Int J Clin Exp Med 2016;9(9):17219-17231 www.ijcem.com /ISSN:1940-5901/IJCEM0019542

Original Article Expression of SIRT1, , and E-cadherin and its correlations with clinicopathological characteristics in gastric cancer patients

Jiajia Xu1, Weijie Wang2, Xueqing Wang1, Lihua Zhang1, Peilin Huang3

1Department of Pathology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China; 2Department of Gynaecology and Obstetrics, Subei People’s Hospital, Yangzhou 225001, China; 3School of Medi- cine, Southeast University, Nanjing 210009, China Received November 10, 2015; Accepted January 23, 2016; Epub September 15, 2016; Published September 30, 2016

Abstract: Aberrant expression of SIRT1, H3K9me3, H3K9Ac and E-cadherin is considered to be associated with tu- morigenesis and survival outcomes. In this study, we aim to investigate the expression of SIRT1, H3K9me3, H3K9Ac and E-cadherin and their associations with clinicopathological features in gastric cancer. A total of 105 gastric can- cer samples were obtained from patients underwent gastrectomy. Twenty corresponding normal adjacent tissues were simultaneously taken as control. The expression of SIRT1, H3K9me3, H3K9Ac and E-cadherin in these tissues was detected by immunohistochemical and their correlations with clinicopathological features were analyzed. Sig- nificant increase of SIRT1 expression was noted in gastric cancer compared with that of normal adjacent tissues (P<0.001). SIRT1 expression was markedly associated with tumor invasion depth (P=0.026), lymph node metasta- sis (P=0.001), clinical stage (P=0.004) and survival time (P=0.029). No significant difference was identified in the expression of H3K9Ac between gastric and normal tissues. Furthermore, the expression of H3K9Ac was unrelated with gender, age, tumor size, differential degree, invasion depth, lymph node metastasis, clinical stage and survival time (P>0.05). However, H3K9me3 expression was significantly higher in gastric cancer than that in normal tissues (P<0.001), and was remarkably correlated with tumor size (P=0.004), invasion depth (P=0.003), clinical stage (P=0.027) and survival time (P=0.013). In contract, the strong-positive expression of E-cadherin was significantly lower in gastric cancer than that in normal tissues (P<0.05), and its expression was markedly associated with tumor size (P=0.015), differential degree (P=0.031), tumor invasion depth (P<0.001), lymph node metastasis (P<0.001), clinical stage (P<0.001) and survival time (P<0.001). Meanwhile, SIRT1 expression was positively correlated with H3K9me3 (r=0.402, P<0.001), while E-cadherin was negatively associated with SIRT1 (r=-0.285, P=0.003) and H3K9me3 (r=-0.303, P=0.002). Aberrant expression of SIRT1, H3K9me3 and E-cadherin was closely associated with the progression and prognosis in patients with gastric cancer. Also, the expression of such factors were mu- tually related, based on which may provide an alternative strategy for the clinical diagnosis and treatment by the regulation of their expression in gastric cancer.

Keywords: Gastric cancer, SIRT1, H3K9me3, E-cadherin, immunohistochemical, prognosis

