High Levels of Nucleolar Expression of Nucleolin Are Associated with Better Prognosis in Patients with Stage II Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death in the United States (1). Only 15% to 20% of patients diagnosed with PDA are surgically resectable and most of the patients who undergo resection have stage II disease. The long-term survival rate of patients who have curative resection is approximately 20%, with a median survival of 18 to 24 months (1). Despite pancreaticoduodenectomy, the disease commonly recurs, with the most common sites of recurrence being the liver, lung, peritoneal cavity, and pancreatic surgery bed; the prognosis is poor (1).

Previous studies have shown that multiple genes are frequently altered in PDAs, including the activation of the K-ras oncogene by point mutation (2) and inactivation of the tumor suppressor genes p16 (3), SMAD4/DPC4 (4, 5), and p53 (6). More recently, it has been shown that telomere shortening occurs at the early stage during the development of PDA. Telomere shortening was detected in 91% of the pancreatic intraepithelial neoplasia 1A (PanIN 1A), the earliest putative precursor lesion for PDAs (7). Telomere length is maintained by the human telomerase complex, which is composed of human telomerase RNA (hTR or TERC), and telomerase-associated protein 1 (hTEP1) and its catalytic subunit-human telomerase reverse transcriptase (hTERT; refs. 8–10). Telomerase activity has been detected in 80% of surgically resected PDAs and is increased in 75% to 95 % of pancreatic juice samples obtained from patients with PDAs (11–13). These data suggest that telomere shortening and telomerase play a role in the development of PDA. Critical shortening in telomere length may lead to progressive accumulation of chromosomal abnormalities and the progression of PanIN lesions to invasive PDA.

The function and intracellular localization of the telomerase complex are regulated in part by nucleolin, which is a multifunctional and mobile protein that can shuttle among the nucleolus, nucleoplasm, cytoplasm, and cytoplasmic membrane (14, 15). Nucleolin is overexpressed in highly proliferative cells and is involved in many aspects of gene expression, including chromatin remodeling, DNA recombination and replication, gene transcription, mRNA stability, etc. (15, 16). Cytosolic nucleolin has been shown to suppress the translation and induction of p53 after DNA damage (17) and to regulate the stability of BCL-2 mRNA in breast cancer cells (18). Nucleolin has also been shown to be expressed on the cell surface of endothelial cells and different types of cancer cells (19–21). Nucleolin expression on cell surface has been used as a marker for angiogenic endothelial cells and functions as a receptor for endostatin, which mediates the antiangiogenic and antitumor effects of endostatin (19, 21). More recently, high levels of nucleolin expression have been shown to correlate with poor survival in patients with cutaneous melanoma and pediatric intracranial ependymoma (22, 23). Collectively, these studies suggest that nucleolin plays a role in human malignancies. However, nucleolin expression and its significance in PDA have not been examined. Because majority of the patients with surgically resectable PDAs have stage II disease and little is known about the prognosis in this group of patients, we examined nucleolin expression in 69 pancreaticoduodenectomy specimens with stage II PDA and their paired nonneoplastic pancreatic ductal epithelial cells. Using univariate and multivariate analysis, we determined whether the expression level of nucleolin was associated with survival and other clinicopathologic features in patients with stage II PDA.

We retrospectively reviewed the medical records and tissue specimens of 69 patients with stage II PDA (25 stage IIA and 44 stage IIB) who had undergone pancreaticoduodenectomy at the University of Texas M. D. Anderson Cancer Center between 1990 and 2005 and who had not received any form of preoperative chemotherapy and/or radiation therapy. Patients who received preoperative chemotherapy and/or radiation and who died from postoperative complications were excluded. Only two patients with stage I disease and three patients with stage IV disease diagnosed by positive peritoneal washing on cytology at the time of surgery, but no patients with stage III disease, were identified in our study group. These five cases were excluded from this study because the number of patients with stage I and IV disease were too small to be representative. The median patient age at time of surgery was 62 years (range, 40-80 years). Fifty-two patients received postoperative chemotherapy and/or radiation therapy due to recurrence or distant metastasis. This study was approved by the Institutional Review Board of the University of Texas M. D. Anderson Cancer Center.

