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DNA methyltransferases 1, 3a, and 3b overexpression and clinical significance in gastroenteropancreatic neuroendocrine tumors
Human Pathology (2010) 41, 1069–1078
www.elsevier.com/locate/humpath
Original contribution
DNA methyltransferases 1, 3a, and 3b overexpression and clinical significance in gastroenteropancreatic neuroendocrine tumors Md. Mustafizur Rahman PhD a,1 , Zhi Rong Qian MD, PhD a,⁎,1 , Elaine Lu Wang MD, PhD a , Katsuhiko Yoshimoto MD, PhD b , Masahiko Nakasono MD, PhD a,c , Razia Sultana MD, PhD a , Tomoyuki Yoshida MD a , Toshitetsu Hayashi MD, PhD d , Reiji Haba MD, PhD d , Mitsuaki Ishida MD, PhD e , Hidetoshi Okabe MD, PhD e , Toshiaki Sano MD, PhD a a
Department of Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan b Department of Medical Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan c Department of Medicine, Tsurugi Municipal Handa Hospital, Tokushima 779-4401, Japan d Faculty of Medicine, Department of Pathology, Kagawa University, Kagawa 761-0793, Japan e Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Shiga 502-2192, Japan Received 2 November 2009; revised 30 December 2009; accepted 5 January 2010
Keywords: GEP NET; DNMT1, DNMT3a, DNMT3b, methylation
Summary The alteration of DNA methylation is one of the most common epigenetic changes in human cancers. Three genes, namely, DNA methyltransferase 1, 3a, and 3b, which code for DNA methyltransferases that affect promoter methylation status, are thought to play an important role in the development of cancers and may be good anticancer therapy targets. The methylation of tumor suppressor genes has been reported in gastroenteropancreatic neuroendocrine tumors; however, there have been no studies about DNA methyltransferase protein expression and its clinical significance in gastroenteropancreatic neuroendocrine tumors. In this study, the expression of DNA methyltransferase 1, 3a, and 3b was studied in 63 gastroenteropancreatic neuroendocrine tumors by immunohistochemistry. The expression of DNA methyltransferase 1, 3a, and 3b was frequently detected in gastroenteropancreatic neuroendocrine tumors (87%, 81%, and 75%, respectively). The DNA methyltransferase 3a expression level was significantly higher in poorly differentiated neuroendocrine carcinomas than in well-differentiated neuroendocrine tumors or well-differentiated neuroendocrine carcinomas (P b .01 and P b .05, respectively). The expression of DNA methyltransferase 1, 3a, and 3b showed significantly higher levels in stage IV tumors than in stage I or II tumors. In addition, the expression levels of DNA methyltransferase 1, 3a, and 3b were positively correlated with the MIB-1 labeling index in gastroenteropancreatic neuroendocrine tumors (R = 0.293, P = .019; R = 0.457, P = .001; and R = 0.249, P = .049; respectively). In addition, the expression levels and positive
⁎ Corresponding author. E-mail address: [email protected] (Z. R. Qian). 1 M. M. Rahman and Z. R. Qian contributed equally to this work. 0046-8177/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2010.01.011
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M. M. Rahman et al. immunostaining frequencies of DNA methyltransferase 3a and 3b were significantly lower in midgut neuroendocrine tumors than in foregut or hindgut neuroendocrine tumors. Our findings suggest that the overexpression of DNA methyltransferase 1, 3a, and 3b is related to tumorigenesis and the progression of gastroenteropancreatic neuroendocrine tumors. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are rare, with an age-adjusted annual incidence of 2.5 to 4.5 per 100 000 [1]. Over the past 3 decades, their incidence has increased because of improvements in diagnostic techniques. However, the survival of patients with GEP NET has not changed appreciably [2]. In particular, patients with poorly differentiated neuroendocrine carcinoma have the worst survival, which ranges from several weeks for untreated patients and from 6 to 12 months for those receiving therapy [3]. As GEP NET frequently demonstrates unpredictable and unusual biological behavior, cases of GEP NET frequently involve a late or delayed diagnosis. However, few effective targeted treatments are available because little is known about the mechanistic regulation of these tumors [2]. DNA hypermethylation of CpG islands is one of the most common epigenetic changes observed in human cancers [4]. Abnormal methylation of CpG islands can efficiently repress transcription of the associated gene, and its DNA methylation is a reversible process [4,5]. Hypermethylation takes place after DNA replication and is catalyzed by DNA methyltransferase (DNMT) using S-adenosylmethionine as the methyl donor. Three distinct families of DNMT genes, DNMT1, DNMT2, and DNMT3, have been identified in mammals [6,7]. DNMT1 is known to maintain methylation [8]; and the DNMT3 family consists of 2 related gene products, DNMT3a and DNMT3b, which function as de novo methyltransferases [9]. However, the function of DNMT2 remains unknown [10]. The overexpression of DNMT1, 3a, and 3b has been reported in various cancers including colorectal, prostate, hepatocelluar, breast, gastric, and lung tumors [11-16] and is significantly correlated with poor histologic differentiation and poor prognosis [14-16]. In addition, the blocking activity of DNMT may inhibit or reverse methylation-induced silencing of tumor suppressor genes (TSGs) [17]. Therefore, DNMT are major drug targets for epigenetic therapy [17]. The molecular mechanism behind the tumorigenesis and progression of GEP NET remains largely unknown. GEP NETs do not show any alterations in the expression levels of oncogenes such as ras, myc, fos, jun, and src [18]. Recently, the down-regulation of TSGs by the hypermethylation of CpG islands was reported as an important event in GEP NET development [19-22] and showed a trend toward worse survival [23]. However, to the best of our knowledge, no study has provided comprehensive and clinical analyses regarding the altered expression levels of DNMT1, 3a, and 3b in GEP NETs.
In the present study, we examined DNMT1, 3a, and 3b protein expression by immunohistochemistry in a series of GEP NETs taken from different anatomical sites. We analyzed the correlations between the expression of DNMT1, 3a, and 3b proteins and the clinicopathologic features and proliferative activity of GET NETs.
2. Materials and methods 2.1. Patients and samples Sixty-three paraffin-embedded surgical or biopsy specimens were collected from the Pathology Departments of the University of Tokushima Faculty of Medicine, Shiga University of Medicine Science, and Kagawa University Faculty of Medicine. These patients were affected by the following GEP NETs from 1982 to 2008: foregut tumors: esophagus (n = 3), stomach (n = 11), pancreas (n = 9), duodenum (n = 7); midgut tumors: ileum (n = 6), appendix (n =3); hindgut tumors: colon (n = 6), rectum (n = 14); and lymph node metastasis (n = 4). Notably, the frequency of midgut NET was low. The frequency of GEP NET in this study is consistent with that found in a report from the Neuroendocrine Tumor Workshop of Japan [24]. To confirm the NE immunophenotype, paraffin sections were stained with pan-NE markers such as chromogranin A and synaptophysin. The diagnosis of NET was made on the basis of morphologic and immunohistochemical findings evaluated by 2 independent pathologists (Z. R. Q. and T. S.) according to the World Health Organization classification [25]. These included well-differentiated NET (WNET, of either benign or uncertain behavior; n = 31), welldifferentiated neuroendocrine carcinoma (WNEC; n = 16), and poorly differentiated neuroendocrine carcinoma (PNEC; n = 16; including 4 mixed exocrine-endocrine carcinomas). The tumor stage was assessed on the basis of the TNM classification system [26]. Thus, 63 tumors included 33 tumors of stage I, 12 tumors of stage II, 14 tumors of stage III, and 4 tumors of stage IV. The clinicopathologic characteristics of the patients are summarized in Table 1.
