J Cutan Pathol 2009: 36: 629–636doi: 10.1111/j.1600-0560.2008.01117.xJohn Wiley & Sons. Printed in Singapore

Copyright # 2009 John Wiley & Sons A/S

Journal of

Cutaneous Pathology

The role of RhoC in growth andmetastatic capacity of melanoma

Background: RhoC overexpression in tumor cells promotes invasiveand metastatic behavior. RhoC expression levels have been correlatedwith tumor progression and metastasis in multiple human cancers. Inmelanoma, RhoC is upregulated in highly metastatic tumors. Inducedexpression in melanoma cell lines resulted in invasion and metastasis,whereas inhibition of RhoC reversed the metastatic phenotype bothin vitro and in vivo.Methods: RhoC mRNA and protein expression in two humanmelanoma cell lines (DX3aza and MeWo) and pooled primarymelanocytes were investigated by means of real-time quantitativepolymerase chain reaction and western blotting. RhoC proteinexpression was evaluated in 123 primary cutaneous melanoma samplesby the use of immunohistochemistry and correlated with knownprognostic features.Results: RhoC upregulation was observed in the highly metastaticDX3aza cell line, whereas in MeWo, only low expression levels couldbe detected. RhoC expression in primary cutaneous melanoma wasstrongly associated with thicker and ulcerated tumors. RhoCexpression was associated with the presence of lymphatic metastases atthe time of diagnosis and shorter disease-free and overall survival rates,without being an independent predictor.Conclusion: These results further support a role for RhoC in growthand metastasis of melanoma.

Boone B, Gele MV, Lambert J, Haspeslagh M, Brochez L. The role ofRhoC in growth and metastatic capacity of melanoma.J Cutan Pathol 2009; 36: 629–636. # 2009 John Wiley & Sons A/S.

Barbara Boone1,2, Mireille VanGele1, Jo Lambert1,2, MarcHaspeslagh2 and LieveBrochez1,2

1Dermatology Research Unit, Department ofDermatology, and2Department of Dermatopathology, GhentUniversity Hospital, Ghent, Belgium

Barbara Boone, Department of Dermatology, GhentUniversitiy Hospital, De Pintelaan 185, 9000 Ghent,BelgiumTel: 132 9 332 58 36Fax: 132 9 332 49 96e-mail: barbara.boone@ugent.be

Accepted for publication June 17, 2008

Melanoma represents a significant health burden indeveloped countries. It mainly affects young people,1

and an efficient therapeutic approach for advancedstage disease is still lacking. The lifetime risk is stillincreasing.2,3 Insights into the molecules and path-ways that are involved inmelanoma development andprogression can contribute to better prognostic infor-mation and the definition of new therapeutic targets.RhoC belongs to the Ras-homologous (Rho)

guanosine triphosphate (GTP)-binding proteins,which act as molecular switches, cycling betweenactive GTP-bound and inactive guanosine diphos-phate (GDP)-bound states. Rho proteins are impor-tant regulators of the organization of actin andmicrotubule filaments. They are also implicated in

the control of cell adhesion, cell motility, cell cycleprogression, gene expression and apoptosis.4,5

Because all these functions are deregulated in cancer,it is not surprising that Rho proteins may beimplicated in malignant transformation and metas-tasis. No activating Rho mutations have beenreported in cancer; however, there is an increasingamount of evidence that overexpression ofRho familyproteins plays a role in tumorigenesis and metastasis.It has been shown by in vitro and in vivo experiments inhumanmammary epithelial cells, murine lung cancercells and esophageal squamous cell carcinomacells thatinduced RhoC overexpression promotes anchorage-independent growth and invasive and metastaticbehavior via the mitogen-activated protein kinase and

629

phosphatidylinositol-3 kinase (PI3K) pathways.6–8

RhoC is overexpressed in multiple human malig-nancies, and high RhoC expression levels have beencorrelated with tumor progression and metastasis invarious cancer types, such as breast cancer,9,10

ovarian cancer,11 bladder cancer,12 hepatocellularcarcinoma,13–15 colorectal cancer,16 esophageal squa-mous cell carcinoma,17–19 pancreatic cancer,20 gastriccancer,21 squamous cell carcinoma of the head andneck22 and cutaneous squamous cell carcinoma.23

