase-8 and -3. This suggests that manumycin-inducedapoptosis involves several pathways. Overall results indi-rectly emphasise the central role of oncogenic Ras in themolecular pathogenesis of MM and provide a frameworkfor the therapeutic evaluation of FTIs in combination withconventional or novel therapies.

Acknowledgements The study was supported by AssociazioneItaliana per la Ricerca sul Cancro (AIRC) and the Ministry forEducation, Universities and Research (MIUR), “MolecularEngineering-C03” funds, Inter-University Funds for BasicResearch (FIRB), Rome, and the “Cassa di Risparmio di Puglia”Foundation, Bari.

References

1. Hideshima T, Bergsagel PL, Kuehl WM, Anderson KC (2004)Advances in biology of multiple myeloma: clinical applica-tions. Blood 104:607–618

2. Hannun YA (1997) Apoptosis and the dilemma of cancerchemotherapy. Blood 89:1845–1853

3. Tu Y, Renner S, Xu F, Fleishman A, Taylor J, Weisz J, VescioR, Rettig M, Berenson J, Krajewski S, Reed JC, LichtensteinA (1998) BCL-X expression in multiple myeloma: possibleindicator of chemoresistance. Cancer Res 58:256–262

4. Landowski TH, Gleason-Guzman MC, Dalton WS (1997)Selection for drug resistance results in resistance to Fas-medi-ated apoptosis. Blood 89:1854–1861

5. Watts GS, Futscher BW, Isett R, Gleason-Guzman M, KunkelMW, Salmon SE (2001) cDNA microarray analysis of mul-

tidrug resistance: doxorubicin selection produces multipledefects in apoptosis signaling pathways. J Pharmacol ExpTher 299:434–441

6. Yoshida NL, Miyashita T, U M, Yamada M, Reed JC, SugitaY, Oshi T (2002) Analysis of gene expression patterns duringglucocorticoid-induced apoptosis using oligonucleotidearrays. Biochem Biophys Res Commun 293:1254–1261

7. Nagata S (1997) Apoptosis by death factor. Cell 88:355–3658. Tschopp J, Irmler M, Thome M (1998) Inhibition of Fas death

signals by FLIPs. Curr Opin Immunol 10:552–5589. Krueger A, Baumann S, Kramme PH, Kirchhoff S (2001)

FLICE-inhibitory proteins: regulators of death receptor-medi-ated apoptosis. Mol Cell Biol 21:8247–8254

10. French LE, Tschopp J (1999) Inhibition of death receptor sig-naling by FLICE-inhibitory protein as a mechanism forimmune escape of tumors. J Exp Med 190:891–894

11. Djerbi M, Screpanti V, Catrina AI, Bogen B, Biberfeld P,Grandien A (1999) The inhibitor of death receptor signaling,FLICE-inhibitory protein defines a new class of tumor pro-gression factors. J Exp Med 190:1025–1032

12. Westendorf JJ, Lammert JM, Jelinek DF (1995) Expressionand function of Fas (APO-1/CD95) in patient myeloma cellsand myeloma cell lines. Blood 85:3566–3576

13. Frassanito MA, Silvestris F, Silvestris N, Cafforio P, CamardaG, Iodice G, Dammacco F (1998) Fas/Fas ligand (FasL)-deregulated apoptosis and IL-6 insensitivity in highly malig-nant myeloma cells. Clin Exp Immunol 114:179–188

14. Chauhan D, Kharbanda S, Ogata A, Urashima M, Teoh G,Robertson M, Kufe DW, Anderson KC (1997) Interleukin-6inhibits Fas-induced apoptosis and stress-activated proteinkinase activation in multiple myeloma cells. Blood89:227–234

15. Frassanito MA, Cusmai A, Iodice G, Dammacco F (2001)Autocrine interleukin-6 production and highly malignant mul-tiple myeloma: relation with resistance to drug-induced apop-tosis. Blood 97:483–489

16. Kim DK, Cho ES, Yoo JH, Um HD (2000) FLIP is constitu-tively hyperexpressed in Fas-resistant U-266 myeloma cells,but is not induced by IL-6 in Fas-sensitive RPMI 18226 cells.Mol Cells 10:552–556

17. Klein B, Zhang XG, Lu ZY, Bataille R (1995) Interleukin-6 inhuman multiple myeloma. Blood 85:863–872

18. Ogata A, Chauhan D, Teoh G, Treon SP, Urashima M,Schlossman RL, Anderson KC (1997) IL-6 triggers cellgrowth via the Ras-dependent mitogen-activated proteinkinase cascade. J Immunol 159:2212–2221

19. Fukada T, Hibi M, Yamanaka Y, Takahashi-Tezuka M,Fujitani Y, Nakajima K, Hirano T (1996) Two signals are nec-essary for cell proliferation induced by a cytokine receptorgp130: involvement of STAT3 in anti-apoptosis. Immunity5:449–460

20. Billadeau D, Liu P, Jelinek D, Shah N, LeBien TW, Van NessB (1997) Activating mutations in the N- and K-ras oncogenesdifferentially affect the growth properties of the IL-6-depen-dent myeloma cell line ANBL6. Cancer Res 57:2268–2275

21. Downward J (1998) Ras signaling and apoptosis. Curr OpinGenet Dev 8:49–54

22. Kazama H, Yonehara S (2000) Oncogenic K-Ras and basicfibroblast growth factor prevent Fas-mediated apoptosis infibroblasts through activation of mitogen-activated proteinkinase. J Cell Biol 148:557–566