2.2. Immunohistochemistry Immunolocalization of DNMT1, 3a, and 3b and Ki-67 antigen based on the streptavidin-biotin labeling method was performed on sections from representative blocks of paraffinembedded tissues for pathologic diagnosis. After
DNMT overexpression in GEP NET Table 1
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Summary of characteristics of 63 GEP NET and expression of DNMT1, DNMT3a and DNMT3b
Number Sex/ Location Organ age (y)
Classification Tumor stage, invasion, and metastasis
Tumor MIB-1 DNMT1 DNMT3a DNMT3b size (cm) LI (%) (%) (%) (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
M/40 M/75 F/70 M/78 F/48 F/61 M/64 M/63 M/63 F/48 M/82 F/57 M/53 F/53 M/26 M/48 F/38 M/61 M/47 F/62 F/62 M/62 M/61 F/67 M/56 M/34 F/51 M/62 M/49 M/35 F/61 M/66 M/56 F/68 M/66 F/63 M/48 F/53 M/61 M/82 M/60 M/62
Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Midgut Midgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Midgut Midgut Midgut Midgut
Stomach Stomach Stomach Duodenum Duodenum Duodenum Duodenum Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Appendix Appendix Colon Colon Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Stomach Duodenum Duodenum Pancreas Pancreas Metastasis Metastasis Ileum-cecum Ileum-cecum Ileum-cecum Ileum-cecum
WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC
0.9 0.5 0.9 0.4 0.4 0.3 0.5 1.1 1.5 1 1.1 1.6 0.6 1.5 0.7 1.4 0.2 0.3 0.4 1.2 1.5 0.9 2.2 0.6 1 0.6 0.8 0.5 0.4 1.4 0.5 6 1.3 1.5 7 3 0.9 1.9 0.4 2.4 0.6 3
0 0 0 3 2 0.5 0 0.5 1 0 0.5 2 0.6 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0.6 0.4 1 5 50 0 5 0.5 3 10 10 0 2 3
43 44 45 46 47 48 49
F/74 F/40 M/53 F/62 M/44 M/69 M/68
Midgut Midgut Hindgut Hindgut Hindgut Foregut Foregut
Appendix Metastasis Metastasis Colon Rectum Esophagus Esophagus
WNEC WNEC WNEC WNEC WNEC PNEC PNEC
1.3 1.5 2.5 3.5 0.8 4.5 5
50 51 52
F/69 M/72 F/60
Foregut Foregut Foregut
Stomach Stomach Stomach
PNEC PNEC PNEC a
0.2 4.5 2.8
Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I, v(+) Stage I Stage IIa Stage Ia Stage Ia Stage Ia Stage Ib Stage Ib Stage Ia, ly(+) Stage IIa Stage Ia Stage Ib, ly(+) Stage Ia Stage Ia Stage Ia Stage Ia Stage Ib Stage Ia Stage IIa Stage IIIb, ly(+), n(+) Stage IIa Stage IIb Stage IIa Stage IV, ly(+), m(+) Stage IIIb, ly(+), n(+) Stage I Stage IIa Stage I Stage IIIb, ly(+), v(+), n(+) Stage IIa Stage IIIb, n(+) Stage IIIb, ly(+), n(+) Stage IV, ly(+), v(+), m(+) Stage Ia Stage IV, m(+) Stage IIIb, ly(+), v(+), n(+) Stage IV, m(+) Stage IIIa, ly(+), v(+) Stage IIIb, ly(+), v(+), n(+)
41 0 5 82 17 0 5 75 86 89 31 80 84 68 76 78 63 30 0 66 69 79 78 7 22 5 41 63 89 7 90 10 0 60 5 0 99 93 88 5 37 90
8 0 0 75 80 5 14 24 69 20 0 79 76 7 0 5 4 40 0 36 71 22 71 32 5 7 17 43 21 0 80 5 0 57 3 55 67 91 38 4 0 55
0 75 12 62 17 12 0 11 6 4 0 12 15 4 4 0 4 95 54 64 71 83 16 12 0 35 43 31 15 0 45 6 32 85 6 60 35 9 0 0 0 5
0 8 10 20 0 50 32
0 0 99 100 60 100 100
0 0 85 65 5 93 80
0 0 67 90 42 82 53
15 1 20
64 0 87
95 0 85
82 0 18
(continued on next page)
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M. M. Rahman et al.
Table 1 (continued) Number Sex/ Location Organ age (y)
Classification Tumor stage, invasion, and metastasis
Tumor MIB-1 DNMT1 DNMT3a DNMT3b size (cm) LI (%) (%) (%) (%)
53
M/67
Foregut
Stomach
PNEC a
7
10
65
28
0
54 55 56 57 58 59 60
M/69 M/74 F/80 M/67 M/63 M/79 M/71
Foregut Foregut Foregut Foregut Foregut Midgut Hindgut
Stomach Stomach Stomach Stomach Duodenum Ileum-cecum Colon
PNEC a PNEC a PNEC PNEC PNEC PNEC PNEC
5.5 5 4.3 3 8 2.5 7
25 50 50 30 50 25 30
26 99 100 80 94 16 66
89 76 62 100 93 0 19
0 35 24 90 29 0 0
61 62 63
F/53 M/57 F/62
Hindgut Colon Hindgut Colon Hindgut Rectum
2.3 6 0.3
50 3 30
36 75 92
67 18 85
66 11 93
PNEC PNEC PNEC
Stage IIIb, ly(+), v(+), n(+) Stage IIIb, ly(+), n(+) Stage IIb, v(+) Stage IIIb, ly(+), n(+) Stage IIIb, ly(+), n(+) Stage IIIa, ly(+), v(+) Stage IIb Stage IIIb, ly(+), v(+), n(+) Stage IIa Stage IIa, ly(+), v(+) Stage Ia
Abbreviations: v(+), vascular invasion; ly(+), lymph vessel invasion; n(+), regional lymph node metastasis; m(+), distant metastasis. a Mixed exocrine-endocrine carcinoma.
deparaffinization and antigen retrieval using an autoclave oven technique, the sections were incubated at 4°C overnight with goat polyclonal anti-DNMT1 antibody (1:100; sc10219, Santa Cruz Biotech, Santa Cruz, CA), goat polyclonal anti-DNMT3a antibody (1:150; sc-10231, Santa Cruz Biotech), goat polyclonal anti-DNMT3b antibody (1:100; sc-10235, Santa Cruz Biotech), or MIB-1 mouse monoclonal antibody (1:100; DakoCytomation, Glostrup, Denmark). Antigen-antibody complexes were detected using the 3amino-9-ethylcarbazole reaction. The slides were counterstained lightly with hematoxylin and mounted for microscopic examination. Colon and breast carcinomas known to be positive for DNMT1, 3a, and 3b were used as a positive control [11,14]. Nuclear immunoreactivity in the proliferative zones of noncancerous foveolar epithelia was used as a positive internal control for some sections. Sections incubated in phosphate-buffered saline without the primary antibody served as a negative control. Furthermore, the specificity of all reactions for DNMT1, 3a, and 3b was verified by replacing the primary antibody with normal serum and preabsorbing each primary antibody with blocking peptide, respectively (sc-10219 P, sc-10231 P, sc-10235P; Santa Cruz Biotech). Because of the absence of tissues wholly composed of neuroendocrine cells, which are by definition part of a diffuse system of cells, several whole normal human organs were used as controls: the esophagus, stomach, duodenum, appendix, small intestine, pancreas, colon, and rectum.
2.3. Evaluation of immunohistochemical findings Each slide was examined by an observer blinded to the diagnosis and clinicopathologic data, whose observation was then confirmed by a second blinded observer. Nuclear staining was considered to represent a positive stain for DNMT1, 3a, 3b, and MIB-1. Several high-power fields (×400) selected
from different staining density regions including high, moderate, low, and negative staining areas were captured with a digital camera (Olympus Q-color 3; Olympus Inc, Center Valley, PA). Photographs were printed on plain paper, and a grid was drawn over them. We counted a mean of 1000 tumor cells per tumor (range, 800-1500), and the results were expressed as percentage of tumor cells with positive nuclei. The percentage of DNMT1-, 3a-, and 3b-stained tumor cells was then scored on a scale of 0 to 3 (0, no expression; 1+, b 20%; 2+, b 50%; and 3+, ≥50%). Furthermore, according to the score, the expression levels of DNMT1, 3a, and 3b were divided into following 3 groups: low (0, 1+), moderate (2+), and high (3+). As most samples stained for DNMT1, 3a, and 3b showed similar color intensity, no evaluation of color intensity was performed in this study.
2.4. Statistical analyses To determine the significance of associations between different variables, data were statistically analyzed by the Mann-Whitney U test, the Kruskal-Wallis test, the χ2 test, the Pearson correlation coefficient, and the Spearman correlation coefficient, using the StatView J-4.5 software (Abacus Concepts, Berkeley, CA). Probability values (P) b .05 were considered statistically significant.
3. Results 3.1. Association between DNMT1, 3a, and 3b expression and tumor differentiation and TNM classification In general, DNMT1, 3a, and 3b were not expressed in normal NE cells, which were identified by choromogranin A
DNMT overexpression in GEP NET staining, in the normal esophagus, stomach, duodenum, appendix, small intestine, pancreas, colon, and rectum (data not shown). In tumor cells, the immunolocalization of
1073 DNMT1, 3a, and 3b proteins was observed in the nuclei with strong staining. Positive cell staining pattern is diffuse or in various sites in each tumor.