Geneexpressionprofiling experiments inmelanomacell lines with low vs. high metastatic capacity revealedRhoC to be upregulated in the latter.24 Infection of thepoorly metastatic A375P melanoma cell line withretroviral particles containing full length humanRhoCresulted into RhoC overexpression and enhancedmetastatic capacity.24,25 Specific inhibition of RhoC inthe highly metastatic A375M human melanoma cellline reversed migration and invasion.26 Analogously,the cholesterol-lowering agent atorvastatin inhibitedRhoC activation and reverted the metastatic pheno-type of humanmelanoma cells in vitro. In addition, oralatorvastatin treatment dramatically inhibited coloni-zation and the formation of metastatic lesions in micethat were injected with A375M melanoma cells.27

The present study wanted to further assess the roleof RhoC in melanoma progression. For this purpose,mRNA and protein expression levels of RhoC in twohuman melanoma cell lines with different prolifera-tive and metastatic capacity were examined. Inaddition, RhoC immunostaining in 123 primarymelanoma tissues was studied and correlated withclinicopathological and follow-up data of the patients.

Materials and methods

Cell culture

Primary human epidermal melanocyte cultures wereestablished as described previously.28 Briefly, foreskinsfromneonatalswere incubatedovernight at 4�Cin10%Dispase II (Roche Diagnostics GmbH, Mannheim,Germany) to separate the epidermal layer (withmelanocytes anchored to the basal membrane) fromthe underlying dermis. Melanocytes were cultured inHam’s F10 medium (Invitrogen Ltd, Paisley, UK)supplemented with 2.5% fetal calf serum, 1%Ultroser-G, 5 ng/ml basic fibroblast growth factor, 10 ng/mlendothelin-1, 0.33 nM cholera toxin, 33 mM isobutyl-methylxanthine, 5.3 nM 12 tetradecanoylphorbol 13-acetate and antibiotics/antimycotics.

DX3aza and Mewo melanoma cell lines weremaintained in Dulbecco’s Modified Eagle’s Mediumsupplemented with 10% heat-inactivated fetal bovineserum, 100 IU/ml penicillin, 100 mg/ml streptomy-cin and 2.5 mg/ml amphotericin until subconfluency(80%). All cells were housed in an incubator at 37�C,99% humidity and 10% CO2.

Both cell lines were routinely tested for myco-plasm infection using the polymerase chain reaction(PCR)-based VenorGeM Mycoplasm Detection kit(Minerva Biolabs GmbH, Berlin, Germany), follow-ing the supplier’s instructions. Both cell lines weremycoplasm free.

RNA isolation and RT quantitative PCR

Total RNA was extracted from pooled primarymelanocytes (control sample) andmelanoma cell linesusing the RNeasy Mini Kit (Qiagen, Venlo, theNetherlands) according to manufacturer’s recommen-dations. All RNA were quantified using a ND-1000spectrophotometer (Isogen Life Science, St-Pieters-leeuw, Belgium). Total RNA (2 mg) was treated withthe RQ1 RNase-free DNase from Promega (Leiden,the Netherlands). In addition, treated RNA sampleswere desalted prior cDNA synthesis using Microcon-100 spin columns (Millipore, Brussels, Belgium). Firststrand cDNAwas synthesized using the iScript cDNASynthesis Kit according to the manufacturer (Bio-Rad, Eke, Belgium) and subsequently diluted withnuclease-free water (Sigma, Bornem, Belgium) to12.5 ng/ml cDNA. In order to quantify the geneexpression level of RhoC, primer sequences weredesigned using Primer Express software (AppliedBiosystems, Forster City, CA, USA). The primer pairused was RhoC (forward: 5#-CCCGTTCGGTCT-GAGGAA-3#; reverse: 5#-GAGCACTCAAGGTA-GCCAAAGG-3#). Relative gene expression levelswere determined using a SYBR Green I real-time(RT)-PCR assay as described by Vandesompeleet al.29 and the comparative CT method was usedfor quantification. PCR reagents were obtained asSYBR Green I mastermixes (Eurogentec, Seraing,Belgium) and used according to the manufacturer’sinstructions. PCR reactionswere run on anABIPrism7000 Sequence Detection System (Applied Biosys-tems). To correct for differences in RNA quantitiesand cDNA synthesis efficiency, relative gene expres-sion levels were normalized using the geometricmeanof three housekeeping genes (ribosomal protein L13a,ubiquitin-C and succinate dehydrogenase complexsubunit A) according to Vandesompele et al.30

