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Aug 5, 2014 - Abstract. Background Changes in expression patterns of the sialyl. Lewis antigens and MUC1 mucin can be considered as markers for the ...
Clin Exp Nephrol (2015) 19:732–737 DOI 10.1007/s10157-014-1013-y

ORIGINAL ARTICLE

Increased expression of MUC1 and sialyl Lewis antigens in different areas of clear renal cell carcinoma Małgorzata Borzym-Kluczyk • Iwona Radziejewska Marzanna Cechowska-Pasko



Received: 4 May 2014 / Accepted: 8 July 2014 / Published online: 5 August 2014 Ó Japanese Society of Nephrology 2014

Abstract Background Changes in expression patterns of the sialyl Lewis antigens and MUC1 mucin can be considered as markers for the diagnosis of various cancers. However, there are no reports which have been devoted to analysis of differences in the sialyl Lewis antigens and MUC1 expression patterns as potential discrimination markers among different areas of clear cell renal cell carcinoma (ccRCC). The aim of this study was to determine the level of MUC1 and specific Lewis antigens on glycoproteins in three different areas: tumor (cancer tissue), intermediate zone (adjacent to tumor tissue) and normal renal cortex/ medulla (uninvolved by tumor). Methods Study was performed on renal tissues taken from 30 patients with clear cell renal cell carcinoma. Relative amounts of sugar structures bound with proteins were determined by ELISA-like test with biotinylated lectins or monoclonal antibodies: anti-MUC1 and anti-sialyl Lewisa/x. The study presented here provides novel information about relationship between MUC1 and sialyl Lewis antigens in the tumor, intermediate zone and noninvolved areas of normal renal tissue distant of tumor. Results We have found statistically significant increase of MUC1 and sialic acid linked by a-2,3 bond with galactose in cancer tissue and in intermediate zone comparing to normal renal tissue distant of tumor. Moreover, we M. Borzym-Kluczyk (&)  M. Cechowska-Pasko Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Mickiewicza 2A, 15-230 Białystok, Poland e-mail: [email protected] I. Radziejewska Department of Medical Chemistry, Medical University of Bialystok, Białystok, Poland

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observed statistically significant increase of sialic acid linked by a-2,6 bond with Gal/GalNAc and sialyl Lewisa/x antigens in cancer tissues only, comparing to normal ones. Conclusions MUC1 and sialylated antigens can be involved in renal tumor development and can be considered as potential markers distinguishing normal renal tissue from intermediate zone and from cancer renal cells during ccRCC development. Keywords Clear renal cell carcinoma  Glycosylation  MUC1  Sialyl Lewisa/x antigens Abbreviations BSA Bovine serum albumin ccRCC Clear cell renal cell carcinoma ELISA Enzyme-linked immunosorbent assay Gal Galactose GalNAc N-acetylgalactosamine GlcNAc N-acetylglucosamine MAA Maackia Amurensis Agglutinin sLea Sialyl Lewisa antigen sLex Sialyl Lewisx antigen SNA Sambucus Nigra Agglutinin

Introduction Altered glycosylation in epithelial cancers cells may play an important role in tumor progression, as it may affect tumor cell migration and antigen presentation by antigenpresenting cells [1]. MUC1 is a membrane-associated and membranesecreted member of the mucin family [2, 3]. MUC1 is

