Oral Maxillofac Surg DOI 10.1007/s10006-014-0459-0

ORIGINAL ARTICLE

Twist expression in dentigerous cyst, odontogenic keratocyst, and ameloblastoma Azadeh Andisheh-Tadbir & Soheil Pardis & Pegah Ranjbaran

Received: 14 May 2014 / Accepted: 25 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Epithelial-mesenchymal transition (EMT) is a process which is associated with a loss of intercellular adhesion, acquired mesenchymal shape, and increased motility by epithelial cells. Twist is one of the key regulators of EMT. In view of the distinct clinical behavior of odontogenic lesions, the objective of the present study was to investigate the immunohistochemical expression of Twist in these lesions. In this study, 70 formalin-fixed, paraffin-embedded tissue blocks of odontogenic lesion consisting of 16 unicystic ameloblastomas (UA), 17 solid ameloblastomas (SA), 18 odontogenic keratocysts (OKC), and 19 dentigerous cysts (DC) were reviewed using immunohistochemistry for Twist staining. In this study, Twist immunostaining was evident in all groups of the specimens except the dentigerous cyst group. Twist expression was seen in 58.8 % (10/17) of SA, 50 % (8/16) of UA, and 44.4 % (8/18) of OKCs. 23.5 % of SA, 18.8 % of UA, and 16.7 % of OKCs showed Twist expression in more than 50 % of cells. Statistical analysis showed that Twist expression levels were significantly higher in ameloblastomas (SA and UA) and OKCs than dentigerous cysts (P=0.002). There were no significant differences between Twist expression in SAs, UAs, and OKCs (P>0.05). The results of this study propose that the high expression rate of Twist plays a role in the pathogenesis of ameloblastomas A. Andisheh-Tadbir (*) : S. Pardis Department of Oral and Maxillofacial Pathology, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran e-mail: [email protected] A. Andisheh-Tadbir e-mail: [email protected] P. Ranjbaran School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran

and OKCs and might be one of the reasons for the aggressive behavior of ameloblastomas and high recurrence of OKCs and could reinforce the classification of OKC as an odontogenic tumor. Keywords Twist . Ameloblastoma . Dentigerous cyst . Odontogenic keratocyst

Introduction Epithelial-mesenchymal transition (EMT) is a process which is associated with a loss of intercellular adhesion, acquired mesenchymal shape, and increased motility by epithelial cells. EMT is necessary for embryonic morphogenesis and for the progression of early primary tumor cells into invasive and metastatic carcinomas [1]. EMT dysregulation has been recognized as a potential mechanism for cancer progression, leading to the acquisition of migratory and invasive phenotypes by tumor cells [2]. Twist protein is a transcription factor of the basic helix loop-helix family [3] which is essential for morphogenesis during embryonic development and is mainly expressed in a subgroup of adult mesenchymal cells [4, 5]. For the first time, Yang et al. explained that Twist plays an essential role in cancer metastasis [6]. Initially, it was discovered that Twist decreases the expression of cell adhesion molecules and E-cadherin, but later it was found that it is involved in the metastatic process through a diverse range of signaling pathways [7]. During early bone development, Twist has a negative regulation that can prevent osteoblast maturation and preserve cells in an osteoprogenitor-like state for preventing premature ossification [8].

Oral Maxillofac Surg Table 1 Score of Twist expression in different groups of lesion Type of lesion

Solid ameloblastoma Unicystic ameloblastoma Odontogenic keratocyst Dentigerous cyst

Score n (%) 1

2

3

4

7 (41.2) 8 (50) 10 (55.6) 19 (100)

2 (11.8) 3 (18.8) 2 (11) 0 (0)

4 (23.5) 2 (12.4) 3 (16.7) 0 (0)

4 (23.5) 3 (18.8) 3 (16.7) 0 (0)

Recent studies have shown that Twist can induce angiogenesis and drug resistance in tumors, has an anti-apoptosis effect, and can inhibit the tumor-suppressor function of P53 [9, 10]. The overexpression of Twist has been demonstrated in several malignant tumors, such as those of breast cancer [11], gastrointestinal tract cancer [12], nasopharyngeal carcinoma [13], melanoma [14], and prostate cancer [15], in which it was closely associated with clinical metastasis and worse outcomes.