To construct the tissue microarray used in this study, formalin-fixed, paraffin-embedded archival tissue blocks and their matching H&E-stained slides were retrieved, reviewed, and screened for representative tumor regions and nonneoplastic pancreatic parenchyma by a pathologist (H. W.). For each patient, two cores of tumor and two cores of paired nonneoplastic pancreatic tissue were sampled from representative areas using a 1.0-mm punch. The tissue microarray was constructed with a tissue microarrayer (Beecher Instruments) as described previously (24).

Immunohistochemical stain for nucleolin was done on 4-μm unstained sections from the tissue microarray blocks using a mouse monoclonal antibody against nucleolin (4E2, Abcam Inc.). To retrieve the antigenicity, the tissue sections were treated at 100°C in a steamer containing 10 mmol citrate buffer (pH 6.0) for 60 minutes. The sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase activity and were incubated in 2.5% blocking serum to reduce nonspecific binding. Sections were incubated for 90 minutes at 37°C with primary antinucleolin antibody at a 1:2,000 dilution. Standard avidin-biotin immunohistochemical analysis of the sections was done according to the manufacturer's recommendations (Vector Laboratories). Diaminobenzidine was used as a chromogen, and hematoxylin was used for counterstaining.

The immunohistochemically stained slides of PDA tissue microarrays were scanned at ×200 magnification with the Ariol 2.1 scanner and digital imaging instrument (Applied Imaging). The staining results were scored quantitatively using the Ariol image analysis system (Applied Imaging). To measure nucleolin expression in PDA cells and nonneoplastic pancreatic ductal epithelial cells, tumor cells or nonneoplastic pancreatic ductal cells were selected and marked with a hand-draw tool. The stromal cells, pancreatic acinar cells, and islet cells were not included for the measurement of nucleolin expression levels. Figure 1 shows representative micrographs of tissue cores and the selected PDA cells or nonneoplastic pancreatic ductal epithelial cells from the corresponding tissue cores for quantitation of nucleolin expression levels. The nucleolin labeling index in tumor cells or nonneoplastic pancreatic ductal epithelial cells was measured as a ratio between the stained nucleolar area and the total nuclear area. The computerized images and the nucleolin labeling index were reviewed independently by two pathologists (L. P. and H.W.). Cases were categorized into two groups based on the median nucleolin labeling index in PDA specimens: nucleolin-high (nucleolin labeling index ≥3.5%) and nucleolin-low (nucleolin labeling index <3.5%).

The pancreatic cancer cell lines Panc-1, AsPC-1, and Capan-1 were purchased from the American Type Culture Collection. The Panc-48, CFPAC-1, Panc-3, Panc-28, Capan-2, MIA PaCa-2, and Hs766T pancreatic cancer cells were generously provided by Dr. Paul Chiao (The University of Texas M. D. Anderson Cancer Center). All cell lines were maintained either in DMEM or in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO₂ at 37°C. The HPDE cell line, an immortalized normal pancreatic ductal cell line, was provided as a generous gift from Dr. Ming-Sound Tsao (Ontario Cancer Institute, Toronto, ON, Canada). Protein expression of nucleolin was analyzed by 10% SDS-PAGE, which was electroblotted onto polyvinylidene difluoride membranes (Novex), blocked in 5% skim milk in 1× TBS, and probed with the antibodies against nucleolin or actin. Proteins were detected using an enhanced chemiluminescence (ECL) kit (Amersham-Pharmacia Biotech).

MPanc-96 cells with 90% confluence were collected and washed with PBS buffer. The cell pellets were resuspended in 800 μL of buffer A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, plus cocktail of protease inhibitors] and lysed on ice for 15 minutes. Then 50 μL of 10% NP-40 were added and the tubes were vortexed briefly and vigorously. The nuclear pellets were collected by centrifugation at 1,500 rpm for 30 seconds. The nuclei were then lysed in 200 μL of buffer C [20 mmol/L HEPES (pH 7.9), 0.4mol/L NaCl, 1 mmol/L EDTA, 1mmol/L DTT, plus cocktail of protease inhibitors] in cold room with vigorous shaking for 15 minutes. Nuclear extracts (300 μg) in 300 μL of protein lysis buffer were incubated with 2 μg of antinucleolin or anti-hTERT antibody (Santa Cruz Biotechnology Inc.) at 4°C for overnight with gentle shaking. Fifteen microliters of protein G for antinucleolin antibody or protein A for anti-hTERT antibody were added and incubated for 2 hours on ice to pull down the immunocomplex. The precipitated proteins were washed three times with protein lysis buffer, separated by 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and detected by Western blotting using anti-hTERT and antinucleolin antibodies.

