Jagged1 and DLL4 expressions in benign and malignant pancreatic lesions and their clinicopathological significance

Sheng-Fu Huang, Zhu-Lin Yang, Dai-Qiang Li, Zhi-Yu Liu, Chun-Wei Wang, Xiong-Ying Miao, Qiong Zou and Yuan Yuan

Changsha, China

Jagged1 and DLL4 expressions in benign and malignant pancreatic lesions and their clinicopathological significance

Sheng-Fu Huang, Zhu-Lin Yang, Dai-Qiang Li, Zhi-Yu Liu, Chun-Wei Wang, Xiong-Ying Miao, Qiong Zou and Yuan Yuan

Changsha, China

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a poor prognosis. Despite intensive research, markers for the early diagnosis, prognosis, and targeting therapy of PDAC are not available. This study aimed to investigate the protein expressions of Jagged1 and DLL4 in PDAC tumor, benign pancreatic and normal pancreatic tissues, and analyze the associations of the two proteins with the clinical and pathological characteristics of PDAC.

METHODS: A total of 106 PDAC tumor tissues and 35 peritumoral tissues were collected from January 2000 to December 2011 at our hospitals. Thirteen normal pancreatic tissues and 55 benign pancreatic specimens were collected at the same period. Immunohistochemical staining was used to measure Jagged1 and DLL4 protein expressions in these tissues.

RESULTS: The percentage of positive Jagged1 and DLL4 was significantly higher in PDAC than in normal pancreatic tissues, benign pancreatic tissues, and peritumoral tissues (P＜0.01). The higher Jagged1 and DLL4 expressions in PDAC were significantly associated with poor differentiation, maximum tumor size ＞5 cm, invasion, regional lymph node metastasis, and TNM III/IV disease (P＜0.05). In PDAC, Jagged1 expression positively correlated with DLL4 expression. Univariate Kaplan-Meier analysis showed that positive Jagged1 and DLL4 expressions were significantly associated with shorter survival in patients with PDAC. Multivariate Cox regression analysis showed that positive Jagged1 and DLL4 expressions were independent prognostic factors for poor prognosis of patients with PDAC.

CONCLUSION: Positive Jagged1 and DLL4 expression is closely correlated with severe clinicopathological characteristics and poor prognosis in patients with PDAC.

(Hepatobiliary Pancreat Dis Int 2016;15:640-646)

Jagged1;

DLL4;

pancreatic ductal adenocarcinoma;

prognosis

Introduction

Pancreatic cancer is a highly malignant tumor. The occurrence of pancreatic cancer is on the rise in the youth population.[1]Early diagnosis of pancreatic cancer is difficult because of the lack of obvious symptoms and signs. Pancreatic tumor cells easily invade surrounding major blood vessels and nerves because of the rich blood and lymph vessels of the pancreas and the lack of capsule of pancreatic acinar cells. Pancreatic cancer, even very small, can cause lymph node and distant metastases. Therefore, at diagnosis, most of the patients with pancreatic tumors have already lost the chance for curative resection.[2]Screening biomarkers for asymptomatic pancreatic tumor patients is a hot research topic currently.[3]Although some progress has been made in finding biomarkers in body fluids, blood, and pancreatic secretions for the diagnosis of pancreatic cancer, such as detecting mutations in the KRAS gene, its clinical value

remains to be further validated. Therefore, exploration of new biomarkers is still important for pancreatic cancer diagnosis and treatment.

The Notch signaling pathway is vital for organ formation and morphogenesis as well as disease progression. Human Notch ligands comprise five members, namely Delta1, Delta3, Delta4, Jagged1 and Jagged2. The Jagged1 gene is located on chromosome 20p12 with a full length of 36 kb. Notch and Jagged1 expressions are significantly increased in breast cancer which is associated with poor prognosis of patients.[4]In Hodgkin’s lymphoma and anaplastic large cell lymphoma, Jagged1-mediated activation of Notch1 signaling was involved in tumor occurrence.[5]Tohda and Nara[6]found that Notch1/Jagged1 signal can inhibit cell differentiation and induce excessive self-renewal of cancer stem cells, resulting in abnormal amplification of leukemia cells, thus promoting the development of acute myeloid leukemia. Recently, many studies also reported that high Jagged1 expression is closely related to the development of a variety of epithelial malignancies, such as non-small cell lung cancer,[7]hepatocellular carcinoma,[8]gastric cancer,[9]colorectal cancer,[10]and prostate cancer,[11]and is associated with high malignancy, rapid progression, and poor prognosis of these cancers. A study[12]showed increased expression of Jagged1 in pancreatic ductal adenocarcinoma (PDAC) tumor tissues compared to that in the normal pancreatic tissues, but no association between Jagged1 immunohistochemical scores and tumor grade or disease stage was observed. They also did not observe the predictive role of Jagged1 in the prognosis of PDAC.

