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doi:10.1111/jgh.12652
GASTROENTEROLOGY
Effect of Twist and Bmi1 on intraductal papillary mucinous neoplasm of the pancreas Daichi Ishikawa, Mitsuo Shimada, Tohru Utsunomiya, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Yusuke Arakawa, Mami Kanamoto, Shuichi Iwahashi, Yu Saito, Shinichiro Yamada and Hidenori Miyake Department of Surgery, The University of Tokushima, Tokushima, Japan
Key words Bmi1, cancers: biology, diagnosis and therapy, epithelial-mesenchymal transition, pancreatic neoplasms: biology and therapy, Twist. Accepted for publication 19 April 2014. Correspondence Prof Mitsuo Shimada, Department of Surgery, The University of Tokushima, 3-18-15 Kuramoto, Tokushima City, Tokushima 770-8503, Japan. Email: [email protected] Declaration of conflict of interest: The authors have no financial or other interests in entities related to the subject of this article.
Abstract Background: Intraductal papillary mucinous neoplasm (IPMN) is a well-established entity among pancreatic neoplasms that ranges from low-grade dysplasia to invasive carcinoma. Epithelial-mesenchymal transition (EMT) contributes to tumor progression in various cancers. Moreover, Notch signaling is one of the important upstream effectors of EMT promotion. Currently, it is unclear whether EMT causes pathological progression of IPMN. Aim: We evaluated the expression of EMT-promoting transcription factors Twist and B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) in IPMN. Methods: Patients who underwent resections at our institute and its affiliated hospital were enrolled in this study (n = 35). Protein expression of EMT markers Twist, Bmi1, Jagged1, and E-cadherin in resected specimens was investigated by immunohistochemistry. Expression of these proteins was compared with the clinicopathological factors and patient survival. Results: Positive expression of Twist and Bmi1 was observed in 40.0% and 42.9% of IPMNs, respectively. Twist and Bmi1 expression was significantly higher in IPMNs with high-grade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) than that in IPMNs with low-grade dysplasia. High expression of Twist was correlated with Jagged1 expression and inversely correlated with expression of E-cadherin (P = 0.06 and P < 0.05, respectively). In survival analyses, the recurrence rate was significantly higher in the group that showed simultaneous high expression of Twist and Bmi1 (P < 0.05). Conclusions: Expression of Twist and Bmi1 is associated with aggressiveness and poor prognoses of IPMN through EMT promotion that might be induced by Notch signaling.
Introduction Intraductal papillary mucinous neoplasm (IPMN) is a pancreatic tumor that grows within the pancreatic duct, and is characterized by mucin-filled cystic dilatation of the main or branch pancreatic duct.1,2 IPMN was first reported in 1982,3 and during the 1980s it was described in various terminologies that caused confusion. In 1996, the term “IPMN” was endorsed by the World Health Organization (WHO),4 and a standardized concept was proposed for the disease. IPMN is thought to be a precursor lesion for a subset of pancreatic carcinomas as well as pancreatic intraepithelial neoplasia.5 The histological spectrum of IPMN ranges broadly from low-grade dysplasia to invasive carcinoma.6 However, compared with invasive ductal adenocarcinoma, IPMN patients have a favorable clinical outcome.7 Accumulated information about IPMN based on the current understanding has been summarized in the international consensus guidelines 2012.8 Over the past decade, 2032
many researchers have investigated the morphological and molecular aspects of IPMN. However, the mechanisms involved in tumor progression of IPMN are not well understood because it is difficult to perform in vitro studies and establish animal models. Epithelial-mesenchymal transition (EMT) is the morphological transformation of epithelial cells to mesenchymal phenotypes that commonly occurs during early embryonic development. EMT confers mesenchymal properties on epithelial cells, and it is the pivotal mechanism contributing to cancer invasion and metastasis.9 A key feature in the initiation and execution of EMT is downregulation of E-cadherin expression.10,11 EMT programs are orchestrated by various transcription factors, including Twist, Snail, and Zeb1.12 Among them, Twist, a basic helix-loop-helix transcription factor, was first recognized for its role in mesodermal development in Drosophila.13 Twist is highly involved in loss of E-cadherin expression and considered to be a master regulator of EMT to facilitate cancer invasion and metastasis.14 The role of
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Twist in EMT regulation has been reported in many cancer types, including breast cancer, gastric cancer, and hepatocellular carcinoma.14–16 Moreover, the Notch signaling pathway has been demonstrated to be one of the upstream effectors of the EMT program.17 Originally, it was reported that the Notch pathway regulates cell fate decisions during embryonic development by facilitating short-range signaling between neighboring cells that are in physical contact.18 In particular, Notch signaling upregulates the Twist pathway and promotes EMT progression in gastric cancer cells.19 B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) is an essential constituent of polycomb repressive complex 1 that has an essential role in the maintenance of chromatin silencing.20,21 It was originally identified as an oncogene that cooperated with c-Myc in the initiation of mouse pre-B cell lymphomas.22 Aberrant Bmi1 expression has been reported in a variety of human cancers, including mantle cell lymphomas, non-small cell lung cancer, B-cell non-Hodgkin’s lymphoma, and breast cancer.23–26 Recently, the roles of Bmi1 have been highlighted in the selfrenewal of stem cells and a link between maintenance of cellular homeostasis and tumorigenesis.27 Because IPMN is gaining attention as a precursor lesion of infiltrating adenocarcinoma, there is an emergent need to investigate the pathological and molecular aspects of this disease. Although the proteins mentioned above are considered to play important roles in cancer progression, there are no reports of their clinicopathological significance in IPMN. Here, we characterized expression of EMT-related transcription factors Twist and Bmi1 in IPMN.