Fig. 1 Nuclear immunostaining of DNMT1 (A-D), DNMT3a (E-H), and DNMT3b (I-L) in GEP NET. DNMT1, 3a, and 3b immunostaining figures in WNET (A, E, I), insulinoma (B, F, J), WNEC (C, G, K), and PNEC (D, H, L) (0, no expression; 1+, b 20%; 2+, b 50%; 3+, ≥50%) are shown. (A-L: original magnification ×200.)
1074 Table 2
M. M. Rahman et al. DNMT1, DNMT3a, and DNMT3b expression levels in 63 GEP NETs and their clinicopathologic associations
Variable
Age (y) Sex Female Male Histologic type WNET WNEC PNEC Tumor stage Stage I Stage II Stage III Stage IV Site Foregut Midgut Hindgut Total
No. of DNMT1 expression cases Low Moderate High (N = 63) (n =18) (n = 8) (n = 37) 61 ± 3.4
59 ± 4.4
59 ± 1.7
DNMT3a expression
DNMT3b expression
Low
Moderate High
Low
Moderate High
(n = 27)
(n = 10)
(n = 26)
(n = 36)
(n = 9)
(n = 18)
57 ± 3
60 ± 2
62 ± 1.6
58 ± 2.1
57 ± 4.8
63 ± 1.8
23 40
7 (30%) 11 (28%)
2 (9%) 14 (61%) 6 (15%) 23 (57%)
7 (30%) 20 (50%)
3 (13%) 13 (57%) 7 (18%) 13 (32%)
12 (52%) 24 (60%)
31 16 16
9 (30%) 7 (44%) 2 (13%)
5 (16%) 17 (54%) 1 (6%) 8 (50%) 2 (13%) 12 (74%)
15 (48%) 8 (50%) 4 (25%)
8 (26%) 8 (26%) ⁎ 20 (65%) 1 (6%) 7 (44%) 9 (56%) 1 (6%) 11 (69%) 7 (44%)
4 (13%) 2 (13%) 3 (18%)
7 (22%) 5 (31%) 6 (38%)
33 12 14 4
9 (27%) 6 (50%) 3 (21%) 0
6 (18%) 18 (55%) 1 (8%) 5 (42%) 1 (7%) 10 (72%) 0 4 (100%)
16 (49%) 7 (58%) 4 (29%) 0
9 (27%) 0 1 (7%) 0
8 (24%) ⁎ 20 (61%) 5 (42%) 8 (67%) 9 (64%) 8 (57%) 4 (100%) 0
5 (15%) 1 (8%) 2 (14%) 1 (25%)
8 (24%) 3 (25%) 4 (29%) 3 (75%)
32 9 22 63
10 (31%) 4 (44.5%) 4 (18%) 18 (29%)
3 (9%) 19 (60%) 11 (34%) 3 (9%) 18 (57%) ⁎ 19 (60%) 1 (11%) 4 (44.5%) 7 (78%) 1 (11%) 1 (11%) 9 (100%) 4 (18%) 14 (64%) 9 (41%) 6 (27%) 7 (32%) 8 (36%) 8 (13%) 37 (58%) 27 (43%) 10 (16%) 26 (41%) 36 (57%)
3 (13%) 8 (35%) 6 (15%) 10 (25%)
4 (12%) 9 (28%) ⁎ 0 0 5 (23%) 9 (41%) 9 (14%) 18 (29%)
NOTE. Low, 0 and 1+; moderate, 2+; high, 3+ (0, no expression; 1+, b20%; 2+, b50%; 3+, ≥50%). ⁎ P b .05.
In GEP NETs, nuclear DNMT1 expression was detected in a high proportion of tumors (55 of 63, 87%) (Fig. 1A-D and Table 1). Thirty-seven (59%) tumors displayed high expression (3+), 8 (13%) tumors showed moderate expression (2+), and 18 (28%) tumors showed low expression (1+ and 0) (Table 2). Although DNMT1 was detected in all tumor categories, there were no significantly statistical differences among WNET, WNEC, and PNEC (Table 2 and Fig. 2A). In addition, high expression of DNMT1 was detected in 18 (55%) of 33 stage I tumors, 5 (42%) of 12 stage II tumors, 10 (72%) of 14 stage III tumors, and 4 (100%) of 4 of stage IV tumors (Table 2). DNMT1 expression was significantly higher in stage IV tumors than in stage I and II tumors (P b .05 and P b .05, respectively; Fig. 2D). DNMT3a nuclear expression was observed in 51 (81%) of 63 GEP NETs (Fig. 1E-H and Table 1): high expression (3+), moderate expression (2+), and low expression (1+ and 0)
were observed in 26 (41%), 10 (16%), and 27 (43%) tumors, respectively (Table 2). High expression of DNMT3a was more frequently detected in PNEC (69%) than in WNET (26%) and WNEC (44%) (P b .05, Table 2). DNMT3a showed the highest levels in PNEC (PNEC versus WNET, P b .01; PNEC WNEC, P b .05; Fig. 2B). In addition, the frequency of high expression of DNMT3a was 24% in stage I tumors, 42% in stage II tumors, 64% in stage III tumors, and 100% in stage IV tumors (P b .05, Table 2). Furthermore, DNMT3a expression level was higher in stage IV and III tumors than in stage I and II tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; stage III versus stage I, P b .05; stage III versus stage II, P b .05; Fig. 2E). Nuclear staining of DNMT3b was detected in 47 (75%) of 63 GEP NETs (Fig. 1I-L and Table 1): high expression (3+), moderate expression (2+), and low expression (1+ and 0) were observed in 29%, 14%, and 57% of tumors,
Fig. 2 The relationships between the expression levels of DNMT1, 3a, and 3b and GEP NET clinicopathologic data. The expression levels of DNMTs in each group were shown as mean ± SD of percentage calculated from the data in Table 1. DNMT1 and DNMT3b protein expression levels were not significantly different among WNETs (n = 31), WNECs (n = 16), and PNECs (n = 16) (A, C). The DNMT3a expression levels increased from WNETs to WNECs and PNECs, and significant differences were observed between WNETs and PNECs (P b .01) and between WNECs and PNECs (P b .05) (B). DNMT1 expression was higher in stage IV tumors (n = 4) than in stage I (n = 33), II (n = 12), and III (n = 14) tumors; but the difference was not statistically significant (D). DNMT3a expression levels were higher in stage IV and III tumors than in stage I and II tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; and stage III versus stage I, P b .05) (E). DNMT3b expression levels were also higher in stage IV tumors than stage I, II, and III tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; and stage IV versus stage III, P b .01) (F). DNMT3a expression levels were positively correlated with the MIB-1 labeling index in GEP NET (R = 0.424, P = 0.001) (H). DNMT1 and DNMT3b expression levels were potentially correlated with the MIB-1 labeling index, but the correlations were not statistically significant (R = 0.225, P = 0.098 and R = 0.241, P = 0.075, respectively) (G, I). DNMT1 expression levels did not show any significant difference among foregut (n = 32), midgut (n = 9), and hindgut (n = 22) GEP NETs (L). The expression levels of DNMT3a and 3b were significantly lower in midgut NETs than in foregut and hindgut NETs (M, N).
DNMT overexpression in GEP NET respectively (Table 2). There were no significant differences in the frequency of high DNMT3b expression among different histopathologic classifications (WNET, 22%; WNEC, 31%; PNEC, 38%; Table 2). DNMT3b showed the highest level in PNEC, although it was not statistically
1075 significant (Fig. 2C). In addition, the frequency of high expression of DNMT3b was 24% in stage I tumors, 25% in stage II tumors, 29% in stage III tumors, and 75% in stage IV tumors (Table 2). DNMT3b expression level was higher in stage IV tumors than in stage I, II, and III tumors (stage IV
1076 versus stage I, P b .01; stage IV versus stage II, P b .01; stage IV versus stage III, P b .01; Fig. 2F).
3.2. Association of DNMT1, 3a, and 3b expression with the MIB-1 labeling index The MIB-1 labeling index, a predictive marker for the prognosis of GEP NET, was significantly correlated with tumor differentiation and tumor stage (data not shown). However, 2 PNECs showed low MIB-1 labeling index. This phenomenon also has been reported in another study [22]. These findings indicate that other molecular marker may predict survival of these cases [22]. In other hand, one WNEC showed extremely high MIB-1 labeling index. This patient may have worse prognosis. Unfortunately, we do not have survival data about this patient. Here, we investigated the correlation between the MIB-1 labeling index and DNMT protein expression. DNMT3a expression was significantly correlated with the MIB-1 labeling index in GEP NET (R = 0.457, P = 0.001; Fig. 2H). DNMT1 and DNMT3b expression levels also were positively correlated with the MIB-1 labeling index (R = 0.293, P = 0.019 and R = 0.249, P = 0.049, respectively; Fig. 2G, I).
