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5-Lipoxygenase, a Marker for Early Pancreatic Intraepithelial Neoplastic Lesions

Departments of ¹ Surgery, ² Pathology, and ³ Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois and ⁴ Department of Surgery, University of Heidelberg, Germany

Requests for reprints: Thomas E. Adrian, Department of Surgery, Northwestern University, Tarry Building, 4-711, 303 East Chicago Avenue, Chicago, IL 60611. Phone: 312-503-3489; Fax: 312-503-3491; E-mail: tadrian{at}northwestern.edu.

	Abstract

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Pancreatic cancer has an abysmal prognosis because of late diagnosis. Therefore, it is important to identify risk factors if we are to be able to prevent and detect this cancer in an early, noninvasive stage. Pancreatic intraepithelial neoplasias (PanIN) are the precursor lesions which could be an ideal target for chemoprevention. This study shows up-regulation of 5-lipoxygenase (5-LOX) in all grades of human PanINs and early lesions of pancreatic cancer in two different animal models (EL-Kras mice and N-nitrosobis(2-oxopropyl)amine–treated hamsters) by immunohistochemistry. The results were consistent in all tissues examined, including seven chronic pancreatitis patients, four pancreatic cancer patients, one multiorgan donor, nine EL-Kras mice, and three N-nitrosobis(2-oxopropyl)amine–treated hamsters, all with PanINs. Overexpression of 5-LOX in NIH3T3 cells resulted in greater sensitivity of these cells to the growth inhibitory effects of the 5-LOX inhibitor Rev5901. These findings provide evidence that 5-LOX plays a key role in the development of pancreatic cancer. Furthermore, the lipoxygenase pathway may be a target for the prevention of this devastating disease.

	Introduction

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Patients diagnosed with pancreatic cancer have to face a disease with an abysmal prognosis and little hope for cure because of late diagnosis and lack of effective therapies. Pancreatic cancer is the fourth leading cause of cancer death in both men (after lung, prostate, and colon) and women (after lung, breast, and colon) in the United States and the incidence of this disease has not declined. Indeed, it has increased in Japanese and African Americans over the last decades (1–4). Mortality almost equals incidence and most patients die within 6 months of diagnosis of this disease (1, 5). Potentially curative surgery can only be done in less than 20% of these patients because of metastatic spread or involvement of major blood vessels (5, 6). However, even in this selected group, 5-year survival rate is less than 20% because of early tumor recurrence or metastatic tumor progression (7). Therefore, it is important to identify risk factors and to be able to prevent or detect this cancer at an early, noninvasive stage. Pancreatic intraepithelial neoplasias (PanIN) are the histologically defined precursor lesions in the small ducts and ductules. PanINs may be an ideal target for chemoprevention (8). However, despite the existence of a well-accepted classification for PanINs, there is often a lack of consensus among pathologists regarding the grading of PanINs which, in turn, leads to differences in histologic diagnosis from the same tissue section (8). A particular challenge is the decision of whether a PanIN-1a lesion exists or whether the duct is normal. A common marker for PanIN lesions would be very helpful in this decision-making process.

The results from epidemiologic and animal studies suggest that a high fat consumption is associated with an increased incidence and growth of tumors at several specific organs sites including pancreas, colon, breast, and prostate (9, 10). A recent review pointed out the important role of cyclooxygenase and lipoxygenase pathways in fat metabolism and in the regulation of pancreatic cancer cell proliferation and survival (11). Cyclooxygenases (COX) are the rate-limiting enzymes in prostaglandin synthesis and it is well accepted that the inducible isoform COX-2 plays an important role in carcinogenesis (11). Indeed, one epidemiologic study suggests that frequent use of aspirin might decrease the risk of pancreatic cancer (12). COX-2 up-regulation has been reported in 56% to 90% of pancreatic adenocarcinomas and recently in 65% of PanINs (11, 13). However, the 5-lipoxygenase (5-LOX) pathway seems to play an even more important role in pancreatic carcinogenesis. Among the products and biological mediators of 5-LOX are 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄), which promote pancreatic cancer cell growth (11). 5-LOX is localized in the nucleoplasm and cytoplasm and translocates to the nuclear envelope on cellular activation (14, 15). Recently, we reported marked expression of 5-LOX and the receptor for its downstream metabolite, LTB₄, in human pancreatic cancer tissues, although little or no expression is seen in normal ducts or in cultured cells derived from normal human ducts (16). However, the expression of 5-LOX in PanIN lesions has not been reported. Therefore, we did this immunohistochemical study to evaluate and compare 5-LOX expression in pancreatic adenocarcinomas, PanIN lesions, and normal pancreatic ducts in tissues from patients with pancreatic cancer, chronic pancreatitis, and from multiorgan donors. In addition, we analyzed 5-LOX expression in PanIN lesions from two animal models of pancreatic adenocarcinoma, N-nitrosobis(2-oxopropyl)amine (BOP)–treated hamsters and EL-Kras transgenic mice.