Methods Patients and tissue samples. This study was approved by the ethics committee of Tokushima University Hospital. Patient information was obtained from the medical records of the institutes. A total of 35 samples of IPMN were investigated in this study. IPMNs were surgically resected at Tokushima University Hospital and its affiliated hospital from 2000 to 2012. Twenty-four patients had IPMNs located in pancreatic head, and 11 patients had IPMNs in pancreatic body and/or tail. Pancreatoduodenectomy, total pancreatectomy and distal pancreatectomy were performed for 22, 2 and 11 cases, respectively (Table 1). All patients underwent R0 resection. For histopathological diagnosis, the tissue specimens were embedded in paraffin, serially sectioned (4 μm in thickness), and stained with HE. Based on the greatest degree of dysplasia present, the lesions were diagnosed as IPMN with lowgrade dysplasia, IPMN with moderate-grade dysplasia, IPMN with high-grade dysplasia, and invasive carcinoma according to the WHO classification 2010. After the operation, the patients were followed up by computed tomography in each 3 months to diagnose the recurrence of IPMN. Immunohistochemistry. Tissue specimens were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Immunostaining was performed as reported previously.28 Briefly, sections were deparaffinized with xylene, followed by rehydration in a graded ethanol series. The sections were treated with 3% hydrogen peroxide in methanol for 10 min to quench the
Twist and Bmi1 on pancreatic tumor
Table 1
Clinicopathological findings of resected IPMNs
Parameters
Number
Age(years, mean ± SD) Gender (male/female) Site (head/body and tail) Type (main duct type/brunch duct type) Cyst (single/multiple) Diameter of cyst (mm ± SD) Mural nodule (absent/present) Histological grade (WHO classification) Low-grade dysplasia Moderate-grade dysplasia High-grade dysplasia Invasive carcinoma Operation Pancreatoduodenectomy Total pancreatectomy Distal pancreatectomy
67.7 ± 9.2 22/13 24/11 21/14 11/24 29.3 ± 15.5 20/15 13 4 7 11 22 2 11
IPMN, intraductal papillary mucinous neoplasm; SD, standard deviation; WHO, World Health Organization.
endogenous peroxidase activity. Antigen retrieval was performed by boiling in 10 mM citrate buffer (pH 6) using a microwave. After incubation with 1% bovine serum albumin to block nonspecific antibody binding, the sections were incubated with primary antibodies against Twist (1:500, ab50581; Abcam, Cambridge, UK), Bmi1 (1:100, ab38295; Abcam), E-cadherin (1:100, sc7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or Jagged1 (1:50, sc6011; Santa Cruz Biotechnology) for 60 min at room temperature. After washing with phosphate-buffered saline, the sections were subjected to the Dako REAL EnVision/HRP detection system (Dako Corporation, Tokyo, Japan) for 60 min at room temperature. The peroxidase reaction was developed with 3, 3′diaminobenzidine as the chromogen. The sections were counterstained with 10% Mayer’s hematoxylin, dehydrated in a graded series of ethanol, treated with xylene, and mounted in a synthetic resin. Evaluation of immunostaining. Immunostaining was evaluated by a pathologist without knowledge of the clinical data as reported previously.29,30 Twist expression was scored by the intensity of staining (0, negative; 1, week; 2, moderate; 3, intense) and the proportion of positive cells (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%), and presented as the sum of the two scores (0–7). Scores of more than 4 were regarded as positive. Bmi1 expression was scored by the proportion of positive cells (0, negative; 1, up to 30%; 2, 30–50%; 3, 50–80%; 4, over 80%), and scores of 2–4 were regarded as positive. E-cadherin and Jagged1 expression were scored by multiplying the intensity (0, negative; 1, week; 2, moderate; 3, intense) and the proportion of positive cells (%). A score of more than 80 was regarded as positive. The samples were evaluated in five different fields at 400×, and at least 1000 tumor cells were analyzed in each sample. Statistical analysis. Statistical analyses were carried out using the JMP 10 statistical software package (SAS Institute Inc,
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Figure 1 Twist and Bmi1 protein expression in intraductal papillary mucinous neoplasm. Representative images of positive and negative expression of Twist and Bmi1 are shown in (a, c) and (b, d), respectively.
Tokyo, Japan). The Student’s t-test was used for comparison of continuous variables. The χ2 test was used to analyze the relationship between Twist or Bmi1 expression and clinicopathological characteristics. Survival curves were plotted by the Kaplan–Meier method and analyzed by the log-rank test to calculate differences between the curves. A P-value of less than 0.05 was considered to be statistically significant.
