Claudin 1 differentiates endometrioid and serous papillary endometrial adenocarcinoma

Gynecologic Oncology 103 (2006) 591 – 598 www.elsevier.com/locate/ygyno

Claudin 1 differentiates endometrioid and serous papillary endometrial adenocarcinoma☆ Gábor Sobel a,1 , Júlia Németh b,1 , András Kiss b , Gabor Lotz b , István Szabó a , Nóra Udvarhelyi c , Zsuzsa Schaff b,⁎, Csilla Páska b a

2nd Department of Obstetrics and Gynecology, Semmelweis University, H-1082 Budapest, Üllõi út 78/a, Hungary b 2nd Department of Pathology, Semmelweis University, H-1091 Budapest, Üllõi út 93, Hungary c National Institute of Oncology, H-1122 Budapest, Ráth György u. 7-9, Hungary Received 1 February 2006 Available online 22 June 2006

Abstract Objective. The expression of claudins, the main tight junction proteins involved in cell adhesion and carcinogenesis, was studied in endometrioid (type I) and seropapillary (type II) endometrial adenocarcinoma. The characteristics and possible diagnostic potential of claudin expression pattern were investigated in the two cancer types having different prognosis. Methods. Protein and mRNA expression of claudins was evaluated in 17 endometrioid carcinomas and 15 seropapillary adenocarcinomas by immunohistochemistry and real-time PCR in comparison with 38 cases of hyperplasia, normal proliferative and secretory endometrium samples. Further, protein expressions used in diagnostics (estrogen and progesterone receptors, p53, PCNA and β-catenin) were also studied. Results. In endometrioid carcinoma and hyperplasia low claudin 1 and high claudin 2 protein contents, whereas in seropapillary adenocarcinoma high claudin 1 and low claudin 2 levels were detected. Intense protein expression was noted for claudins 3, 4, 5, and 7, without significantly different patterns in carcinoma, hyperplasia, secretory, and proliferative endometrium. Real-time PCR results confirmed differences in claudin 1 but not claudin 2 mRNA expression, whereas some minor discrepancies were observed in comparison with immunohistochemistry patterns. Conclusion. The two types of endometrial adenocarcinomas were well distinguished by claudins 1 and 2 by immunohistochemistry, claudins 3, 4, and 7, however, did not prove useful in distinguishing the two entities. The similar claudin pattern seen in hyperplasia and endometrioid carcinoma and the differences regarding seropapillary adenocarcinoma support the dualistic model of endometrial carcinogenesis. The claudin pattern of the two tumor types might reflect a different cellular or pathogenetic pathway as well as a different cell adhesion behavior explaining the invasive properties. © 2006 Elsevier Inc. All rights reserved. Keywords: Claudins; Endometrioid carcinoma; Seropapillary endometrial adenocarcinoma

Introduction Endometrial carcinoma (EC) is the most prevalent malignancy of the female genital tract in the developed ☆

The research was supported by the following grants: NKFP1/00009/2005 by the Hungarian Ministry of Education, National Research Development Projects and OTKA T049559 by the Hungarian National Scientific Research Fund. ⁎ Corresponding author. Fax: +36 1 215 6921. E-mail address: [email protected] (Z. Schaff). 1 Equally contributed. 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.04.005

countries [1–5]. EC can be classified biologically and histologically into two diverse groups with different pathogenesis [5]. The most common form which accounts for > 80% of ECs is called endometrioid or type I EC, is low grade, estrogen-dependent, usually associated with complex and atypical endometrial hyperplasia, with a better prognosis [5]. In contrast, the more aggressive nonestrogenrelated cancers (type II) occur in older postmenopausal women, are high grade, and lack an association with hyperplasia [6]. However, paradigmatic cases also exist with overlapping clinical, morphological, immunohistochemical,