	Materials and Methods

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Materials. The monoclonal 5-LOX antibody was purchased from BD PharMingen (San Diego, CA). Normal goat serum, streptavidin-peroxidase, and 3,3'-diaminobenzidine (DAB) reagent set were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The biotinylated secondary antibody (Multilink) was purchased from BioGenex (San Ramon, CA).

Immunocytochemistry for 5-lipoxygenase. Eleven surgical pancreatic specimens showing PanIN lesions from patients with pancreatic adenocarcinoma or chronic pancreatitis were examined. Six specimens showed PanIN-1a and PanIN-1b lesions, four presented PanIN-2, and four PanIN-3 lesions. Five pancreatic specimens from multiorgan donors were included as controls. However, one of them contained PanIN-1a lesions. Immunostaining was also carried out in pancreatic specimens from two models of pancreatic adenocarcinoma, carcinogen (BOP)-treated hamsters (n = 3) and EL-Kras transgenic mice (mutant Kras; n = 9), as well as from normal hamster (n = 3) and from other six normal mouse pancreatic tissues obtained for the pancreas irrelevant transgenic backgrounds. All specimens were fixed in 10% buffered formalin, paraffin embedded, and processed for histology by conventional methods. Sections (4 µm thick) were prepared from the paraffin blocks. After deparaffinization, the slides were submerged in methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature to inhibit endogenous peroxidase activity. Antigen retrieval was done for 5-LOX by incubating the sections in 10 mmol/L citrate buffer (pH 6) in a microwave oven for 12 minutes (2 minutes high power, 10 minutes medium low power). Thereafter, slides were cooled to room temperature and then washed in TBS (0.1 mol/L, pH 7.4). The slides were incubated with normal goat serum for 30 minutes at room temperature and then with the primary antibody directed against 5-LOX (mouse monoclonal, 1:250 in TBS containing 1% bovine serum albumin) for 18 hours at 4°C. The slides were then washed again in TBS and incubated with biotinylated secondary anti-mouse antibody (Multilink) for 10 minutes at 37°C. Detection of the antibody complex was done by the streptavidin-peroxidase reaction kit using DAB as chromogen. To ensure the specificity of the primary antibody, control tissue sections were incubated in the absence of primary antibody. Counterstaining was done with hematoxylin Gil no. 2. The stained tissue samples were verified by two pathologists.

