Immunohistochemical profiling of Wnt, NF-κB, Stat3 and Notch signaling in human epidermal tumors
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LETTER TO THE EDITOR Immunohistochemical profiling of Wnt, NF-kB, Stat3 and Notch signaling in human epidermal tumors Multiple oncogenic factors such as cyclin D1, c-Myc, survivin and VEGF promote skin cancer formation, and their expression can be triggered by more than one pathway including NF-kB, Wnt, Stat3 and/or Notch signaling. The associations of those signaling pathways with epidermal tumors were proposed previously [1—3], but the knowledge about their roles in epidermal carcinogenesis remains incoher- ent because no correlative study has been per- formed on them using an identical experimental system. The current study thus aimed to assess the overall statuses of Wnt, NF-kB, Stat3 and Notch signaling in epidermal tumors by tissue microarray- based immunohistochemical staining. 21 basal cell carcinomas (BCCs), 19 squamous cell carcinomas (SCCs), 16 Bowen 0 s diseases, 12 Paget 0 s diseases, 10 actinic keratoses and 5 cutaneous horns were used in this study. Eight normal skin specimens obtained from plastic surgery were cited as normal control. The tissue samples were fixed with 10% formaldehyde for paraffin sections and pathological diagnosis. Tissue microarrays (TMAs) were con- structed by the method described previously [4] and sectioned to 5 mm thickness for immunohisto- chemical staining (IHC). The antibodies used were the goat anti-human Wnt2, mouse anti-human b- catenin, rabbit anti-human TCF4, mouse anti- human NF-kB p65, rabbit anti-human Stat3, mouse anti-human phospho-(Tyr705) Stat3, mouse anti- human Notch1, rabbit anti-human Notch2 antibo- dies purchased from Santa Cruz, CA, USA and a rabbit anti-human Hes1 antibody (a generous gift of Dr. Tetsuo Sudo, Toray Industries, Tokyo, Japan). The staining results were evaluated by two researchers, with the labeling density scored as negative (), weakly positive (+), moderately posi- tive (++) or strong positive (+++). Immunocytochem- ical (ICC) and immunofluorescent (IF) staining were performed on the cell-bearing coverslips of human skin SCC cell line, Colo16 and human immortalized keratinocyte line, HaCat, using the same antibodies mentioned above and an anti-human E-cadherin monoclonal antibody (NCH-38; Dako North America, Inc., CA, USA). Kruskal—Wallis and Mann—Whiteney tests were used for statistical analyses. Tumorigenic roles of Wnt signaling have been reported in many cancer types but less mentioned for epidermal tumors. As shown in Fig. 1a and summarized in Table 1, Wnt2 was detected in low incidence (37.5%; 3/8) and low level in normal epidermal tissues, while in 60% (6/10) of actinic keratoses, 62.5% (10/16) Bowen 0 s diseases, 68.42% (13/19) SCCs, 91.67% (11/12) Paget 0 s diseases and 100% BCCs (21/21) and cutaneous horns (5/5), sug- gesting a close association of Wnt2 expression with skin cancer formation. This notion was further sup- ported because of the absence of nuclear TCF4 and b-catenin staining in normal epithelial cells, and their strong nuclear labeling in 68.42% of SCCs, 95.24% BCCs, 62.5% Bowen 0 s diseases, 100% Paget 0 s s diseases, 40% actinic keratoses and 80% cutaneous horns. Interestingly, b-catenin/TCF4 nuclear co- translocation was absent in three Wnt2-expressing normal epidermis and HaCat cells that exhibit intact membranous E-cadherin, indicating the importance of membranous E-cadherin in preventing intracel- lular b-catenin accumulation and nuclear transmis- sion of Wnt signaling. The appearance of nuclear immuno-labeling of p65 and/or p50 NF-kB subunit reflects NF-kB activa- tion. Although NF-kB protein was detected in the cytoplasm of epidermal tissues irrespective to their morphologies, p65 nuclear translocation was unde- tectable in normal