592

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598

and molecular features, sharing characteristics of both type I and II endometrial carcinomas [7]. The most common histological subtypes in the second group are the serous and clear cell types. Different genetic anomalies have been detected in ECs as microsatellite instability, mutations of the PTEN, KRAS, βcatenin, TP53 genes and loss of heterozygosity on several chromosomes [7,8], and a dualistic model of endometrial carcinogenesis has been proposed [9,10]. Endometrioid carcinoma is associated with a progressive, estrogen-driven model of carcinogenesis leading to cancer through a stepwise progression from normal – to atypical complex hyperplasia – to carcinoma. In contrast, serous adenocarcinomas are not estrogen-driven tumors [8]. However, the molecular basis for endometrial tumorigenesis has not been clearly elucidated [11]. Recent studies dealt with the differences in several markers distinguishing type I and II ECs. Besides estrogen/progesterone receptors, p53 may have role in differentiation, giving a nuclear/ cytoplasmic positivity mainly in EC type II, however, neither C-erbB-2 nor PTEN has been found useful in differentiation [1]. Other features as architectural pattern, nuclear grade, mitotic index, play role in grading systems and are regarded as predictors of patient outcome [12]. Because of the poor outcome of patients with type II ECs, a better understanding of the more aggressive biological behavior of this EC type remains high priority. Differentiation of the two types of ECs, detailed characterization of the cellular characteristics, and histogenesis of the tumor types may even facilitate more effective treatment. The recently described claudins, the main transmembrane proteins of tight junctions (TJs) [13,14], have been suggested to be involved in carcinogenesis and cancer progression in several tumors, including gynecological cancers [2,15–17]. Some of the 24 known members of the claudin family might even be a target of cancer therapy [18] as in the case of ovarian [16,17], endometrial [2] and pancreatic [19] cancers, but the list of claudins usable as potential targets for pancancer therapy is expanding [20]. Tumors overexpressing certain types of claudins on their surface and having upregulated mRNA are the primary targets for this type of antibody-related selective chemotherapy, similarly to other treatment modalities [2,21]. For this reason, more detailed knowledge on the expressional pattern of claudins in different cells and tissues during carcinogenesis seems important not only for differential diagnostic purposes, but from the viewpoint of treatment, too. The goals mentioned above initiated the present study, based on our previous observations of several other tumors, including cervical premalignant and malignant neoplasias [15]. The intent was to define claudin pattern in endometrial tissue and to make comparisons with the changes occurring in endometrial carcinomas. Our aim was to find differences in expression at protein and mRNA levels between normal and altered endometrial glands, to obtain data providing further information about the molecular basis of endometrial carcinogenesis and further, to define the molecular background underlying the morphological alterations detected in different types of endometrial tumors.

Material and methods Patient samples Samples were selected from the archives of the 2nd Department of Pathology and the National Institute of Oncology with the permission of the local ethical committee of the Semmelweis Medical University (No. 172/ 2003). Total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed in all patients, neither of whom had received chemo- or radiotherapy prior to surgery. Hematoxylin- and eosin-stained slides from all cases were reviewed and the diagnosis and typing confirmed. The present study comprises tumor specimens in which the predominant architectural pattern contained more than 90% of the tumor (“pure” forms) and included at least 80% of viable tumor tissue. Cases with mixed subtypes were excluded from the study. The cases fulfilling the above criteria between the period of 2001–2005 were as follows: 17 endometrioid (type I, average age 55.3 ± 8.6 years) and 15 nonendometrioid, serous (type II, average age 63.8 ± 5.6 years) carcinomas. The endometrioid carcinoma (type I) included 10 G1 and 7 G2 adenocarcinomas. The serous adenocarcinoma group consisted of 10 cases showing well defined papillary architecture with typical fibrovascular cores and 5 cases in which the papillae were less defined (solid growth pattern predominated and focal necroses were common). The nuclei of all 15 cases were poorly differentiated, mitoses were frequent. Twenty-four cases of normal endometria (12 of proliferative and 12 of secretory phase, average age 46.2 ± 3.6 and 46 ± 4.3 years respectively) and 14 samples of complex endometrial hyperplasia (average age 45 ± 6.3 years) were studied for comparison.

Histopathology Tissue blocks were fixed in 10% neutral buffered formalin in PBS (pH 7.0) for 24 h and embedded in paraffin. 3- to 5-μm-thick sections were routinely stained with hematoxylin and eosin (HE).

Immunohistochemistry 3- to 4-μm-thick deparaffinized sections were used for the reactions. The following primary antibodies were used for 60 min at room temperature: mouse monoclonal antibodies against claudin 2 (Cat #187363 Zymed Inc, San Francisco, CA, USA), claudin 4 (Cat #187341, Zymed), claudin 5 (Cat #187364, Zymed), estrogen-receptor (Novocastra NCL-ER 6F11, Newcastle, UK), progesteron-receptor (Novocastra NCL-PGR 312), p53 (Dako M 7001, Glostrup, Denmark), PCNA (Dako), β-catenin (BD Transduction, San Diego, CA, USA) and rabbit polyclonal antibodies against claudin 1 (Cat #519000, Zymed), claudin 3 (Cat #341700, Zymed), claudin 7 (Cat #349100, Zymed). Reactions were carried out in Ventana ES automatic immunostainer (Ventana Medical System Inc, Tucson, Arizona, USA) using avidin–biotin peroxidase technique and diaminobenzidine as chromogen, with the reagents provided by the manufacturer. Antigen retrieval solution (Dako K5205) was applied in microwave oven for 30 min prior to primary antibodies. Tissues were blocked for endogenous peroxidase activity with 3% H2O2. Negative controls for nonspecific binding, incubated with secondary antibodies only, were processed and revealed no signals. Positive controls recommended by manufacturer (Zymed) were used to confirm correct immunohistochemical staining for claudins, that is normal skin for claudin 1, normal colon for claudins 2, 3, 4, 5, and normal breast tissue for claudin 7. For positive control of β-catenin immunoreaction human hepatoblastoma tissue was used, previously reported to show nuclear positivity and analyzed for β-catenin mutation [22]. Immunoreactions were evaluated by two independent pathologists, then discussed and presented through a 10 head Olympus BX51 multidiscussion microscope. In all, 10 randomly selected areas of each slide were analyzed using high power fields objective (× 40) with 100 cells counted per field and the positively stained cells were determined in respect to the total number of cells. For semiquantitative evaluation, tumorous and nontumorous epithelia were considered negative if less than 5% of the cells reacted. The following further values were given: 1 (6–20% positivity), 2 (21–40% positivity), 3 (41–60% positivity), 4 (61–80% positivity), 5 (81–100% positivity). Claudins 1, 3, 4, 5,