Determination of the effect of 5-lipoxygenase up-regulation on cell proliferation by transfection of NIH3T3 cells with 5-lipoxygenase cDNA. We first attempted to investigate the effect of overexpression of 5-LOX on proliferation of human pancreatic ductal-derived cells (17) as well as in immortalized bovine pancreatic ductal cells (18); however, these cells proved to be very difficult to efficiently transfect. Therefore, we instead investigated the effect of 5-LOX overexpression in NIH3T3 cells that are readily transfected. NIH3T3 cells (American type Culture Collection, Rockville, MD) were grown in DMEM media with 10% fetal bovine serum (FBS) as monolayers in a humidified atmosphere at 37°C with 5% CO₂ in T75 cm² flasks. The 5-LOX expression vector and the control pcDNA3.1 vector were kindly provided by Dr. C.D. Funk (University of Pennsylvania, Philadelphia, PA). Both pcDNA3.1 control vector and 5-LOX–expressing vector were transfected into NIH3T3 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the protocol provided by the company. The cells were grown in T25 flasks in DMEM media with 10% FBS for 48 hours following transfection. Cells were then treated with 800 µg/mL G418 for 3 weeks to establish the stable transfected cell lines. To confirm the expression of 5-LOX in the stable cell lines, cell lysates from both empty vector– and 5-LOX expressing vector–transfected NIH3T3 cells were prepared with a cell lysis buffer [20 mmol/L Tris-HCl (pH 7.4), 2 mmol/L sodium vanadate, 1.0 mmol/L sodium fluoride, 100 mmol/L NaCl, 2.0 mmol/L phosphate substrate, 1% NP40, 0.5% sodium deoxycholate, 25 g/mL each aprotinin, leupeptin, and pepstatin, 2.0 mmol/L each EDTA and EGTA]. Cell lysates were clarified by microcentrifugation at 12,000 x g following incubation at 4°C for 25 minutes. Supernatants were recovered and their protein concentrations measured using a protein assay reagent (Bio-Rad, Hercules, CA). Samples of each lysate containing 20 µg protein were resolved by SDS-PAGE (10%). Proteins were then transferred to nitrocellulose membranes by electroblotting using a mini semidry transfer blotting apparatus (Bio-Rad). The 5-LOX protein was detected on the membrane using a monoclonal 5-LOX antibody as previously described (16).

The effect of 5-LOX expression on the proliferation of NIH3T3 cells was investigated using both [³H]thymidine incorporation and cell counting (19). To examine the responsiveness of the 5-LOX–overexpressing cells to 5-LOX inhibition, cells were treated with a 5-LOX inhibitor, Rev5901 (Calbiochem, San Diego, CA), for 24 hours and cell proliferation was determined by [³H]thymidine incorporation.

Statistical analysis. Data on 5-LOX expression by immunocytochemistry in humans and thymidine incorporation assays in 5-LOX–overexpressing NIH3T3 cells were analyzed by ANOVA with the Student-Newman-Keuls post hoc test for multiple comparisons. Immunostaining data from the hamster and mouse tissues were analyzed by the Mann-Whitney rank sum test.

	Results and Discussion

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We recently reported that 5-LOX is overexpressed in 9 of 10 human pancreatic adenocarcinomas with an intense staining in cancer cells, ductal cells, and adjacent islets (16). Moreover, strong staining of ductal cells was also seen in seven of nine chronic pancreatitis specimens and in six of seven pancreatic tissues obtained from the resection margins of pancreatic adenocarcinomas. In contrast, 5-LOX stained less than 7.5% of ductal cells in 10 tissues obtained from multiorgan donors (16). The observation that 5-LOX is up-regulated in cancer cells as well as ductal cells from primary and secondary chronic pancreatitis specimens led us speculate that 5-LOX may be important for the development of pancreatic cancer. Therefore, we did the present study to investigate the expression of 5-LOX in all grades of PanIN lesions.

5-Lipoxygenase is expressed in all grades of human pancreatic intraepithelial neoplastic lesions. In addition to the previously stained tissue sections, all of the new examined PanIN-1a, PanIN-1b, PanIN-2, and PanIN-3 lesions from 11 human pancreatic specimens showed strong positive staining for 5-LOX in more than 90% of the ductal cells (Fig. 1). No difference in staining intensity between the different grades was evident. We incidentally found PanIN-1a lesions in one pancreatic specimen from a multiorgan donor. This also showed marked expression of 5-LOX. Moreover, intense staining of islet cells was shown in four pancreatic specimens, all from patients with pancreatic adenocarcinoma. The positive staining was evident in the cytoplasm and at higher intensity in the nucleoplasm and nuclear envelope. Consistent with our previous study, 5-LOX expression was detected in 0% to 7.5% of ductal cells in the five pancreatic specimens from multiorgan donors (Fig. 1). The difference in staining of PanIN lesions in pancreas from patients with chronic pancreatitis or pancreatic cancer compared with ductal cells in normal pancreas was highly statistically significant (both P < 0.01). Controls with the first antibody omitted showed no staining. Detailed information about PanIN grades and staining intensity is shown in Table 1. As reported in our previous study, 5-LOX staining was also prominent in islets from tissue surrounding pancreatic cancers, but not in islets from normal pancreas or chronic pancreatitis.