Results Expression of Twist and Bmi1 is upregulated during IPMN progression. The clinicopathological characteristics of the patients are shown in Table 1. IPMNs with lowgrade, moderate-grade, or high-grade dysplasia, and invasive carcinoma were found in 13, 4, 7, and 11 cases, respectively. Representative immunohistochemical results are shown in Figure 1. Among the 35 patients, positive expression of Twist and Bmi1 was observed in 14 cases and 15 cases, respectively. Compared with IPMNs with low-grade dysplasia, Twist and Bmi1 expression was significantly upregulated in IPMNs with highgrade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) (Fig. 2).
Twist and Bmi1 expression are inversely correlated with E-cadherin expression and positively correlated with Jagged1 expression. To confirm that EMT-related transcription factors downregulate E-cadherin expression, we investigated the relationship between expression of E-cadherin, Twist, and Bmi1. As shown in Figure 3a, Twist expression was inversely correlated with E-cadherin expression (P < 0.05). For Bmi1, there was a tendency of negative correlation with the expression of E-cadherin (P = 0.06; Fig. 3b). By 2034
Figure 2 Twist and Bmi1 expression are upregulated as intraductal papillary mucinous neoplasm progresses pathologically.
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Twist and Bmi1 on pancreatic tumor
Figure 3 Twist expression is inversely correlated with E-cadherin expression and tends to be positively correlated with Jagged1 expression. , E-cadherin negative; , E-cadherin positive.
investigating the relationship between Twist and the putative upstream Notch signaling, Twist expression tended to be correlated with Jagged1 expression (P = 0.06; Fig. 3c). Simultaneous expression of Twist and Bmi1 is a prognostic factor. To perform the survival analysis, we excluded low-grade and moderate-grade dysplasias that were considered to be a benign lesion. For overall survival, there was no significant difference between positive and negative groups of both Twist and Bmi1 expression (data not shown). For disease-free survival (DFS), positive expression of Twist or Bmi1 alone did not show a significant difference. However, simultaneous high expression of Twist and Bmi1 demonstrated a significant difference (P < 0.05; Fig. 4). Furthermore, only simultaneous expression of Twist and Bmi1 emerged as a significant prognostic factor of 3-year prognostic survival (Table 2, 50.0% vs 81.5%). In cases with high expression of both Twist and Bmi1, there was a higher rate of single cysts, more malignant histology, and a lower level of E-cadherin expression (P < 0.05).
Discussion IPMN is an intriguing and increasingly recognized pancreatic neoplasm. Its molecular biology is relatively less investigated than that of pancreatic ductal adenocarcinoma. However, in recent years, a number of studies have identified molecular events that might play important roles in the tumorigenesis and pathological aggressiveness of IPMN. Initially, similar to the adenoma-carcinoma sequence, K-ras mutation was reported to show a stepwise increase from IPMN with low-grade dysplasia to high-grade dysplasia/ invasive carcinoma.31,32 Phosphoinositide 3-kinase/protein kinase
Figure 4 Kaplan–Meier disease-free survival analysis of intraductal papillary mucinous neoplasm. Simultaneous expression of both Twist , Twist negative or Bmi1 and Bmi1 predicts poor clinical outcomes. negative (n = 12); , Twist and Bmi1 co-over expression (n = 6).
B/Akt, CDKN2A/p16/MTS1, and p53 pathways are associated with the pathological aggressiveness of IPMNs.31,33,34 Investigation of these molecular pathways has provided a better understanding of the characteristics of IPMN. However, little is known about the epidemiological risk factors and molecular mechanisms leading to the carcinogenesis of IPMNs. During cancer progression, induction of EMT is associated with acquisition of motility that contributes to cancer cell invasion and metastasis.8 In this study, we focused on Twist, a master regulator of EMT, which directly represses E-cadherin expression,35 and
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Table 2
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Univariate analysis of 3-year DFS
Factors
3-year DFS (%)
P-value
Age(< 70/≥ 70) Gender (male/female) Type (main duct type/brunch duct type) Single cyst/multiple cysts Diameter of cyst (< 30 mm/≥ 30 mm) Mural nodule (absent/present) E-cadherin (negative/positive) Twist and Bmi1 (non-overexpression/ co-overexpression)
70.0/70.0 84.6/40.0 67.3/80.0 62.5/78.7 77.1/62.5 100/57.1 66.7/83.3 81.5/50.0
0.56 0.11 0.46 0.59 0.45 0.19 0.42 < 0.05
References
DFS, disease-free survival.