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598

593

Fig. 1. Immunohistochemical detection of claudin 1 (A, B) and claudin 2 (C, D) in endometrioid carcinoma (type I) (A, C) and seropapillary carcinoma (type II) (B, D). Magnification: 600× and 1000× for inserts. and 7 exhibited membranous, whereas claudin 2 cytoplasmic staining. For βcatenin the membranous, cytoplasmic and/or nuclear staining, for the estrogen/ progesterone receptors, p53 and PCNA the positive nuclei were counted and expressed in percentage.

For the statistical analysis of the immunohistochemical scores, the Mann–Whitney U test was used to compare the expression of proteins in the different groups. Probability values of P < 0.05 were accepted as being significant.

Fig. 2. Comparison of immunohistochemistry positivity scores for claudins in carcinoma type I (endometrioid) and II (seropapillary), hyperplasia, proliferative and secretory phase endometrium.

594

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598 Real-time RT-PCR Each PCR was carried out in a 25 μl volume of 1xSybr Green PCR puffer (BIORAD, Hercules, CA, USA) with 500 nM primers for 2 min at 95°C for initial denaturing, followed by 40 cycles at 95°C for 20 s, at 63°C for 30 s and at 72°C for 60 s, after which melting analysis was performed from 55–95°C in AB 7000 Real-time PCR System (Applied Biosystems) with claudins, and β-actin housekeeping gene primers. The following primer pairs were used: Claudin 1-F: GCG CGA TAT TTC TTC TTG CAG G, Claudin 1-R: TTC GTA CCT GGC ATT GAC TGG, Claudin 2-F: CTC CCT GGC CTG CAT TAT CTC, Claudin 2R: ACC TGC TAC CGC CAC TCT GT, Claudin 3-F: CTG CTC TGC TGC TCG TGT CC, Claudin 3-R: TTA GAC GTA GTC CTT GCG GTC GTA G, Claudin 4-F: GGC TGC TTT GCT GCA ACT GTC, Claudin 4-R: GAG CCG TGG CAC CTT ACA CG, Claudin 5-F: TTC CTG AAG TGG TGT CAC CTG AAC, Claudin 5-R: TGG CAG CTC TCA ATC TTC ACA G, Claudin 7-F: CAT CGT GGC AGG TCT TGC C, Claudin 7-R: GAT GGC AGG GCC AAA CTC ATA C, β actin-F: CCT GGC ACC CAG CAC AAT, β actin-R: GGG CCG GAC TCG TCA TAC.

Statistical analysis Statistical differences between real-time PCR groups and data evaluation were calculated by the program described by Pfaffl et al. [23], using Pair Wise Fixed Reallocation Randomisation Test. Relative quantification was performed using β-actin as internal control. Real-time PCR results were carried out by 3 replicate measurements of each sample.

Results Fig. 3. Comparison of individual immunohistochemistry positivity scores for claudins 1 and 2 in carcinoma type I (endometrioid) and II (seropapillary). Right lines indicate the mean value of the group.

Real-time PCR RNA extraction Five 10-μm sections were cut from each paraffin block (necrosis and bleeding excluded) and placed in 1.5 ml RNase free centrifuge tubes. Total RNA was extracted using High Pure RNA Paraffin kit (Roche 3270289, Mannheim, Germany) in accordance with the manufacturer's instructions and stored at − 80°C. Reverse transcription of RNA 1 μg aliquot of total RNA was reverse transcribed using random hexamers and MuLv reverse transcriptase (Applied Biosystems N8080127 and N8080018, Foster City, CA, USA), for 10 min at 25°C, 50 min at 42°C, and 5 min at 95°C.

Immunohistochemistry Positive membranous linear reactions along the cell surface were detected for claudins 1 (Fig. 1B), 3, 4, 5, and 7. A granular pattern mainly at the periphery associated with the cell membranes, occasionally intracytoplasmatically, was seen for claudin 2 (Fig. 1C). Significant differences were observed, however, in the number of positive cells among the samples studied (Figs. 1–3, Table 1). Claudin 1 definitely showed the highest reaction in seropapillary (type II) adenocarcinoma (Figs. 1B, 2, 3, Table 1). The most significant difference was found by comparison of the seropapillary and endometrioid cancers (Figs. 1A, B). Significant differences were detected in the nontumorous

Table 1 Immunohistochemistry significancy levels of differential expression of claudins in endometrium samples Versus

