IMMUNOHISTOCHEMICAL ASSESSMENT OF E-CADHERIN AND β-catenin IN TRICHOFOLLICULOMAS AND TRICHOEPITHELIOMAS

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1 Biomed Pp Med Fc Univ Plcky Olomouc Czech Repu. 2007, 151(2): M. Bezdekov, S. Brychtov, E. Sedlkov, J. Steigerov, A. Hloilkov, M. Bienov, R. Kucerov, T. Brycht, V. Krejci, Z. Kolr 251 IMMUNOHISTOCHEMICAL ASSESSMENT OF E-CADHERIN AND β-catenin IN TRICHOFOLLICULOMAS AND TRICHOEPITHELIOMAS Michl Bezdekov *, Svetln Brychtov, Ev Sedlkov, Jn Steigerov, Alice Hloilkov, Mrtin Bienov, Rent Kucerov, Toms Brycht c, Veronik Krejci, Zdenek Kolr Lortory of Moleculr Pthology nd Deprtment of Pthology, Fculty of Medicine nd Dentistry, Plcky University, Hnevotinsk 3, Olomouc, Czech Repulic Deprtment of Dermtovenerology, Fculty of Medicine nd Dentistry, Plcky University nd University Hospitl, I. P. Pvlov 6, Olomouc, Czech Repulic c Deprtment of Internl Medicine, SPEA Olomouc, nm. Hrdinu 2, Olomouc, Czech Repulic e-mil: mfiurskov@seznm.cz Received: Septemer 5, 2007; Accepted: Novemer 1, 2007 Key words: E-cdherin/β-ctenin/Immunohistochemistry/Pthogenesis/Trichoepitheliom/Trichofolliculom Bckground: Trichofolliculoms nd trichoepithelioms re enign skin neoplsms originting from hir follicle cells. They result from defects in the signling pthwys tht regulte hir follicle morphogenesis nd regenertion. Thus they seem to e n excellent model of these processes. It is known tht the E-cdherin/β-ctenin system of dhesion molecules plys crucil role in the mintennce of tissue rchitecture. Aim: The im of the present study ws to investigte their involvement in enign hir follicle tumor development. Methods: Semiquntittive intensity of expression were exmined in formlin-fixed nd prffin-emedded tissue sections of 53 trichoepithelioms, 15 trichofolliculoms nd 19 norml skin smples y indirect immunohistochemistry. Results: The intensity of E-cdherin/β-ctenin expression in tumor cells did not differ from controls. However, norml hir follicles cells exhiited memrnous E-cdherin/β-ctenin expression, wheres oth types of tumors, prticulrly trichoepithelioms, showed E-cdherin/β-ctenin expression with predominntly cytoplsmic locliztion. Conclusions:We suggest tht this dystopic distriution of the E-cdherin/β-ctenin complex in hir follicle tumor cells my e mrker of cell-cell dhesion disruption which my contriute to the tumor formtion. INTRODUCTION Trichofolliculoms (TF) nd trichoepithelioms (TE) re enign tumors derived from the hir follicle (hir follicle tumors, HFT) (ref. 1 ). According to recent dt, disruption of norml derml-epiderml interctions in HFT resulting in ltered hir follicle cell morphogenesis is ssumed 2. In norml epithelil structures, cell-cell junctions ply n importnt role in the mintennce, integrity nd morphology of the epithelium 3. It hs een reported tht the E-cdherin/β-ctenin system of dhesion molecules plys crucil role in this processes 4. E-cdherin is cell dhesion trnsmemrne molecule which is memer of fmily of functionlly relted trnsmemrne glycoproteins tht medite C 2+ -dependent intercellulr cellulr dhesion 5. E-cdherin is importnt for epiderml intercellulr dherence ecuse it is required for the dhesive properties of kertinocytes nd skin differentition nd loss of E-cdherin-medited cell dhesion is one rte-limiting step in the progression from denom to crcinom 6. Furthermore, it determines the morphogenesis nd hir follicle cycling 7. The dhesion is medited vi interction with djcent cells through their N-terminl ectodomins nd the cytoplsmic terminl