Bmi1 which was recently demonstrated to act downstream of Twist.36 Both Twist and Bmi1 are associated with the prognosis and malignancy of various types of tumors.14–16,19,23–26 We demonstrated that Twist and Bmi1 exhibit significantly higher expression in IPMNs with high-grade dysplasia (four out of seven cases) and invasive carcinoma (seven and eight out of eleven cases, respectively; P < 0.05) than that in IPMNs with low-grade dysplasia (two out of thirteen cases). Moreover, Twist and Bmi1 expression was inversely correlated with E-cadherin expression, suggesting that EMT is induced in IPMN. Chetty et al. reported that Wnt/βcatenin signaling occurs early in the tumorigenesis of IPMN.37 In their study, E-cadherin showed low expression in pathologically more aggressive IPMNs, suggesting that translocation of cytoplasmic β-catenin to the nucleus causes a loss of E-cadherin expression. Using laser microdissection and RNA array analysis, Jury et al. demonstrated that EMT-related genes, such as transforming growth factor-β (TGF-β), are highly expressed in pathologically aggressive IPMNs.38 Interestingly, Bmi1 is more intensely stained in the invading fronts of primary invasive breast cancers compared with that in the central portions, which is correlated with axillary lymph node metastasis.39,40 In our study, patients with IPMN expressing Twist and Bmi1 simultaneously showed a decrease of DFS. Interestingly, a strong connection between Twist1 and Bmi1 has been demonstrated by Yang et al.41 Their results presented the first molecular demonstration that Twist1 and Bmi1 are simultaneously required to repress E-cadherin expression. Some factors, including Notch1, TGF-β, and hypoxia-inducible factor 1, have been reported to be the triggering signals of EMT.17,41,42 Therefore, we investigated the relationship between Twist and Jagged1, one of the Notch1 ligands. Our results showed a tendency of correlation between Twist and Jagged1 protein expression, suggesting that EMT occurring in IPMN might be partly induced by Notch signaling. A previous report has demonstrated that gastric cancer progression induced by the Notch/Twist axis is mediated by STAT3 phosphorylation.17 The limitation of our study is that immunohistochemistry is not quantitative, and we did not show direct interactions among Twist, Bmi1, and Jagged. Because of the difficulty of performing in vitro experiments of IPMN, a further study using pancreatic cancer cells or normal pancreatic cells is needed to validate the tissue-specific molecular events. The results demonstrated here may be helpful in designing future studies to understand the molecular development of IPMNs. 2036
Patients with IPMN exhibiting simultaneously elevated expression of Twist and Bmi1 might require adjuvant chemotherapy and intensive follow-up. In conclusion, the results of this study suggest that Twist and Bmi1 are associated with the pathological aggressiveness of IPMN, which may be caused by EMT. Initiation of such EMT might be induced by Notch signaling, and simultaneous high expression of Twist and Bmi1 may be a prognostic factor of poor clinical outcomes.
1 Itai Y, Ohhashi K, Nagai H et al. “Ductectatic” mucinous cystadenoma and cystadenocarcinoma of the pancreas. Radiology 1986; 161: 697–700. 2 Tanaka M, Kobayashi K, Mizomoto K, Yamaguchi K. Clinical aspects of intraductal papillary mucinous neoplasm of the pancreas. J. Gastroenterol. 2005; 40: 669–75. 3 Ohhashi K, Murakami M, Murayama M et al. Four cases of mucin producing cancer of the pancreas on specific findings of the papilla of Vater. Prog. Dig. Endosc. 1982; 20: 348–51. 4 Kloppoel G, Solcia E, Longnecker DS et al. Histologic typing of tumors of the exocrine pancreas. In: Lee CS, ed. World Health Organization, Histologic Typing of Tumors of the Exocrine Pancreas. Geneva: Springer Verlag, 1996; 1–55. 5 Tanaka M. Intraductal papillary mucinous neoplasm of the pancreas: diagnosis and treatment. Pancreas 2004; 28: 282–8. 6 Adsay NV, Longnecker DS, Klimstra DS. Pancreatic tumors with cystic dilatation of the ducts: intraductal papillary mucinous neoplasms and intraductal oncocytic papillary neoplasms. Semin. Diagn. Pathol. 2000; 17: 16–30. 7 Loftus EV Jr, Olivares-Pakzad BA, Btts KP et al. Intraductal papillary mucinous tumors of the pancreas: clinicopathologic features, outcome, and nomenclature. Members of the pancreas clinic and pancreatic surgeons of Mayo Clinic. Gastroenterology 1996; 110: 1909–18. 8 Tanaka M, Fernandez-del Castilio C, Adsay V et al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas. Pancreatology 2012; 12: 183–97. 9 Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transition in development and disease. Cell 2009; 139: 871–90. 10 Bates RC, Mercurio AM. The epithelial-mesenchymal transition (EMT) and colorectal cancer progression. Cancer Biol. Ther. 2005; 4: 365–70. 11 Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr. Opin. Cell Biol. 2003; 15: 740–6. 12 Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal state: acquisition of malignant and stem cell traits. Nat. Rev. Cancer 2009; 9: 265–73. 13 Wieschaus E, Nüsslein-Volhard C, Jürgens G. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Roux’s Arch. Der. Biol. 1984; 193: 267–82. 14 Yan J, Mani SA, Donaher JL et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004; 117: 927–39. 15 Lee TK, Poon RT, Yuen AP et al. Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition. Clin. Cancer Res. 2006; 12: 5369–76. 16 Yang Z, Zhang X, Gang H et al. Up-regulation of gastric cancer cell invasion by Twist is accompanied by N-cadherin and fibronectin expression. Biochem. Biophys. Res. Commun. 2007; 358: 925–30.