Western blotting

Total cell lysates were solubilized and denaturated byboiling in 1 3/4 3 Laemmli sample buffer, contain-ing 5% b-mercapto-ethanol and 0.25%bromophenolblue. Proteins were separated by SDS-PAGE (12%gel) and electroblotted onto nitrocellulosemembranes(Amersham Biosciences UK Ltd, Buckinghamshire,UK). Membranes were blocked for 1 h at roomtemperature in 5% non-fat dry milk dissolved in

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Tris-buffered saline (137 mMNaCl, 20 mMTris, pH7.6) with 0.1% Tween-20 (TTBS), followed by over-night incubation at 4�C with anti-RhoC primaryantibody (sc-26481; Santa Cruz, Boechout, Belgium)at 1 : 100 dilution. Following several washing steps withTTBS,membraneswere incubatedwithahorseradishperoxidase-conjugated anti-goat immunoglobulin G(IgG) secondary antibody (sc-2020; Santa Cruz) atdilution 1 : 5000 during 1 h at room temperature.After final washing steps with TTBS, protein bandswere visualized with the enhanced chemilumines-cence detection kit (Amersham Biosciences UK Ltd)according to the manufacturer’s protocol and themembrane was exposed to X-ray films.

Patient and biopsy selection

Patients were identified retrospectively from themelanoma unit database, Department of Dermatol-ogy, University Hospital Ghent, Ghent, Belgium.In total, 123 patients, who were diagnosed with mel-anoma between January 1996 and August 2005 andwere subjected to a sentinel node biopsy, agreed uponparticipation. The collection of skin biopsies wasperformed after written informed consent. The studywas approved by the Institutional Ethical Committee(project number: 2004/041) and was conducted inaccordance with institutional guidelines on theDeclaration of Helsinki Principles.

Immunohistochemistry

Four micrometer sections were prepared fromparaffin-embedded melanoma specimens. Sectionswere dewaxed in toluene and hydrated with a gradedseries of ethanol concentrations and water. Sub-sequent antigen retrieval was obtained using hotwater bath incubation of sections in pH9 ethyl-enediaminetetraacetic acid or EDTA buffer (PP20-0226; Prosan, Merelbeke, Belgium) during 30 min.Endogenous peroxidase activity was quenched byincubating the section for 10 min with 3% hydrogenperoxide. In order to block specific binding sites, thesections were incubated with normal rabbit serum(sc-2338; Santa Cruz, TebuBio, Boechout, Belgium).Using a DAKO Autostainer device (Dako, Heverlee,Belgium), sections were incubated at room tempera-ture during 60 minwith anti-RhoCprimary antibody(sc-26481; Santa Cruz, TebuBio) diluted at 1 : 100 inAntibody Diluent (S0809; Dako). Subsequently, ananti-goat secondary antibody (E0466; Dako) dilutedat 1 : 400 in Antibody Diluent was applied during30 min. 3-amino-9-ethylcarbazole was used as chro-mogen. Finally, nuclei were counterstained withhematoxylin. Negative controls were performed bysubstituting the primary antibody with non-immune

goat IgG (sc-2028; Santa Cruz, TebuBio). RhoCimmunostaining in breast cancer tissue was used asa positive control.