Introduction than 25% [4]. Furthermore, patients are gener- ally poor response to surgical resection as the Gastric cancer is one of the most common consequence of advanced presentation, toge- malignant tumors accounting for about 10% of ther with its aggressive nature involving malig- invasive cancer worldwide [1]. It remains the nant proliferation, extensive invasion and lym- second leading cause of cancer death followed phatic metastasis [5]. Therefore, early diagno- by lung cancer [2]. In general, the incidence of sis and treatment is thus important for the such cancer is approximately twice as common reduction of gastric cancer mortality. in males as females [3]. Current advances of new therapeutic strategies and supportive care The aggressive nature of metastatic gastric contribute to prolonged survival. However, the cancer is closely associated with various molec- outcome is still not satisfactory as the 5-ye- ular and genetic alterations, which adversely ar survival rate of gastric cancer remains less interfere with the downstream signal transduc- Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Figure 1. Immunohistochemical staining of SIRT1in gastric cancer tissue (×400). A. Negative expression; B. Positive expression. tion pathways involved in the cell proliferation, mediated DNA and H3K9me3 differentiation and survival [6]. SIRT1 (silent could restore E-cadherin expression and inhibit mating-type information regulation 2 homo- cell migration, invasion and metastasis in bas- logue 1) is a kind of deacetylase playing al-like breast cancer [14]. Moreover, SIRT1 was an important role in cellular metabolism, lon- considered to be associated with the expres- gevity and stress response [7]. H3K9me3 and sion of E-cadherin responsible for the regula- H3K9Ac refer to the methylation and acetyla- tion of tumor-suppressor genes and the pro- tion of 9 involving in the gene gression of gastroesophageal junction cancer repression and gene activation [8]. E-cadherin, [15]. Taken together, we speculated that there a single-span transmembrane protein, plays a also may be some correlations of such factors crucial role in the establishment of epithelial and their expression with clinicopathological architecture and maintenance of cell polarity features in gastric cancer. and differentiation [9]. Currently, massive re- ports have clearly indicated that aberrant exp- In this study, we determined the expression of ression of these factors is responsible for the SIRT1, H3K9me3, H3K9Ac and E-cadherin in development and progression of various malig- gastric cancer tissues, and analyzed their asso- nant tumors. For instance, elevated expression ciations with clinicopathological characteristics of SIRT1 has been observed in multiple human and prognostic impacts. Meanwhile, we ana- tumors such as breast, colon, prostate and lyzed the correlations of SIRT1, H3K9me3 and gastric cancer [10]. High H3K9me3 and low E-cadherin to explore their potential roles in the H3K9Ac expression were correlated with the occurrence and development of gastric cancer. solid tumor growth pattern of salivary adenoid cystic carcinoma and low overall survival [11]. Materials and methods Further, down-regulation of E-cadherin appe- ared to be associated with aggressive, poorly Patients and samples differentiated carcinomas and poor patient sur- vival [12]. A total of 105 patients diagnosed with gastric adenocarcinoma and underwent gastrectomy As is known, tumor-related factors involved in at Zhongda Hospital affiliated to Southeast tumorigenesis may be mutually related and fur- University between January 2007 and Dece- ther influence the biological behavior of malig- mber 2009 were enrolled in this study. The nant tumors. Similarly, that is considered to diagnosis of gastric adenocarcinoma was per- exist in the above mentioned factors. For in- formed according to the pathological analysis. stance, SIRT1 could direct recruit histone meth- Patients with any prior history of chemotherapy, yl-transferase Suv39H1 (suppressor of variega- radiotherapy and other treatments were exclud- tion 3 to 9 homologue 1) resulting in the tri- ed. Pathologic staging was classified based on methylation of H3K9 [13]. Blocking Suv39H1- tumor node metastasis (TNM) staging system

17220 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Figure 2. Immunohistochemical staining of H3K9Ac in gastric cancer tissue (×400). A. Negative expression; B. Posi- tive expression.

Figure 3. Immunohistochemical staining of H3K9me3 in gastric cancer tissue (×400). A. Weak-positive expression; B. Strong-positive expression. of the American Joint Committee on Cancer Immunohistochemistry (AJCC, the 6th edition) [16]. All subjects were grouped according to their age, gender, maxi- EnVision assay was used for immunohisto- mum diameter of tumor, histological type, inva- chemical staining adhering to the manufactur- er’s instructions. All obtained specimens were sion depth, metastatic lesions identified in ly- fixed with 10% neutral buffered formalin for 48 mph nodes and TNM stage. The patients were h and embedded in the paraffin. Consecutive followed up until December 31, 2013 by tele- sections (4 μm thick) were used for hematoxylin phone calls and e-mails. To compare the ex- and eosin (HE) staining and immunohistochem- pression of SIRT1, H3K9Ac, H3K9me3 and E- ical staining. After deparaffinization and rehy- cadherin in gastric cancer and normal gastric dration, sections were heated in a microwave tissue, a control group of 20 normal tissues at oven for 15 minutes in 10 mM citrate buffer more than 5 cm away from lesions were recruit- (pH=6.0) and treated with 3% hydrogen perox- ed in this study. Written informed consent was ide (XiYa JinQiao Biological Technology Co. Ltd, obtained from each patient. The study was car- Beijing, China) for 10 minutes. Then sections ried out with the approval from the Ethics were incubated at 4°C overnight with the fol- Committee of Zhongda Hospital affiliated to lowing primary antibodies: SIRT1 (clone: E104, Southeast University. 1:200, Epitomics, USA), H3K9Ac (clone: A4022,

17221 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Figure 4. Immunohistochemical staining of E-cadherin in gastric cancer tissue (×400). A. Weak-positive expression; B. Strong-positive expression.