til of E-cdherin links specificlly to β-ctenin tht inds directly to the cytoskeletl ctin 5. A deficiency in E-cdherin cuses loss of dherent junctions which leds to impired intercellulr signling, ut not to direct tumor trnsformtion 8. It hs een reported tht dysfunction or disruption of cell dhesion molecules ccompny the invsiveness nd metsttic ehvior of mlignnt cells 9 nd tht loss or decresed expression of E-cdherin is frequently oserved minly in dvnced, poorly differentited crcinoms 10. β-ctenin is moleculr sensor tht integrtes cellcell junctions nd cytoskeletl dynmics with signl ing pthwys ffecting morphogenesis, tissue homeostsis, nd intercellulr communiction within tissues 11. It hs een shown tht differentition of the hir follicle is regulted nd linked to the sme pthwys tht initite its development 12. Generlly, β-ctenin hs dul function. It plys key role in cell-cell dhesion y linking cdherins to α-ctenin nd cytoskeletl ctin 4. In the sence of Wnt signl, β-ctenin is constitutively down-regulted y multicomponent destruction complex contining GSK3β (glycogen synthse kinse 3β), xin

2 252 M. Bezdekov, S. Brychtov, E. Sedlkov, J. Steigerov, A. Hloilkov, M. Bienov, R. Kucerov, T. Brycht, V. Krejci, Z. Kolr nd tumor suppressor APC (denomtous polyposis coli). These proteins promote the phosphoryltion of serine nd threonine residues in the NH 2 -terminl region of β-ctenin. β-ctenin is then degrded y csein kinse CK1 nd protein phosphtses PP2A nd PP2C through the uiquitin protesome pthwy. Wnt signling inhiits this process, leding to n ccumultion of β-ctenin in the nucleus which promotes the formtion of trnscriptionlly ctive complexes with memers of the Tcf/lef fmily (T-cell fctor/lymphoid enhncer fctor). The Tcf/β-ctenin heterodimers ct s iprtite trnscription fctors nd ctivte expression of the specific Wnt responsive genes tht encode proteins regulting cell cycle, e.g. c-myc, Cyclin D1 nd Pitx Altertions in β-ctenin medited regultion hve een demonstrted minly during cncer development, where muttions of the β-ctenin gene CTNNB.1 result in disruption of lrge numer of cellulr functions leding to loss of growth control nd neoplstic chnge However, β-ctenin muttions lso induce enign tumor growth, s hs een demonstrted for exmple in pilomtricoms 7. This study is focused on study of the E-cdherin/ β-ctenin expression in trichofolliculoms (TF) nd trichoepithelioms (TE), enign dnexl neoplsms originting from the hir follicle 20, 21. We hypothesize tht errnt ctivtion of these cell dhesion molecules my e implicted in hir follicle tumor development nd cn contriute to etter understnding of hir follicle cell regultion. MATERIAL AND METHODS Smples Formlin-fixed, prffin-emedded, routine iopsy specimens were tken from the tissue rchive of Deprtment of Pthology, Fculty of Medicine nd Dentistry, Plcký University, Olomouc. Ech smple ws reexmined y n experienced pthologist in order to confirm the dignosis. Fifty-three trichoepithelioms nd fifteen trichofolliculoms were nlyzed. Nineteen specimens of helthy sclp skin tken from uthopsy (9 smples) nd from iopsy (10 smples) were used s controls. Immunohistochemistry Indirect immnunohistochemicl stining of 5 μm thick tissue sections ws performed using the immunostiner Benchmrk (Ventn Medicl Systems S.A.). Sections were incuted with mouse monoclonl ntiodies nti-e-cdherin (clone 36B5, dilution 1 : 25, Novocstr, UK), nti-β-ctenin (clone E-5, dilution 1 : 50, Snt Cruz Biotechnology, CA, USA). Colon crcinom nd ductl rest crcinom tissues were used s positive