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17 Timmerman LA, Gregg-Bess J, Raya A et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 2004; 18: 99–115. 18 Bray SJ. Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 2006; 7: 678–89. 19 Hsu KW, Hsieh RH, Huang KH et al. Activation of the Notch/STAT3/Twist signaling axis promotes gastric cancer progression. Carcinogenesis 2012; 33: 1459–67. 20 Valk-Lingbeek ME, Bruggeman SW, van Lohuizen M. Stem cells and cancer: the polycomb connection. Cell 2004; 118: 409–18. 21 Widschwendter M, Fiegl H, Egle D et al. Epigenetic stem cell signature in cancer. Nat. Genet. 2007; 39: 157–8. 22 Haupt Y, Alexander W, Barri G et al. Novel zing finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice. Cell 1991; 64: 753–63. 23 Bea S, Tort F, Pinyol M et al. BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer Res. 2001; 61: 2409–12. 24 Vonlanthen S, Heighway D, Altermatt HJ et al. The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression. Br. J. Cancer 2001; 84: 1372–6. 25 van Kemenade FJ, Raaphorst FM, Blokzijl T et al. Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma. Blood 2001; 97: 3896–901. 26 Silva J, Garcia JM, Pena C et al. Implication of polycomb members Bmi-1, Mel-18, and Hpc-2 in the regulation of p16INK4a, p14ARF, h-TERT, and c-Myc expression in primary breast carcinomas. Clin. Cancer Res. 2006; 12: 6929–36. 27 Liu S, Dontu G, Mantle I et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006; 66: 6063–71. 28 Yao X, Wang X, Wang Z et al. Clinicopathological and prognostic significance of epithelial mesenchymal transition-related protein expression in intrahepatic cholangiocarcinoma. Onco. Targets Ther. 2012; 5: 255–61. 29 Yuen HF, Chaua CW, Chan YP et al. Significance of Twist and E-cadherin expression in the metastatic progression of prostatic. Histopathology 2007; 50: 648–58.
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30 Hayry V, Makinen LK, Atula T et al. Bmi-1 expression predicts prognosis in squamous cell carcinoma of the tongue. Br. J. Cancer 2010; 102: 892–7. 31 Satoh K, Shimosegawa T, Moriizumi S et al. K-ras mutation and p53 protein accumulation in intraductal mucin-hypersecreting neoplasms of the pancreas. Pancreas 1996; 12: 362–8. 32 Kitago M, Ueda M, Aiura K et al. Comparison of K-ras point mutation distributions in intraductal papillary-mucinous tumors and ductal adenocarcinoma of pancreas. Int. J. Cancer 2004; 110: 177–82. 33 Semba S, Moriya T, Kimura W et al. Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreas. Pancreas 2003; 26: 250–7. 34 Furukawa T, Fujisaki R, Yoshida Y et al. Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas. Mod Pathol. 2005; 18: 1034–42. 35 Ansieau S, Bastid J, Doreau A et al. Induction of EMT by twist proteins as a collateral effect tumor-promoting inactivation of premature senescence. Cancer Cell 2008; 14: 79–89. 36 Keith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell 2007; 129: 465–72. 37 Chetty R, Serra S, Salahshor S et al. Expression of Wnt-signaling pathway proteins in intraductal papillary mucinous neoplasms of the pancreas: a tissue microarray analysis. Hum. Pathol. 2006; 37: 212–17. 38 Jury RP, Tribodeau BJ, Fortier LE et al. Gene expression changes associated with the progression of intraductal papillary mucinous neoplasms. Pancreas 2012; 41: 611–18. 39 Kim J, Yoon S, Jeong S et al. Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastasis in invasive ductal breast cancer. Breast 2004; 13: 383–8. 40 Kim J, Yoon S, Kim C et al. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlated with the reduced p16INK4a/p14ARF proteins. Cancer Lett. 2004; 203: 217–24. 41 Yang MH, Hsu DS, Wang HW et al. Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat. Cell Biol. 2010; 12: 982–92. 42 Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005; 24: 5764–74.
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doi:10.1111/jgh.12652
GASTROENTEROLOGY
Effect of Twist and Bmi1 on intraductal papillary mucinous neoplasm of the pancreas Daichi Ishikawa, Mitsuo Shimada, Tohru Utsunomiya, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Yusuke Arakawa, Mami Kanamoto, Shuichi Iwahashi, Yu Saito, Shinichiro Yamada and Hidenori Miyake Department of Surgery, The University of Tokushima, Tokushima, Japan
Key words Bmi1, cancers: biology, diagnosis and therapy, epithelial-mesenchymal transition, pancreatic neoplasms: biology and therapy, Twist. Accepted for publication 19 April 2014. Correspondence Prof Mitsuo Shimada, Department of Surgery, The University of Tokushima, 3-18-15 Kuramoto, Tokushima City, Tokushima 770-8503, Japan. Email: [email protected] Declaration of conflict of interest: The authors have no financial or other interests in entities related to the subject of this article.
Abstract Background: Intraductal papillary mucinous neoplasm (IPMN) is a well-established entity among pancreatic neoplasms that ranges from low-grade dysplasia to invasive carcinoma. Epithelial-mesenchymal transition (EMT) contributes to tumor progression in various cancers. Moreover, Notch signaling is one of the important upstream effectors of EMT promotion. Currently, it is unclear whether EMT causes pathological progression of IPMN. Aim: We evaluated the expression of EMT-promoting transcription factors Twist and B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) in IPMN. Methods: Patients who underwent resections at our institute and its affiliated hospital were enrolled in this study (n = 35). Protein expression of EMT markers Twist, Bmi1, Jagged1, and E-cadherin in resected specimens was investigated by immunohistochemistry. Expression of these proteins was compared with the clinicopathological factors and patient survival. Results: Positive expression of Twist and Bmi1 was observed in 40.0% and 42.9% of IPMNs, respectively. Twist and Bmi1 expression was significantly higher in IPMNs with high-grade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) than that in IPMNs with low-grade dysplasia. High expression of Twist was correlated with Jagged1 expression and inversely correlated with expression of E-cadherin (P = 0.06 and P < 0.05, respectively). In survival analyses, the recurrence rate was significantly higher in the group that showed simultaneous high expression of Twist and Bmi1 (P < 0.05). Conclusions: Expression of Twist and Bmi1 is associated with aggressiveness and poor prognoses of IPMN through EMT promotion that might be induced by Notch signaling.
