Expression of fibrogenic cytokines in desmoplastic malignant melanoma

Expression of fibrogenic cytokines in desmoplasticmalignant melanoma

M.KUBO, K.KIKUCHI,* K.NASHIRO,* T.KAKINUMA, N.HAYASHI, H.NANKO† ANDK.TAMAKIDepartment of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113–8655, Japan*Department of Dermatology, Tokyo University Branch Hospital, Tokyo, Japan†Department of Dermatology, Tokyo Koseinenkin Hospital, Tokyo, Japan

Accepted for publication 19 February 1998

Summary Desmoplastic malignant melanoma (DMM) consists of amelanotic spindle-shaped melanoma cellsand is accompanied by desmoplasia with fibrous stromata. It has a strong tendency for localinfiltrative growth and recurrence and a propensity for neurotropism. It is not yet known whichcytokine is responsible for the desmoplasia in DMM. In the present study, we investigated the roles ofseveral fibrogenic cytokines and cytokine receptors in DMM: basic fibroblast growth factor (bFGF),connective tissue growth factor (CTGF), transforming growth factor-b, platelet-derived growth factor(PDGF) and PDGF receptors. Immunostaining and in situ hybridization were conducted in four casesof DMM and four cases of amelanotic malignant melanoma (AMM) as negative controls fordesmoplasia. PDGF-b receptor, bFGF and CTGF were intensely expressed in the DMM specimens incomparison with the AMM specimens. The reaction of PDGF-B ligand and CTGF to PDGF-b receptor,in addition to the expression of bFGF, may contribute to the desmoplasia in DMM.

Desmoplastic malignant melanoma (DMM) is a rareclinicopathological entity of spindle cell melanoma pro-posed by Conley et al. in 1971.1 Histopathologically,DMM consists of amelanotic spindle-shaped melanomacells; it is accompanied by desmoplasia with a fibrousstroma, and has a strong tendency for local infiltrativegrowth and recurrence and a propensity for neurotrop-ism.2,3 DMM is thought to be the result of dedifferentia-tion of melanoma cells due to mechanical irritation.However, it is unclear which cytokine is responsible forthe desmoplasia in DMM.

Transforming growth factor (TGF)-b is a family ofmultifunctional regulatory peptides which act on fibro-blasts to promote the production of collagen or fibro-nectin comprising the extracellular matrix and whichinhibit the proteinases of the extracellular matrix.4

Three isotypes of TGF-b (TGF-b1, TGF-b2 and TGF-b3) have been identified in humans.

Platelet-derived growth factor (PDGF) is a disulphide-linked dimer consisting of two related polypeptidechains (designated A and B) encoded by separategenes, and biologically active PDGF occurs either as ahomodimer (AA and BB) or as a heterodimer (AB).5,6

Two kinds of PDGF receptors (PDGF-a receptor and

PDGF-b receptor) are presently known. PDGF-b recep-tor shows a high affinity to only PDGF-BB and PDGF-AB, while PDGF-a receptor shows a high affinity toPDGF-AA, PDGF-BB and PDGF-AB. Recent findingsindicate that both chemotaxis and proliferation aremediated by signal transduction via either PDGF-areceptor or PDGF-b receptor.7,8

Many kinds of malignant tumours includingmalignant melanoma have been reported to expressPDGF.9–11 PDGF acts as a potent mitogen in variouskinds of cells and acts as a growth factor in fibroblasts inwound healing.6 Thus, PDGF is thought to contribute tothe fibrosis of the dermis. Additionally, the angiogenicpotency of PDGF has been suggested to proliferatecapillaries in damaged tissues while a wound is heal-ing.6 The proliferation of capillaries around malignantmelanoma may therefore be induced by the presence ofPDGF.

Basic fibroblast growth factor (bFGF) is a member ofa homologous polypeptide gene family and functionsas a mitogen, morphogen and angiogenesis promoterfor a variety of mesenchymal and neuroectodermal-derived cells.12,13 bFGF is also found as a potentinducer of PDGF receptors.14 Al-Alousi et al. recentlyrevealed the expression of bFGF in DMM.15 In thisstudy, we investigated the expressions of TGF-b, PDGF

British Journal of Dermatology 1998; 139: 192–197.

192 q 1998 British Association of Dermatologists

Correspondence: M.Kubo. E-mail: [email protected]

and other fibrogenic cytokines to clarify their roles inDMM.

Materials and methods

Specimens

Human DMM specimens and human amelanotic malig-nant melanoma (AMM) specimens were collectedduring surgical procedures. The specimens includedfour primary DMMs, three primary AMMs and onemetastatic AMM. The tumour cells were observed byimmunohistochemistry to have S-100 protein in eachcase of DMM and AMM. All specimens were fixed by10% formaldehyde and embedded in paraffin. The dataon the patients in this study are shown in Table 1.