DLL4 (Delta-like ligand 4) is a Notch signaling pathway ligand. Recent studies[13,14]demonstrated that DLL4 plays an inhibitory role in tumor blood vessel sprouting and angiogenesis by affecting the proliferation of endothelial cells. Also, DLL4 can inhibit tumor cell apoptosis by indirectly affecting NF-κB signaling and reducing cancer stem cell activity.[15-17]In recent years, high DLL4 expression was found in some epithelial malignancies, such as breast cancer,[18]non-small cell lung cancer,[19]gastric cancer,[20]colon cancer,[21]ovarian cancer[22]and prostate cancer,[23]and DLL4 expression was associated with fast progression, high malignancy and poor prognosis of these cancers. A recent study[24]showed that high DLL4 expression correlated with the poor clinical outcome and overall survival in PDAC patients. Chen et al[25]revealed that high DLL4 expression is significantly associated with advanced tumor stage, lymph node metastasis, and poor prognosis in patients with surgically resected PDAC. The present study investigated the protein expressions of Jagged1 and DLL4 in PDAC tumor, benign pancreatic tissues, and normal pancreatic tissues, as well as the associations of these two proteins with the clinical and pathological characteristics of PDAC.

Methods

Specimens

One hundred and six PDAC tumor tissues and 35 peritumoral tissues were collected from January 2000 to December 2011 at the Second and Third Xiangya Hospitals, Central South University. The peritumoral specimens were collected ≥2 cm from the edge of tumors. Thirteen normal pancreatic tissues and 55 benign pancreatic specimens were collected at the same period. The survival information was collected by telephone and mail for two years.

The 106 PDAC patients consisted of 45 females and 61 males. Their mean age was 54.50 years. The histological staining showed well, moderately, and poorly differentiated tumors in 38, 35 and 33 PDACs, respectively. Thirteen, 68 and 25 of the 106 PDAC patients had maximum tumor size ＜3 cm, 3-5 cm, and ＞5 cm, respectively. Twenty-nine PDAC patients had regional lymph node metastasis and 64 had invasion to the surrounding tissues and organs. Of the 106 PDACs, 11, 42, 37 and 16 were classified into TNM stage I, II, III and IV disease, respectively. No patients received preoperative radiotherapy and chemotherapy. Twelve peritumoral tissues exhibited normal dysplasia, while 10, 8 and 5 exhibited mild, moderate and severe dysplasia, respectively. The 55 benign pancreatic lesions included 20 adenomas, 15 intraepithelial neoplasias, and 20 chronic pancreatitis. Among the 20 adenomas, 5 were mucinous adenomas and 15, serous adenomas. Six, 5 and 4 of the 15 intraepithelial neoplasias were classified into grade I, grade II, and grade III, respectively.

Immunohistochemistry

The tissues were paraffin-embedded and sectioned at 4 μm. Immunohistochemistry was carried out using EnVision detection kit (Dako Laboratories, USA). The rabbit anti-human Jagged1 and DLL4 antibodies were purchased from Abgent Company (San Diego, CA, USA). Briefly, the sections were deparaffinized and treated with 3% H2O2for 15 minutes, followed by incubation with Jagged1 or DLL4 antibody for 1 hour at room temperature. After washing with PBS, the sections were then incubated with secondary antibody for 1 hour. After washing with PBS for 3 times and developing color shortly, 500 cells were counted from 10 random fields. Cases were defined as positive Jagged1 or DLL4 expression when ≥25% cells were positively stained with anti-

Jagged1 or DLL4 antibody, respectively.[26]Positive controls were provided by the EnVision detection kit. GFP antibody was used for negative control of the staining.

Statistical analysis

SPSS17.0 software (Chicago, IL, USA) was used for data analysis. The Chi-square test or Fisher’s exact test was used for analyzing the relationships between the Jagged1 and DLL4 protein expressions and clinicohistological factors. Univariate survival analysis was performed using the Kaplan-Meier method (log-rank test). Cox proportional hazard model was used for multivariate analysis and determination of 95% CI. A P＜0.05 was considered statistically significant.

Results

Jagged1 and DLL4 protein expression

Positive Jagged1 (Fig. 1) and DLL4 (Fig. 2) staining were mainly observed in the cytoplasm. Jagged1 and DLL4 were negative in all normal pancreatic tissues. Jagged1 was positive in 10.0% of chronic pancreatitis tissues, 25.0% of adenomas, and 26.7% of intraepithelial neoplasias. DLL4 was positive in 10.0% of chronic pancreatitis tissues, 15.0% of adenomas, and 26.7% of intraepithelial neoplasias. In benign and peritumoral pancreatic tissues, positive Jagged1 or DLL4 staining was observed with grade II/III intraepithelial neoplasia or dysplasia (Table 1). The percentage of positive Jagged1 and DLL4 staining in PDAC was 51.9% and 50.0%, respectively, which was significantly higher than that in normal pancreatic tissues, peritumoral tissues, and benign lesions (P＜0.01), suggesting that Jagged1 and DLL4 may be involved in the tumorigenesis of PDAC. In contrast, no significant differences were observed in the positive Jagged1 and DLL4 expression between benign lesions, peritumoral tissues, and normal pancreatic tissues (P＞0.05).