Categorical data were compared by χ² analysis or Fisher's exact tests. Overall survival curves were constructed using the Kaplan-Meier method, and the log-rank test was used to evaluate the statistical significance of differences. The patients' follow-up information through August of 2009 was extracted from the medical records and, if necessary, updated by review of the U.S. Social Security Index. The recurrence information was updated every time a patient came to the clinic/institution for a follow-up visit. Overall survival was calculated from the time of surgery to the time of death from any cause or to the time of last follow-up, at which point the data were censored. The prognostic significance of clinical and pathologic characteristics was determined using univariate Cox regression analysis. Cox proportional hazards models were fitted for multivariate analysis. After interactions between the variables were examined, a backward stepwise procedure was used to derive the best-fitting model. Statistical analysis was done using Statistical Package for Social Sciences software (for Windows 12.0, SPSS Inc.). We used a two-sided significance level of 0.05 for all statistical analyses.

Immunohistochemical stain for nucleolin showed nucleolar staining pattern in both PDA cells and benign pancreatic ductal epithelial cells. No cytoplasmic staining or membranous staining was detected in either PDA cells or benign pancreatic ductal epithelial cells. Representative micrographs showing nucleolin staining of nonneoplastic pancreatic ductal epithelium, and nucleolin-low and nucleolin-high tumors are shown in Fig. 2A to C. Nucleolar staining for nucleolin was also present in all 10 PDA cell lines included in our tissue microarray, and the nucleolin staining in Panc-1 cells is shown in Fig. 2D. The mean nucleolin labeling index in PDA cells was 5.8% ± 3.4% (median, 3.5%) in 69 samples. The nucleolar expression of nucleolin was higher in PDAs compared with that in paired samples of nonneoplastic pancreatic ducts (0.7% ± 0.5%, P < 0.0001, Fig. 3A). None of benign pancreatic ducts showed a nucleolin labeling index of ≥3.5% (the median nucleolin labeling index in PDAs). In contrast, 34 of 69 (49%) PDAs showed a nucleolin labeling index of ≥3.5% (nucleolin-high). Similar results were obtained using another mouse monoclonal antinucleolin antibody (clone 44F12, Novocastra; data not shown; ref. 25). To confirm our immunohistochemical staining results, we did Western blot analysis to measure the expression levels of nucleolin in 10 different PDA cell line and HPDE cells, an immortalized normal pancreatic ductal cell line. Consistent with our immunohistochemical staining results, the expression levels of nucleolin was significant higher in all 10 PDA cell lines compared with HPDE cells (Fig. 3B).

To examine the function of nucleolin in PDA cells, we carried out coimmunoprecipitation assay using the nuclear extracts prepared from MPanc-96 cells and antinucleolin or anti-hTERT antibodies. We found that antinucleolin antibody could coprecipitate endogenous hTERT and anti-hTERT antibody could coprecipitate endogenous nucleolin (Fig. 3C). These data showed that nucleolin interacted with nuclear hTERT in MPanc-96 cells and may regulate hTERT activity in pancreatic cancer cells.

Table 1 summarizes the clinicopathologic characteristics of the study population. There were 47 men and 22 women, ranging from 40 to 80 years of age. According to the WHO classification, 10 (15%) tumors were well-differentiated, 41 (59%) moderately differentiated, and 18 (26%) poorly differentiated adenocarcinomas. Twenty-five (36%) patients had stage IIA tumors and 44 (64%) had stage IIB tumors. Fifty-five (80%) patients had R0 resection (defined as all margins negative microscopically), 14 (20%) had R1 resection, and no patient had a grossly positive margin of resection. No significant associations were identified between nucleolin expression and patient's age, gender, tumor size, differentiation, margin status, regional lymph node metastasis, or recurrence.