The primary antibodies used are listed in Table 2.Polyclonal antibodies to human PDGF-A chain andPDGF-B chain and monoclonal antibody to humanPDGF-b receptor were purchased from Genzyme (Cam-bridge, MA, U.S.A.). Monoclonal antibody to humanPDGF-a receptor was purchased from Biogenesis (San-down, NH, U.S.A.). Polyclonal antibody to bFGF was

kindly provided by Kaken Pharmaceutical Corporation(Tokyo, Japan).

Antisense oligonucleotide probes were purchasedfrom R&D Systems (Abingdon, U.K.) (TGF-b1) andOncogene Research Products (Cambridge, MA, U.S.A.)(TGF-b2). We made the 40-mer antisense oligonucleo-tide probe of TGF-b3 from the cDNA of exon 1 whichhad no codes similar to any known sequences of humancDNA. The probes not labelled with digoxigenin (DIG)were labelled by a DIG oligonucleotide tailing kit (Boeh-ringer Mannheim, Mannheim, Germany). The cDNA ofconnective tissue growth factor (CTGF) was a kind giftfrom Dr G.R.Grotendorst, and the antisense probe fromthe cDNA was labelled with DIG by a DIG labelling kit(Boehringer Mannheim).

In situ hybridization

The in situ hybridization of CTGF was performed using aslight modification of the non-radioactive in situ hybri-dization technique with DIG-labelled RNA probes aspreviously reported.16

Investigations of the in situ hybridization of TGF-b1,TGF-b2 and TGF-b3 were performed using an in situhybridization kit (R&D Systems) according to the manu-facturer’s recommendations. Briefly, the sections weredewaxed by xylene and rehydrated through gradedethyl alcohol and phosphate-buffered saline (PBS). Thesections were then incubated for 30 min in 5 mg/mLproteinase K in 0·05 mol/L hydroxymethyl aminomethane(Tris) pH 7·6 to improve the penetration of the labelledDNA-probes. Proteinase K digestion was stopped by fixa-tion in cooled 0·4% paraformaldehyde/PBS. The tissuesections were incubated in prehybridization buffer con-sisting of 50% deionized formamide/2 × standard salinecitrate buffer (SSC) at 37 8C for at least 30 min. DIG-labelled DNA probes in hybridization buffer were hybri-dized at 37 8C overnight. The hybridized tissue sections

FIBROGENIC CYTOKINES AND MALIGNANT MELANOMA 193

q 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 192–197

Table 1. Details of the patients with desmoplastic malignantmelanoma (DMM) and amelanotic malignant melanoma (AMM)examined in this study

Age(years) Sex Primary or metastatic Site of the tumour

DMM 1 86 F Primary Right footDMM 2 68 M Primary Oral mucosaDMM 3 43 M Primary Left footDMM 4 76 F Primary ScalpAMM 1 58 M Primary Left legAMM 2 59 M Metastatic AbdomenAMM 3 88 M Primary Left palmAMM 4 41 F Primary Right foot

Table 2. Origin and working dilution of the antibodies

Designation Cell identified Origin Working dilution

Polyclonal rabbit antihuman PDGF-AA PDGF-A chain presenting cells Genzyme 1 : 50Polyclonal rabbit antihuman PDGF-BB PDGF-B chain presenting cells Genzyme 1 : 50Monoclonal mouse antihuman PDGF receptor b-subunit PDGF receptor b-subunit presenting cells Genzyme 1 : 100Monoclonal antibody to PDGF receptor a-subunit PDGF receptor a-subunit presenting cells Biogenesis 1 : 100Polyclonal antibody to human bFGF bFGF presenting cells Kaken Pharmaceutical 1 : 1000

Corporation

PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor.

were washed in graded SSC in 30% formamide at 37 8Cand modified Tris-buffered saline. The hybridized messen-ger RNAs (mRNAs) were then visualized by a DIG nucleicacid detection kit (Boehringer Mannheim) which usedthe alkaline phosphatase reaction to 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.

Immunohistochemistry

Immunostaining on paraffin-embedded sections wasperformed using a Vectastain ABC kit (Vector Labora-tories, Burlingame, CA, U.S.A.) according to the manu-facturer’s recommendations. Five-micrometre thicksections were mounted on APES-coated slides, thendeparaffinized by xylene and rehydrated through agraded series of ethyl alcohol and PBS. The sectionswere then incubated with each polyclonal or mono-clonal antibody diluted in PBS (see Table 2) or controlnormal mouse IgG overnight at 37 8C. The immuno-reactivity was visualized by diaminobenzidine. Thesections were then counterstained with haematoxylin.