tissues, SCCs and actinic kera- toses, and in low rates in BCCs (14.29%), Bowen 0 s diseases (12.5%), Paget 0 s diseases (25%) and cuta- neous horns (20%). These results implicate the unlikelihood of involvement of this signaling in epi- dermal carcinogenesis or in regulating epidermal cancer-associated gene expression. Stat3 activation via p-Stat3 nuclear transloca- tion had been detected in cutaneous SCCs [2]. In this study, cytoplasmic distribution and nuclear translocation of Stat3 were observed in either normal (62.5%; 5/8) or the tissues with different neoplastic changes. However, only the cells located in the basal layer of normal epidermis were positive in p-Stat3, whereas p-Stat3 positive cells distribute diffusely in tumor tissues, especially in SCCs (Fig. 1a). Since normal basal cells keep constant regeneration to remedy the aged epithelial cells [5], and Stat3 is required for survival and pro- liferation of skin cancers [6], it would be reason- able to find p-Stat3 restrictively in the regenerative normal basal cells and diffusely in epidermal cancer tissues. Notch1 and Notch2 show equivalent or reverse biological effects in cell type-dependent fashions [7]. Impaired Notch signaling promotes de novo SCC formation [3], and Notch1, as a p53 target gene, involved in human keratinocyte tumor suppression [8]. Nevertheless, the data obtained from a good collection of skin tumors have not yet been avail- Letters to the Editor 133
Transcript of Immunohistochemical profiling of Wnt, NF-κB, Stat3 and Notch signaling in human epidermal tumors
LETTER TO THE EDITOR
Letters to the Editor 133
Immunohistochemical profiling of Wnt,NF-kB, Stat3 and Notch signaling inhuman epidermal tumors
Multiple oncogenic factors such as cyclin D1, c-Myc,survivin and VEGF promote skin cancer formation,and their expression can be triggered by more thanone pathway including NF-kB, Wnt, Stat3 and/orNotch signaling. The associations of those signalingpathways with epidermal tumors were proposedpreviously [1—3], but the knowledge about theirroles in epidermal carcinogenesis remains incoher-ent because no correlative study has been per-formed on them using an identical experimentalsystem. The current study thus aimed to assessthe overall statuses of Wnt, NF-kB, Stat3 and Notchsignaling in epidermal tumors by tissue microarray-based immunohistochemical staining.
21 basal cell carcinomas (BCCs), 19 squamous cellcarcinomas (SCCs), 16 Bowen0s diseases, 12 Paget0sdiseases, 10 actinic keratoses and 5 cutaneous hornswere used in this study. Eight normal skin specimensobtained from plastic surgery were cited as normalcontrol. The tissue samples were fixed with 10%formaldehyde for paraffin sections and pathologicaldiagnosis. Tissue microarrays (TMAs) were con-structed by the method described previously [4]and sectioned to 5 mm thickness for immunohisto-chemical staining (IHC). The antibodies used werethe goat anti-human Wnt2, mouse anti-human b-catenin, rabbit anti-human TCF4, mouse anti-human NF-kB p65, rabbit anti-human Stat3, mouseanti-human phospho-(Tyr705) Stat3, mouse anti-human Notch1, rabbit anti-human Notch2 antibo-dies purchased from Santa Cruz, CA, USA and arabbit anti-human Hes1 antibody (a generous giftof Dr. Tetsuo Sudo, Toray Industries, Tokyo, Japan).The staining results were evaluated by tworesearchers, with the labeling density scored asnegative (�), weakly positive (+), moderately posi-tive (++) or strong positive (+++). Immunocytochem-ical (ICC) and immunofluorescent (IF) staining wereperformed on the cell-bearing coverslips of humanskin SCC cell line, Colo16 and human immortalizedkeratinocyte line, HaCat, using the same antibodiesmentioned above and an anti-human E-cadherinmonoclonal antibody (NCH-38; Dako North America,Inc., CA, USA). Kruskal—Wallis and Mann—Whiteneytests were used for statistical analyses.