3.3. Association of DNMT1, 3a, and 3b expression with tumor site and other clinicopathologic characteristics The expression levels and frequencies of positive staining of DNMT3a and 3b were significantly lower in midgut NETs than in foregut and hindgut NETs (Fig. 2K, L; Table 2). DNMT1 did not show significantly different expression levels among 3 anatomical sites (Fig. 2J). Between 7 gastrointestinal WNETs with uncertain behavior (N1 cm in size or vascular invasion) and 17 gastrointestinal WNETs with benign behavior, the expression levels of DNMT1, 3a, and 3b did not show any significant differences (data not shown). Although the expression levels of DNMT1, 3a, and 3b were potentially high in WNECs, no significant correlations between DNMT expression levels and tumor malignancy were detected (data not shown). In WNECs and PNECs, each expression level of DNMT1, 3a, and 3b was higher in metastatic tumors than in tumors without metastasis. However, these tendencies were not statistically significant (data not shown). We could not find any significant differences in the expression levels of DNMT1, 3a, and 3b between PNEC areas and adenocarcinoma areas in several PNECs (data not shown). In addition, the expression levels of DNMT1, 3a, and 3b were found to be unrelated to the patient's age or sex.
4. Discussion The development of effective drugs for GEP NET requires an understanding of GEP NET tumor biology and the
M. M. Rahman et al. discovery of molecular targets specific to all subtypes of GEP NET. Gene transcription silencing by DNA promoter hypermethylation is a crucial event in cancer initiation and progression [4]. Aberrant methylation-induced silencing of TSGs was recognized as an important mechanism contributing to GEP NET tumorigenesis. Hypermethylation of several TSGs, including RASSF1A, CDKN2A(p14), CDKN2A(p16), HIC1, and MGMT, has been reported in GEP NET [19-23]. DNMT1 overexpression plays an important role in the mechanism of methylation [27] and is associated with aberrant methylation of CpG islands [28]. In many studies, DNMT1 overexpression has been shown in tumors; and forced overexpression of DNMT1 results in transformation and de novo methylation of CpG islands. Similar to DNMT1, DNMT3a and 3b were also overexpressed in cancerous tissues [29]. This study, for the first time, revealed the overexpression of DNMT1, 3a, and 3b in GEP NET. We found that the overexpression of DNMT1, 3a, and 3b is a common event in all histologic types of GEP NET. Our findings suggested that DNMT1, 3a, and 3b are essential enzymes for tumorigenesis in all types of GEP NET. Several studies have suggested that the overexpression of DNMT1, 3a, and 3b is associated with poorer tumor differentiation [13,30,31]. Recently, the overexpression of DNMT1, 3a, and 3b and their activity were also investigated in benign endocrine tumors such as pituitary adenomas [32,33]. In this study, although DNMT3a expression was found to be highest in PNEC, high expression levels of DNMT1, 3a, and 3b were frequently observed in WNET, WNEC, and PNEC. Our findings suggested that DNMT contributes to tumorigenesis in benign tumors as well as in malignant tumors of GEP. It has been shown that high expression of DNMT1, 3a, and 3b is associated with portal vein involvement and is indicative of a poor prognosis. In addition, DNMT1 protein expression was greater in the advanced stages of hepatocellular cancer [13]. In this study, the expression of all DNMTs was significantly related to tumor stage. DNMT1 showed a higher expression level in stage IV tumors than in stage I and II tumors; DNMT3a showed a higher expression level in stage III and IV tumors than in stage I and II tumors; and DNMT3b showed the highest expression level in stage IV tumors. Furthermore, high expression of DNMT1, 3a, and 3b was possibly related to tumor metastasis in WNEC and PNEC. DNMT may contribute to cancer progress by silencing metastasis-related TSGs such as CDH1 and RASSF1A. A previous study suggested that RASSF1A methylation in GEP NET is associated with lymph node metastasis [21]. However, the exact mechanisms behind these associations remain to be elucidated. DNMT1 is expressed mainly during the S-phase in normal cells. As tumor tissues presumably contain a greater proportion of dividing cells than normal tissues, whether increased DNMT1 expression is due to an increase in the proportion of dividing cells or to an acute increase in DNMT1 expression per individual cell remains unclear [34].
DNMT overexpression in GEP NET The relationship between DNMT1 and the proliferation cell nuclear antigens has been documented in several cancers [11,30,35,36]. This study revealed that the MIB-1 labeling index was correlated with tumor stage and tumor differentiation. Interestingly, overexpression of DNMT1, DNMT3a, and DNMT3b was significantly associated with the MIB-1 labeling index. These findings are not consistent with previous several studies [30,35,36]. Thus, the interaction of DNMT with other nuclear proteins involved in the cell cycle or proliferation needs further investigation. The current literature suggests that genetic events such as MEN1 gene mutations and deletions, 18q losses, and X-chromosome losses are involved in GEP NET tumorigenesis, with significant differences in their contributions among different embryologic tumors, that is, foregut, midgut, and hindgut tumors [37]. In the present study, GEP NET in midgut showed the lowest level and lower frequency of DNMT3a and 3b expression; however, DNMT1 expression did not show this tendency in tumor anatomical sites. RASSF1A methylation was observed frequently in the foregut but not in the midgut or hindgut [38]; however, only a few hindgut NETs were investigated. Our study included only 9 midgut NETs because of their low incidence in Japan. Thus, the differences in DNMT protein expression and TSG methylation pattern among tumor anatomical sites need further identification. GEP NETs involving DNMT1, 3a, and 3b overexpression are potentially treatable with currently used anticancer therapies. Hypermethylated gene promoters have the potential to be reactivated by nucleoside analogues, such as 5-azacytidine and 5-aza-2-deoxycytidine (decitabine), both of which have been found to be particularly effective in inducing DNA demethylation and epigenetic gene reactivation in vitro [39,40]. These effects are caused by covalent trapping of DNMTs through DNA-incorporated azacytosine bases and subsequent degradation of the trapped enzymes [41,42]. Azacytidine and decitabine are currently used to treat myelodysplastic syndrome and acute myeloid leukemia [17]. Furthermore, clinical trials of the effectiveness of decitabine against solid tumors are currently in the early stages [17]. In addition, hydralazine, a nonnucleoside DNA methylation inhibitor depending on its ability to inhibit DNMT1 activity and/or decrease DNMT1 and 3a expression, was reported to be able to reactivate the expression of TSGs in cancers in 2 clinical trial studies [43,44]. Our data suggest that GEP NETs displaying overexpression of DNMT1, 3a, and 3b would be good candidates for epigenetic therapy. As PNECs showed the worst prognosis and frequently expressed a high level of DNMT, a clinical trial to test the effectiveness of decitabine and hydralazine against PNECs is considerable. In summary, analysis of DNMT1, 3a, and 3b expression in GEP NET showed a higher frequency of DNMT overexpression. In particular, DNMT3a demonstrated greater clinical significance in GEP NET. These results suggest that DNMT plays important roles in the tumori-
1077 genesis and development of GEP NET and that epigenetic therapy directed against DNMT would be an effective therapeutic approach.
Acknowledgments The authors declare that they received no specific grants for this project and that no conflicts of interest that could be perceived as prejudicing the impartiality of the research exist. We thank Ms Noriko Amo and Mr Akihito Amo for their excellent technical assistance.