Jagged1 and DLL4 are associated with the clinicopathological characteristics of PDAC

Fig. 1. Immunohistochemical staining of Jagged1 expression (original magnification ×200). Cells with brown granules in the cytoplasm were identified as Jagged1 positive. A: Representative staining of positive Jagged1 expression in poorly differentiated PDAC; B: Representative staining of negative Jagged1 expression in moderately differentiated PDAC; C: Representative staining of Jagged1 positive expression in peritumoral tissues; D: Representative staining of Jagged1 positive expression in stage II intraepithelial neoplasia.

Fig. 2. Immunohistochemical staining of DLL4 expression (original magnification ×200). Cells with brown granules in the cytoplasm were identified as DLL4 positive. A: Representative staining DLL4 positive expression in moderately differentiated PDAC; B: Representative staining of DLL4 negative expression in well differentiated PDAC; C: Representative staining of DLL4 positive expression in adenoma; D: Representative staining of DLL4 positive expression in chronic pancreatitis.

Table 1. Comparison of positive Jagged1 and DLL4 staining in PDAC, peritumoral tissues, benign and normal pancreatic tissues

A significantly higher percentage of positive Jagged1 and DLL4 expression was observed in PDAC patients with TNM stage III/IV disease, invasion, regional lymph node metastasis, and poorly differentiated tumors compared with PDAC patients with TNM stage I/II disease,

no invasion, no lymph node metastasis, and well differentiated tumors (P＜0.05) (Table 2). These findings suggest that Jagged1 and DLL4 expressions were associated with disease severity. No significant association was observed between Jagged1 and DLL4 expressions and age and gender in PDAC patients (Table 2). Among the 55 PDAC tumors with positive Jagged1 staining, 39 had positive DLL4 staining. Among the 51 PDAC tumors with negative Jagged1 staining, 37 had negative DLL4 staining. Jagged1 protein expression positively correlated with DLL4 protein expression in PDAC (χ2=23.795, P＜0.001).

Prognostic effects of Jagged1 and DLL4 protein expression and clinicopathological characteristics in PDAC patients

The patients with PDAC were followed up for two years. In total, 77 PDAC patients died in 1 year, 29 survived longer than 1 year. Kaplan-Meier survival analysis showed that PDAC patients with poor differentiation, tumor size ＞5 cm, regional lymph node metastasis, invasion to surrounding organs and tissues, and TNM stage III/IV disease survived shorter than those with well differentiated tumors, small tumor size, no metastasis and invasion, and TNM stage I/II (P＜0.05). Positive Jagged1 or DLL4 protein expression correlated with a significantly shorter survival in PDAC patients (P＜0.001) (Table 3, Fig. 3). Cox multivariate analysis revealed that poor differentiation, tumor size ＞5 cm, regional lymph node metastasis, invasion, and high TNM stage III/IV are poor survival factors for PDAC patients. Positive Jagged1 (Table 4) and DLL4 (Table 5) protein expressions were independent negative prognostic factors for PDAC patients.

Table 2. Association of Jagged1 and DLL4 protein expression with the clinicopathological characteristics in PDAC patients (n, %)

Table 3. The effects of clinicopathological characteristics, Jagged1 and DLL4 expression on the mean survival of PDAC patients

Fig. 3. Kaplan-Meier plots of mean survival in PDAC patients with positive or negative Jagged1 (A) and DLL4 (B) expression.

Table 4. Multivariate Cox regression analysis of survival rate and clinicopathological characteristics, Jagged1 expression in patients with PDAC

Table 5. Multivariate Cox regression analysis of survival rate and clinicopathological characteristics, DLL4 expression in patients with PDAC

Discussion

PDAC is a highly aggressive cancer. A biological marker for the progression and poor prognosis of PDAC remains to be identified. Ligand-activated Notch signaling has been demonstrated to play a crucial role in various aspects of cancer biology and the associated ligands are attractive targets for cancer therapy.[5]Jagged1 and DLL4 are two of the Notch ligands, but their expressions and clinical and pathological significance in PDAC have not been addressed. The present study demonstrated that positive Jagged1 and DLL4 expressions were significantly associated with disease severity and poor prognosis of PDAC.