Table 1. Expression of SIRT1 in gastric cancer and correlations with cadherin (clone: 36B2, 1: clinicopathological characteristics 200, Novocastra, England). SIRT1 expression After washing with PBS, rab- bit anti-mouse antibody was Demographic features N Negative Positive P value (14) (91) added and incubated for 30 min at room temperature. Gender Hematoxylin was used for Male 76 9 67 0.467 contrast staining after the Female 29 5 24 DAB color reaction. Mean- Age (year) while, reactions with PBS re- ≥60 66 8 58 0.635 placing the first antibody we- <60 39 6 33 re performed and served as Tumor size (cm) negative control. ≥5 53 4 49 0.078 Evaluation of immunostain- <5 52 10 42 ing Histological type Well differentiated adenocarcinoma 4 1 3 0.781 Semiquantitative analysis Moderately differentiated adenocarcinoma 56 7 49 was performed to evaluate Poorly differentiated adenocarcinoma 32 4 28 the immunohistochemical Signet ring cell carcinoma 9 2 7 values based on the per- Mucinous adenocarcinoma 4 0 4 centage of positively sta- Invasion depth ined cells. For each secti- T1-T2 (mucosal and muscular) 32 9 23 0.003* on, ten microscopic fields (×400) were randomly se- T3-T4 (serosa and out of serosa) 73 5 68 lected. Positive staining was Lymph node metastasis assessed in the presence of Positive 64 3 61 0.001* brown staining granules in Negative 41 11 30 the cytoplasm and cellular TNM stage membrane. Staining was sc- I 25 8 17 0.004* ored on a scale of 0~4 ac- II 21 4 17 cording to the percentage III 48 1 47 of positive cells involved: 0 IV 11 1 10 score, 0~1/100 positive ce- lls; 1 score, 1/100~1/10 positive cells; 2 score, 1/ 1:200, Epigentek, USA), H3K9me3 antibody 10~1/3 positive cells; 3 score, 1/3~2/3 posi- (clone: A4036, 1:200, Epigentek, USA) and E- tive cells; 4 score, >2/3 positive cells. A score

17222 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Table 2. Expression of H3K9Ac in gastric cancer and correlations to determine the associati- with clinicopathological characteristics ons between SIRT1, H3K9- H3K9Ac expression me3 and E-cadherin. P<0.05 was considered statistical di- Demographic features N Negative Positive P (97) (8) value fference. Gender Results Male 76 70 6 0.863 Female 29 27 2 Demographic information of Age (year) the patients ≥60 66 61 5 0.983 <60 39 36 3 A total of 105 patients (76 males, 29 females) were en- Tumor size (cm) rolled in this study, among ≥5 53 47 6 0.149 which 66 cases were aged <5 52 50 2 ≥60 years. Histological types Histological type of gastric cancer included Well differentiated adenocarcinoma 4 3 1 0.516 well differentiated adenocar- Moderately differentiated adenocarcinoma 56 51 5 cinoma (n=4), moderately dif- Poorly differentiated adenocarcinoma 32 31 1 ferentiated adenocarcinoma Signet ring cell carcinoma 9 8 1 (n=56), poorly differentiated Mucinous adenocarcinoma 4 4 0 adenocarcinoma (n=32), sig- Invasion depth net ring cell carcinoma (n=9) and mucinous adenocarcino- T1-T2 (mucosal and muscular) 32 31 1 0.25 ma (n=4). For the invasion T3-T4 (serosa and out of serosa) 73 66 7 depth, 32 cases were identi- Lymph node metastasis fied as mucosal and muscul- Positive 64 59 5 0.926 ar layer infiltration, while the Negative 41 38 3 tumors infiltrated serosa layer TNM stage and out of serosa in 73 cases. I 25 24 1 0.887 A total of 64 patients were II 21 19 2 identified as metastatic carci- III 48 44 4 noma in lymph nodes. Pa- IV 11 10 1 thologic staging was catego- rized based on TNM classifi- cation, including I stage (n= of zero was estimated as negative expression, 25), II stage (n=21), III stage (n=48) and IV while the scores of ≥1 indicated positive ex- stage (n=11). Sixty-eight patients accomplished pression. Further, the score of 1~2 was con- the follow-up, thirty-seven patients lost during sidered as weak positive staining, and 3~4 the follow up. points was strong positive staining. Expression of SIRT1, H3K9Ac, H3K9me3 and E-cadherin in gastric cancer and adjacent tis- Statistical analysis sues