Immunostained sections were investigated by twoobservers (B. B. and L. B.) who were blinded to theclinicopathological information of the patient. In caseof disagreement between the observers, sections werediscussed with a third observer (M. H.) until concor-dance was reached. RhoC immunoreactivity wasscored as a percentage of the total tumor area.Attention was paid to different staining patterns andintracellular distribution.

Statistical analysis

Associations between RhoC staining and clinico-pathological parameters were analyzed using theMann-Whitney U-test and Pearson chi-squared test.Kaplan-Meier survival analysis was used to obtainoverall survival and disease-free survival curves thatwere compared using the log rank test. Cox propor-tional hazards model and logistic regression analysiswere used to assess odds and hazards ratios and toevaluate whether RhoC was an independent predictor.

Results

RhoC mRNA and protein expression in melanoma cell lines

Because RhoC expression in melanocytes has beendescribed,27,31 we pooled cultured primary melano-cytes from 10 patients and used it as a control sample.Further, we cultured two melanoma cell lines withdifferent biological behavior. DX3aza has beendescribed as amelanoma cell linewith highmetastaticcapacity,32 whereas MeWo is known to have weakproliferative and metastatic capacity.33 The mRNAand protein expression levels of RhoC were deter-mined by use of RT quantitative PCR and westernblotting, respectively. The obtained results of twoseparate experiments were reproducible, and therelative mRNA expression values represent the mean(and SEM) of these two independent experiments.The mRNA expression in melanoma cell lines wasexpressed relative to that of pooled melanocytes forwhich the expression level was set as 1.

RhoC mRNA expression levels in MeWo (mean ¼0.60; SEM ¼ 0.30) were comparable with that ofpooledmelanocytes, whereas theywere upregulated inDX3aza (mean ¼ 5.84; SEM ¼ 1.52). Analogously,RhoC protein expression appeared to be upregulatedin DX3aza compared with MeWo (Fig. 1).

Clinicopathological characteristics

In total, 123 patients treated for primary cutaneousmelanoma with known sentinel lymphnode status

Role of RhoC in melanoma

631

gave informed consent to be included. There were 48(39%) males and 75 (61%) females with a mean age of50 years at time of diagnosis. According to thetumour-node-metastasis (TNM) classification,34 13(10.6%) melanomas presented as pT1 (Breslow� 1.0 mm), 67 (54.5%) melanomas as pT2 (Breslow1.01–2.0 mm), 34 (27.6%) melanomas as pT3(Breslow 2.01–4 mm) and 9 (7.3%) melanomas aspT4 (Breslow . 4.0 mm). Ulceration was present in33 (27.3%) patients. Lymphatic metastasis at the timeof diagnosis was present in 30 (24.4%) patients.Sentinel lymphnode biopsy was performed in allpatients and revealed metastatic invasion in 27 (22%)patients. Subsequent radical lymphadenectomyshowed additional lymphnodes to be involved in10/27 (37%) of these patients. Three patientspresented with in-transit metastasis at the time ofdiagnosis without the presence of a metastaticallyinvaded sentinel lymphnode (N2c). Disease progres-sion was observed in 21 (17.2%) patients with a meanfollow-up time of 34 months. At the end of the follow-up period, 12 (9.8%) patients had died (Table 1).

RhoC immunoreactivity in melanoma tissue

RhoC immunoreactivity in melanoma cells wasobserved in 46 (37.4%) of the primary melanomatissues. RhoC staining was mainly cytoplasmic

sometimes associated with a nuclear pattern. In mostpositive cases, RhoC staining was observed diffusethroughout the tumor with a weak-to-moderatestaining intensity (Fig. 2).RhoC expression was observed not only in

melanoma cells but also in hair follicle bulbs andsebaceous glands, whereas immunoreactivity wasconsistently absent in blood vessel endothelium andmuscle tissue.