control for β-ctenin nd E-cdherin, respectively. The primry ntiodies were omitted in the cse of negtive controls. Semiquntittive ssessment of protein expression ws performed using modified H-score in which oth intensity nd proportion of stining ws ctegorized. Ctegory A indicted the proportion of positive cell stining throughout the section nd ws ssigned scle from 0 to 3 (0 = 0 4 %; 1 = 5 24 %; 2 = %; 3 = %). Ctegory B represented the verge intensity; the presence of negtive, wek, intermedite nd strong stining ws given score from 0 to 3. Ctegory A ws multiplied y ctegory B to form multiplictive score. The cses were sorted into three sugroups; H-score 0 referred to negtive expression; H-score 1-2 to wek expression; H-score 3-9 to moderte/strong expression. Sttisticl nlysis The protein expressions were evluted using Chi-squre test (p < 0.05). The cellulr locliztions of proteins were sttisticlly nlyzed using Fisher s exct test with Ytes correction (p < 0.05). RESULTS E-cdherin E-cdherin positivity ws found in the memrnes of epiderml kertinocytes nd norml hir follicle cells nd seceous cells (Fig. 1) where moderte to strong intensity of the protein expression previled (Fig. 2). When compring norml epithelil/hir follicle/seceous cells to tumorous ones, mrked differences in protein expression or locliztion were found. In TF, sucellulr relocliztion of the protein ws oserved (Fig. 1); prt from typicl memrnous, lso cytoplsmic (42.9 %) or oth cytoplsmic nd memrnous locliztion of protein (21.4 %) were seen (p = 0.003) (Fig. 2). The intensity of E-cdherin expression in tumor cells did not differ from control cells. TE exhiited totl loss of purely memrnous E-cdherin locliztion; oth cytoplsmic (68.4 %) nd comined memrnous nd cytoplsmic (28.9 %) expression ws oserved (p = ) (Fig. 2). The protein expression in tumor cells ws significntly decresed in comprison to norml control kertinocytes (p = 0.01). Furthermore, TE exprimed E-cdherin in lower levels thn TF (p = 0.05). β-ctenin The protein β-ctenin ws present in high to moderte levels in memrnes of epiderml kertinocytes nd in norml hir follicle cells (Fig. 3, 4). In TF, significnt chnges in protein locliztion were oserved in contrst to control epithelil cells nd cells of hir follicle nd seceous glnds (p = 0.001); 50 % of the tumor smples hd cytoplsmic only or comined memrnous/cytoplsmic coexpression (Fig. 3). TF showed similr intensities of β-ctenin expression s control skin cells. In TE, signifi cnt loss of memrne-type expression ws oserved in contrst to control epithelium (p < ); 81.3 % of tumor smples demonstrted cytoplsmic nd 9.4 % memrnous/cytoplsmic β-ctenin stining. The difference in β-ctenin locliztion ws pprent etween TE nd TF tumor cells (p = 0.002) wheres memrneous pttern of stining previled in

3 Immunohistochemicl ssessment of E-cdherin nd β-ctenin in trichofolliculoms nd trichoepithelioms 253 Fig. 1. Memrnous stining of E-cdherin in the hir follicle of norml skin, originl mgnifiction 200 (); Strong cytoplsmic stining of E-cdherin in enign trichofolliculom cells, originl mgnifiction 400 (). Fig. 2. Comprison of E-cdherin expression in the epithelium, dnex nd tumor of trichoepithelioms (TE), trichofolliculoms (TF) nd control skin (C) (); E-cdherin distriution in the tumor smples (M=memrnous, C=cytoplsmic, N=nucler) (). Fig. 3. Memrnous stining of β-ctenin in the control norml epidermis, originl mgnifiction 400 (); β-ctenin positivity on the memrne nd in the cytoplsm of trichoepitheliom tumor cells, originl mgnifiction 320 ().