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distributed widely on the apical membrane of many glandular epithelial cells such organs as: breast, colon, pancreas, lung, and kidney [3–5]. The MUC1 gene is located on chromosome 1q21-24 [6]. MUC1 is among major constituents of glycocalyx. In the kidney, MUC1 is expressed in normal distal convoluted tubules, collecting ducts, and clear cell renal cell carcinoma (ccRCC) [7]. Mucin glycoproteins consist of a protein backbone with a large amount of O-linked carbohydrate side chains. The protein backbone of mucins is composed of a variable number of tandem repeat (VNTR) regions rich in threonine and serine residues. The composition, sequence and length of protein core are unique for each particular mucin. MUC1 is heavily glycosylated in normal tissues. Carbohydrates contribute 50–90 % of its molecular mass [8]. Usually, O-linked mucins are constructed from 1 to 20 sugar residues, which can form linear or branched structures. They are negatively charged due to the presence of numerous sialic acid or sulfate groups, attached to galactose or N-acetylglucosamine sugar chain. MUC1 contains a large extracellular domain, which forms a long filamentous structure that appears to interact with extracellular matrix components and other cells [9]. MUC1 is associated with the cell membrane by an integral transmembrane domain and possesses short cytoplasmic tail that associates with cell cytoskeletal proteins [10]. Changes in the protein or sugar component of mucins are often associated with tumorigenesis. Alteration in the protein may involve both reduction and increase in the expression of the apomucin. Changes in sugar chains can be divided into three categories: (1) Changes in the structure of the O-glycosidic chains; (2). O-glycans incomplete synthesis, which is accompanied by the accumulation of the precursor structure; (3) Reduction in the amount of O-glycoside chains attached to the apomucin [11]. Mucins are involved in cell protection, adhesion, and signaling [12]. Many studies have defined the patterns of mucin expression in carcinomas of different organs. Moreover, few have emphasized analysis of differential mucin expression patterns as potential discrimination markers among tumors. Since sialic acids are located at the terminal positions of glycans attached to the glycoproteins, sialylation determines the half-lives of many glycoproteins and plays critical roles in cell–cell interactions, cell adhesion, and protein targeting [13, 14]. Altered sialylation has been found to be involved in critical pathological events during cancer progression, including cancer transformation and metastasis [15]. Abnormal expressions of sialylated glycoproteins have been discovered in many cancers such as pancreatic [16], brain [17], colon [18] and breast cancers [19].

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Increased Lewis antigen expression is a common feature of many epithelial cancers. When presented on mucin-like molecules, like MUC1, some Lewis structures can form ligands for other cell adhesion molecules involved in the development and metastasis of cancer cells. It is of interest, the expression and relationship of MUC1 mucin and sialyl Lewis antigens in the clear cell renal carcinoma, and especially in the intermediate zone. It has not been studied before. We decided to evaluate the expression of MUC1 mucin and sialyl Lewis antigens in three different areas: the tumor (clear cell renal cell carcinoma (ccRCC) cancer tissue), intermediate zone (adjacent to tumor tissue) and normal renal cortex/medulla (remote from tumor).

Materials and methods Patients Study was performed on renal tissues taken from 30 patients with clear cell renal cell carcinoma kidney cancer, hospitalized in the Department of Urology, Medical University of Bialystok, Poland. Diagnosis was confirmed by histopathological assessment in the Department of Pathological Anatomy, Medical University of Bialystok. The age of patients, 15 females and 15 males, ranged from 50 to 74 years (mean age = 60). Kidneys with clear cell renal cell carcinoma were subjected to complete nephrectomy, and then sectioned in a way to have a cross-section plane passing through tumor center and long axis of the tumor. Fuhrman nuclear grade and TNM stage are presented in Table 1. Tissue samples (2 9 2 9 2 cm) were taken from tumor (C), intermediate zone which was adjacent to tumor tissue (I) and normal (N) renal cortex/medulla which was uninvolved by tumor. Samples were rinsed with saline, dried with blotting paper and stored at -70 °C. The study was approved by the Institutional Ethical Committee and conducted according to the principles of the Declaration of Helsinki (R-I-003/216/2003). Preparation of tissue extract Renal tissues were defrosted and immersed in 0.1 M citric buffer (pH 4.3) to prepare 10 % (w/v) tissue suspensions. Then tissues were homogenized in ULTRA-TURRAX T8 Table 1 Number by Fuhrman nuclear grade, and TNM tumor stage Patients

