Claudin11 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin, but Uncommon in Nevus Cell Nevi
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Cancers 2015, 7, 1233-1243; doi:10.3390/cancers7030834 cancersOPEN ACCESS ISSN 2072-6694 www.mdpi.com/journal/cancers Article Claudin11 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin, but Uncommon in Nevus Cell Nevi Sara K. Walesch 1, Antje M. Richter 1, Peter Helmbold 2 and Reinhard H. Dammann 1;* 1 Institute for Genetics, Justus-Liebig-University Giessen, Giessen D-35392, Germany; E-Mails: [email protected] (S.K.W.); [email protected] (A.M.R.) 2 Department of Dermatology, University of Heidelberg, Heidelberg D-69120, Germany; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-641-99-35462; Fax: +49-641-993-5469. Academic Editor: Chyi-Chia Richard Lee Received: 6 May 2015 / Accepted: 1 July 2015 / Published: 7 July 2015 Abstract: Epigenetic inactivation of tumor-related genes is an important characteristic in the pathology of human cancers, including melanomagenesis. We analyzed the epigenetic inactivation of Claudin 11 (CLDN11) in malignant melanoma (MM) of the skin, including six melanoma cell lines, 39 primary melanoma, 41 metastases of MM and 52 nevus cell nevi (NCN). CLDN11 promoter hypermethylation was found in 19 out of 39 (49%) of the primary MM and in 21 out of 41 (51%) of the MM metastases, but only in eight out of 52 (15%) of NCN (p = 0.001 and p = 0.0003, respectively). Moreover, a significant increase in the methylation level of CLDN11 from primary melanomas to MM metastases was revealed (p = 0.003). Methylation of CLDN11 was significantly more frequent in skin metastases (79%) compared to brain metastases (31%; p = 0.007). CLDN11 methylation was also found in five out of six MM cell lines (83%) and its promoter hypermethylation correlated with a reduced expression. Treatment of MM cell lines with a DNA methylation inhibitor reactivated CLDN11 transcription by its promoter demethylation. In summary, CLDN11 proved to be an epigenetically inactivated tumor related gene in melanomagenesis, and analysis of CLDN11 methylation level represents a potential tool for assisting in the discrimination between malignant melanoma and nevus cell nevi. Cancers 2015, 7 1234 Keywords: malignant melanoma; Claudin 11; tumor suppressor gene; epigenetics; DNA methylation 1. Introduction Malignant melanoma is a malignant skin cancer showing a rising incidence worldwide [1]. Several molecular pathways have been found altered in melanocytic tumors including the MAPK pathway, the p16INK4a/RB pathway and the Hippo/Ras Association Domain Family (RASSF) pathway [2–4]. Aberrant regulation of these pathways is accomplished through inactivation of tumor suppressor genes (e.g., RASSF10) and activation of proto-oncogenes (e.g., BRAF)[5,6]. Apart from mutation, the epigenetic silencing of tumor suppressor genes is a frequent and fundamental event in the pathogenesis of cancer, including melanomagenesis [7,8]. This inactivation is achieved by hypermethylation of CpG island promoters in malignant melanoma. In this context, methylation markers may serve as important tools to distinguish between benign lesions and aggressive tumors. Recently, it has been suggested that Claudin 11 (CLDN11) could be a useful epigenetic biomarker for identifying malignant melanoma [9]. CLDN11 is a member of the claudin family that encodes integral membrane proteins and is involved in the formation of the paracellular tight junction seal in tissues [10,11]. Thus CLDN11 harbors a Claudin_2/PMP22 domain (Figure1a) that is also found in the peripheral myelin protein PMP22 and the epithelial membrane proteins (e.g., EMP1) [12]. So far, 27 members of the CLDN family (CLDN1 to 27) have been identified in the human genome [13]. Expressional analysis suggests that several claudin genes exhibit decreased transcript levels in cancer. However, CLDN3, CLDN4 and CLDN7 levels are elevated in certain tumor entities [10]. For CLDN11 it has been reported that it is silenced in gastric cancer by promoter hypermethylation and its inactivation is associated with invasiveness of this cancer [14]. A genome-wide analysis has identified the methylation of CLDN11 in primary cutaneous melanoma [15]. However, the epigenetic regulation (e.g., expression) in melanoma has not been analyzed. The aim of our study was to illuminate the epigenetic inactivation of CLDN11 in malignant melanomas (MM) in more detail. Here, we report a significant increase in the methylation level of CLDN11 in MM metastases compared to primary MM and nevus cell nevi. 2. Results 2.1. Epigenetic Inactivation of CLDN11 in Malignant Melanoma Recently hypermethylation of Claudin 11 (CLDN11) has been reported in primary melanomas [9], however its epigenetic