Method and material This retrospective study was performed using 70 formalinfixed, paraffin-embedded tissue blocks of odontogenic lesion consisting of 16 unicystic ameloblastomas (luminal type) (UA), 17 solid ameloblastomas (SA), 18 odontogenic keratocysts (OKC), and 19 dentigerous cysts (DC) obtained from the Department of Oral and Maxillofacial Pathology, School of Dentistry, Shiraz University of Medical Sciences. H & E slides of available blocks were reviewed, and then, cases with a definite diagnosis and adequate tissue were selected for immunohistochemical staining (IHC). Cases with severe inflammation were excluded from the study. IHC staining and analysis IHC staining was performed using the EnVision Labeled Peroxides System (DAKO, Carpentaria, CA, USA). All samples were fixed in 10 % buffered formalin and embedded in paraffin. Sections were prepared with 4μ thickness, Fig. 1 Cytoplasmic twist expression in ameloblast-like and stellate reticulum-like cell in solid ameloblastoma (score 4) (×200)

deparaffinized in xylene, rehydrated in graded alcohol, and washed with distilled water. Antigen retrieval was performed using DAKO cytomation target retrieval solution with PH=9 for 20 min. Internal peroxidase activity was inhibited by 3 % H2O2. Tissue sections were then incubated for 30 min with the anti-Twist polyclonal antibody (Abcam company, ab49254) at a 1/100 dilution. Omission of the primary antibody was employed as negative control, while tissue of breast carcinoma was used as positive control. Brown cytoplasmic staining was considered positive. The Twist-immunostained cells were evaluated in 5 randomly selected fields per section at _400 magnification and expressed as a percentage. According to the percentage of positive cells, all specimens were scored as follows: score 1, 10 % or fewer cells positive; score 2, 11 to 25 % cells positive; score 3, 26 to 50 % cells positive; and score 4, more than 50 % cells positive. A tumor was designated as overall Twistpositive if greater than 10 % of cells (score of 2, 3, or 4) exhibited immunoreactivity [16]. Statistical analysis The Mann-Whitney and Kruskal-Wallis tests were used to compare results. A p value less than 0.05 was considered significant.

Result In this study, Twist immunostaining was evident in all groups of the specimen except dentigerous cysts. As shown in Table 1, Twist expression was seen in 58.8 % (10/17) of SA, 50 % (8/16) of UA, and 44.4 % (8/18) of OKCs. 23.5 % of SA,18.8 % of UA, and 16.7 % of OKCs showed Twist expression in more than 50 % of cells. In TWIST-positive cases, protein localization was mostly cytoplasmic and/or nuclear. Twist expression was seen in both peripheral ameloblast-like and stellate reticulum-like cells of ameloblastoma and in all layers of OKCs (Figs. 1, 2, and 3). Stromal components were negative to weakly positive for Twist. Statistical analysis showed

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Fig. 2 Cytoplasmic twist expression in unicystic ameloblastoma (score 40) (×200)

that Twist expression levels were significantly higher in ameloblastomas (SA and UA) and OKCs than in dentigerous cysts (P=0.002), but no significant differences were observed in Twist expression between SAs, UAs, and OKCs (P>0.05).

Discussion “Invasion” means the infiltration of tumor cells into the adjacent environment and their occupation [17]. The process of invasion is not specific to cancer; it is also found in embryonic development, in healthy organs, and in many noncancerous diseases [18]. Ameloblastoma is the most common odontogenic neoplasm with slow-growing and locally invasive behavior which leads to a high postoperative recurrence rate [19]. The keratocystic odontogenic tumor (KCOT) has recently been suggested to describe the lesion previously named odontogenic keratocyst. Clinical and immunohistological research questioned the cystic nature of this lesion, since the clinical course showed a high recurrence rate, and local invasion suggests a neoplastic entity [20]. Histological evaluation of this entity may therefore attract clinical consideration, since in contrast to the dentigerous cyst, various radical procedures are recommended for such lesions [21]. Most investigators suggest that the epithelial lining plays a role in the pathogenesis of OKC and is responsible for the Fig. 3 Cytoplasmic twist expression in odontogenic keratocyst (score 4)