Fuhrman nuclear grade

TNM tumor stage

N = 30

G1

G2

G3

G4

T1

T2

T3

T4

4

18

3

5

14

12

2

2

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homogenizer for 2 min. The homogenate was centrifuged for 20 min, at 100009g, at 4 °C. The supernatants were concentrated in concentrators Centriprep C30 (Amicon, Millipore, Bedford, MA, USA). Concentrated samples were applied on a Sephadex G-10 column and eluted with water. Collected 2 ml fractions were assayed for absorbance at 280 nm. Aliquots were centrifuged several times for 60 min, at 15009g and supernatants were taken to further analysis. The concentration of protein in all the samples was 40–60 mg of protein/mL. The protein concentration was determined by the method of Smith [20] using BCA protein assay kit (Thermo Scientific). Elisa assay In-plate lectin-based ELISA assay was developed to analyze sialylation changes of glycoproteins. The levels of sugar structures of isolated proteins were analyzed using ELISA-like test with biotinylated lectins (Vector, Burlingame, USA) and monoclonal antibodies (Millipore, CA, USA). The sources and dilutions of all antibodies were presented in Table 2. Samples of renal tissue were diluted in the PBS buffer (phosphate-buffered saline) to achieve protein concentration equal 0.005 mg/mL. Aliquots (50 lL) were coated onto microtiter plates (NUNC F96; Maxisorp, Roskilde, Denmark) overnight at room temperature. The plates were washed three times with 100 lL of PBS, pH 7.4, containing 0.05 % Tween 20 (PBS-T), between all ensuing steps. Unbound sites were blocked for 1 h with 100 lL of 1 % blocking reagent for ELISA (Roche Diagnostics, Mannheim, Germany). The microtiter plates were incubated for 1 h with 100 lL of biotinylated lectins diluted to 0.5 lg/mL in PBS-T and 1 % bovine serum albumin, (BSA-Sigma, St Luis, MO, USA). Lectin solutions were supplemented with metal cations: SNA with 0.1 mmol/L CaCl2; MAA with 0.1 mmol/L CaCl2 and 0.01 mmol/L MnCl2. Plates were then incubated with 100 lL of horseradish peroxidase avidin D (Vector, Burlingame, CA, USA) (1:2,500) for 1 h in PBS-T and 1 % BSA. Next, the plates were washed four

times in PBS, and the colored reaction was developed by incubating with 100 lL of 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)-liquid substrate for horseradish peroxidase (Sigma, St Luis, MO, USA). Spectrophotometric measurements were performed after 45 min at 405 nm using an Infinite M200 microplate reader (Tecan, Salzburg, Austria). For analysis with specific anti-MUC1 and anti-sialyl Lewisa/x monoclonal antibodies (diluted in PBS-T and 1 % BSA), the plates were coated for 1 h with 100 lL of antibody. Next, the plates were incubated with secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (for anti-sialyl Lewisa and anti-MUC1) or anti-mouse IgM (for anti-sialyl Lewisx). The colored reaction was developed as mentioned.

Statistical analysis Statistical analysis was carried out using the Statistica 11 software (StatSoft Cracow, Poland) according to ANOVA and post hoc tests. Results are expressed as mean ± SD. Statistical significance was defined as p \ 0.05. The correlations between MUC1 level and all examined sugar structures in three analyzed areas (normal, intermediate and cancer) of renal tissue were also calculated.

Results Antigens expression in examined areas of normal (N), intermediate (I) and cancer (C) renal tissue was determined by the OD value (405 nm). The levels of sialic acid linked by a-2,3 bond to Gal and sialic acid linked by a-2,6 bond to

Table 2 The sources of antibodies used in this study Antibodies

Clone

Source

Final dilution

Sialyl Lewisa

KM231

Millipore

1:200

Sialyl Lewisx

KM93

Millipore

1:500

MUC1 (anti-human episialin-EMA)

GP1.4

Sigma

1:400

Horseradish peroxidase-conjugated rabbit anti-mouse IgG

Sigma

1:1.500

Horseradish peroxidase-conjugated rabbit anti-mouse IgM

Sigma

1:1.500

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Fig. 1 Comparison of reactivity of lectin SNA (Sambucus Nigra) and MAA (Maackia Amurensis) with glycoproteins of normal (N), intermediate (I), and cancer (C) renal tissue