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M. M. Rahman et al. [32] Zhu X, Mao X, Hurren R, Schimmer AD, Ezzat S, Asa SLJ. Deoxyribonucleic acid methyltransferase 3B promotes epigenetic silencing through histone 3 chromatin modifications in pituitary cells. Clin Endocrinol Metab 2008;93:3610-7. [33] Dudley KJ, Revill K, Whitby P, Clayton RN, Farrell WE. Genomewide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors. Mol Cancer Res 2008;6:1567-74. [34] Lee PJ, Washer LL, Law DJ, Boland CR, Horon IL, Feinberg AP. Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation. Proc Natl Acad Sci USA 1996;93:10366-70. [35] Nakagawa T, Kanai Y, Saito Y, Kitamura T, Kakizoe T, Hirohashi S. Increased DNA methyltransferase 1 protein expression in human transitional cell carcinoma of the bladder. J Urol 2003;170:2463-6. [36] Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S. DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis. J Urol 2005;173:1767-71. [37] Lubensky IA, Zhuang Z. Molecular genetic events in gastrointestinal and pancreatic neuroendocrine tumours. Endocr Pethol 2007;18: 156-62. [38] Pizzi S, Azzoni C, Bottarelli L, et al. RASSF1A promoter methylation and 3p21.3 loss of heterozygosity are features of foregut, but not midgut and hindgut, malignant endocrine tumours. J Pathol 2005;206: 409-16. [39] Chuang JC, Yoo CB, Kwan JM, et al. Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2'deoxycytidine. Mol Cancer Ther 2005;4:1515-20. [40] Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F. Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res 2006;66:2794-800. [41] Santi DV, Norment A, Garrett CE. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A 1984;81:6993-7. [42] Weisenberger DJ, Velicescu M, Cheng JC, Gonzales FA, Liang G, Jones PA. Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation. Mol Cancer Res 2004;2:62-72. [43] Zambrano P, Segura-Pacheco B, Perez-Cardenas E, et al. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer 2005;5:44. [44] Candelaria M, Gallardo-Rincón D, Arce C, et al. A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors. Ann Oncol 2007;18:1529-38.
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Original contribution
DNA methyltransferases 1, 3a, and 3b overexpression and clinical significance in gastroenteropancreatic neuroendocrine tumors Md. Mustafizur Rahman PhD a,1 , Zhi Rong Qian MD, PhD a,⁎,1 , Elaine Lu Wang MD, PhD a , Katsuhiko Yoshimoto MD, PhD b , Masahiko Nakasono MD, PhD a,c , Razia Sultana MD, PhD a , Tomoyuki Yoshida MD a , Toshitetsu Hayashi MD, PhD d , Reiji Haba MD, PhD d , Mitsuaki Ishida MD, PhD e , Hidetoshi Okabe MD, PhD e , Toshiaki Sano MD, PhD a a
Department of Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan b Department of Medical Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan c Department of Medicine, Tsurugi Municipal Handa Hospital, Tokushima 779-4401, Japan d Faculty of Medicine, Department of Pathology, Kagawa University, Kagawa 761-0793, Japan e Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Shiga 502-2192, Japan Received 2 November 2009; revised 30 December 2009; accepted 5 January 2010
Keywords: GEP NET; DNMT1, DNMT3a, DNMT3b, methylation
Summary The alteration of DNA methylation is one of the most common epigenetic changes in human cancers. Three genes, namely, DNA methyltransferase 1, 3a, and 3b, which code for DNA methyltransferases that affect promoter methylation status, are thought to play an important role in the development of cancers and may be good anticancer therapy targets. The methylation of tumor suppressor genes has been reported in gastroenteropancreatic neuroendocrine tumors; however, there have been no studies about DNA methyltransferase protein expression and its clinical significance in gastroenteropancreatic neuroendocrine tumors. In this study, the expression of DNA methyltransferase 1, 3a, and 3b was studied in 63 gastroenteropancreatic neuroendocrine tumors by immunohistochemistry. The expression of DNA methyltransferase 1, 3a, and 3b was frequently detected in gastroenteropancreatic neuroendocrine tumors (87%, 81%, and 75%, respectively). The DNA methyltransferase 3a expression level was significantly higher in poorly differentiated neuroendocrine carcinomas than in well-differentiated neuroendocrine tumors or well-differentiated neuroendocrine carcinomas (P b .01 and P b .05, respectively). The expression of DNA methyltransferase 1, 3a, and 3b showed significantly higher levels in stage IV tumors than in stage I or II tumors. In addition, the expression levels of DNA methyltransferase 1, 3a, and 3b were positively correlated with the MIB-1 labeling index in gastroenteropancreatic neuroendocrine tumors (R = 0.293, P = .019; R = 0.457, P = .001; and R = 0.249, P = .049; respectively). In addition, the expression levels and positive
⁎ Corresponding author. E-mail address: [email protected] (Z. R. Qian). 1 M. M. Rahman and Z. R. Qian contributed equally to this work. 0046-8177/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2010.01.011
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M. M. Rahman et al. immunostaining frequencies of DNA methyltransferase 3a and 3b were significantly lower in midgut neuroendocrine tumors than in foregut or hindgut neuroendocrine tumors. Our findings suggest that the overexpression of DNA methyltransferase 1, 3a, and 3b is related to tumorigenesis and the progression of gastroenteropancreatic neuroendocrine tumors. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are rare, with an age-adjusted annual incidence of 2.5 to 4.5 per 100 000 [1]. Over the past 3 decades, their incidence has increased because of improvements in diagnostic techniques. However, the survival of patients with GEP NET has not changed appreciably [2]. In particular, patients with poorly differentiated neuroendocrine carcinoma have the worst survival, which ranges from several weeks for untreated patients and from 6 to 12 months for those receiving therapy [3]. As GEP NET frequently demonstrates unpredictable and unusual biological behavior, cases of GEP NET frequently involve a late or delayed diagnosis. However, few effective targeted treatments are available because little is known about the mechanistic regulation of these tumors [2]. DNA hypermethylation of CpG islands is one of the most common epigenetic changes observed in human cancers [4]. Abnormal methylation of CpG islands can efficiently repress transcription of the associated gene, and its DNA methylation is a reversible process [4,5]. Hypermethylation takes place after DNA replication and is catalyzed by DNA methyltransferase (DNMT) using S-adenosylmethionine as the methyl donor. Three distinct families of DNMT genes, DNMT1, DNMT2, and DNMT3, have been identified in mammals [6,7]. DNMT1 is known to maintain methylation [8]; and the DNMT3 family consists of 2 related gene products, DNMT3a and DNMT3b, which function as de novo methyltransferases [9]. However, the function of DNMT2 remains unknown [10]. The overexpression of DNMT1, 3a, and 3b has been reported in various cancers including colorectal, prostate, hepatocelluar, breast, gastric, and lung tumors [11-16] and is significantly correlated with poor histologic differentiation and poor prognosis [14-16]. In addition, the blocking activity of DNMT may inhibit or reverse methylation-induced silencing of tumor suppressor genes (TSGs) [17]. Therefore, DNMT are major drug targets for epigenetic therapy [17]. The molecular mechanism behind the tumorigenesis and progression of GEP NET remains largely unknown. GEP NETs do not show any alterations in the expression levels of oncogenes such as ras, myc, fos, jun, and src [18]. Recently, the down-regulation of TSGs by the hypermethylation of CpG islands was reported as an important event in GEP NET development [19-22] and showed a trend toward worse survival [23]. However, to the best of our knowledge, no study has provided comprehensive and clinical analyses regarding the altered expression levels of DNMT1, 3a, and 3b in GEP NETs.
In the present study, we examined DNMT1, 3a, and 3b protein expression by immunohistochemistry in a series of GEP NETs taken from different anatomical sites. We analyzed the correlations between the expression of DNMT1, 3a, and 3b proteins and the clinicopathologic features and proliferative activity of GET NETs.
2. Materials and methods 2.1. Patients and samples Sixty-three paraffin-embedded surgical or biopsy specimens were collected from the Pathology Departments of the University of Tokushima Faculty of Medicine, Shiga University of Medicine Science, and Kagawa University Faculty of Medicine. These patients were affected by the following GEP NETs from 1982 to 2008: foregut tumors: esophagus (n = 3), stomach (n = 11), pancreas (n = 9), duodenum (n = 7); midgut tumors: ileum (n = 6), appendix (n =3); hindgut tumors: colon (n = 6), rectum (n = 14); and lymph node metastasis (n = 4). Notably, the frequency of midgut NET was low. The frequency of GEP NET in this study is consistent with that found in a report from the Neuroendocrine Tumor Workshop of Japan [24]. To confirm the NE immunophenotype, paraffin sections were stained with pan-NE markers such as chromogranin A and synaptophysin. The diagnosis of NET was made on the basis of morphologic and immunohistochemical findings evaluated by 2 independent pathologists (Z. R. Q. and T. S.) according to the World Health Organization classification [25]. These included well-differentiated NET (WNET, of either benign or uncertain behavior; n = 31), welldifferentiated neuroendocrine carcinoma (WNEC; n = 16), and poorly differentiated neuroendocrine carcinoma (PNEC; n = 16; including 4 mixed exocrine-endocrine carcinomas). The tumor stage was assessed on the basis of the TNM classification system [26]. Thus, 63 tumors included 33 tumors of stage I, 12 tumors of stage II, 14 tumors of stage III, and 4 tumors of stage IV. The clinicopathologic characteristics of the patients are summarized in Table 1.