Claudin 1

Claudin 2

Claudin 3

Claudin 4

Claudin 5

Claudin 7

Secretory–proliferative phase Secretory phase–Hyperplasia Secretory phase–Carcinoma I Secretory phase–Carcinoma II Proliferative phase–Hyperplasia Proliferative phase–Carcinoma I Proliferative phase–Carcinoma II Hyperplasia–Carcinoma I Hyperplasia–Carcinoma II Carcinoma I–II

⁎ ⁎⁎ ns ⁎⁎⁎ ns ns ⁎⁎⁎ ⁎ ⁎⁎⁎ ⁎⁎⁎

ns ns ns ⁎⁎ ns ⁎ ⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

⁎⁎ ns ns ns ns ⁎ ⁎ ns ns ns

ns ⁎ ⁎⁎ ns ns ⁎ ns ns ⁎ ⁎⁎

⁎⁎ ⁎⁎⁎ ⁎⁎ ns ⁎ ns ns ⁎⁎⁎ ⁎⁎ ns

ns ns ns ns ns ns ns ns ns ns

ns = non significant. ⁎ P < 0.05. ⁎⁎ P < 0.01. ⁎⁎⁎ P < 0.001.

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598

595

Table 2 Comparison of immunohistochemistry results in carcinoma type I and II

Carcinoma I Carcinoma II Carcinoma I vs II

Oestrogen R

Progesterone R

p 53

PCNA

β-catenin

66,47 ± 5,9 27,27 ± 9,5 ⁎⁎

69,38 ± 7,1 9,09 ± 6,7 ⁎⁎⁎

11,31 ± 5,7 63,18 ± 12,1 ⁎⁎

71,76 ± 3,2 100 ± 0 ⁎⁎⁎

4,7 ± 0,1 3,68 ± 0,5 Ns

Differences are expressed as percentages of positively stained nuclei for oestrogen receptor (R), progesterone receptor (R), p53, and proliferating cell nuclear antigen (PCNA) and positivity scores for β membranous catenin. Four cases with intensive cytoplasmic reaction for β-catenin in carcinoma type I were not evaluated statistically. Values are expressed as mean ± s.e. Significancy levels were: **P < 0.01, ***P < 0.001, ns = non significant.

endometrial samples too; claudin 1 expression was the highest in the secretory endometrium, as compared with the proliferative phase and hyperplasia (Fig. 2, Table 1). Highly significant, although “reversed” differences characterized the claudin 2 expression (Figs. 1C, D, 2, 3, Table 1). Similarly to the nontumorous samples, endometrioid (type I) cancer (Fig. 1C) stained strongly for claudin 2, in contrast to seropapillary (type II) carcinoma (Fig. 1D). No or only slight differences were detected for claudins 3, 4, and 7 (Fig. 2, Table 1), while claudin 5 was significantly higher in the secretory phase than in any other sample (Fig. 2, Table 1). There were, however, no significant differences between the two carcinoma types in claudin 5 expression. Estrogen and progesterone receptors, p53 and PCNA exhibited nuclear localization in the positive samples. Endometrioid carcinoma expressed significantly higher estrogen and progesterone receptor levels than seropapillary carcinoma (Table 2) (P < 0.01 and P < 0.0001, respectively). Seropapillary carcinoma showed a significant increase of p53 and PCNA (Table 2, P < 0.01 and P < 0.0001) as compared with endometrioid carcinoma. Membranous β-catenin was detected in both types of carcinomas (Table 2), both groups, however, lacked nuclear localization of β-catenin using cases of hepatoblastomas with strong nuclear reaction for β-catenin as

positive control. Strong diffuse cytoplasmic reaction without nuclear positivity was observed in 4 endometrioid carcinoma cases. Real-time PCR Claudin 1 mRNA expression was upregulated in seropapillary carcinoma (type II) as compared with endometrioid carcinoma (type I), proliferative and hyperplastic endometria (Fig. 4). No significant differences were detected, however, for claudin 2 except between the secretory and hyperplastic endometria (Fig. 4). The secretory and proliferative phases differed only in claudin 4, which was 3.2-fold (P < 0.05) higher in the secretion phase. In hyperplasia, claudins 2 and 4 decreased significantly (7.5- and 3.7-folds; P < 0.05 and P < 0.01, respectively) as compared with the secretion phase, whereas claudin 7 showed an increase (2.9-fold) when correlated with the proliferative phase. Endometrioid carcinoma could not be distinguished from hyperplasia by claudin expression, some decrease regarding the proliferative phase was notable in case of claudin 5. Seropapillary carcinoma differed in claudin expression from normal and hyperplastic tissues. From the samples compared,

Fig. 4. Real-time PCR results. Values are expressed as ratio of claudin up(U)- or down(D)regulation between groups. (Example: claudin 1 is upregulated in the carcinoma type II as compared with the carcinoma type I group 6756 times). Significant differences were *P < 0.05, **P < 0.01.