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Fig. 2 Comparison of reactivity of monoclonal antibody anti-MUC1 and antisialyl Lewisa/x with glycoproteins of normal (N), intermediate (I), and cancer (C) renal tissue

Gal/GalNAc were examined by MAA (Maackia Amurensis) and SNA (Sambucus Nigra) lectins, respectively. Figure 1 shows that the level of sialic acid linked by a2,3 bond to Gal (MAA lectin) was significantly higher in cancer and intermediate tissue when compared to normal tissue. Significant increase for sialic acid linked by a-2,6 bond to Gal/GalNAc (SNA lectin) was revealed only for cancer tissue in comparison to normal one. It is interesting that the higher amount of sialic acid linked by a-2,6 bond than a-2,3 was observed in all examined areas. Figure 2 shows the interactions of MUC1 glycoforms in different areas of renal tissue with specific anti-MUC1 and anti-sialyl Lewisa/x antibodies. Significantly higher expression of sialyl Lewisa (NeuAca2-3Galb1-4GlcNAca1-4Fuc) and sialyl Lewisx (NeuAca2-3Galb1-4GlcNAca1-3Fuc) was observed in cancer tissues in comparison to normal. Moreover, we observed significant increase in expression of MUC1 in intermediate and cancer tissue, comparing to normal renal tissue. Table 3 shows correlations of two variables—the relative expression of MUC1 in all examined areas of renal tissue versus the expression of the sialic acid linked by a2,3 a-2,6 bonds and sialyl Lewisa/x antigens. We revealed moderate correlations values for MUC1 level in normal renal tissue versus sialic acid linked by a-2,6 bonds to Gal/ GalNAc, (r = 0.34). Moderate Spearman’s coefficient value was calculated also for MUC1 antigen in cancer renal tissue versus the level of sialyl Lewisx antigen (r = 0.34; p \ 0.05). Low Spearman’s coefficient value was calculated also for MUC1 antigen in cancer renal tissue versus the level of sialic acid linked by a-2,3 bonds to Gal (r = 0.12; p \ 0.05). The rest of correlation coefficient values were low or negative.

Table 3 Spearman’s coefficients calculated for the relative amounts of MUC1 in normal, intermediate and cancer renal tissues versus the level of MAA, SNA, sialyl Lewisa, sialyl Lewisx in cancer tissue Coefficient value Sialic acid linked by a-2,3 to Gal (MAA)A

Sialic acid linked by a-2,6 to Gal/ GalNAc (SNA)A

Sialyl Lewisa (sLea)B

Sialyl Lewisx (sLex)B

MUC1 antigen in normal renal tissue

r = 0.11

r = 0.34

r = -0.28

r = 0.14

MUC1 antigen in intermediate renal tissue

r = -0.21

r = 0.06

r = 0.16

r = 0.21

MUC1 antigen in cancer renal tissue

r = 0.12*

r = 0.09

r = -0.13

r = 0.34*

SNA, Sambucus Nigra agglutinin; MAA, Maackia Amurensis agglutinin; sLea, sialyl Lewisa; sLex, sialyl Lewisx * p \ 0.05 A B

Determined by lectins Determined by monoclonal antibodies

Discussion Glycosylation is an important post-translational modification and has been reported to play an important role in cancer progression. The process of malignant transformation is often accompanied by abnormal glycosylation of glycoconjugates, resulting in the altered expression or incomplete structure of oligosaccharide chains. These