regulation was not analyzed in detail. The schematic promoter region of CLDN11 and according CpGs are shown in Figure1a. The promoter lies within a CpG island of 1644 bp on chromosome 3q26.2 from position 17011361243 to 17011371886 (UCSC genome browser). To reveal the epigenetic status of CLDN11 in malignant melanoma (MM) cell lines, we have analyzed its aberrant methylation in buf1280, C8161, IGR1, MeWo, SKMEL13, SKMEL28, lung cancer (A549), cervix cancer (HeLa) and human fibroblast (HF) by COBRA (Figure1b). Fragmentation of the PCR product by TaqI indicates an underlying methylated CLDN11 promoter. In five MM cell lines (buf1280, C8161, Cancers 2015, 7 1235 Cancers 2015, 7 3 IGR1,IGR1, MeWo, MeWo, SKMEL28) SKMEL28) hypermethylation hypermethylation of of CLDN11CLDN11 waswas detected detected (Figure (Figure 11b).b). CLDN11CLDN11 waswas unmethylatedunmethylated in in normal normal human human fibroblast fibroblast (HF) (HF) and and melanoma melanoma cell cell line line SKMEL13 SKMEL13.. Methylation Methylation of of CLDN11CLDN11 waswas also also observed observed in in A549 A549 and and HeLa HeLa cancer cancer cells cells (Figure (Figure 1b).1b). Previously, Previously, we we analyzed analyzed the the BRAFBRAF mutational mutational status status in in MM MM cell cell lines lines [5] [5.]. There There was was no no obvious obvious correlation correlation between between CLDN11CLDN11 methylationmethylation and and BRAF BRAF mutation mutation stat statusus in in MM MM cell cell lines lines (Figure (Figure 1b1b).). FigureFigure 1. 1. EpigeneticEpigenetic regulation regulation of of ClaudinClaudin 1111 (CLDN11(CLDN11)) in in malignant malignant melanoma. melanoma. (a ) (aStructure) Structure of of the theCLDN11 CLDN11CpG CpG island island promoter promoter on chromosome on chromosome 3 and 3 theand CLDN11 the CLDN11 protein protein(207 aa).(207 Arrows aa). Arrows mark transcriptionalmark transcriptional (+1) start (+1) site start for siteCLDN11. for CLDN11.Vertical Vertic linesal indicate lines indicateCpGs. CpG Thes. 157 The bp 157 PCR bp productPCR product with respective with respective primers primers and the andTaq Ithe site Taq areI site depicted. are depicted.The conserved The conserved Claudin_2 Claudin_2 superfamily superfamily domain of CLDN11domain of is marked;CLDN11 (b )is Methylation marked; (banalysis) Methylation of CLDN11 analysisby of CLDN11 COBRA. by Bisulfite-treated COBRA. Bisulfite DNA-treated from DNA MM from cell linesMM cell (buf1280, lines (buf1280,C8161, IGR1,C8161, MeWo, IGR1, MeWo, SKMEL13 SKMEL13 and SKMEL28), and SKMEL28), lung cancer lung cancer A549, A549 HeLa, HeLa and human and humanfibroblasts fibroblast (HF)s was(HF amplified,) was amplified, digested di withgestedTaq withI (+) Taq or mockI (+) digestedor mock ( ´digest) anded resolved (−) and on resolved2% gels on with 2% a 100gels bpwith marker a 100 (M). bp Themarker methylation (M). The status methylation of CLDN11 status(meth./methylated of CLDN11 (meth./meand unmeth./unmethylated)thylated and unmeth./unmethylated) and BRAF status (WT/wild and BRAF type, V600E/Codonstatus (WT/wild 600 mutationtype, V600E/Codonand nd/not determined) 600 mutation are and indicated. nd/not determined) are indicated. SubsequentlySubsequently,, we we analyzed analyzed the the expression expression of of CLDN11CLDN11 inin six six MM MM cell cell lines lines and and normal normal human human epidermalepidermal melanocytes melanocytes (NHEM) (NHEM) by by RT RT-PCR-PCR (Figure (Figure 22aa).). CLDN11CLDN11 mRNAmRNA levels levels w wereere reduced reduced in in buf1280,buf1280, C8161 C8161,, IGR1 IGR1 and and MeWo MeWo compared compared to to NHEM NHEM (Figure (Figure 2a2a).). Treatment Treatment of of these these four four cell cell lines lines withwith 5- 5-aza-2aza-2′-deoxycyticine1-deoxycytidine (A (Aza),za), a asubstance substance that that inhi inhibitsbits DNA DNA methylation, methylation, resulted resulted in in increased increased CLDN11CLDN11 expressionexpression (Figure (Figure 2a2a).). In In SKMEL13 SKMEL13 cells, cells, which which harbor harbor an an unmethylated unmethylated promoter, promoter, CLDN11CLDN11 expressionexpression was was observerd observerd in in untreated untreated cells cells (Figure (Figure 2a2a).). In In SKMEL28 SKMEL28 cells cells with with a apartially partially methylated methylated CLDN11CLDN11 promoterpromoter,, there there was was no noinduction induction of CLDN11 of CLDN11 expressionexpression after after Aza Azatreatment treatment.. To analyze To analyze the impactthe impact of Aza oftreatment Aza treatment on DNA onmethylation, DNA methylation, quantitative quantitative bisulfite sequencing bisulfite was sequencing performed was (Figure performed 2b). For(Figure all four2b). MM For cell all lines four that MM exhibited