Introduction Intraductal papillary mucinous neoplasm (IPMN) is a pancreatic tumor that grows within the pancreatic duct, and is characterized by mucin-filled cystic dilatation of the main or branch pancreatic duct.1,2 IPMN was first reported in 1982,3 and during the 1980s it was described in various terminologies that caused confusion. In 1996, the term “IPMN” was endorsed by the World Health Organization (WHO),4 and a standardized concept was proposed for the disease. IPMN is thought to be a precursor lesion for a subset of pancreatic carcinomas as well as pancreatic intraepithelial neoplasia.5 The histological spectrum of IPMN ranges broadly from low-grade dysplasia to invasive carcinoma.6 However, compared with invasive ductal adenocarcinoma, IPMN patients have a favorable clinical outcome.7 Accumulated information about IPMN based on the current understanding has been summarized in the international consensus guidelines 2012.8 Over the past decade, 2032
many researchers have investigated the morphological and molecular aspects of IPMN. However, the mechanisms involved in tumor progression of IPMN are not well understood because it is difficult to perform in vitro studies and establish animal models. Epithelial-mesenchymal transition (EMT) is the morphological transformation of epithelial cells to mesenchymal phenotypes that commonly occurs during early embryonic development. EMT confers mesenchymal properties on epithelial cells, and it is the pivotal mechanism contributing to cancer invasion and metastasis.9 A key feature in the initiation and execution of EMT is downregulation of E-cadherin expression.10,11 EMT programs are orchestrated by various transcription factors, including Twist, Snail, and Zeb1.12 Among them, Twist, a basic helix-loop-helix transcription factor, was first recognized for its role in mesodermal development in Drosophila.13 Twist is highly involved in loss of E-cadherin expression and considered to be a master regulator of EMT to facilitate cancer invasion and metastasis.14 The role of
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Twist in EMT regulation has been reported in many cancer types, including breast cancer, gastric cancer, and hepatocellular carcinoma.14–16 Moreover, the Notch signaling pathway has been demonstrated to be one of the upstream effectors of the EMT program.17 Originally, it was reported that the Notch pathway regulates cell fate decisions during embryonic development by facilitating short-range signaling between neighboring cells that are in physical contact.18 In particular, Notch signaling upregulates the Twist pathway and promotes EMT progression in gastric cancer cells.19 B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) is an essential constituent of polycomb repressive complex 1 that has an essential role in the maintenance of chromatin silencing.20,21 It was originally identified as an oncogene that cooperated with c-Myc in the initiation of mouse pre-B cell lymphomas.22 Aberrant Bmi1 expression has been reported in a variety of human cancers, including mantle cell lymphomas, non-small cell lung cancer, B-cell non-Hodgkin’s lymphoma, and breast cancer.23–26 Recently, the roles of Bmi1 have been highlighted in the selfrenewal of stem cells and a link between maintenance of cellular homeostasis and tumorigenesis.27 Because IPMN is gaining attention as a precursor lesion of infiltrating adenocarcinoma, there is an emergent need to investigate the pathological and molecular aspects of this disease. Although the proteins mentioned above are considered to play important roles in cancer progression, there are no reports of their clinicopathological significance in IPMN. Here, we characterized expression of EMT-related transcription factors Twist and Bmi1 in IPMN.
Methods Patients and tissue samples. This study was approved by the ethics committee of Tokushima University Hospital. Patient information was obtained from the medical records of the institutes. A total of 35 samples of IPMN were investigated in this study. IPMNs were surgically resected at Tokushima University Hospital and its affiliated hospital from 2000 to 2012. Twenty-four patients had IPMNs located in pancreatic head, and 11 patients had IPMNs in pancreatic body and/or tail. Pancreatoduodenectomy, total pancreatectomy and distal pancreatectomy were performed for 22, 2 and 11 cases, respectively (Table 1). All patients underwent R0 resection. For histopathological diagnosis, the tissue specimens were embedded in paraffin, serially sectioned (4 μm in thickness), and stained with HE. Based on the greatest degree of dysplasia present, the lesions were diagnosed as IPMN with lowgrade dysplasia, IPMN with moderate-grade dysplasia, IPMN with high-grade dysplasia, and invasive carcinoma according to the WHO classification 2010. After the operation, the patients were followed up by computed tomography in each 3 months to diagnose the recurrence of IPMN. Immunohistochemistry. Tissue specimens were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Immunostaining was performed as reported previously.28 Briefly, sections were deparaffinized with xylene, followed by rehydration in a graded ethanol series. The sections were treated with 3% hydrogen peroxide in methanol for 10 min to quench the
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Table 1
Clinicopathological findings of resected IPMNs
Parameters
Number
Age(years, mean ± SD) Gender (male/female) Site (head/body and tail) Type (main duct type/brunch duct type) Cyst (single/multiple) Diameter of cyst (mm ± SD) Mural nodule (absent/present) Histological grade (WHO classification) Low-grade dysplasia Moderate-grade dysplasia High-grade dysplasia Invasive carcinoma Operation Pancreatoduodenectomy Total pancreatectomy Distal pancreatectomy
67.7 ± 9.2 22/13 24/11 21/14 11/24 29.3 ± 15.5 20/15 13 4 7 11 22 2 11
IPMN, intraductal papillary mucinous neoplasm; SD, standard deviation; WHO, World Health Organization.