Tumorigenic roles of Wnt signaling have beenreported in many cancer types but less mentionedfor epidermal tumors. As shown in Fig. 1a andsummarized in Table 1, Wnt2 was detected in low
incidence (37.5%; 3/8) and low level in normalepidermal tissues, while in 60% (6/10) of actinickeratoses, 62.5% (10/16) Bowen0s diseases, 68.42%(13/19) SCCs, 91.67% (11/12) Paget0s diseases and100% BCCs (21/21) and cutaneous horns (5/5), sug-gesting a close association of Wnt2 expression withskin cancer formation. This notion was further sup-ported because of the absence of nuclear TCF4 andb-catenin staining in normal epithelial cells, andtheir strong nuclear labeling in 68.42% of SCCs,95.24% BCCs, 62.5% Bowen0s diseases, 100% Paget0ss diseases, 40% actinic keratoses and 80% cutaneoushorns. Interestingly, b-catenin/TCF4 nuclear co-translocation was absent in three Wnt2-expressingnormal epidermis and HaCat cells that exhibit intactmembranous E-cadherin, indicating the importanceof membranous E-cadherin in preventing intracel-lular b-catenin accumulation and nuclear transmis-sion of Wnt signaling.
The appearance of nuclear immuno-labeling ofp65 and/or p50 NF-kB subunit reflects NF-kB activa-tion. Although NF-kB protein was detected in thecytoplasm of epidermal tissues irrespective to theirmorphologies, p65 nuclear translocation was unde-tectable in normal tissues, SCCs and actinic kera-toses, and in low rates in BCCs (14.29%), Bowen0sdiseases (12.5%), Paget0s diseases (25%) and cuta-neous horns (20%). These results implicate theunlikelihood of involvement of this signaling in epi-dermal carcinogenesis or in regulating epidermalcancer-associated gene expression.
Stat3 activation via p-Stat3 nuclear transloca-tion had been detected in cutaneous SCCs [2]. Inthis study, cytoplasmic distribution and nucleartranslocation of Stat3 were observed in eithernormal (62.5%; 5/8) or the tissues with differentneoplastic changes. However, only the cells locatedin the basal layer of normal epidermis were positivein p-Stat3, whereas p-Stat3 positive cells distributediffusely in tumor tissues, especially in SCCs(Fig. 1a). Since normal basal cells keep constantregeneration to remedy the aged epithelial cells[5], and Stat3 is required for survival and pro-liferation of skin cancers [6], it would be reason-able to find p-Stat3 restrictively in theregenerative normal basal cells and diffusely inepidermal cancer tissues.
Notch1 and Notch2 show equivalent or reversebiological effects in cell type-dependent fashions[7]. Impaired Notch signaling promotes de novo SCCformation [3], and Notch1, as a p53 target gene,involved in human keratinocyte tumor suppression[8]. Nevertheless, the data obtained from a goodcollection of skin tumors have not yet been avail-
134 Letters to the Editor
Fig. 1 (a) Immunohistochemical illustration (�20) of Wnt, NF-kB, Stat3 and Notch signaling statuses in an epidermalsquamous cell carcinoma (SCC) and its normal counterpart (N). The arrows indicate the sites corresponding to the insetsand shown in higher magnification (�40). b,b-Catenin; T, TCF4. Both normal and SCC tissues are negative in Notch1 (datanot shown). (b) Immunocytochemical illustration (�40) of Wnt2, b-catenin, TCF4, E-cadherin, p-Stat3, Notch2 and Hes1expression and intracellular distribution in a human SCC (Colo16) and immortalized karatinocyte (HaCat) lines. NeitherColo16 nor HaCat cells show NF-kB p65/p50 nuclear labeling and Notch1 expression (data not shown).
able. We found that Notch1 was absent in normalepidermis, actinic keratoses, cutaneous horns andBCCs, and in a low rates among Bowen0s diseases(6.25%), SCCs (15.79%) and Paget0s diseases(33.33%). Notch2 and its target gene Hes1 couldbe detected in both cytoplasm and nuclei of normalepithelia (8/8), while their detection rates reducedin SCCs, BCCs, Bowen0s diseases and Paget0s diseasetissues, accompanied with attenuated expressionlevel (Table 1). These results indicate (1) that Notchsignaling in normal epidermis may be mediatedmainly by Notch2, and (2) that Notch signaling mighthave twofold bioactivities: it benefits normal main-tenance/renewal of epidermal cells, while itsattenuation may favor epidermal tumor growthdue to its tumor inhibitory effects [9].