Clinical significance of RhoC immunoreactivity inmelanoma tissues

RhoC expression in primary cutaneous melanomawas strongly associated with thicker tumors andulceration. The median Breslow thickness in RhoC-negative sectionswas 1.46 mmvs. 2.20 mm inRhoC-positive sections (Mann-Whitney U-test, p ¼ 0.000).

Fig. 1. A) Relative RhoC mRNA expression in melanoma cell lines

compared with pooled primary melanocytes (level 1). B) RhoC protein

expression in melanoma cell lines and pooled primary melanocytes.

Tubulin was used as a loading control. MC, melanocytes.

Table 1. Clinicopathological parameters

Number(%) Mean (95% CI)

Clinical characteristicsSex (n ¼ 123)Male 48 (39)Female 75 (61)

Age at time of diagnosis(years) (n ¼ 123)

50.06 (47.27–52.84)

Location of primary melanoma(n ¼ 123)Head and neck 10 (8.1)Trunk 41 (33.3)Extremities 72 (58.5)

Metastasis development duringfollow up (n ¼ 122)Absent 101 (82.8)Present 21 (17.2)

Mortality (n ¼ 123)Alive 111 (90.2)Dead 12 (9.8)

Disease-free survival time(months) (n ¼ 122)

32.72 (28.33–37.11)

Follow-up time (months)(n ¼ 122)

34.29 (30.05–38.52)

Pathological characteristicsBreslow (mm) (n ¼ 123)pT1 � 1.0 13 (10.6)pT2 1.01–2.0 67 (54.5)pT3 2.01–4.0 34 (27.6)pT4 . 4.0 9 (7.3)

Clark (n ¼ 119)Level II, III 65 (54.6)Level IV, V 54 (45.4)

Ulceration (n ¼ 121)Absent 88 (72.7)Present 33 (27.3)

Sentinel lymphnode (n ¼ 123)Negative 96 (78)Positive 27 (22)

Lymphatic metastasis attime of diagnosis(n ¼ 123)Absent 93 (75.6)Presence of positive sentinellymphnode

27 (22)

Presence of in-transitmetastases (N2c)

3 (2.4)

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RhoC expression was present in 57.6% (19/33) ofulcerated tumors vs. 29.6% (26/88) of non-ulceratedtumors (Pearson chi-squared test, p ¼ 0.004). RhoC

positivity occurred more frequently in patients withlymphatic dissemination at the time of diagnosis (16/30 or 53%) comparedwith patientswithout lymphaticmetastasis (30/93 or 32%) (Pearson chi-squared test,p ¼ 0.038). The relative risk of lymphatic dissemina-tion at the time of diagnosis in melanoma sampleswith positive RhoC staining was 2.4 (odds ratio ¼2.4; 95% CI ¼ 1.04–5.55). Multivariate analysisshowed RhoC not to be an independent predictor ofthe presence of lymphatic metastasis; this associationis probably explained by its strong correlation withtumor thickness and ulceration.

Overall survival tended to be higher in patientswithout RhoC expression in their primary melanoma(log rank test, p ¼ 0.04). The presence of RhoCexpression was significantly associated with reduceddisease-free survival (log rank test, p ¼ 0.023). Therelative risk of a shorter disease-free survival inpatients with RhoC expression compared withpatients without RhoC expression in their primarymelanoma is 2.6 (hazards ratio ¼ 2.6, 95% CI ¼1.1–6.229). However, after adjusting for Breslowthickness and ulceration, these associations were nolonger significant (Table 2, Fig. 3).