endogenous peroxidase activity. Antigen retrieval was performed by boiling in 10 mM citrate buffer (pH 6) using a microwave. After incubation with 1% bovine serum albumin to block nonspecific antibody binding, the sections were incubated with primary antibodies against Twist (1:500, ab50581; Abcam, Cambridge, UK), Bmi1 (1:100, ab38295; Abcam), E-cadherin (1:100, sc7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or Jagged1 (1:50, sc6011; Santa Cruz Biotechnology) for 60 min at room temperature. After washing with phosphate-buffered saline, the sections were subjected to the Dako REAL EnVision/HRP detection system (Dako Corporation, Tokyo, Japan) for 60 min at room temperature. The peroxidase reaction was developed with 3, 3′diaminobenzidine as the chromogen. The sections were counterstained with 10% Mayer’s hematoxylin, dehydrated in a graded series of ethanol, treated with xylene, and mounted in a synthetic resin. Evaluation of immunostaining. Immunostaining was evaluated by a pathologist without knowledge of the clinical data as reported previously.29,30 Twist expression was scored by the intensity of staining (0, negative; 1, week; 2, moderate; 3, intense) and the proportion of positive cells (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%), and presented as the sum of the two scores (0–7). Scores of more than 4 were regarded as positive. Bmi1 expression was scored by the proportion of positive cells (0, negative; 1, up to 30%; 2, 30–50%; 3, 50–80%; 4, over 80%), and scores of 2–4 were regarded as positive. E-cadherin and Jagged1 expression were scored by multiplying the intensity (0, negative; 1, week; 2, moderate; 3, intense) and the proportion of positive cells (%). A score of more than 80 was regarded as positive. The samples were evaluated in five different fields at 400×, and at least 1000 tumor cells were analyzed in each sample. Statistical analysis. Statistical analyses were carried out using the JMP 10 statistical software package (SAS Institute Inc,
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Figure 1 Twist and Bmi1 protein expression in intraductal papillary mucinous neoplasm. Representative images of positive and negative expression of Twist and Bmi1 are shown in (a, c) and (b, d), respectively.
Tokyo, Japan). The Student’s t-test was used for comparison of continuous variables. The χ2 test was used to analyze the relationship between Twist or Bmi1 expression and clinicopathological characteristics. Survival curves were plotted by the Kaplan–Meier method and analyzed by the log-rank test to calculate differences between the curves. A P-value of less than 0.05 was considered to be statistically significant.
Results Expression of Twist and Bmi1 is upregulated during IPMN progression. The clinicopathological characteristics of the patients are shown in Table 1. IPMNs with lowgrade, moderate-grade, or high-grade dysplasia, and invasive carcinoma were found in 13, 4, 7, and 11 cases, respectively. Representative immunohistochemical results are shown in Figure 1. Among the 35 patients, positive expression of Twist and Bmi1 was observed in 14 cases and 15 cases, respectively. Compared with IPMNs with low-grade dysplasia, Twist and Bmi1 expression was significantly upregulated in IPMNs with highgrade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) (Fig. 2).
Twist and Bmi1 expression are inversely correlated with E-cadherin expression and positively correlated with Jagged1 expression. To confirm that EMT-related transcription factors downregulate E-cadherin expression, we investigated the relationship between expression of E-cadherin, Twist, and Bmi1. As shown in Figure 3a, Twist expression was inversely correlated with E-cadherin expression (P < 0.05). For Bmi1, there was a tendency of negative correlation with the expression of E-cadherin (P = 0.06; Fig. 3b). By 2034
Figure 2 Twist and Bmi1 expression are upregulated as intraductal papillary mucinous neoplasm progresses pathologically.
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Figure 3 Twist expression is inversely correlated with E-cadherin expression and tends to be positively correlated with Jagged1 expression. , E-cadherin negative; , E-cadherin positive.
investigating the relationship between Twist and the putative upstream Notch signaling, Twist expression tended to be correlated with Jagged1 expression (P = 0.06; Fig. 3c). Simultaneous expression of Twist and Bmi1 is a prognostic factor. To perform the survival analysis, we excluded low-grade and moderate-grade dysplasias that were considered to be a benign lesion. For overall survival, there was no significant difference between positive and negative groups of both Twist and Bmi1 expression (data not shown). For disease-free survival (DFS), positive expression of Twist or Bmi1 alone did not show a significant difference. However, simultaneous high expression of Twist and Bmi1 demonstrated a significant difference (P < 0.05; Fig. 4). Furthermore, only simultaneous expression of Twist and Bmi1 emerged as a significant prognostic factor of 3-year prognostic survival (Table 2, 50.0% vs 81.5%). In cases with high expression of both Twist and Bmi1, there was a higher rate of single cysts, more malignant histology, and a lower level of E-cadherin expression (P < 0.05).