2.2. Immunohistochemistry Immunolocalization of DNMT1, 3a, and 3b and Ki-67 antigen based on the streptavidin-biotin labeling method was performed on sections from representative blocks of paraffinembedded tissues for pathologic diagnosis. After
DNMT overexpression in GEP NET Table 1
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Summary of characteristics of 63 GEP NET and expression of DNMT1, DNMT3a and DNMT3b
Number Sex/ Location Organ age (y)
Classification Tumor stage, invasion, and metastasis
Tumor MIB-1 DNMT1 DNMT3a DNMT3b size (cm) LI (%) (%) (%) (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
M/40 M/75 F/70 M/78 F/48 F/61 M/64 M/63 M/63 F/48 M/82 F/57 M/53 F/53 M/26 M/48 F/38 M/61 M/47 F/62 F/62 M/62 M/61 F/67 M/56 M/34 F/51 M/62 M/49 M/35 F/61 M/66 M/56 F/68 M/66 F/63 M/48 F/53 M/61 M/82 M/60 M/62
Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Midgut Midgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut Foregut Foregut Foregut Foregut Foregut Foregut Foregut Midgut Midgut Midgut Midgut
Stomach Stomach Stomach Duodenum Duodenum Duodenum Duodenum Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Appendix Appendix Colon Colon Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Stomach Duodenum Duodenum Pancreas Pancreas Metastasis Metastasis Ileum-cecum Ileum-cecum Ileum-cecum Ileum-cecum
WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNET WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC WNEC
0.9 0.5 0.9 0.4 0.4 0.3 0.5 1.1 1.5 1 1.1 1.6 0.6 1.5 0.7 1.4 0.2 0.3 0.4 1.2 1.5 0.9 2.2 0.6 1 0.6 0.8 0.5 0.4 1.4 0.5 6 1.3 1.5 7 3 0.9 1.9 0.4 2.4 0.6 3
0 0 0 3 2 0.5 0 0.5 1 0 0.5 2 0.6 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0.6 0.4 1 5 50 0 5 0.5 3 10 10 0 2 3
43 44 45 46 47 48 49
F/74 F/40 M/53 F/62 M/44 M/69 M/68
Midgut Midgut Hindgut Hindgut Hindgut Foregut Foregut
Appendix Metastasis Metastasis Colon Rectum Esophagus Esophagus
WNEC WNEC WNEC WNEC WNEC PNEC PNEC
1.3 1.5 2.5 3.5 0.8 4.5 5
50 51 52
F/69 M/72 F/60
Foregut Foregut Foregut
Stomach Stomach Stomach
PNEC PNEC PNEC a
0.2 4.5 2.8
Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I Stage I, v(+) Stage I Stage IIa Stage Ia Stage Ia Stage Ia Stage Ib Stage Ib Stage Ia, ly(+) Stage IIa Stage Ia Stage Ib, ly(+) Stage Ia Stage Ia Stage Ia Stage Ia Stage Ib Stage Ia Stage IIa Stage IIIb, ly(+), n(+) Stage IIa Stage IIb Stage IIa Stage IV, ly(+), m(+) Stage IIIb, ly(+), n(+) Stage I Stage IIa Stage I Stage IIIb, ly(+), v(+), n(+) Stage IIa Stage IIIb, n(+) Stage IIIb, ly(+), n(+) Stage IV, ly(+), v(+), m(+) Stage Ia Stage IV, m(+) Stage IIIb, ly(+), v(+), n(+) Stage IV, m(+) Stage IIIa, ly(+), v(+) Stage IIIb, ly(+), v(+), n(+)
41 0 5 82 17 0 5 75 86 89 31 80 84 68 76 78 63 30 0 66 69 79 78 7 22 5 41 63 89 7 90 10 0 60 5 0 99 93 88 5 37 90
8 0 0 75 80 5 14 24 69 20 0 79 76 7 0 5 4 40 0 36 71 22 71 32 5 7 17 43 21 0 80 5 0 57 3 55 67 91 38 4 0 55
0 75 12 62 17 12 0 11 6 4 0 12 15 4 4 0 4 95 54 64 71 83 16 12 0 35 43 31 15 0 45 6 32 85 6 60 35 9 0 0 0 5
0 8 10 20 0 50 32
0 0 99 100 60 100 100
0 0 85 65 5 93 80
0 0 67 90 42 82 53
15 1 20
64 0 87
95 0 85
82 0 18
(continued on next page)
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M. M. Rahman et al.
Table 1 (continued) Number Sex/ Location Organ age (y)
Classification Tumor stage, invasion, and metastasis
Tumor MIB-1 DNMT1 DNMT3a DNMT3b size (cm) LI (%) (%) (%) (%)
53
M/67
Foregut
Stomach
PNEC a
7
10
65
28
0
54 55 56 57 58 59 60
M/69 M/74 F/80 M/67 M/63 M/79 M/71
Foregut Foregut Foregut Foregut Foregut Midgut Hindgut
Stomach Stomach Stomach Stomach Duodenum Ileum-cecum Colon
PNEC a PNEC a PNEC PNEC PNEC PNEC PNEC
5.5 5 4.3 3 8 2.5 7
25 50 50 30 50 25 30
26 99 100 80 94 16 66
89 76 62 100 93 0 19
0 35 24 90 29 0 0
61 62 63
F/53 M/57 F/62
Hindgut Colon Hindgut Colon Hindgut Rectum
2.3 6 0.3
50 3 30
36 75 92
67 18 85
66 11 93
PNEC PNEC PNEC
Stage IIIb, ly(+), v(+), n(+) Stage IIIb, ly(+), n(+) Stage IIb, v(+) Stage IIIb, ly(+), n(+) Stage IIIb, ly(+), n(+) Stage IIIa, ly(+), v(+) Stage IIb Stage IIIb, ly(+), v(+), n(+) Stage IIa Stage IIa, ly(+), v(+) Stage Ia
Abbreviations: v(+), vascular invasion; ly(+), lymph vessel invasion; n(+), regional lymph node metastasis; m(+), distant metastasis. a Mixed exocrine-endocrine carcinoma.
deparaffinization and antigen retrieval using an autoclave oven technique, the sections were incubated at 4°C overnight with goat polyclonal anti-DNMT1 antibody (1:100; sc10219, Santa Cruz Biotech, Santa Cruz, CA), goat polyclonal anti-DNMT3a antibody (1:150; sc-10231, Santa Cruz Biotech), goat polyclonal anti-DNMT3b antibody (1:100; sc-10235, Santa Cruz Biotech), or MIB-1 mouse monoclonal antibody (1:100; DakoCytomation, Glostrup, Denmark). Antigen-antibody complexes were detected using the 3amino-9-ethylcarbazole reaction. The slides were counterstained lightly with hematoxylin and mounted for microscopic examination. Colon and breast carcinomas known to be positive for DNMT1, 3a, and 3b were used as a positive control [11,14]. Nuclear immunoreactivity in the proliferative zones of noncancerous foveolar epithelia was used as a positive internal control for some sections. Sections incubated in phosphate-buffered saline without the primary antibody served as a negative control. Furthermore, the specificity of all reactions for DNMT1, 3a, and 3b was verified by replacing the primary antibody with normal serum and preabsorbing each primary antibody with blocking peptide, respectively (sc-10219 P, sc-10231 P, sc-10235P; Santa Cruz Biotech). Because of the absence of tissues wholly composed of neuroendocrine cells, which are by definition part of a diffuse system of cells, several whole normal human organs were used as controls: the esophagus, stomach, duodenum, appendix, small intestine, pancreas, colon, and rectum.
2.3. Evaluation of immunohistochemical findings Each slide was examined by an observer blinded to the diagnosis and clinicopathologic data, whose observation was then confirmed by a second blinded observer. Nuclear staining was considered to represent a positive stain for DNMT1, 3a, 3b, and MIB-1. Several high-power fields (×400) selected
from different staining density regions including high, moderate, low, and negative staining areas were captured with a digital camera (Olympus Q-color 3; Olympus Inc, Center Valley, PA). Photographs were printed on plain paper, and a grid was drawn over them. We counted a mean of 1000 tumor cells per tumor (range, 800-1500), and the results were expressed as percentage of tumor cells with positive nuclei. The percentage of DNMT1-, 3a-, and 3b-stained tumor cells was then scored on a scale of 0 to 3 (0, no expression; 1+, b 20%; 2+, b 50%; and 3+, ≥50%). Furthermore, according to the score, the expression levels of DNMT1, 3a, and 3b were divided into following 3 groups: low (0, 1+), moderate (2+), and high (3+). As most samples stained for DNMT1, 3a, and 3b showed similar color intensity, no evaluation of color intensity was performed in this study.