Discussion

Because RhoC is mainly known as a protein organiz-ing cytoskeletal structures and thereby involved in cellmotility and polarity, its specific role in tumormigration, invasion and metastasis has been wellestablished. In vitro migration and invasion assaysshowed inhibition of RhoC by means of the smallinterfering RNA (siRNA) technique or by the use ofgeranylgeranyl transferase inhibitors or C3 exotrans-ferase to result in a significant decrease of themigratory and invasive capacity of cell lines derivedfrom breast cancer,35–37 gastric cancer,21,38 prostatecancer39 and lung cancer.7 These in vitro findings were

Fig. 2. RhoC immunoreactivity in three primary cutaneous mela-

noma tissues. A and B) This picture shows a weak-to-moderate

cytoplasmic RhoC immunoreactivity in melanoma cells with an

increasing intensity toward the infiltrating border of the tumor. RhoC

expression can also be observed in a sebaceous gland (yellow arrow),

whereas it is absent in muscle tissue (green arrow). Original

magnification 325 (A), 3200 (B). C) In this primary melanoma

tissue, a mixed cytoplasmic and nuclear (yellow arrow) staining

pattern in melanoma cells is present. Original magnification 3200.

Table 2. RhoC immunoreactivity in primary cutaneous melanoma andprognostic pathological parameters

RhoC expression

Absent Present p Value

Breslow (mm)Median 1.46 2.20 0.000�

P25–P75 1.10–2.0 1.56–3.16Clark

I, III 50 15 0.001†

IV, V 25 29Ulceration

Absent 62 26 0.004†

Present 14 19Lymphatic metastasis (at time of diagnosis)

Absent 63 30 0.038†

Present 14 16

�Mann-Whitney U-test, Significance , 0.05.†Pearson chi-squared test, Significance , 0.05.

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confirmed byexperiments performed in tumormousemodels.7,8,36,40 Moreover, immunohistochemicalstudies investigating RhoC expression in tissuesamples from stomach cancer,21,41 esophageal can-cer,17,18 squamous cell carcinoma of the head andneck,22 primary hepatocellular carcinoma,15 coloncarcinoma16 and breast cancer9,10 showed RhoCimmunoreactivity to be associated with the presenceof metastatic disease.

Current insights in the role of RhoC in melanomaprogression are mainly based on experiments inmelanoma cell lines. Gene expression profilingrevealed an increased expression of RhoC inmelanoma cell lines with high compared with lowmetastatic potential. Infection of poorly metastaticmelanoma cell lines with retroviral particles over-expressingRhoC increased their motility and invasivecapacity both in in vitro assays and in animal tumormodels. Conversely, inhibition of RhoC activity bymeans of a dominant inhibitory Rho mutantmarkedly suppressed the generation of metastases.24

Selective inhibition of RhoC in a highly metastaticmelanoma cell line was found to reduce bothmigration and invasion activities.26 Inhibition ofRhoC isoprenylation by atorvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase,reverted the metastatic phenotype of human mela-noma cell lines in vitro and in a tumor animal model.27

RhoC promotion of invasion of melanoma cell lineswas recently shown to be driven through separatePI3K and Rho-kinase (ROCK) pathways.25 Immu-nohistochemistry in five primary cutaneous melano-mas and five metastatic melanoma lymphnodesshowed a lower staining intensity in the first group.27

Triggered by these findings, we aimed at furtherassessment of the role of RhoC in melanomaprogression. For this purpose, mRNA and proteinexpression levels of RhoC in two human melanomacell lines with different proliferative and metastatic

capacity were examined. In addition,RhoC immuno-staining in 123 primary melanoma tissues wasstudied and correlated with clinicopathological andfollow-up data of the patients.In this study, we compared RhoC mRNA and

protein expression in a melanoma cell line with highmetastatic (DX3aza) and low metastatic (MeWo)potential. DX3aza was derived from the humanmelanomacell lineDX3, established fromacutaneousmelanoma metastasis, treated with the nucleosideanalogue 5-azacytidine (5-azaC) resulting in a highlymetastatic cell line.32 MeWo was derived froma metastatic melanoma lymphnode of a white, 78-year-old male and has been found to be a cell linewith weak proliferative42 and metastatic capacity.33

Because RhoC expression has been reported inhumanmelanocytes,27,31 we pooled primary culturedmelanocytes of 10 patients and used this as a positivecontrol for the in vitro experiments. RhoCmRNA andprotein expression was upregulated in the highlymetastatic DX3aza cell line. These results are inagreement with the previously reported involvementofRhoC in highlymetastaticmelanoma cell lines.24,26