Jagged1 is widely expressed in a variety of human tissues[27]and involved in the regulation of tissue growth and organ development, the maintenance of normal hematopoietic precursor cells and their proliferation.[28]Jagged1 binds to Notch1, Notch2 and Notch3. Abnormalities in both Notch receptor and ligand expressions and abnormal activation of Notch signal were observed in numerous malignant tumors. Studies[4,29,30]found that Jagged1 and Notch1 expressions were upregulated in human breast cancer and they had a synergistic effect in tumor differentiation, lymph node metastasis, and prognosis. Jagged1 can function alone or together with other signaling pathways to control cell proliferation, apoptosis, cell adhesion, transformation, and tumor angiogenesis.[31]Jagged1/Notch signals are also involved in the transformation of endothelial cells to mesenchymal cells, and stromal cells to vascular smooth muscle cells.[32,33]However, a study[12]did not find the association between Jagged1 expression and the tumor grade, disease stage, and recurrence of PDAC in a small sample size of PDAC. The expression and clinical significance of Jagged1 were

also investigated in other malignant pancreatic tumors. Ikemoto and coworkers[34]found that Jagged1 expression reflects the aggressiveness in intraductal papillary mucinous neoplasm, but another study[35]did not find the association between the aggressiveness and poor prognoses of intraductal papillary mucinous neoplasm. A study[36]using a pancreatic cancer tissue microarray showed that Jagged1 is overexpressed in pancreatic tumors. Moreover, upregulation of Jagged1 was observed in pancreatic adenocarcinoma[37]and the epithelial component of mucinous cystic neoplasms.[38]

As a ligand of Notch signaling, DLL4 can bind to Notch1 and Notch4 receptors on the membranes of adjacent cells and initiate Notch signaling.[34]DLL4 was revealed to be a downstream regulator of vascular endothelial growth factor, play a role in the negative regulation of vascular endothelial growth factor activity, and subsequently inhibit tumor blood vessel sprouting and growth.[13]Recent studies demonstrated that DLL4 plays a critical role in tumor cell carcinogenesis, progression, and prognosis in several cancers, such as lymphoma,[39]leukemias,[40]etc. High DLL4 expression in PDAC was recently reported together with an angiogenic molecule, and the study proposed that the role of DLL4 in the poor prognosis of PDAC is associated with regulation of tumor angiogenesis.[24,25]In contrast, DLL4 expression in other malignant pancreatic tumors has not been reported. The present study compared the clinicopathological significance of two ligands of Notch signaling in PDAC. The results of our study demonstrated that positive DLL4 protein expression was significantly associated with clinicopathological characteristics of PDAC, such as poor differentiation, large tumor size, lymph node metastasis, invasion to surrounding tissues and organs, and high TNM stage. Although positive DLL4 expression was also observed in 16.4% of benign pancreatic tissues, in PDAC tumors, positive DLL4 expression correlated with the severity of disease. Also, positive DLL4 protein expression is a predictive factor of poor prognosis in PDAC patients. In our study, positive Jagged1 protein expression was significantly associated with poor differentiation, large tumor size, lymph node metastasis, invasion, and high TNM stage in PDAC patients. The Notch pathway is widely considered as a key player in cancer stem cell self-renewal and cancer cell survival.[22]Therefore, this study suggests that DLL4 and Jagged1 may be ideal targets for the therapy of PDAC, and high DLL4 and Jagged1 protein expressions are biological markers for identifying high risk subgroups of PDAC patients.

In conclusion, positive Jagged1 and DLL4 protein expressions were associated with disease severity of PDAC and are independent markers for poor prognosis in PDAC patients. Our study highlighted the significance of the Notch signaling pathway in the tumorigenesis, progression and prognosis of PDAC, and in developing therapeutic agents for PDAC.

Contributors: YZL and MXY proposed the study. HSF, LDQ, LZY, WCW, ZQ and YY performed the study. HSF and MXY wrote the first draft. YZL critically revised the manuscript. All authors have read and approved the final manuscript. YZL is the guarantor.

Funding: None.

Ethical approval: This study was approved by the Ethics Committee of the Second and Third Xiangya Hospitals of Central South University, Changsha, China.

Competing interest: The authors do not choose to declare any conflict of interest related directly or indirectly to the subject of this article.

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Received September 20, 2015

Accepted after revision April 6, 2016

Author Affiliations: Research Laboratory of Hepatobiliary Diseases (Huang SF, Yang ZL, Liu ZY, Wang CW and Miao XY), and Department of Pathology (Li DQ), Second Xiangya Hospital, Central South University, Changsha 410011, China; Department of Pathology, Third Xiangya Hospital, Central South University, Changsha 410013, China (Zou Q and Yuan Y)

Zhu-Lin Yang, MD, Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tel: +86-731-88187376; Fax: +86-731-84898168; Email: yangzhulin8@sina.com)

? 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(16)60110-8

Published online June 14, 2016.