2.4. Statistical analyses To determine the significance of associations between different variables, data were statistically analyzed by the Mann-Whitney U test, the Kruskal-Wallis test, the χ2 test, the Pearson correlation coefficient, and the Spearman correlation coefficient, using the StatView J-4.5 software (Abacus Concepts, Berkeley, CA). Probability values (P) b .05 were considered statistically significant.
3. Results 3.1. Association between DNMT1, 3a, and 3b expression and tumor differentiation and TNM classification In general, DNMT1, 3a, and 3b were not expressed in normal NE cells, which were identified by choromogranin A
DNMT overexpression in GEP NET staining, in the normal esophagus, stomach, duodenum, appendix, small intestine, pancreas, colon, and rectum (data not shown). In tumor cells, the immunolocalization of
1073 DNMT1, 3a, and 3b proteins was observed in the nuclei with strong staining. Positive cell staining pattern is diffuse or in various sites in each tumor.
Fig. 1 Nuclear immunostaining of DNMT1 (A-D), DNMT3a (E-H), and DNMT3b (I-L) in GEP NET. DNMT1, 3a, and 3b immunostaining figures in WNET (A, E, I), insulinoma (B, F, J), WNEC (C, G, K), and PNEC (D, H, L) (0, no expression; 1+, b 20%; 2+, b 50%; 3+, ≥50%) are shown. (A-L: original magnification ×200.)
1074 Table 2
M. M. Rahman et al. DNMT1, DNMT3a, and DNMT3b expression levels in 63 GEP NETs and their clinicopathologic associations
Variable
Age (y) Sex Female Male Histologic type WNET WNEC PNEC Tumor stage Stage I Stage II Stage III Stage IV Site Foregut Midgut Hindgut Total
No. of DNMT1 expression cases Low Moderate High (N = 63) (n =18) (n = 8) (n = 37) 61 ± 3.4
59 ± 4.4
59 ± 1.7
DNMT3a expression
DNMT3b expression
Low
Moderate High
Low
Moderate High
(n = 27)
(n = 10)
(n = 26)
(n = 36)
(n = 9)
(n = 18)
57 ± 3
60 ± 2
62 ± 1.6
58 ± 2.1
57 ± 4.8
63 ± 1.8
23 40
7 (30%) 11 (28%)
2 (9%) 14 (61%) 6 (15%) 23 (57%)
7 (30%) 20 (50%)
3 (13%) 13 (57%) 7 (18%) 13 (32%)
12 (52%) 24 (60%)
31 16 16
9 (30%) 7 (44%) 2 (13%)
5 (16%) 17 (54%) 1 (6%) 8 (50%) 2 (13%) 12 (74%)
15 (48%) 8 (50%) 4 (25%)
8 (26%) 8 (26%) ⁎ 20 (65%) 1 (6%) 7 (44%) 9 (56%) 1 (6%) 11 (69%) 7 (44%)
4 (13%) 2 (13%) 3 (18%)
7 (22%) 5 (31%) 6 (38%)
33 12 14 4
9 (27%) 6 (50%) 3 (21%) 0
6 (18%) 18 (55%) 1 (8%) 5 (42%) 1 (7%) 10 (72%) 0 4 (100%)
16 (49%) 7 (58%) 4 (29%) 0
9 (27%) 0 1 (7%) 0
8 (24%) ⁎ 20 (61%) 5 (42%) 8 (67%) 9 (64%) 8 (57%) 4 (100%) 0
5 (15%) 1 (8%) 2 (14%) 1 (25%)
8 (24%) 3 (25%) 4 (29%) 3 (75%)
32 9 22 63
10 (31%) 4 (44.5%) 4 (18%) 18 (29%)
3 (9%) 19 (60%) 11 (34%) 3 (9%) 18 (57%) ⁎ 19 (60%) 1 (11%) 4 (44.5%) 7 (78%) 1 (11%) 1 (11%) 9 (100%) 4 (18%) 14 (64%) 9 (41%) 6 (27%) 7 (32%) 8 (36%) 8 (13%) 37 (58%) 27 (43%) 10 (16%) 26 (41%) 36 (57%)
3 (13%) 8 (35%) 6 (15%) 10 (25%)
4 (12%) 9 (28%) ⁎ 0 0 5 (23%) 9 (41%) 9 (14%) 18 (29%)
NOTE. Low, 0 and 1+; moderate, 2+; high, 3+ (0, no expression; 1+, b20%; 2+, b50%; 3+, ≥50%). ⁎ P b .05.
In GEP NETs, nuclear DNMT1 expression was detected in a high proportion of tumors (55 of 63, 87%) (Fig. 1A-D and Table 1). Thirty-seven (59%) tumors displayed high expression (3+), 8 (13%) tumors showed moderate expression (2+), and 18 (28%) tumors showed low expression (1+ and 0) (Table 2). Although DNMT1 was detected in all tumor categories, there were no significantly statistical differences among WNET, WNEC, and PNEC (Table 2 and Fig. 2A). In addition, high expression of DNMT1 was detected in 18 (55%) of 33 stage I tumors, 5 (42%) of 12 stage II tumors, 10 (72%) of 14 stage III tumors, and 4 (100%) of 4 of stage IV tumors (Table 2). DNMT1 expression was significantly higher in stage IV tumors than in stage I and II tumors (P b .05 and P b .05, respectively; Fig. 2D). DNMT3a nuclear expression was observed in 51 (81%) of 63 GEP NETs (Fig. 1E-H and Table 1): high expression (3+), moderate expression (2+), and low expression (1+ and 0)
were observed in 26 (41%), 10 (16%), and 27 (43%) tumors, respectively (Table 2). High expression of DNMT3a was more frequently detected in PNEC (69%) than in WNET (26%) and WNEC (44%) (P b .05, Table 2). DNMT3a showed the highest levels in PNEC (PNEC versus WNET, P b .01; PNEC WNEC, P b .05; Fig. 2B). In addition, the frequency of high expression of DNMT3a was 24% in stage I tumors, 42% in stage II tumors, 64% in stage III tumors, and 100% in stage IV tumors (P b .05, Table 2). Furthermore, DNMT3a expression level was higher in stage IV and III tumors than in stage I and II tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; stage III versus stage I, P b .05; stage III versus stage II, P b .05; Fig. 2E). Nuclear staining of DNMT3b was detected in 47 (75%) of 63 GEP NETs (Fig. 1I-L and Table 1): high expression (3+), moderate expression (2+), and low expression (1+ and 0) were observed in 29%, 14%, and 57% of tumors,
Fig. 2 The relationships between the expression levels of DNMT1, 3a, and 3b and GEP NET clinicopathologic data. The expression levels of DNMTs in each group were shown as mean ± SD of percentage calculated from the data in Table 1. DNMT1 and DNMT3b protein expression levels were not significantly different among WNETs (n = 31), WNECs (n = 16), and PNECs (n = 16) (A, C). The DNMT3a expression levels increased from WNETs to WNECs and PNECs, and significant differences were observed between WNETs and PNECs (P b .01) and between WNECs and PNECs (P b .05) (B). DNMT1 expression was higher in stage IV tumors (n = 4) than in stage I (n = 33), II (n = 12), and III (n = 14) tumors; but the difference was not statistically significant (D). DNMT3a expression levels were higher in stage IV and III tumors than in stage I and II tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; and stage III versus stage I, P b .05) (E). DNMT3b expression levels were also higher in stage IV tumors than stage I, II, and III tumors (stage IV versus stage I, P b .01; stage IV versus stage II, P b .02; and stage IV versus stage III, P b .01) (F). DNMT3a expression levels were positively correlated with the MIB-1 labeling index in GEP NET (R = 0.424, P = 0.001) (H). DNMT1 and DNMT3b expression levels were potentially correlated with the MIB-1 labeling index, but the correlations were not statistically significant (R = 0.225, P = 0.098 and R = 0.241, P = 0.075, respectively) (G, I). DNMT1 expression levels did not show any significant difference among foregut (n = 32), midgut (n = 9), and hindgut (n = 22) GEP NETs (L). The expression levels of DNMT3a and 3b were significantly lower in midgut NETs than in foregut and hindgut NETs (M, N).
DNMT overexpression in GEP NET respectively (Table 2). There were no significant differences in the frequency of high DNMT3b expression among different histopathologic classifications (WNET, 22%; WNEC, 31%; PNEC, 38%; Table 2). DNMT3b showed the highest level in PNEC, although it was not statistically
1075 significant (Fig. 2C). In addition, the frequency of high expression of DNMT3b was 24% in stage I tumors, 25% in stage II tumors, 29% in stage III tumors, and 75% in stage IV tumors (Table 2). DNMT3b expression level was higher in stage IV tumors than in stage I, II, and III tumors (stage IV
1076 versus stage I, P b .01; stage IV versus stage II, P b .01; stage IV versus stage III, P b .01; Fig. 2F).