Contrary to its well-established role in tumormetastasis, the role of RhoC in tumor growth hasbeen investigated less extensively and the results arenot univocal. Induced RhoC expression did not affecttumor growth in orthotopic lung cancer in mice.7 Atransgenic mouse model showed no significant dif-ferences between the development and the growthof mammary adenocarcinomas in RhoC-deficientmice vs. RhoC-competent mice.40 However, inhibi-tion of RhoC by means of anti-RhoC siRNA or C3exotransferase resulted in a substantial reductionof the proliferation and the anchorage-independentgrowth in soft agar of stomach cancer cells,21 chol-angiocarcinoma cells43 and breast cancer cells.36,44 Innude mouse models, intratumoral injection of RhoCsiRNAhas been shown to inhibit the growth of gastric

Fig. 3. RhoC expression vs. overall (A) and

disease-free (B) survival.

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carcinoma and breast cancer.36,38 This study is thefirst to investigate the correlation between RhoCexpression in primary melanoma tissue and clinico-pathological follow-up data. We found RhoC immu-nostaining to be strongly associated with tumorthickness and the presence of ulceration, which areparameters correlating with growth of a primarycutaneous melanoma. This study is therefore thefirst to indicate a possible role of RhoC in tumorgrowth. RhoC expression was also associated withthe presence of lymphatic metastases at the time ofdiagnosis and with reduced disease-free survivalrates.Multivariate analysis revealed these associationsto be linked to the strong association of RhoC ex-pression with tumor thickness and ulceration. How-ever, because the number of patients developingmetastases was limited, this study could have beenunderpowered to show an independent role of RhoCin metastasis.Because we only analyzed primary cutaneous

melanoma samples of patients who have undergonea sentinel lymphnode biopsy, it is possible that ourseries has a bias toward cases with worse prognosis.The emerging role of RhoC in growth and

metastasis of melanoma and other human cancerssuggests that this molecule might be a target for noveltherapeutic options. To become biologically active,RhoC must be isoprenylated by geranylgeranyltransferase. Statins or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, which are widelyused in the treatment of hypercholesterolemia, blockgeranylgeranyl transferase activity leading to inacti-vation of RhoC. Atorvastatin was found to inhibitRhoC isoprenylation and metastasis in a mousemodel of human melanoma.27 A case control studyin 1318 cases and 6786 controls showed statin use tobe associated with reduced melanoma Breslowthickness,45 further supporting a possible role ofRhoC in melanoma growth. In addition, it has beenshown in The Air Force/Texas Coronary Atheroscle-rosis Prevention Study that the incidence of newmelanomas in the lovastatin arm was significantlydecreased comparedwith the placebo arm.46All theseobservations implicate a possible role for statins as anadjuvant and perhaps even chemopreventive strategyin melanoma, as has been suggested by others.47

Because statins also act on other Rho proteins andbecause the inhibitory effect on RhoC may not beequal for all statins,48 further studies investigating thespecific effect of different statins are needed.This study is the first to implicate a role for RhoC

protein in melanoma growth. Themelanoma cell linedata further support a possible role of RhoC inmelanoma metastasis. Other studies in several tumortypes have underscored the potentially crucialfunction of RhoC in tumor metastasis and perhaps

proliferation. This offers a possible target for newtherapeutic interventions.

Acknowledgements

The authors thank Mrs MC Herteleer, Mrs De Mil, Mrs R. Heyse

and Mrs S. D’Hont for their technical support and assistance in

completing this study. They also thank Prof. Dr D De Bacquer for

advice in the statistical analysis of the data and Prof. Dr M Bracke

(Laboratory of Experimental Cancerology, Ghent University

Hospital, Ghent, Belgium) for providing DX3aza melanoma cell

line. B. B. and M. V. G. are respectively research and postdoctoral

fellows of the Fund for Scientific Research – Flanders. This work

was also financially supported by the Fund for Scientific Research –

Flanders (Grant number G.0128.06).

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