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changes can affect interactions between tumor cell-surface glycans and endogenous lectins, which may determine the metastatic potential of the tumor cells [21]. However, establishing how changes in glycosylation affect the properties of tumor cells requires a more detailed understanding of how glycans are presented at the cell surface. Normal, physiological function of MUC1 mucin is designed to provide a protective barrier on the outer surface of epithelial cells. MUC1 has long been viewed as a tumorassociated molecule because of its frequent overexpression and aberrant glycosylation in most carcinomas. By characteristic pattern of glycosylation, mucins can modulate immunological response, facilitate cell adhesion during tumor metastasis, and also alter the functions of proteins interacting with the mucin carbohydrate moieties [22]. MUC1 is overexpressed in over 90 % of breast carcinomas and frequently in other types of cancer, including ovarian, lung, colon, and pancreatic carcinomas [23]. The overexpression of MUC1 is associated with a loss of polarity and circumferential distribution in tumor cells [24]. However, the exact mechanism of MUC1 action in carcinogenesis remains unclear. The mucin-type O-glycans have several cancer-associated structures, including the T and Tn antigens, and certain sialyl Lewis antigens. All of them can be presented on mucin MUC1. These structural changes can alter the function of the cell, and its antigenic and adhesive properties, as well as its potential to invade and metastasize [25]. In the normal kidney, MUC1 is expressed in the distal convoluted tubules and in the collecting ducts with a cellular apical expression but was not detected in proximal tubules [26]. Some authors suggested that the genesis of renal cell carcinoma in the proximal nephron is based on the expression of Lewisa and Lewisx antigens. These authors also showed: (1) an apparent deletion, down-regulation or structural modification of Lewisx determinant in most of the metastatic tumors; (2) undetectable levels of ABH specificities in tumor cells of primary renal cell carcinoma; and (3) enhanced expression and/or neosynthesis of precursor type 1 structure and Lewisy determinant in some renal cell carcinomas [27]. The majority of reports concerning tumors glycosylation profiles refer the various changes of tumor tissue and areas not located within the tumor, but there is little information about intermediate zone, i.e., between tumor and histologically normal tissue. In the available literature, we did not find any information on changes in the expression of MUC1 mucin and sialyl Lewis antigens occurring in tumor (cancer tissue), intermediate zone (adjacent to tumor tissue) and normal renal cortex/ medulla (uninvolved by tumor) in the kidney holding clear cell renal cell carcinoma (ccRCC). Therefore, we

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included to our research samples of ccRCC tissue, tissue from intermediate zone, and histologically normal kidney area, to obtain additional information on changes in glycosylation pattern connected with presence of clear cell renal carcinoma (ccRCC) tumor in human renal tissue. We believe that our results support the idea about involvement of MUC1 and sialylated antigens in cancer development, and opinion that sialylated forms of MUC1 are typical in many tumors. Thus, we decided to check whether in examined renal cancer tissues there is any correlation between MUC1 and examined antigens. We revealed rather moderate, low or even negative coefficient values. Upon our results, we could only suggest that sialyl Lewisx antigen in cancer tissue and sialic acid linked by a2,6 to Gal/NAcGal in normal tissue could be presented on MUC1. Low or even a lack of correlations between MUC1 expression and expression of remaining examined sialylated structures may suggest that they are also presented on glycoforms different from MUC1. It is possible because examined renal homogenates were not thoroughly purified. Summing up, we conclude that MUC1 and examined sialylated antigens can be involved in renal tumor development. Their overexpression in cancer tissues could mean that they can play a special role in neoplasia. From our results, we can conclude that sialylation changes are associated with clear cell renal carcinoma progression and altered protein sialylations and could be potentially used to detect ccRCC. The increased expression of MUC1 and sialic acid linked by a-2,3 bond to Gal in intermediate zone may be considered as the result of infiltration of intermediate zone by some cancer cells or inflammatory cells responding to the tumors, so upon this assumption their higher expression rather do not reflect the tumor development. Much more detailed examinations should be performed to assess the exact role of MUC1 and its sialylation in cancerogenesis of renal tissues. Our results indicate on usefulness of future research devoted to detection and quantitative determination of MUC1 and sialic acid bearing structures in urine, as screening markers in detection of ccRCC or markers of ccRCC therapy efficiency. Conflict of interest interest exists.

The authors have declared that no conflict of

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