596

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598

seropapillary carcinoma showed the highest values of claudin 1, which was upregulated when compared with both normal tissues (11.8-fold in the secretory and 8.3-fold in the proliferative phase, P < 0.01) and hyperplasia (5-fold, P < 0.01). In addition, seropapillary carcinoma showed higher claudin 7 expression (3.5-fold) than the proliferative phase and higher claudin 4 expression (3.8-fold) than hyperplasia. Regarding claudin 1 expression, real-time PCR results were in agreement with the immunohistochemistry evaluation in the two subtypes of endometrial cancers. As regards claudin 2 expression, however, no significant downregulation was detected at mRNA level in the seropapillary compared with the endometrioid cancers, as shown by immunohistochemistry; the mRNA and protein expression levels did not correspond completely. Discussion Our group has recently described altered claudin expression in several tumors [20,22,24,25], including cervical premalignant and malignant neoplasias [15]. Specific expression patterns detected in different tumors can raise the possibility of using the characteristic claudin expression for the differentiation of tumors of diverse histogenesis, as suggested by the results of Soini [26]. The differences in claudin 1 and 2 expressions in endometrioid and seropapillary adenocarcinomas reported in the present study further support this view. Claudins are the main transmembrane protein components of tight junctions (TJs) [27]. They play an essential role in the tight sealing of the epithelial cellular sheets and function as selective permeability barriers to the diffusion of solutes and ions. [13,27–29]. Recently, their role in cell signaling and transcriptional regulation has also emerged [30]. So far, 24 members of the family have been discovered. This considerable choice of similar molecules in TJs permits the fine tuning of solute filtration by size exclusion and charge selection in the intercellular space, adaptation to various internal and external signals. The TJ permeability barrier plays role during embryo implantation [31] and influences endometrial receptivity [32]. Claudins in fact exhibit a tissue- as well as a tumor-specific distribution as revealed in several findings (reviewed by Turksen and Troy [33]). In some tumors, under- or overexpression combinations of claudins are suggested to play role in carcinogenesis by loosening or altering cell adhesion, leaving passage to larger molecules regarding normal tissues like growth hormones, etc. [34]. This characteristic pattern might also be utilized to differentiate tumors of various histogenesis and may well be associated with aggressiveness, too. Some claudins are known to be influenced by signaling pathways: claudin 1 by wnt β-catenin pathway [35], claudin 4 by transforming growth factor β (TGF-β) [19], claudin 2 by IL1β [36] and discoveries of further connections related to signaling pathways are to be expected. In the present study, claudins 1 and 2 immunohistochemistry allows good distinction of the seropapillary tumor from endometrioid cancer, showing considerably diverse expression in the two clinicopathological types of ECs. In contrast,

however, our real-time PCR results showed differences only in claudin 1 mRNA expression. The regulation of claudins is certainly not a simple matter, sometimes even being controversial. Low claudin 1 level was found associated with invasive properties in colorectal [37], as well as breast tumors [24]. Miwa et al. [35] reported that claudin 1 is regulated by the βcatenin/tcf signal transduction pathway, Dhawan [30] demonstrated the contrary, i.e., claudin 1 should be the regulator of βcatenin through E cadherin expression, finding upregulated claudin 1 in colorectal cancer. De Oliviera et al. [38] have noted the upregulation of claudins 1, 3, and 4 in colon cancer and increased paracellular permeability. Others [25] also detected upregulated claudin 1 expression in esophageal squamous cell carcinoma and in uterine cervical tumors [15,39]. Endometrial carcinomas seem to belong to the group of tumors where claudin 1 expression is rising with aggressive behavior. It is to be noted that β-catenin was found frequently mutated in endometrioid (type I) carcinomas in contrast to type II [7]. The reported frequency of the nuclear accumulation of βcatenin representing mutation in exon 3 of the gene (CTNNB1) ranges from 14% to 44% in different series [7,40], and all positive cases were identified in endometrioid carcinomas characteristically in the early stages [40–42]. No nuclear accumulation of β-catenin was detected in the 17 endometrioid carcinoma cases in our study. This might be associated with the selection of cases involved, however, the lack of β-catenin nuclear staining and/or gene mutation in a certain proportion of endometrioid cancer cases suggests that alterations in other genes may be responsible for the altered wnt/β-catenin pathway in these tumors [7]. Four cases of endometrioid carcinomas, however, presented intensive cytoplasmic immunoreaction with lower membrane reaction, which is also considered as altered βcatenin distribution [43]. The regulation of claudin expression is not entirely explored yet, there are factors influencing both claudins 1 and 2 expression [35,44–46], whereas other factors only act on claudin 1 or 2 [45,47]. Protein level related differences revealed by immunohistochemistry could be explained by their antagonistic physical characteristics; claudin 1 is a tightener of the epithelial barrier [48,49], whereas claudin 2 renders tight junctions leaky [28,50], they therefore exert their action in a reciprocal manner. Altered protein levels with unvaried mRNA expression levels were observed in relation to claudin expression [51]. Further, Escaffit et al. [45] suggest the indirect influence of GATA4 and other regulators on claudin 2 transcript or protein turnover, which would explain the discrepancy of the mRNA and immunohistochemistry results. The minor discrepancies observed in relation to other claudin expression data might be explained by yet unproven regulatory mechanisms at the protein level. Variances between protein and mRNA levels could also be specific characteristics accompanying the pathological processes [41]. Still, the different immunohistochemical staining of claudins 1 and 2 in endometrial cancers both types I and II are of satisfactory diagnostic value, owing to their apparent, distinct and homogenous features. Santin et al. [2] identified claudins 3 and 4 as novel markers for uterine seropapillary cancer by chip technology. According