We use the following grading system: þ for slightstaining, þþþ for strong staining, and þþ for stainingbetween þ and þþþ.

Results

The results of the in situ hybridization and immunohis-tochemistry are shown in Table 3.

In situ hybridization

We investigated the expression of the mRNAs of TGF-b1, TGF-b2 and TGF-b3. No expression of these mRNAs

was found in any of the eight cases of DMM or AMM, ineither the tumour cells or the surrounding fibroblasts.

Expression of CTGF mRNA was found on the fibro-blasts in fibrotic areas of specimens from two of theDMM patients (Fig. 1a,b). We did not detect the expres-sion of CTGF in the other two DMM cases or in any ofthe four AMM cases (Fig. 1c). CTGF mRNA was notexpressed on the fibroblast-like tumour cells of the DMMtissues or the ordinary (not desmoplastic) melanomacells of the AMM tissues.

Immunohistochemistry

In all eight cases of AMM or DMM, PDGF-A chain andPDGF-B chain were slightly stained on the small vesselsin and around the tumour nests. There were no sig-nificant differences in their intensity between the DMMsamples and the AMM samples. However, we did notdetect the expression of PDGF-A chain or PDGF-B chainon the tumour cells themselves in the DMM or AMMcases.

Expression of PDGF-a receptor was not found in anyof the DMM or AMM cases. We found intense expressionof PDGF-b receptor in the fibroblast-like tumour cells inthree of the four DMM cases (Fig. 2a). In the other caseof DMM, expression of PDGF-b receptor was only slightin the fibroblast-like tumour cells. In contrast, in AMM,slight expression was found on the tumour cells of onlyone case (Fig. 2b). In the other three cases of AMM, noexpression of PDGF-b receptor was observed on thetumour cells. Weak expression of PDGF-b receptor wasobserved in the fibroblasts of all four AMM cases, but theintensity of the expression was much weaker than the

194 M.KUBO et al.


Table 3. The results of in situ hybridization and immunohistochemistry examining the expression of various cytokines in desmoplastic malignantmelanoma (DMM) and amelanotic malignant melanoma (AMM). Expression of connective tissue growth factor (CTGF) was seen in the fibroblastsaround the tumour. Expression of platelet-derived growth factor (PDGF)-A chain and PDGF-B chain was found around the tumour cells. Theexpression in tumour cells of PDGF-b receptor and basic fibroblast growth factor (bFGF) is shown in this table

In situ hybridization Immunohistochemistry

TGF-b1 TGF-b2 TGF-b3 CTGF PDGF-A chain PDGF-B chain PDGF-a receptor PDGF-b receptor bFGF

DMM 1 – – – þ þ þ – þþþ þþþ

DMM 2 – – – – þþ þ – þþ þþ

DMM 3 – – – þ þ þ – þþþ þþ

DMM 4 – – – – þ þ – þ þ

AMM 1 – – – – þ þ – – þ

AMM 2 – – – – þ þ – – þ

AMM 3 – – – – þ þ – – –AMM 4 – – – – þ þ – þ –

TGF-b, transforming growth factor-b; –, negative staining; þ, slight staining; þþ, moderate staining; þþþ, strong staining.

intensity on the fibroblast-like tumour cells of the threecases of DMM.

Expression of bFGF was intense in the spindle cells ofthree of the DMM cases and weak in the other DMM case(Fig. 3a). In two of the AMM cases, only a weakexpression in comparison with that in the DMM tissues

was found in limited areas of the tumour cells (Fig. 3b).In the other two cases of AMM, no expression wasobserved on the tumour cells. We could detect noexpression of bFGF in the fibroblasts surrounding theAMMs.

Discussion

In recent years much interest has been shown in thepathogenesis of the unique morphology of collageniza-tion in DMM.2 In DMM, collagenization has been seen inboth the spindle-shaped tumour cells and the fibroblastsmigrated from the surrounding dermis. Several cyto-kines may be responsible for the differentiation of thetumour cells into adaptive fibroblasts by metaplasia andthe migration of the fibroblasts of the surroundingdermis into the tumour, but it has been unclear as towhich cytokines cause the prevalence of fibroblasts andthe spindle-shaped tumour cell formation.

A model for human DMM in nude mice was recently



Figure 1. Expression of connective tissue growth factor (CTGF) mRNAwas seen in the fibrotic area around the desmoplastic malignantmelanoma (DMM) in in situ hybridization in two cases. The expressionwas seen in the fibroblasts but not in the tumour cells (a: originalmagnification × 200; b: original magnification × 400). However, noexpression of CTGF mRNA was found in the other cases of DMM (c:original magnification × 200) or any of the cases of amelanoticmalignant melanoma.