3.2. Association of DNMT1, 3a, and 3b expression with the MIB-1 labeling index The MIB-1 labeling index, a predictive marker for the prognosis of GEP NET, was significantly correlated with tumor differentiation and tumor stage (data not shown). However, 2 PNECs showed low MIB-1 labeling index. This phenomenon also has been reported in another study [22]. These findings indicate that other molecular marker may predict survival of these cases [22]. In other hand, one WNEC showed extremely high MIB-1 labeling index. This patient may have worse prognosis. Unfortunately, we do not have survival data about this patient. Here, we investigated the correlation between the MIB-1 labeling index and DNMT protein expression. DNMT3a expression was significantly correlated with the MIB-1 labeling index in GEP NET (R = 0.457, P = 0.001; Fig. 2H). DNMT1 and DNMT3b expression levels also were positively correlated with the MIB-1 labeling index (R = 0.293, P = 0.019 and R = 0.249, P = 0.049, respectively; Fig. 2G, I).
3.3. Association of DNMT1, 3a, and 3b expression with tumor site and other clinicopathologic characteristics The expression levels and frequencies of positive staining of DNMT3a and 3b were significantly lower in midgut NETs than in foregut and hindgut NETs (Fig. 2K, L; Table 2). DNMT1 did not show significantly different expression levels among 3 anatomical sites (Fig. 2J). Between 7 gastrointestinal WNETs with uncertain behavior (N1 cm in size or vascular invasion) and 17 gastrointestinal WNETs with benign behavior, the expression levels of DNMT1, 3a, and 3b did not show any significant differences (data not shown). Although the expression levels of DNMT1, 3a, and 3b were potentially high in WNECs, no significant correlations between DNMT expression levels and tumor malignancy were detected (data not shown). In WNECs and PNECs, each expression level of DNMT1, 3a, and 3b was higher in metastatic tumors than in tumors without metastasis. However, these tendencies were not statistically significant (data not shown). We could not find any significant differences in the expression levels of DNMT1, 3a, and 3b between PNEC areas and adenocarcinoma areas in several PNECs (data not shown). In addition, the expression levels of DNMT1, 3a, and 3b were found to be unrelated to the patient's age or sex.
4. Discussion The development of effective drugs for GEP NET requires an understanding of GEP NET tumor biology and the
M. M. Rahman et al. discovery of molecular targets specific to all subtypes of GEP NET. Gene transcription silencing by DNA promoter hypermethylation is a crucial event in cancer initiation and progression [4]. Aberrant methylation-induced silencing of TSGs was recognized as an important mechanism contributing to GEP NET tumorigenesis. Hypermethylation of several TSGs, including RASSF1A, CDKN2A(p14), CDKN2A(p16), HIC1, and MGMT, has been reported in GEP NET [19-23]. DNMT1 overexpression plays an important role in the mechanism of methylation [27] and is associated with aberrant methylation of CpG islands [28]. In many studies, DNMT1 overexpression has been shown in tumors; and forced overexpression of DNMT1 results in transformation and de novo methylation of CpG islands. Similar to DNMT1, DNMT3a and 3b were also overexpressed in cancerous tissues [29]. This study, for the first time, revealed the overexpression of DNMT1, 3a, and 3b in GEP NET. We found that the overexpression of DNMT1, 3a, and 3b is a common event in all histologic types of GEP NET. Our findings suggested that DNMT1, 3a, and 3b are essential enzymes for tumorigenesis in all types of GEP NET. Several studies have suggested that the overexpression of DNMT1, 3a, and 3b is associated with poorer tumor differentiation [13,30,31]. Recently, the overexpression of DNMT1, 3a, and 3b and their activity were also investigated in benign endocrine tumors such as pituitary adenomas [32,33]. In this study, although DNMT3a expression was found to be highest in PNEC, high expression levels of DNMT1, 3a, and 3b were frequently observed in WNET, WNEC, and PNEC. Our findings suggested that DNMT contributes to tumorigenesis in benign tumors as well as in malignant tumors of GEP. It has been shown that high expression of DNMT1, 3a, and 3b is associated with portal vein involvement and is indicative of a poor prognosis. In addition, DNMT1 protein expression was greater in the advanced stages of hepatocellular cancer [13]. In this study, the expression of all DNMTs was significantly related to tumor stage. DNMT1 showed a higher expression level in stage IV tumors than in stage I and II tumors; DNMT3a showed a higher expression level in stage III and IV tumors than in stage I and II tumors; and DNMT3b showed the highest expression level in stage IV tumors. Furthermore, high expression of DNMT1, 3a, and 3b was possibly related to tumor metastasis in WNEC and PNEC. DNMT may contribute to cancer progress by silencing metastasis-related TSGs such as CDH1 and RASSF1A. A previous study suggested that RASSF1A methylation in GEP NET is associated with lymph node metastasis [21]. However, the exact mechanisms behind these associations remain to be elucidated. DNMT1 is expressed mainly during the S-phase in normal cells. As tumor tissues presumably contain a greater proportion of dividing cells than normal tissues, whether increased DNMT1 expression is due to an increase in the proportion of dividing cells or to an acute increase in DNMT1 expression per individual cell remains unclear [34].
DNMT overexpression in GEP NET The relationship between DNMT1 and the proliferation cell nuclear antigens has been documented in several cancers [11,30,35,36]. This study revealed that the MIB-1 labeling index was correlated with tumor stage and tumor differentiation. Interestingly, overexpression of DNMT1, DNMT3a, and DNMT3b was significantly associated with the MIB-1 labeling index. These findings are not consistent with previous several studies [30,35,36]. Thus, the interaction of DNMT with other nuclear proteins involved in the cell cycle or proliferation needs further investigation. The current literature suggests that genetic events such as MEN1 gene mutations and deletions, 18q losses, and X-chromosome losses are involved in GEP NET tumorigenesis, with significant differences in their contributions among different embryologic tumors, that is, foregut, midgut, and hindgut tumors [37]. In the present study, GEP NET in midgut showed the lowest level and lower frequency of DNMT3a and 3b expression; however, DNMT1 expression did not show this tendency in tumor anatomical sites. RASSF1A methylation was observed frequently in the foregut but not in the midgut or hindgut [38]; however, only a few hindgut NETs were investigated. Our study included only 9 midgut NETs because of their low incidence in Japan. Thus, the differences in DNMT protein expression and TSG methylation pattern among tumor anatomical sites need further identification. GEP NETs involving DNMT1, 3a, and 3b overexpression are potentially treatable with currently used anticancer therapies. Hypermethylated gene promoters have the potential to be reactivated by nucleoside analogues, such as 5-azacytidine and 5-aza-2-deoxycytidine (decitabine), both of which have been found to be particularly effective in inducing DNA demethylation and epigenetic gene reactivation in vitro [39,40]. These effects are caused by covalent trapping of DNMTs through DNA-incorporated azacytosine bases and subsequent degradation of the trapped enzymes [41,42]. Azacytidine and decitabine are currently used to treat myelodysplastic syndrome and acute myeloid leukemia [17]. Furthermore, clinical trials of the effectiveness of decitabine against solid tumors are currently in the early stages [17]. In addition, hydralazine, a nonnucleoside DNA methylation inhibitor depending on its ability to inhibit DNMT1 activity and/or decrease DNMT1 and 3a expression, was reported to be able to reactivate the expression of TSGs in cancers in 2 clinical trial studies [43,44]. Our data suggest that GEP NETs displaying overexpression of DNMT1, 3a, and 3b would be good candidates for epigenetic therapy. As PNECs showed the worst prognosis and frequently expressed a high level of DNMT, a clinical trial to test the effectiveness of decitabine and hydralazine against PNECs is considerable. In summary, analysis of DNMT1, 3a, and 3b expression in GEP NET showed a higher frequency of DNMT overexpression. In particular, DNMT3a demonstrated greater clinical significance in GEP NET. These results suggest that DNMT plays important roles in the tumori-
1077 genesis and development of GEP NET and that epigenetic therapy directed against DNMT would be an effective therapeutic approach.
Acknowledgments The authors declare that they received no specific grants for this project and that no conflicts of interest that could be perceived as prejudicing the impartiality of the research exist. We thank Ms Noriko Amo and Mr Akihito Amo for their excellent technical assistance.
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