Data analysis was performed with SPSS 16.0 The expression of SIRT1, H3K9Ac and H3K9- software (SPSS Inc., Chicago, IL, USA). Chi- me3 was primarily manifested in nucleus and square test was performed for the inter-group cytoplasm, while expression of E-cadherin was comparison of SIRT1, H3K9Ac, H3K9me3 and noticed in epithelial membrane or the parts E-cadherin and clinicopathologic features. Ov- nearby. No positive expression of SIRT1 was erall survival was calculated as the time from identified in all normal adjacent tissues, while the date of surgery to the date of death. Survi- the expression rate yielded to 86.7% (91/105) val curves were estimated using Kaplan-Meier in gastric cancer tissues (Figure 1). The expres- method. Log-rank test was carried out to com- sion of SIRT1 was significantly up-regulated in pare the survival rate between groups. Spe- gastric cancer tissues compared with that of arman correlation coefficients were calculated normal adjacent tissues (P<0.001).

17223 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Table 3. Expression of H3K9me3 in gastric cancer and correlations with cancer tissues than clinicopathological characteristics that of normal adjacent H3K9me3 expression tissues (P<0.05). Demographic features N Weak-posi- Strong-pos- P value Correlations between tive (40) itive (65) SIRT1, H3K9Ac, Gender H3K9me3 and E- Male 76 29 47 0.983 cadherin expression, Female 29 11 18 and clinicopathologic Age (year) characteristics ≥60 66 27 39 0.44 <60 39 13 26 In this study, we also determined the correla- Tumor size (cm) tion between the demo- ≥5 53 13 40 0.004* graphic features of the <5 52 27 25 patients and the ex- Histological type pression of SIRT1 (Ta- Well differentiated adenocarcinoma 4 2 2 0.653 ble 1), H3K9Ac (Table Moderately differentiated adenocarcinoma 56 4 32 2), H3K9me3 (Table 3) Poorly differentiated adenocarcinoma 32 9 23 and E-cadherin (Table Signet ring cell carcinoma 9 3 6 4), respectively. SIRT1 Mucinous adenocarcinoma 4 2 2 expression was unrelat- Invasion depth ed to age, gender, tu- mor size and differenti- T1-T2 (mucosal and muscular) 32 19 13 0.003* ation degree (P>0.05). T3-T4 (serosa and out of serosa) 73 21 52 However, it was signi- Lymph node metastasis ficantly associated wi- Positive 64 21 43 0.164 th infiltration depth (P= Negative 41 19 22 0.026), lymph node me- TNM stage tastasis (P=0.001) and I 25 12 13 0.027* TNM stage (P=0.004), II 21 12 9 respectively. No asso- III 48 15 33 ciation was noted be- IV 11 1 10 tween H3K9Ac expres- sion and all involved clinicopathological fea- Expression of H3K9Ac was observed in 8 tures, including gender, age, tumor size, infiltra- patients, including 7 with weak positive and 1 tion depth, differentiation degree, lymph node with strong positive (Figure 2). Weak position metastasis and TNM stage. Expression of H3- expression of H3K9Ac was observed in three K9me3 was remarkably correlated with tumor adjacent tissues. On this basis, no significant size (P=0.004), infiltration depth (P=0.003) and difference was noted in the expression of TNM stage (P=0.027). However, it showed no H3K9Ac between gastric cancer and adjacent correlation with the age, gender, differentiation tissues (P=0.121). degree, and lymph node metastasis. Further, significant association was identified in the H3K9me3 was expressed in all gastric cancer expression of E-cadherin and tumor size (P= tissues, however, no expression of H3K9me3 0.015), tumor differentiation (P=0.031), inva- was noticed in the adjacent tissues (Figure 3). sion depth (P<0.001), lymph node metastasis The expression of H3K9me3 was significantly (P<0.001) and TNM stage (P<0.001). higher in gastric cancer tissues than that of normal adjacent tissues (P<0.001). Roles of SIRT1, H3K9Ac, H3K9me3 and E- cadherin in the prognosis of gastric cancer Interestingly, strong E-cadherin expression was patients observed in all of normal tissues, while its expression was also positive in all gastric can- Based on the follow-up information, 8 cases cer tissues (Figure 4). However, the expression showed negative SIRT1 expression and 60 pre- of E-cadherin was markedly lower in gastric sented positive SIRT1. The 4-year survival rate