Figure 2. Intense expression of platelet-derived growth factor (PDGF)-b receptor was found in fibroblast-like tumour cells of desmoplasticmalignant melanoma (a: original magnification × 200), whereasslight expression of PDGF-b receptor was found on the tumour cellsof only one case of amelanotic malignant melanoma (b: originalmagnification × 200).

proposed, and observations of the model suggest thattumoral fibroblasts might be the predominant cell typesynthesizing collagen in these tumours.17 Additionally,a human melanoma cell line (UCT mel-7) consistentlyproduces a desmoplastic tumour reminiscent of desmo-plastic melanoma when injected into nude mice. Whenthis cell line is cocultured with fibroblasts, the latter cellsare stimulated to synthesize collagen at a twofoldgreater rate than when cultured alone.18 The UCTmel-7 line, in contrast, does not then produce collagenat a significantly greater rate.18 Based on these accu-mulated findings, we can suggest that some cytokinesexpressed on the tumour cells of DMM may play a partin collagenization.

In human malignant melanoma, the expression ofPDGF ligands and PDGF receptors has been investi-gated11,19,20 Barnhill et al. detected the expression ofPDGF-A chain, PDGF-B chain and PDGF-a receptor inhuman malignant melanomas by in situ hybridizationand immunohistochemistry, but the expression ofPDGF-b receptor was not found.11 The non-detection

of PDGF-a receptor and the detection of PDGF-b recep-tor in the present study probably occurred because ofthe different sensitivities of the immunohistochemicaltests. In our study, expression of PDGF-b receptor in theDMM specimens was evident in comparison withthe AMM specimens on the fibroblast-like cells and thetumour cells. These results suggest that reaction ofthe PDGF-B chain to the PDGF-b receptor induces thedesmoplasia in DMM. There are several reports of theexpression of PDGF-b receptor in vascular endotheliumassociated with inflammation and tumours other thanmalignant melanoma.21,22 These findings are in accordwith the strong tendency of DMM to metastasize andwith the clinical inflammatory phenotype of DMM.

The relationships between PDGF receptors and bFGFfound in our study have previously been reported.14,23

In these reports, bFGF was thought to induce PDGF-areceptor, but not PDGF-b receptor.14,23 However, it wasPDGF-b receptor and not PDGF-a receptor that wasstained intensely in the present DMM cases. From theseresults, three hypotheses can be proposed: (i) in contrastto the previous suggestions,14,23 PDGF-b receptor isinduced by long-term autocrine stimulation by bFGFin vivo; (ii) PDGF-b receptor and bFGF are independentlyexpressed in DMM and both support the clinical man-ifestation of desmoplasia; and (iii) the reaction betweenPDGF-b receptor and PDGF induces bFGF. In malignanttumours, it is known that various cytokines and tumourmarkers can be expressed simultaneously, and we thinkthat it is most likely that bFGF and PDGF-b receptor areexpressed independently, although we cannot concludethis from the results of only this study.

In this study, expression of mRNA of CTGF wasrevealed in DMM and not in AMM. The expression ofmRNA of CTGF in the DMM tissues was found only atthe fibroblasts in the fibrous tissue which showed noatypia, and not in the tumour cells. CTGF has also beenfound not in the lymphocytes but in the fibroblasts inthe fibrous tissue of systemic sclerosis and localizedscleroderma.16,24 Thus, we think that CTGF is expressedin the course of the fibrosis of DMM and that CTGF isinduced by some other cytokines including bFGF. CTGFalso shows biological behaviour quite similar to that ofPDGF, and CTGF has been reported to compete with thePDGF-BB ligand in PDGF cell surface receptor bindingcompetition assay.25 Thus, CTGF can also react withPDGF-b receptor, although the receptor protein of CTGFhas been unclear. The reaction between CTGF andPDGF-b receptor may contribute to the desmoplasia inDMM.

In conclusion, expression of CTGF mRNA and intense

196 M.KUBO et al.


Figure 3. Strong expression of basic fibroblast growth factor (bFGF)was found in the spindle-shaped cells of desmoplastic malignantmelanoma (a: original magnification × 200), whereas a weak andfocal expression of bFGF was found in melanoma cells of amelanoticmalignant melanoma (b: original magnification × 200).

expression of bFGF and PDGF-b receptor were found inDMM tissues in this study. The reaction of PDGF or CTGFto PDGF-b receptor may also contribute to the forma-tion of the unique morphology of DMM in addition tothe expression of bFGF, which was previously found inDMM.15

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Expression of fibrogenic cytokines in desmoplastic malignant melanoma

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