23. Peli J, Schroter M, Rudaz C, Hahne M, Meyer C, ReichmannE, Tschopp J (1999) Oncogenic Ras inhibits Fas ligand-medi-ated apoptosis by downregulating the expression of Fas.EMBO J 18:1824–1831

24. Fenton RG, Hixon JA, Wright PW, Brooks AD, Sayers TJ(1998) Inhibition of Fas (CD95) expression and Fas-mediatedapoptosis by oncogenic Ras. Cancer Res 58:3391–3400

25. Reuter CW, Morgan MA, Bergmann L (2000) Targeting theRas signaling pathway: a rational, mechanism-based treat-ment for hematologic malignancies? Blood 96:1655–1669

26. Frassanito MA, Cusmai A, Piccoli C, Dammacco F (2002)Manumycin inhibits farnesyltransferase and induces apopto-sis of drug-resistant interleukin 6-producing myeloma cells.Br J Haematol 118:157–165

27. Le Gouill S, Pellat-Deceunynck C, Harousseau JL, Rapp MJ,Robillard N, Bataille R, Amiot M (2002) Farnesyl transferaseinhibitor R115777 induces apoptosis of human myelomacells. Leukemia 16:1664–1667

28. Shi Y, Gera J, Hsu JH, Van Ness B, Lichtenstein A (2003)Cytoreductive effects of farnesyl transferase inhibitors onmultiple myeloma tumor cells. Mol Cancer Ther 2:563–572

29. Micheau O, Lens S, Gaide O, Alevizopoulos K, Tschopp J(2001) NF-kappaB signals induce the expression of c-FLIP.Mol Cell Biol 21:5299–5305

30. Bharti AC, Shishodia S, Reuben JM, Weber D, Alexanian R,Raj-Vadhan S, Estrov Z, Talpaz M, Aggarwal BB (2004)Nuclear factor-kappaB and STAT3 are constitutively active inCD138+ cells derived from multiple myeloma patients, andsuppression of these transcription factors leads to apoptosis.Blood 103:3175–3184

31. Debatin KM, Krammer PH (2004) Death receptors inchemotherapy and cancer. Oncogene 23:2950–2966

32. Herr I, Debatin KM (2001) Cellular stress response and apop-tosis in cancer therapy. Blood 298:2603–2614

33. Nguyen JT, Wells JA (2003) Direct activation of the apopto-sis machinery as a mechanism to target cancer cells. Proc NatlAcad Sci USA 100:7533–7538

34. Shima Y, Nishimoto N, Ogata A, Fujii Y, Yoshizaki K,Kishimoto T (1995) Myeloma cells express Fas antigen/APO-1 (CD95) but only some are sensitive to anti-Fas antibodyresulting in apoptosis. Blood 85:757–764

35. Mitsiades N, Mitsiades CS, Poulaki V, Anderson KC, TreonSP (2002) Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in humanmultiple myeloma cells. Blood 99:2162–2171

36. Los M, Herr I, Friesen C, Fulda S, Schulze-Osthoff K,Debatin KM (1997) Cross-resistance of CD95- and drug-induced apoptosis as a consequence of deficient activation ofcaspases (ICE/Ced-3 proteases). Blood 90:3118–3129

37. Munshi NC, Hideshima T, Carrasco D, Shammas M, AuclairD, Davies F, Mitsiades N, Mitsiades C, Kim RS, Li C,Rajkumar SV, Fonseca R, Bergsagel L, Chauhan D, AndersonKC (2004) Identification of genes modulated in multiplemyeloma using genetically identical twin samples. Blood103:1799–1806

38. Hu L, Shi Y, Hsu JH, Gera J, Van Ness B, Lichtenstein A(2003) Downstream effectors of oncogenic ras in multiplemyeloma cells. Blood 101:3126–3135

39. Zhang B, Prendergast GC, Fenton RG (2002) Farnesyltran-sferase inhibitors reverse Ras-mediated inhibition of Fas geneexpression. Cancer Res 62:450–458

40. Hsu JH, Shi Y, Frost P, Yan H, Hoang B, Sharma S, Gera J,Lichtenstein A (2004) Interleukin-6 activates phosphoinosi-tol-3´ kinase in multiple myeloma tumor cells by signalingthrough RAS-dependent and, separately, through p85-depen-dent pathways. Oncogene 23:3368–3375

41. Rowley M, Van Ness B (2002) Activation of N-ras and K-rasinduced by interleukin-6 in a myeloma cell line: implicationsfor disease progression and therapeutic response. Oncogene21:8769–8775

42. Mayo MW, Baldwin AS (2000) The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance.Biochim Biophys Acta 1470:M55–M62

43. Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, RichardsonPG, Hideshima T, Munshi N, Treon SP, Anderson KC (2002)Biologic sequelae of nuclear factor-kappaB blockade in multi-ple myeloma: therapeutic applications. Blood 99:4079–4086

44. Ni H, Ergin M, Huang Q, Qin JZ, Amin HM, Martinez RL,Saeed S, Barton K, Alkan S (2001) Analysis of expression ofnuclear factor kappa B (NF-kappa B) in multiple myeloma:downregulation of NF-kappa B induces apoptosis. Br JHaematol 115:279–286

blockade of the ras pathway by manumycin, a farnesyltransferase inhibitor, overcomes the resistance...

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