17224 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

Table 4. Expression of E-cadherin in gastric cancer and correlations with more favorable surviv- clinicopathological characteristics al (4-year survival ra- E-cadherin expression te: 74.3%, mean sur- vival time: 40.43 mo- Demographic features N Weak-posi- Strong-pos- P value tive (57) itive (48) nths), while the strong H3K9me3 expression Gender (n=40) was associat- Male 76 43 33 0.445 ed with a poor 4-year Female 29 14 15 survival rate of 45.5% Age (year) (mean survival time: ≥60 66 36 30 0.945 30.52 months) More- <60 39 21 18 over, remarkable dif- Tumor size (cm) ference was identifi- ≥5 53 35 18 0.015 ed in the survival rate <5 52 22 30 between patients wi- th strong-positive and Histological type weak-positive H3K9- Well differentiated adenocarcinoma 4 0 4 0.0031* me3 expression (P= Moderately differentiated adenocarcinoma 56 27 29 0.013). In contract, Poorly differentiated adenocarcinoma 32 21 11 patients with weak- Signet ring cell carcinoma 9 5 4 positive E-cadherin ex- Mucinous adenocarcinoma 4 4 0 pression (n=33) were Invasion depth linked to a poor prog- T1-T2 (mucosal and muscular) 32 5 27 0.000* nosis with a 4-year T3-T4 (serosa and out of serosa) 73 52 21 survival rate of 27.3% and mean survival ti- Lymph node metastasis me of 25.12 months, Positive 64 49 15 0.000* whereas those with Negative 41 8 33 strong-positive E-cad- TNM stage herin expression (n= I 25 4 21 0.000* 35) showed a favor- II 21 3 18 able prognosis with III 48 40 8 the 4-year survival ra- IV 11 10 1 te of 91.4% and mean survival time of 45.51 months. Obviously, si- was up to 100% in patients with negative SIRT1 gnificant elevation was identified of overall sur- expression. Nevertheless, for the patients with vival in the cohort with strong E-cadherin ex- positive SIRT1 expression, the 4-year survival pression than those with weak E-cadherin ex- rate was 55.0% with mean survival time of pression (P<0.001). Further, the survival curves 33.97 months. Significant difference was noted for the expression of SIRT1, H3K9Ac, H3K9me3 in the survival rate between SIRT1-positive and and E-cadherin in patients with gastric cancer -negative patients as revealed by log-rank test were provided as shown in Figures 5-8, re- (P=0.029). For the provided follow-up informa- spectively. tion, 3 cases showed positive H3K9Ac expres- Correlation between SIRT1, H3K9me3 and sion and 65 cases were negative. In H3K9Ac E-cadherin positive group, 4-year survival rate was 66.7% (mean survival time: 35 months). The 4-year Table 5 showed the correlations between the survival rate was 60.0% with the mean survival expression of SIRT1, H3K9me3 and E-cadherin. time of 35.64 months in negative H3K9Ac The expression of SIRT1 was positively corre- expression group. Log-rank test revealed no lated with H3K9me3 in patients with gastric statistical difference in the survival rate be- cancer (r=0.402, P<0.001). Whereas, it was tween two groups (P=0.923). Patients with negatively correlated to E-cadherin (r=-0.285, weak H3K9me3 expression (n=28) presented a P=0.003). Besides, the expression of H3K9me3

17225 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

was negatively correlated wi- th E-cadherin expression (r=- 0.303, P=0.002).

Discussion

Increasing evidence reveals th- at multiple molecular and ge- netic alterations contribute to the malignant transformation in the pathogenesis of gastric can- cer [17]. It has been well ac- knowledged that alternations in specific genes/proteins play crucial roles in regulating vari- ous cellular functions, including Figure 5. Kaplan-Meier survival curves for SIRT1-positive and -negative cell differentiation and metas- groups in gastric cancer patients. tasis, signal transduction, DNA repair and cell adhesion [18]. Therefore, identification of aber- rant expression of tumor-relat- ed factors is thus important for developing prognostic molecu- lar markers and evaluating gas- tric carcinogenesis. In this st- udy, we found that up-regulati- on of SIRT1 and H3K9me3 con- current with down-regulation of E-cadherin in the gastric cancer tissues. All these factors were markedly associated with tumor infiltration depth, TNM stage and survival time of patients. Meanwhile, the expression of such factors was mutually relat- Figure 6. Kaplan-Meier survival curves for H3K9Ac-positive and -negative ed in gastric cancer. These find- groups in gastric cancer patients. ings suggested that SIRT1, H3- K9me3 and E-cadherin may be interrelated in the tumorigene- sis and progression of gastric cancer.