ICC and IF evaluations of Wnt, Stat3, and Notchsignalings in Colo16 cells and HaCat cells revealed
similar findings (Fig. 1b) in terms ofWnt2 expressionin both cell lines but only Colo16 cells with b-catenin/TCF4 nuclear co-translocation, intactmembranous E-cadherin in HaCat but not in Colo16cells, nuclear p-Stat3 labeling in Colo16 rather thanHaCat cells, absence of Notch1 and nuclear NF-kB/p65 in both cell lines, and more distinct Notch2and Hes1 reduction in Colo16 cells. In summary, ourdata demonstrate the potential correlations ofepidermal carcinogenesis with Wnt activation,attenuated tendency of Notch signaling during epi-dermal cancer formation, the favorable roles ofStat3 activation in epidermal cell regenerationand tumor formation, and the coexistence of dif-ferent signaling alterations during epidermal car-cinogenesis. On the basis of current study, thedetailed functions of Wnt, Notch and Stat3 andthe potential involvements of other signaling such
Letters to the Editor 135
Table 1 Statuses of Wnt, NF-kB, Stat3 and Notch signaling and intracellular distribution of b-catenin and Hes1 indifferent histological groups of epidermal tissues
β-CateninWnt2
Membrane NucleusNF-κb* p-Stat3*# Notch1 Notch2 Hes1*
Group n
- + >++ - + >++ - + >++ - + >++ - + >++ - + >++ - + >++ - + >++
N 8 5 2 1 0 0 8 8 0 0 8 0 0 3 2 3 8 0 0 0 0 8 0 0 8
SCC 19 6 7 61
44 1 6 8 5 19 0 0 7 6 6 16 3 0 6 8 5 4 10 5
BCC 21 0 16 51
34 4 1 6 14 18 1 2
1
32 6 21 0 0 8 8 5 3 10 8
Bowen 16 6 10 0 4 7 5 6 6 4 14 2 0 6 7 3 15 1 0 4 5 7 3 8 5
Paget 12 1 3 8 2 6 4 0 8 4 9 1 2 8 3 1 8 2 2 5 3 4 4 3 5
AK 10 4 0 6 1 3 6 6 3 1 10 0 0 8 0 2 10 0 0 0 1 9 0 0 10
CH 5 0 3 2 2 2 1 1 2 2 4 1 0 2 1 2 5 0 0 0 0 5 0 0 5
*Nuclear labeling. #Restricted p-Stat3 nuclear labeling was found in the basal layer of five normal epidermal and two cutaneous horntissues. SCC: squamous cell carcinoma; BCC: basal cell carcinoma; AK: actinic keratosis; CH: cutaneous horn.
as sonic hedgehog and TGF-beta in epidermal tumorformation will be investigated.
Acknowledgments
We thank Dr. Tetsuo Sudo (Toray Industries, Tokyo,Japan) for providing anti-Hes1 antibody. This work isfunded by Natural Science Foundation of China(30527002, 30670946 and 30700365) and LiaoningDepartment of Education (2006-0193; 2007-7026).
References
[1] Kobielak A, Fuchs E. Links between alpha-catenin, NF-kap-paB, and squamous cell carcinoma in skin. Proc Natl Acad SciUSA 2006;103:2322—7.
[2] Suiqing C, Min Z, Lirong C. Overexpression of phosphorylated-STAT3 correlated with the invasion and metastasisof cutaneous squamous cell carcinoma. J Dermatol2005;32:354—60.
[3] Proweller A, Tu L, Lepore JJ, Cheng L, Lu MM, Seykora J, et al.Impaired notch signaling promotes de novo squamous cellcarcinoma formation. Cancer Res 2006;66:7438—44.
[4] Li H, Sun Y, Kong QY, Zhang KL, Wang XW, Chen XY, et al.Combination of nucleic acid and protein isolation with tissuearray construction: using defined histologic regions in singlefrozen tissue blocks for multiple research purposes. Int J MolMed 2003;12:299—304.