After pancreaticoduodenectomy, the median and mean follow-up times were 22.9 months and 38.0 months, respectively. No patient was lost to follow-up. High levels of nucleolin expression correlated with better overall survival in patients with stage II PDA. The median overall survival was 65.2 ± 16.3 months for patients who had nucleolin-high PDAs compared with 19.5 ± 3.3 months for patients whose tumors were nucleolin-low (P = 0.03, log-rank method; Fig. 4A and Table 2). There was no significant difference in recurrence-free survival between the patients whose tumors were nucleolin-high and those with nucleolin-low tumors (P = 0.17; Fig. 4B). In multivariate analysis, nucleolin-high expression and tumor differentiation were prognostic factors for overall survival in patients with stage II PDA independent of patient's age, gender, tumor size, and lymph node status (Table 2).

In this study, we found that nucleolar expression of nucleolin was significantly higher in PDA tumor samples and PDA cell lines than in nonneoplastic pancreatic ductal epithelial cells or HPDE cells. A high level of nucleolar expression of nucleolin was associated with better survival and was an independent prognostic factor in stage II PDAs. Our results suggested that nucleolar expression of nucleolin may play a role in the progression of pancreatic cancer.

Nucleolin is a major nucleolar protein and has been shown to be overexpressed in rapidly dividing cells and cancer cell lines. Nucleolin plays an essential role in cell proliferation and survival (16, 26). Conditional knockout of nucleolin markedly inhibits cell proliferation in DT40 avian B-cell lymphoma cells (27). In this study, we found that nucleolin was expressed at high levels only in the nucleolus of PDA cells. The nonneoplastic pancreatic ductal epithelial cells also showed nucleolar expression of nucleolin, but at significantly lower levels than PDA cells. Consistent with our immunohistochemical staining results, all 10 PDA cell lines examined in this study showed significantly higher levels of nucleolin expression compared with HPDE cells, an immortalized normal pancreatic ductal epithelial cell line. Although nucleolin has been reported previously to be expressed in the cytoplasm and cytoplasmic membrane, we did not detect cytoplasmic or membranous expression of nucleolin either in PDA cells or in nonneoplastic pancreatic ductal epithelial cells. Given the fact that nucleolin plays a critical role in the nucleolar localization and the function of hTERT (14, 15), it is possible that nucleolin colocalizes with hTERT to the nucleolus in PDA cells and regulates the formation of active telomerase complex and biological functions of hTERT (28–30). Consistent with this hypothesis, we showed that endogenous nucleolin interacted with hTERT in the nuclei of PDA cells. Therefore, our findings support the notion that telomere shortening with the resultant chromosome instability, upregulation of nucleolar expression of nucleolin, and activation of telomerase plays a role in the development of PDA.

Using a mouse monoclonal anti-hTERT antibody (clone 44F12, Novocastra), recent studies indicated that nucleolar expression of hTERT is associated with worse survival and is a better predictor than established clinical indicators in patients undergoing curative hepatic resection for colorectal metastases (31, 32), whereas others showed that nucleolar hTERT expression is associated with better survival of patients with urothelial bladder cancer (33). However, this antibody (clone 44F12) has been shown to recognize nucleolin, but not hTERT (25). Using a monoclonal antibody against nucleolin, we found that a high level of nucleolar expression of nucleolin in stage II PDAs was associated with better overall survival and was an independent prognostic factor in patients with stage II PDAs. Similar results were obtained using the mouse monoclonal antibody (clone 44F12, data not shown). However, we did not observe significant correlation between nucleolar expression of nucleolin and other clinicopathologic parameters in stage II PDAs. Better survival in the nucleolin-high tumor group may be due to the fact that nucleolin increases telomerase activity, which may protect against genetic instability and prevent evolution of more aggressive tumor clones leading to more aggressive behavior and distant metastasis (33, 34). Consistent with this hypothesis, it was recently shown that low expression levels of hTERT mRNA in PDAs are associated with worse prognosis and poor overall survival when compared with the patients whose tumors express high levels of hTERT mRNA (35).

In conclusion, our results showed that nucleolar expression of nucleolin was markedly increased in PDAs compared with nonneoplastic pancreatic ductal epithelial cells. A high level of nucleolar expression of nucleolin in stage II PDAs was associated with a better prognosis and was an independent prognostic marker for stage II PDAs.

No potential conflicts of interest were disclosed.

We thank Dr. Jinsong Liu for providing valuable comments.

Grant Support: AACR-Pancreatic Cancer Action Network Career Development Award in Pancreatic Cancer Research and the Institutional Research Grant from The University of Texas M. D. Anderson Cancer Center.

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