SIRT1 refers to a class III, nico- tinamide adenine dinucleotide (NAD+)-dependent histone de- acetylase [19] playing an impor- tant role in the cell growth, apoptosis, cell metabolism and tumorigenesis. To date, mas- sive reports have been demon- strated the role of SIRT1 in the development and progression of carcinomas. However, the sp- Figure 7. Kaplan-Meier survival curves for patients with H3K9me3-strong ecific mechanism of SIRT1 in positive and weak positive expression. tumor pathogenesis is still un-

17226 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

of SIRT1 was significantly ele- vated in gastric cancer com- pared with that in adjacent tis- sues (55.56% vs 24.44%, P< 0.05). The expression of SIRT1 was markedly associated with TNM stage and lymph node metastasis [23]. Also, Cha et al. showed that SIRT1 expres- sion was remarkably higher in gastric cancer than that in norm- al gastric mucosa. Meanwhi- le, SIRT1 was closely correlat- ed with differentiation degree, TNM stage and the depth of Figure 8. Kaplan-Meier survival curves for patients with E-cadherin-strong positive and weak positive expression. tumor infiltration, as well as the shorter overall survival and relapse-free survival [2]. Table 5. Correlation assay of SIRT1, H3K9me3 and Consistent with previous reports, we found E-cadherin SIRT1 expression was significantly increased Item SIRT1 H3K9me3 E-cadherin in 105 gastric cancer tissues in comparison SIRT1 - r=0.402, P<0.001 r=-0.285, P=0.003 with that of normal adjacent tissues. Higher expression of SIRT1 was not correlated with H3K9me3 - - r=-0.303, P=0.002 gender, age, tumor size and differentiation degree, but with invasion depth, lymph node clear and its role in human malignant tumors is metastasis and TNM stage. Furthermore, ele- controversial. Nowadays, SIRT1 is reported to vated SIRT1 expression was significantly asso- serve as both a tumor promoter and a tumor ciated with shorter 4-year survival rate. All suppressor. The promotional effect of SIRT1 these indicated that SIRT1 might be a clinically may present by stimulating tumor signal path- significant indicator for evaluating the progres- ways and repressing tumor suppressor factors sion and prognosis of gastric cancer. including p53, NF-κB, KU70, and the FOXO fam- ily proteins [7], while its adverse role may be H3K9Ac is a histone that occurs in determined by inhibiting several oncoproteins the N-terminal of lysine residues with the spe- and oncogenes such as surviving and β-catenin cific regulation by histone acetyl transfera- [20]. Striking increase of SIRT1expression has ses (HATs) and histone deacetylases (HDACs). been observed in human prostate cancer, pri- Histone acetylation is known to regulate gene mary colon cancer and acute myeloid leukemia transcription via the alteration of [21]. Also, up-regulated expression of SIRT1 structure, among which H3K9Ac is considered was generally identified in all kinds of non-mel- to bring about gene activation [24]. H3K9me3, anoma skin carcinomas [10]. In contract, some an marker, has been found reports indicated that the expression of SIRT1 to be associated with silencing of gene tran- was down-regulated in many other types of scription playing a crucial role in many biologi- cancers such as ovarian cancer, glioblastoma, cal behaviors such as cellular differentiation, bladder and hepatic carcinoma [22]. Thus, the cell-cycle, DNA damage and stress response specific intervention of SIRT1on the oncogenes [8]. Recently, emerging studies have demon- and suppressor genes remains elusive. The dif- strated the aberrant expression of such histone ferent action of SIRT1 may primarily depend on modifications are frequent events during the the oncogenic pathways specific to particular development and progression of carcinomas. tumors. For instance, down-regulated H3K9Ac expres- Currently, only a few studies are available con- sion was identified in several cancers, including cerning the research of SIRT1 expression and prostate, bladder, lung and kidney cancer [25- its relationship with clinicopathological featu- 27]. The expression of H3K9me3 was elevated res and prognostic significance in gastric can- in patients with salivary adenoid cystic carci- cer. Zhang et al. indicated that the positive rate noma and myeloma [11, 28].