[5] Houben E, De Paepe K, Rogiers V. A keratinocyte0s course oflife. Skin Pharmacol Physiol 2007;20:122—32.
[6] Chan KS, Sano S, Kiguchi K, Anders J, Komazawa N, Takeda J,et al. Disruption of Stat3 reveals a critical role in both theinitiation and the promotion stages of epithelial carcinogen-esis. J Clin Invest 2004;114:720—8.
[7] Weng AP, Aster JC. Multiple niches for Notch in cancer:context is everything. Curr Opin Genet Dev 2004;14:48—54.
[8] Lefort K, Mandinova A, Ostano P, Kolev V, Calpini V, KolfschotenI, et al. Notch1 is a p53 target gene involved in human kera-tinocyte tumor suppression through negative regulationof ROCK1/2 and MRCKalpha kinases. Genes Dev 2007;21:562—77.
[9] Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling indevelopment and cancer. Endocr Rev 2007;28:339—63.
Zhi-Li Liu,
Liaoning Laboratory of Cancer Genomicsand Department of Cell Biology,
College of Basic Medical Sciences,Dalian Medical University,116044 Dalian, PR China
Department of Dermatology,Dalian Hospital for Skin Diseases,
116021 Dalian, PR China
Yan LiQing-You Kong
Liaoning Laboratory of Cancer Genomicsand Department of Cell Biology,
College of Basic Medical Sciences,Dalian Medical University,116044 Dalian, PR China
Cheng ZhanDepartment of Pathology,
Dalian Hospital for Skin Diseases,116021 Dalian, PR China
136 Letters to the Editor
Qian WangXiao-Yan Chen
Yuan SunSu Wen
Cai-Xia TuJia Liu
Hong Li*Liaoning Laboratory of Cancer Genomics
and Department of Cell Biology,College of Basic Medical Sciences,
KEYWORDSSjogren-Larsson syndrome; Fatty aldehyde dehydrogenase;ALDH3A2; Mutation; Ichthyosis
Dalian Medical University,116044 Dalian, PR China
*Corresponding author. Tel.: +86 411 86110316E-mail address: [email protected]
(H. Li)
22 February 2008
doi:10.1016/j.jdermsci.2008.06.011
LETTER TO THE EDITOR
A novel homozygous missense mutationin the fatty aldehyde dehydrogenasegene causes Sjogren-Larsson syndrome
Fig. 1 Clinical features and pedigree of the patient.Generalized xerotic skin with fine scales was observed andirregular linear scaling was predominately seen on thearms (a) and the thighs (b). The pedigree shows that theproband was the only affected individual. Arrow, proband;oblique line, deceased (c).
Sjogren-Larsson syndrome (SLS: OMIM number270200) is an autosomal recessive neurocutaneousdisorder characterized by congenital ichthyosis,mental retardation, and spastic diplegia or tetra-plegia. This genodermatosis is currently consideredto be categorized in the ‘‘lamellar granule diseases’’[1]. The disease is caused by mutations in ALDH3A2,which encodes fatty aldehyde dehydrogenase(FALDH: E.C. 1.2.1.48). This enzyme catalyzes theNAD-dependent oxidation of long-chain aldehydesderived from lipid metabolism. To date, more than70 mutations in ALDH3A2 have been reported world-wide. Whereas several common mutations havebeen identified, the majority of the mutations arescattered throughout ALDH3A2. Here, we describea novel missense mutation in a Japanese patientwith SLS.
A 54-year-old Japanese man was referred to ourdepartment for a genetic diagnosis of his SLS. He hadbeen clinically diagnosed with SLS based on typicalclinical manifestations, consisting of congenitalgeneralized ichthyosis, spastic tetraplegia, andmental retardation. At the time of the referral tous, the patient showed marked ichthyosis withsevere pruritus on his entire body, including his face,trunk, and extremities (Fig. 1a and b). Moderatehyperkeratosis was noted on the palms and soles.His functional status was poor (grade 4). Spastictetraplegia was associated with inability of gait.