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598

to our findings, neither claudin 3 nor 4 shows really highly distinctive features in the different endometrium samples, even though high levels of their expression were found. Several studies describe investigations of endometrial tumors and endometrial tumor cell lines by chip analysis, without mentioning significantly altered claudin expression [11,52– 56]. These chip results are, however, quite inconsistent, and the same expressional alterations can hardly be reproduced on the same type of disease, most likely dependent on the technology used, the varying pathogenesis and the expressional differences between tumor cells and the cell lines they originated from. Our results showed claudins 3 and 4 stainings to be rather strong on every human endometrium sample studied, independent of the pathology. Claudin 5, so far mainly regarded as having endothelial appearance [14], is probably not limited to the endothelium. In vascular tumor types, in some epithelial tumors and in nonneoplastic epithelial cells it might also give intensive staining [26]. Besides the common endothelial appearance of claudin 5, a strong expression was detected in a certain percentage of the tumorous and normal cells in our study. This characteristic feature which may distinguish endometrial cells from other epithelial cell types also refers to a particular cellular origin, although having little significance in identifying the different endometrial pathologies. Highly expressed claudin 7 is a characteristic feature of a large spectrum of tumors [26], so its lack in certain malignancies is more distinctive than its presence in others. Sometimes normal and tumorous tissues of the same organ differ regarding claudin 7 content with more elevated levels in tumors [15,25], whereas other times this protein is equally expressed in both [24]. The latter was manifest in the present study, with claudin 7 giving intense staining in nontumorous and tumorous endometrial cells alike. Hence, the possible role and function of claudin 7 is yet to be investigated. Claudins 3, 4, and 7 have no diagnostic value in distinguishing endometrial pathologies, however, their intense expression can be a distinctive feature regarding other organs and the cells derived from them. Seropapillary carcinoma seems to follow another pathway than hyperplasia and endometrioid carcinoma, as indicated by the increase noted in claudin 1 mRNA and protein content when correlated with the low claudin 1 expression in the hyperplasia and endometrioid carcinoma cases. The results delineate distinct pathways leading to the two tumor types; hyperplasiaendometrioid carcinoma pathogenesis contra the diverse pathway of seropapillary carcinoma. The similar claudin patterns link hyperplasia and endometrioid carcinoma, as contrasted to seropapillary carcinoma. These findings further support the dualistic model of endometrial carcinogenesis [9,10]. The two subtypes of endometrial cancers may reflect upon a different cellular origin or pathogenetic pathway as well as different behavior in cell adhesion behind invasive properties. The claudin pattern – mainly the marked differences in claudin 1 and 2 expression – could be used as a differentiating marker of the two endometrial cancer types and could also be utilized in