17227 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer

To date, definite role of H3K9 modification in tion of the transcriptional repressors such as gastric cancer remains unknown as only spo- Slug, Snail, and Twist [32, 37]. radic reports are accessible. Young et al. sug- gested that higher H3K9me3 expression was E-cadherin mediated adhesion system is con- positively associated with tumor stage, lympho- sidered to be suppressor in the tumor invasive- vascular invasion, cancer recurrence, as well as ness and metastatic dissemination. Among the poor survival rate in 206 gastric cancer sam- different activities of intercellular adhesion, the ples, while H3K9Ac expression was uncorrelat- adhesion of same cell type is associated wi- ed with the above clinicopathological factors th the cellular detachment from the primary [29]. In this study, no significant difference was tumors, while the adhesion of different cell noted in the expression of H3K9Ac between types is involved in neoplastic cellular implan- gastric cancer and normal adjacent tissues. tation in vessel wall and tumor-killing by host Meanwhile, H3K9Ac expression was unrelated immune cells. After exposure to tumor cells, to survival rate and all pathological features intercellular adhesion appears to attenuate involved in this study. Whereas, H3K9me3 and thus contribute to the tumor metastasis. expression was markedly up-regulated in gas- Currently, abnormal expression of E-cadherin tric cancer compared with that of normal tis- has been generally identified in gastric cancer. sues, and was significantly associated with Dysregulation of E-cadherin/catenin complex tumor size, invasion depth and TNM stage. appears to occur at an early stage of gastric Moreover, significant decrease was revealed of carcinogenesis [38]. Tanaka et al. indicated the 4-year survival rate in patients with strong- that aberrant E-cadherin expression was a positive H3K9me3 as compared to that with potential marker of submucosal invasion in weak-positive H3K9me3 expression (23.08% early-differentiated gastric cancer and the neg- vs 65%). That was, higher expression of H3- ative β-catenin expression might be served as K9me3 corresponded to a worse prognosis in a predictor of lymph node metastasis in the gastric cancer. On this basis, H3K9me3 may be intestinal and diffuse types of gastric cancer served as a potential biological parameter to [39]. Somatic mutation of E-cadherin have assess malignant degree and prognosis of gas- been shown in 50% patients with diffuse-type tric cancer. gastric cancer [40]. Furthermore, down-regulat-

2+ ed expression of E-cadherin was shown to be E-cadherin, a Ca - dependent cell-cell adhe- associated with lymph node metastasis, lym- sion molecule, was mainly located within adher- phatic and venous invasion, as well as poor ent junctions. It is a single-pass transmem- prognosis [41]. In our study, we found the brane protein containing extracellular, trans- strong-positive expression of E-cadherin was membrane and cytoplasmic domain [30, 31]. significantly decreased in gastric cancer com- E-cadherin exist widely in all kinds of epithelial pared with that of corresponding normal adja- cells playing an important role in the mainte- cent tissues. E-cadherin expression appeared nance of cell polarity and structural integrity of to markedly correlated with tumor size, tumor epithelial tissues. In light of the well-estab- differentiation, invasion depth, lymph node lished cell-cell adhesions of E-cadherin, its loss metastasis and TNM stage. Meanwhile, pa- appears to promote the invasive and metastat- tients with strong-positive E-cadherin expres- ic behavior in various epithelial tumors [32]. sion exhibited significant superiority in the Massive reports have demonstrated that E- 4-year survival rate as compared to that with cadherin loss is frequently observed in many weak-positive expression (91.43% vs 27.27%), malignant tumors such as gastric cancer [33], which was in line with previous report [42]. All colon cancer [34], breast cancer [35] and these findings indicated E-cadherin may be a esophageal carcinoma [36]. The absent or causal factor in the invasion and metastasis of reduced expression of E-cadherin is common- gastric cancer. ly caused by several molecular mechanisms summarized as follows: (i) E-cadherin-mediated In addition, the correlations among SIRT1, genetic lesion of cellular adhesion caused by H3K9me3 and E-cadherin were further ana- the gene deletion or mutation of E-cadherin; (ii) lyzed in the present study. Significant positive cell-cell adhesion failure due to the altered pro- correlation was noted between the expression tein composition (e.g. as α- or β-catenin) in the of SIRT1 and H3K9me3. E-cadherin displayed E-cadherin/catenin complexs; (iii) hypermethyl- significant negative correlation with H3K9me3 ation of the E-cadherin promoter; (iv) upregula- and SIRT1 in gastric cancer. Recently, emerging