distinctive behavior of OKC [22], but comprehension of both its pathogenesis and invasive growth remains incomplete. Epithelial cells lose their polarity and cohesiveness during the EMT process, and they obtain mesenchymal properties and a migratory capability [23]. Such gain of migratory phenotype triggers the development of invasive and metastatic tumors [24]. Unlike human cancers [25–27], the potential role of EMT in inducing invasiveness in ameloblastomas and OKCs has not been well investigated. Feng et al. [16] were among the first to report on the role of Twist in mediating local invasiveness in ameloblastomas, and Siar and Ng suggested that four transcription factors (Snail, Slug, SIPI, and Twist) probably play differential roles in mediating local invasiveness in ameloblastomas [28]. In the present study, Twist immunoreactivity was higher in ameloblastomas (both solid and unicystic) and OKCs than in dentigerous cysts. This result indicates that Twist plays a role in the pathogenesis of ameloblastomas and OKCs, but not in dentigerous cysts. Recently, increased attention has been paid to the function of Twist in the development and progression of human cancer [29]. Patients with Twist overexpression in primary ovarian cancer had unfavorable clinical outcomes, which may be related to the capability of Twist to promote the invasion and metastatic process of cancer cells by inducing an EMT [30]. Hosono et al. reported that the expression of Twist increased the risk of recurrence in epithelial ovarian carcinoma patients [30]. Tran et al. showed that Twist expression was found to correlate with survival and is highly predictive of recurrent disease [31]. Shen et al. suggested that Twist may increase the risk of postoperative recurrence in salivary adenoid cystic carcinoma patients [32]. The difference in Twist expression in the present study might explain the variable behavior of these lesions and the highly aggressive and invasive behavior of ameloblastomas and OKCs, and it supported the concept that OKC has a neoplastic nature. In accordance with Siar and Ng [28], the present study found no significant difference in regard to Twist expression between SA and UA; however, Feng et al. [16] reported a significant difference between these two types (a higher expression was found in SA). The lack of a statistically significant difference in our study may suggest that

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Twist-mediated EMT activity might not be a major factor influencing the differential growth characteristics of these two subtypes. Pereira et al. found no statistically significant difference in the expression of E-cadherin (which is negatively regulated by Twist) between SA and UA. Their results indirectly support our findings [32]. The reduction in E-cadherin expression is a characteristic feature of EMT [33]. Twist can inhibit the transcription level of E-cadherin by binding to its promoter [6]. Hakim et al. in their study showed a loss of E-cadherin staining in OKCs compared with dentigerous cysts [34]. Their findings support our results, which show the overexpression of Twist in OKC compared with DC and support the role of Twist in downregulating E-cadherin. Twist is negatively associated with P53 protein, and so, it can inhibit apoptosis [35] and promote proliferation [36]. In addition to interfering with P53, Twist is positively associated with anti-apoptotic protein [37]. Therefore, Twist overexpression in ameloblastomas and OKCs may cause increased cellular proliferation of these lesions and lead to their aggressive behavior.

Conclusion The results of this study propose that the high expression rate of Twist plays a role in the pathogenesis of ameloblastomas and OKCs and might be one of the reasons for the aggressive behavior of ameloblastomas and the high recurrence of OKCs and reinforce the classification of OKC as an odontogenic tumor. Acknowledgments The authors thank the Vice-Chancellery of Shiraz University of Medical Sciences for supporting this research (Grant No. 91-01-03-4478). This manuscript is based on the thesis of Pegah Ranjbaran for partial fulfillment of DDS degree. The authors are also grateful to Dr. M. Vossoughi of the Dental Research Development Center of the Dental School, for the statistical analysis.

References 1. Micalizzi DS, Farabaugh SM, Ford HL (2010) Epithelialmesenchymal transition in cancer: parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia 15(2):117–134 2. Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6):442–454 3. Kang Y, Massagué J (2004) Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 118(3):277–279 4. Castanon I, Baylies MK (2002) A Twist in fate: evolutionary comparison of Twist structure and function. Gene 287(1–2):11–22 5. Wang SM, Coljee VW, Pignolo RJ, Rotenberg MO, Cristofalo VJ, Sierra F (1997) Cloning of the human twist gene: its expression is retained in adult mesodermally-derived tissues. Gene 187(1):83–92

6. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C et al (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939 7. Franco HL, Casasnovas J, Rodríguez-Medina JR, Cadilla CL (2011) Redundant or separate entities?—roles of Twist1 and Twist2 as molecular switches during gene transcription. Nucleic Acids Res 39(4):1177–1186 8. Lee MS, Lowe GN, Strong DD, Wergedal JE, Glackin CA (1999) TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem 75(4):566–577 9. Wang XH, Ling MT, Guan XY, Tsao SW, Cheung HW, Lee DT et al (2004) Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene 23:474–482 10. Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E, Kidani A et al (2008) Twist and p53 reciprocally regulate target genes via direct interaction. Oncogene 27(42):5543–5553 11. Mironchik Y, Winnard PT Jr, Vesuna F, Kato Y, Wildes F, Pathak AP et al (2005) Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer. Cancer Res 65(23):10801–10809 12. Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R et al (2002) Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol 161(5):1881–1891 13. Song LB, Liao WT, Mai HQ, Zhang HZ, Zhang L, Li MZ et al (2006) The clinical significance of twist expression in nasopharyngeal carcinoma. Cancer Lett 242(2):258–265 14. Hoek K, Rimm DL, Williams KR, Zhao H, Ariyan S, Lin A et al (2004) Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res 64(15):5270–5282 15. Kwok WK, Ling MT, Lee TW, Lau TC, Zhou C, Zhang X et al (2005) Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res 65(12):5153–5162 16. Feng Y, Zhou YM, Hua CG, Tang XF, He DQ (2009) Expression of Twist in different subtype of ameloblastomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108(4):565–570 17. Ilan N, Elkin M, Vlodavsky I (2006) Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38(12):2018–2039 18. Mareel M, Leroy A (2003) Clinical, cellular and molecular aspects of cancer invasion. Physiol Rev 83:337–376 19. Florescu A, Mărgăritescu C, Simionescu CE, Stepan A (2012) Immunohistochemical expression of MMP-9, TIMP-2, E-cadherin and vimentin in ameloblastomas and their implication in the local aggressive behavior of these tumors. Rom J Morphol Embryol 53(4): 975–984 20. Barnes L, Eveson J, Reichart P, Sidransky D (2005) World Health Organization classification of tumours: pathology and genetics of head and neck tumours. IARC Press, Lyon 21. Gosau M, Draenert FG, Müller S, Frerich B, Bürgers R, Reichert TE e t a l ( 2 0 1 0 ) Tw o m o d i f i c a t i o n s i n t h e t r e a t m e n t o f keratocysticodontogenic tumors (KCOT) and the use of Carnoy’s solution (CS)—a retrospective study lasting between 2 and 10 years. Clin Oral Investig 14(1):27–34 22. Tsuneki M, Cheng J, Maruyama S, Ida-Yonemochi H, Nakajima M, Saku T (2008) Perlecan-rich epithelial linings as a background of proliferative potentials of keratocystic odontogenic tumor. J Oral Pathol Med 37(5):287–293 23. Hay ED (2005) The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn 233(3): 706–720 24. Wells WA (2003) Movement in Colorado: the keystone symposium on cell migration and invasion. J Cell Biol 160(7):985–988 25. Thiery JP (2003) Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15(6):740–746

Oral Maxillofac Surg 26. Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132(14):3151–3161 27. Hotz B, Arndt M, Dullat S, Bhargava S, Buhr HJ, Hotz HG (2007) Epithelial to mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer. Clin Cancer Res 13(16): 4769–4776 28. Siar CH, Ng KH (2014) Differential expression of transcription factors Snail, Slug, SIP1, and Twist in ameloblastoma. J Oral Pathol Med 43(1):45–52 29. Shen M, Wen Y, Hua C, Xiao J (2010) The expression of Twist in salivary adenoid cystic carcinoma and its clinicopathologic significance. Chinese-German J Clin Oncol 9(4):187– 192 30. Hosono S, Kajiyama H, Terauchi M, Shibata K, Ino K, Nawa A et al (2007) Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients. Br J Cancer 96(2):314–320 31. Tran DD, Corsa CA, Biswas H, Aft RL, Longmore GD (2011) Temporal and spatial cooperation of Snail1 and Twist1 during epithelial-mesenchymal transition predicts for human breast cancer recurrence. Mol Cancer Res 9(12):1644–1657

32. Alves Pereira KM, do Amaral BA, dos Santos BR, Galvão HC, Freitas Rde A, de Souza LB (2010) Immunohistochemical expression of E-cadherin and b-cateninin ameloblastomas and tooth germs. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 109:425–431 33. Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S et al (2010) Epithelial-mesenchymal transition in cancer development and its clinical significance. Cancer 101(2):293–299 34. Hakim SG, Kosmehl H, Sieg P, Trenkle T, Jacobsen HC, Attila Benedek G et al (2011) Altered expression of cell-cell adhesion molecules β-catenin/E-cadherin and related Wnt-signaling pathway in sporadic and syndromal keratocystic odontogenic tumors. Clin Oral Investig 15(3):321–328 35. Li B, Han Q, Zhu Y, Yu Y, Wang J, Jiang X (2012) Down-regulation of miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist. FEBS J 279(13):2393–2398 36. Paliwal P, Arora D, Mishra AK (2012) Epithelial mesenchymal transition in urothelial carcinoma: twist in the tale. Indian J Pathol Microbiol 55(4):443–449 37. Sahlin P, Windh P, Lauritzen C, Emanuelsson M, Grönberg H, Stenman G (2007) Women with Saethre-Chotzen syndrome are at increased risk of breast cancer. Genes Chromosom Cancer 46(7): 656–660