597

promoting an earlier aggressive therapy, based on the more aggressive behavior of tumors with increased claudin 1 protein and mRNA expression. Acknowledgments Authors wish to thank M. Pekár and M. Tóth for their expert technical assistance. References [1] Macwhinnie N, Monaghan H. The use of P53, PTEN, and CerbB-2 to differentiate uterine serous papillary carcinoma from endometrioid endometrial carcinoma. Int J Gynecol Cancer 2004;14:938–46. [2] Santin AD, Zhan F, Cane S, Bellone S, Palmieri M, Thomas M, et al. Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy. Br J Cancer 2005;92:1561–73. [3] Leslie KK, Stein M-P, Kumar NS, Dai D, Stephens J, Wandinger-Ness A, et al. Progesterone receptor isoform identification and subcellular localization in endometrial cancer. Gynecol Oncol 2005;96:32–41. [4] Bonatz G, Frahm SO, Klapper W, Helfenstein A, Heidorn K, Jonat W, et al. High telomerase activity is associated with cell cycle deregulation and rapid progression in endometrioid adenocarcinoma of the uterus. Hum Pathol 2001;32:605–14. [5] Silverberg SG, Kurman RJ, Nogales F, Mutter GL, Kubik-Huch RA, Tavassoli FA. Epithelial tumours and related lesions. In: Tavassoli FA, Devilee P, editors. Tumours of the Breast and Femal Genital organs. World Health Organization Classification of Tumours. Lyon: IARC Press; 2003. p. 221–32. [6] Goff BA. Uterine papillary serous carcinoma: what have we learned over the past quarter century? Gynecol Oncol 2005;98:341–3. [7] Matias-Guiu X, Catasus L, Bussaglia E, Lagarda H, Garcia A, Pons C, et al. Molecular pathology of endometrial hyperplasia and carcinoma. Hum Pathol 2001;32:569–77. [8] Wu W, Slomovitz BM, Celestino J, Chung L, Thornton A, Lu KH. Coordinate expression of Cdc25B and ER-α is frequent in low-grade endometrioid endometrial carcinoma but uncommon in high-grade endometrioid and nonendometrioid carcinomas. Cancer Res 2003;63:6195–9. [9] Sherman ME, Bur ME, Kurman RJ. p53 in endometrial cancer and its putative precursors: evidence for diverse pathways of tumorigenesis. Hum Pathol 1995;26:1268–74. [10] Lax SF, Kurman RJ. A dualistic model for endometrial carcinogenesis based on immunohistochemical and molecular genetic analyses. Verh Dtsch Ges Pathol 1997;81:228–32. [11] Planaguma J, Diaz-Fuertes M, Gil-Moreno A, Abal M, Monge M, Garcia A, et al. A differential gene expression profile reveals overexpression of RUNX1/AML1 in invasive endometrioid carcinoma. Cancer Res 2004;64:8846–53. [12] Alkushi A, Abdul-Rahman ZH, Lim P, Schulzer M, Coldman A, Kalloger SE, et al. Description of a novel system for grading of endometrial carcinoma and comparison with existing grading systems. Am J Surg Pathol 2005;29:295–304. [13] Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogenous claudin species within and between tight junction strands. J Cell Biol 1999;147:891–903. [14] Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial claudin: claudin-5/ TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 1999;147:185–94. [15] Sobel G, Páska C, Szabó I, Kiss A, Kádár A, Schaff Z. Increased expression of claudins in cervical squamous intraepithelial neoplasia and invasive carcinoma. Hum Pathol 2005;36:162–9. [16] Rangel LB, Agarwal R, D'Souza T, Pizer ES, Alo PL, Lancaster WD, et al. Tight junction proteins claudin-3 and claudin-4 are frequently

598

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26] [27]

[28]

[29] [30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

G. Sobel et al. / Gynecologic Oncology 103 (2006) 591–598 overexpressed in ovarian cancer but not in ovarian cystadenomas. Clin Cancer Res 2003;9:2567–75. Heinzelmann-Schwarz VA, Gardiner-Garden M, Henshall SM, Scurry J, Scolyer RA, Davies MJ, et al. Overexpression of the cell adhesion molecules DDR1, Claudin 3, Ep-CAM in metaplastic ovarian epithelium and ovarian cancer. Clin Cancer Res 2004;10:4427–36. Ebihara C, Kondoh M, Hasnike N, Harad M, Mizuguchi H, Horiguchi Y, et al. Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin. J Pharmacol Exp Ther 2005; 316:255–60. Michl P, Buchholz M, Rolke M, Kunsch S, Lohr M, McClane B, et al. Claudin 4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin. Gastroenterology 2001;121:678–84. Lódi Cs, Szabó E, Holczbauer Á, Batmunkh E, Szijártó A, Kupcsulik P, et al. Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas. Mod Pathol 2006;19:460–9. Morin PJ. Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res 2005;65:9603–6. Halász J, Holczbauer Á, Páska Cs, Kovács M, Benyó G, Verebély T, Schaff Zs, Kiss A. Claudin-1 and claudin-2 differentiate fetal and embryonal components in human hepatoblastoma. Human Pathol. (in press) [Electronic publication ahead of print May, 2006]. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acid Res 2002;30:e36. Tõkés AM, Kulka J, Paku S, Szik Á, Páska Cs, Kaposi Novák P, et al. Claudin -1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study. Breast Cancer Res 2005;7: R296–305. Gyõrffy H, Holczbauer Á, Nagy P, Szabó Zs, Kupcsulik P, Páska Cs, et al. Claudin expression in Barrett's esophagus and adenocarcinoma. Virchows Arch 2005;447:961–8. Soini Y. Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology 2005;46:551–60. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 1998;141:1539–50. Furuse M, Furuse K, Sasaki H, Tsukita S. Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin 2 into Madin-Darby canine kidney cells. J Cell Biol 2001;153:263–72. Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE. Tight junction proteins. Prog Biophys Mol Biol 2003;81:1–44. Dhawan P, Singh AB, Deane NG, No Y, Shiou SR, Schmidt C, et al. Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer. J Clin Invest 2005;115:1765–76. Wang X, Matsumoto H, Zhao X, Das SK, Paria BC. Embryonic signals direct the formation of tight junctional permeability barrier in the decidualizing stroma during embryo implantation. J Cell Sci 2004;117:53–62. Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, et al. Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology. Mol Hum Reprod 2003;9:253–64. Turksen K, Troy TC. Barriers built on claudins. J Cell Sci 2004;117:2435–47. Soler AP, Miller RD, Laughlin KV, Carp NZ, Klurfeld DM, Mullin JM. Increased tight junctional permeability is associated with the development of colon cancer. Carcinogenesis 1999;20:1425–31. Miwa N, Furuse M, Tsukita S, Niikawa N, Nakamura Y, Furukawa Y. Involvement of claudin 1 in the β catenin/tcf signaling pathway and its frequent upregulation in human colorectal cancers. Oncog Res 2001; 12:469–76. Yamamoto T, Kojima T, Murata M, Takano K, Go M, Chiba H, et al. IL-1β regulates expression of Cx32, occludin and claudin 2 of rat hepatocytes via distinct signal transduction pathways. Exp Cell Res 2004;299:427–41. Resnick MB, Konkin T, Routhier J, Sabo E, Pricolo VE. Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study. Mod Pathol 2005;18:511–8.