17228 Int J Clin Exp Med 2016;9(9):17219-17231 Role of SIRT1, H3K9me3, H3K9Ac and E-cadherin in gastric cancer reports have been conducted to investigate the Address correspondence to: Dr. Lihua Zhang, De- associations of these factors. For instance, partment of Pathology, Zhongda Hospital, School of SIRT1 contributed to the elevation H3K9me3 Medicine, Southeast University, No. 87, Dingjiaqi- expression by recruiting the methyltransferase ao Street, Nanjing 210009, China. E-mail: lihua- Suv39H1 [13]. Also, Li et al. showed that DBC1 [email protected]; Dr. Peilin Huang, School of (deleted in breast cancer 1) expression disrupt- Medicine, Southeast University, No. 87, Dingjiaqiao ed the interaction between SIRT1 and Suv39H1 Street, Nanjing 210009, China. E-mail: plhuang- resulting in the reduction of H3K9me3 expres- [email protected] sion both in vitro and vivo [43]. Dong et al. found that the recruitment of Suv39H1 inter- References acted with Snail on the E-cadherin promoter [1] Roder DM. The epidemiology of gastric cancer. significantly improve the expression of H3K9- Gastric Cancer 2002; 5: 5-11. me3, together with the gene silence of E- [2] Cha EJ, Noh SJ, Kwon KS, Kim CY, Park BH, cadherin. Further, knockdown of Suv39H1 do- Park HS, Lee H, Chung MJ, Kang MJ and Lee wn-regulated H3K9me3 expression accompa- DG. Expression of DBC1 and SIRT1 is associ- nied by the activation of E-cadherin expression ated with poor prognosis of gastric carcinoma. in basal-like breast cancer [14]. Byles et al. indi- Clin Cancer Res 2009; 15: 4453-4459. cated that silencing SIRT1 could restore cell- [3] Kelley JR and Duggan JM. Gastric cancer epi- cell adhesion and induced the elevation of demiology and risk factors. J Clin Epidemiol E-cadherin expression in metastatic prostate 2003; 56: 1-9. tumor cells [44]. Therefore, we supposed that [4] Amedei A, Benagiano M, della Bella C, Niccolai there might be a biological pathway between E and D’Elios MM. Novel immunotherapeutic strategies of gastric cancer treatment. Biomed these factors responsible for the further influ- Res Int 2011; 2011: 437348. ence on cellular behavior, of which Suv39H1 [5] Dassen A, Lemmens VE, Van de Poll-Franse LV, may be an important transferase in their asso- Creemers GJ, Brenninkmeijer S, Lips DJ, vd ciations and biological function. Wurff AA, Bosscha K and Coebergh JW. Trends in incidence, treatment and survival of gastric In conclusion, over-expression of SIRT1 and adenocarcinoma between 1990 and 2007: a H3K9me3 and the down-regulation of E-ca- population-based study in the Netherlands. dherin are identified in patients with gastric Eur J Cancer 2010; 46: 1101-1110. cancer, and these aberrant expression are sig- [6] Fiocca R, Luinetti O, Villani L, Mastracci L, nificantly associated with tumor invasion, ly- Quilici P, Grillo F and Ranzani G. Molecular mph node metastasis, clinical stage and poor mechanisms involved in the pathogenesis of survival, based on which suggest the important gastric carcinoma: interactions between ge- activity of such factors in the development and netic alterations, cellular phenotype and can- prognosis of gastric cancer. Meanwhile, the cer histotype. Hepatogastroenterology 2000; expression of these factors are mutually relat- 48: 1523-1530. [7] Noguchi A, Kikuchi K, Zheng H, Takahashi H, ed, indicating that there may be a potential Miyagi Y, Aoki I and Takano Y. SIRT1 expression regulatory pathway in response to the related is associated with a poor prognosis, whereas biological behavior. Further studies are needed DBC1 is associated with favorable outcomes in to illustrate the molecular mechanism underly- gastric cancer. Cancer Med 2014; 3: 1553- ing how to mutually regulate among SIRT1, 1561. H3K9me3 and E-cadherin. [8] Leszinski G, Gezer U, Siegele B, Stoetzer O and Holdenrieder S. Relevance of histone marks Acknowledgements H3K9me3 and H4K20me3 in cancer. Antican- cer Res 2012; 32: 2199-2205. This study was supported by Jiangsu Provincial [9] Van Roy F and Berx G. The cell-cell adhesion Natural Science Fund (No. BK2012750); the molecule E-cadherin. Cell Mol Life Sci 2008; Fundamental Research Funds for the Central 65: 3756-3788. Universities and the College Graduate Research [10] Deng CX. SIRT1, is it a tumor promoter or tu- and Innovation program of Jiangsu Province mor suppressor? Int J Biol Sci 2009; 5: 147. (No. KYLX15_0179). [11] Xia R, Zhou R, Tian D, Zhang C, Wang L, Hu Y, Han J and Li J. High expression of H3K9me3 is Disclosure of conflict of interest a strong predictor of poor survival in patients with salivary adenoid cystic carcinoma. Arch None. Pathol Lab Med 2013; 137: 1761.

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