Discussion IPMN is an intriguing and increasingly recognized pancreatic neoplasm. Its molecular biology is relatively less investigated than that of pancreatic ductal adenocarcinoma. However, in recent years, a number of studies have identified molecular events that might play important roles in the tumorigenesis and pathological aggressiveness of IPMN. Initially, similar to the adenoma-carcinoma sequence, K-ras mutation was reported to show a stepwise increase from IPMN with low-grade dysplasia to high-grade dysplasia/ invasive carcinoma.31,32 Phosphoinositide 3-kinase/protein kinase
Figure 4 Kaplan–Meier disease-free survival analysis of intraductal papillary mucinous neoplasm. Simultaneous expression of both Twist , Twist negative or Bmi1 and Bmi1 predicts poor clinical outcomes. negative (n = 12); , Twist and Bmi1 co-over expression (n = 6).
B/Akt, CDKN2A/p16/MTS1, and p53 pathways are associated with the pathological aggressiveness of IPMNs.31,33,34 Investigation of these molecular pathways has provided a better understanding of the characteristics of IPMN. However, little is known about the epidemiological risk factors and molecular mechanisms leading to the carcinogenesis of IPMNs. During cancer progression, induction of EMT is associated with acquisition of motility that contributes to cancer cell invasion and metastasis.8 In this study, we focused on Twist, a master regulator of EMT, which directly represses E-cadherin expression,35 and
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Table 2
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Univariate analysis of 3-year DFS
Factors
3-year DFS (%)
P-value
Age(< 70/≥ 70) Gender (male/female) Type (main duct type/brunch duct type) Single cyst/multiple cysts Diameter of cyst (< 30 mm/≥ 30 mm) Mural nodule (absent/present) E-cadherin (negative/positive) Twist and Bmi1 (non-overexpression/ co-overexpression)
70.0/70.0 84.6/40.0 67.3/80.0 62.5/78.7 77.1/62.5 100/57.1 66.7/83.3 81.5/50.0
0.56 0.11 0.46 0.59 0.45 0.19 0.42 < 0.05
References
DFS, disease-free survival.
Bmi1 which was recently demonstrated to act downstream of Twist.36 Both Twist and Bmi1 are associated with the prognosis and malignancy of various types of tumors.14–16,19,23–26 We demonstrated that Twist and Bmi1 exhibit significantly higher expression in IPMNs with high-grade dysplasia (four out of seven cases) and invasive carcinoma (seven and eight out of eleven cases, respectively; P < 0.05) than that in IPMNs with low-grade dysplasia (two out of thirteen cases). Moreover, Twist and Bmi1 expression was inversely correlated with E-cadherin expression, suggesting that EMT is induced in IPMN. Chetty et al. reported that Wnt/βcatenin signaling occurs early in the tumorigenesis of IPMN.37 In their study, E-cadherin showed low expression in pathologically more aggressive IPMNs, suggesting that translocation of cytoplasmic β-catenin to the nucleus causes a loss of E-cadherin expression. Using laser microdissection and RNA array analysis, Jury et al. demonstrated that EMT-related genes, such as transforming growth factor-β (TGF-β), are highly expressed in pathologically aggressive IPMNs.38 Interestingly, Bmi1 is more intensely stained in the invading fronts of primary invasive breast cancers compared with that in the central portions, which is correlated with axillary lymph node metastasis.39,40 In our study, patients with IPMN expressing Twist and Bmi1 simultaneously showed a decrease of DFS. Interestingly, a strong connection between Twist1 and Bmi1 has been demonstrated by Yang et al.41 Their results presented the first molecular demonstration that Twist1 and Bmi1 are simultaneously required to repress E-cadherin expression. Some factors, including Notch1, TGF-β, and hypoxia-inducible factor 1, have been reported to be the triggering signals of EMT.17,41,42 Therefore, we investigated the relationship between Twist and Jagged1, one of the Notch1 ligands. Our results showed a tendency of correlation between Twist and Jagged1 protein expression, suggesting that EMT occurring in IPMN might be partly induced by Notch signaling. A previous report has demonstrated that gastric cancer progression induced by the Notch/Twist axis is mediated by STAT3 phosphorylation.17 The limitation of our study is that immunohistochemistry is not quantitative, and we did not show direct interactions among Twist, Bmi1, and Jagged. Because of the difficulty of performing in vitro experiments of IPMN, a further study using pancreatic cancer cells or normal pancreatic cells is needed to validate the tissue-specific molecular events. The results demonstrated here may be helpful in designing future studies to understand the molecular development of IPMNs. 2036
Patients with IPMN exhibiting simultaneously elevated expression of Twist and Bmi1 might require adjuvant chemotherapy and intensive follow-up. In conclusion, the results of this study suggest that Twist and Bmi1 are associated with the pathological aggressiveness of IPMN, which may be caused by EMT. Initiation of such EMT might be induced by Notch signaling, and simultaneous high expression of Twist and Bmi1 may be a prognostic factor of poor clinical outcomes.
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