[38] de Oliveira SS, de Oliveira IM, De Souza W, Morgado-Diaz JA. Claudin upregulation in human colorectal cancer. FEBS Lett 2005;579:6179–85. [39] Vazquez-Ortiz G, Ciudad CJ, Pina P, Vazquez K, Hidalgo A, Alatorre B, et al. Gene identification by cDNA arrays in HPV-positive cervical cancer. Arch Med Res 2005;36:448–58. [40] Irving JA, Catasus L, Gallardo A, Bussaglia E, Romero M, Matias-Guiu X, et al. Synchronous endometrioid carcinomas of the uterine corpus and ovary: alterations in the β-catenin (CTNNB1) pathway are associated with independent primary tumors and favorable prognosis. Hum Pathol 2005;36:605–19. [41] Saegusa M, Hashimura M, Yoshida T, Okayasu I. β-Catenin mutations and aberrant nuclear expression during endometrial tumorigenesis. Br J Cancer 2001;84:209–17. [42] Scholten AN, Creutzberg CL, van den Broek LJCM, Noordijk EM, Smit VTHBM. Nuclear β-catenin is a molecular feature of type I endometrial carcinoma. J Pathol 2003;201:460–5. [43] Klymkowsky MW. β-Catenin and its regulatory network. Hum Pathol 2005;36:225–7. [44] Mankertz J, Hillenbrand B, Tavalali S, Huber O, Fromm M, Schulzke JD. Functional crosstalk between Wnt signaling and Cdx related transcriptional activation in the regulation of the claudin promoter activity. Biochem Biophys Res Commun 2004;314:1001–7. [45] Escaffit F, Boudreau F, Beaulieu JF. Differential expression of claudin 2 along the human intestine: implication of GATA-4 in the maintenance of claudin 2 in differentiating cells. J Cell Biol 2005; 203:15–26. [46] Ikenouchi J, Matsuda M, Furuse M, Tsukita S. Regulation of tight junctions during the epithelium mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci 2003;116:1959–67. [47] Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC. Cloning of the human claudin-2 5′-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudalrelated homeodomain proteins and hepatocyte nuclear factor-1alpha. J Biol Chem 2002;277:21361–70. [48] Inai T, Kobayashi J, Shibata Y. Claudin-1 contributes to the epithelial barrier function in MDCK cells. Eur J Cell Biol 1999;78:849–55. [49] Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, et al. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 2002;8: 1099–111. [50] Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, et al. Claudin-2 expression induces cation selective channels in tight junctions of epithelial cells. J Cell Sci 2002;115:4969–76. [51] Swisshelm K, Macek R, Kubbies M. Role of claudins in tumorigenesis. Adv Drug Delivery Rev 2005;57:919–28. [52] Smid-Koopman E, Blok LJ, Chadha-Ajwani S, Helmerhorst TJ, Brinkmann AO, Huikeshoven FJ. Gene expression profiles of human endometrial cancer samples using a cDNA-expression array technique: assessment of an analysis method. Br J Cancer 2000;83:246–51. [53] Matsushima-Nishiu M, Unoki M, Ono K, Tsunoda T, Minaguchi T, Kuramoto H. Growth and gene expression profile analyses of endometrial cancer cells expressing exogenous PTEN. Cancer Res 2001;61:3741–9. [54] Moreno-Bueno G, Sanchez-Estevez C, Cassia R, Rodriguez-Perales S, Diaz-Uriarte R, Dominguez O, et al. Differential gene expression profile in endometrioid and nonendometrioid endometrial carcinoma: STK15 is frequently overexpressed and amplified in nonendometrioid carcinomas. Cancer Res 2003;15:5697–702. [55] Risinger JI, Maxwell GL, Chandramouli GV, Jazaeri A, Aprelikova O, Patterson T, et al. Microarray analysis reveals distinct gene expression profiles among different histologic types of endometrial cancer. Cancer Res 2003;63:6–11. [56] Maxwell GL, Chandramouli GV, Dainty L, Litzi TJ, Berchuck A, Barrett JC, et al. Microarray analysis of endometrial carcinomas and mixed mullerian tumors reveals distinct gene expression profiles associated with different histologic types of uterine cancer. Clin Cancer Res 2005;11: 4056–66.