4 254 M. Bezdekov, S. Brychtov, E. Sedlkov, J. Steigerov, A. Hloilkov, M. Bienov, R. Kucerov, T. Brycht, V. Krejci, Z. Kolr Fig. 4. Comprison of β-ctenin expression in the epithelium, dnex nd tumor of trichoepithelioms (TE), trichofolliculoms (TF) nd control skin (C) (); β-ctenin locliztion in trichofolliculoms nd trichoepithelioms (M=memrnous, C=cytoplsmic, N=nucler) (). TF (Fig. 4). No nucler β-ctenin stining ws found either in TE or TF. TE hd only slightly reduced β-ctenin expression compred to control cells. However, TE reveled significnt decrese in β-ctenin in comprison to TF (p = 0.021). DISCUSSION In tumors, we reveled loss of the memrnous β-ctenin immunostining, which is usully oserved in the norml kertinocytes, hir follicles cells nd seceous cells, ccompnied y trnsloction of the protein to the cytoplsmic comprtment. Trichoepithelioms exhiited minly cytoplsmic, less frequently comined memrnous/cytoplsmic stining. On the other hnd third of trichofolliculoms showed preserved memrnous protein expression. Memrnous locliztion of the protein is known to e ssocited with its physiologicl function s modultor of intercellulr communiction, tissue morphogenesis nd homeostsis in different cell types. It hs een proposed tht β-ctenin nucler nd/or cytoplsmic locliztion my correlte with β-ctenin gene muttion 22, 23. In generl, chnges in β-ctenin regultion hve een demonstrted minly during cncer development, where muttions of the β-ctenin gene CTNNB.1 result in disruption of numer of its cellulr functions leding to loss of growth control. Recent studies hve found tht errnt ctivtion of β-ctenin is lso involved in the regultion of enign tumor growth. Exon 3 β-ctenin-ctivting muttions were found in ll cses of pilomtricom; the tumors disply oth nucler nd cytoplsmic, ut only focl memrnous protein expression in the sloid cells 24, 25. The nucler protein ccumultion hs een demonstrted to e ssocited with its stiliztion which is induced y inhiition of GSK3β nd results in reduced β-ctenin phosphoryltion. However, in our study we demonstrted no nucler β-ctenin distriution. On this sis, we suggest tht the Wnt signling pthwy is not involved in the trichofolliculom nd trichoepitheliom development. These dt re prlleled y Demirkn et l. who detected no β-ctenin gene muttion in other enign trichogenic tumors except of pilomtrixoms 26. To support this hypothesis, c-myc nd cyclin D1 expressions were nlyzed in tumors (dt not shown). The levels of c-myc nd cyclin D did not differ from the protein expression in control hir follicle nd seceous cells. Therefore, we consider cytoplsmic distriution of β-ctenin to e importnt for the TE nd TF formtion. Similrly, E-cdherin showed loss of memrne-type expression with relocliztion into the cytoplsm of tumor cells, predominntly in trichoepithelioms. No chnges in intensity of expression however were found. While loss of E-cdherin expression hs een oserved in mny mlignncies s n indictor of the protein muttion ssocited with its enhnced trnscriptionl ctivity 9, 27 29, the cytoplsmic or nucler protein locliztion is considered to e mrker of ltertions in its intercellulr signling. It hs een reported tht the decresed tyrosine phosphoryltion of E-cdherin my reflect loss of the E-cdherin/ β-ctenin complex function nd my e implicted in tumor invsion 30. When we compred TE nd TF, difference in protein locliztion ws noted. In lrge percentge of TF, memrnous stining ws preserved. This suggests tht norml intercellulr communiction is, t lest prtly mintined. From histogenetic point of view, trichofolliculoms re considered to e hmrtoms of hir follicles wheres trichoepithelioms demonstrte clssicl enign dnexl neoplsms, whose tumor cells, like in mny other tumors, proliferte in the sence of stimulting norml signls 31. We conclude tht disturnce in the norml cell-cell dherent junction is proly the key event in the trichofolliculom nd trichoepitheliom formtion.

5 Immunohistochemicl ssessment of E-cdherin nd β-ctenin in trichofolliculoms nd trichoepithelioms 255 ACKNOWLEDGEMENTS This work ws supported y the Ministry of Helth of the Czech Repulic (grnt IGA MZ CR NR8386 3) nd y the Ministry of Eduction of the Czech Repulic (grnt MSM ). REFERENCES 1. Doglioni C, Piccinin S, Demontis S, Cngi MG, Peccirini L, Chirelli C, Armellin M, Vukosvljevic T, Boiocchi M, Mestro R. Altertions of et-ctenin pthwy in non-melnom skin tumors: loss of lph-abc nucler rectivity correltes with the presence of et-ctenin gene muttion. Am J Pthol 2003; 163: Millr SE. Moleculr mechnisms regulting hir follicle development. J Invest Dermtol 2002; 118: Almi J, Willims BR, Yeger H. Differentil expression of E-cdherin nd et-ctenin in primry nd metsttic Wilms's tumours. Mol Pthol 2003; 56: Weis WI, Nelson WJ. Re-solving the cdherin-ctenin-ctin conundrum. J Biol Chem 2006; 281: Young P, Boussdi O, Hlfter H, Grose R, Berger P, Leone DP, Roenek H, Chrny P, Kemler R, Suter U. 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