View larger version (106K): [in this window] [in a new window]   Figure 1. 5-LOX expression in human, mouse, and hamster pancreatic tissues. Immunohistochemistry of 5-LOX in human (A-J), mouse (K-M), and hamster (N-P) tissues. A, normal human pancreas with an unstained main duct with the exception of two ductal cells showing nuclear staining (x200); B, unstained small ducts in normal pancreas (x400); C and D, PanIN-1a with strong nuclear staining (x200, x400); E and F, PanIN-2 lesions with strong positive staining in cytoplasm, nucleus, and nuclear membrane (x200, x400); G and H, PanIN-3 with intense staining in nuclei (x200, x400); I and J, PanIN-3/cancer with strong staining for 5-LOX in the nuclear envelope (x400); K, normal mouse pancreas with no staining in ducts or acini (x400); L and M, pancreas from EL-Kras transgenic mouse with strong positive staining in PanIN-like lesions and surrounding acinar cells; N, normal hamster pancreas with unstained ductal cells and weak cytoplasmic staining in acinar cells (x400); O and P, PanIN-like lesions after treatment with the carcinogen BOP showing intense cytoplasmic and nuclear envelope staining of PanINs (x200, x400). The immunohistochemistry for 5-LOX used mouse monoclonal antibody, 1:250, microwave citrate buffer pretreatment, incubation overnight at 4°C, and DAB. Hematoxylin was used for counterstaining all sections.

5-Lipoxygenase is expressed in EL-Kras transgenic mice. All of the PanIN-like lesions in 9 tissues obtained from EL-Kras transgenic mice showed marked expression of 5-LOX, with the strongest staining in the cytoplasm and nuclear envelope (Fig. 1). The difference in staining between the mouse PanIN lesions and normal ductal cells was highly significant (P = 0.002). Ductal cells in six normal mouse pancreatic tissues showed no staining. Whereas constitutive expression of 5-LOX was seen in acinar cells in normal hamster specimens, it was not or in two cases only weakly seen in acinar cells in normal mouse pancreas. However, EL-Kras transgenic mice with PanIN-like lesions showed strong cytoplasmic 5-LOX staining in acinar cells (Fig. 1). This difference was statistically significant (P < 0.002). This is of interest regarding the cellular origin of PanIN lesions because the elastase promoter used induces expression of mutant Kras in the acinar cells of these animals. The Kras transgenic mice express mutant Kras and develop mouse PanINs after about 1 year without progression to invasive tumors during their lifetime. However, this presented an additional opportunity to study 5-LOX expression in early ductal lesions. Detailed information about PanIN-like lesion grading and staining intensity is shown in Table 1.

Significance and consequences of 5-lipoxygenase expression in pancreatic cancer. Cytosolic staining represents the constitutive expression of 5-LOX, whereas staining of the nuclear envelope results from Ca²⁺-dependent translocation and activation of the enzyme (14, 15). From the present study, we conclude that 5-LOX is markedly up-regulated in early (PanIN) lesions as well as in pancreatic cancers. Marked expression is also seen in the PanIN-like lesions in BOP-treated hamsters and EL-Kras transgenic mice, strongly supporting our findings in human tissues. It is tempting to speculate that expression and activation of 5-LOX is a very early event in pancreatic carcinogenesis which, in turn, may play a key role in the development of this devastating disease. Because of the lack of effective treatment options, late diagnosis, and poor prognosis for pancreatic cancer patients, it is crucial to understand the risk factors involved and to develop preventive strategies for this disease. Maitra et al. (13) reported that COX-2 is up-regulated in pancreatic adenocarcinomas and PanINs, with expression relatively increased from normal ducts through PanINs to adenocarcinoma. COX-2 was expressed in 77% of pancreatic adenocarcinomas, 65% of PanINs, and 40% of normal ducts, where at least 20% of cells showed positive staining. This group suggested that COX-2 could be a potential target for chemoprevention in patients at risk (e.g., members of families with pancreatic cancer, patients with germ line mutations, and patients with hereditary pancreatitis; ref. 13). In contrast to lack of expression in normal ductal cells, 5-LOX is already markedly up-regulated in PanIN-1a lesions, suggesting that 5-LOX overexpression is an earlier event than COX-2 up-regulation. Moreover, 5-LOX expression was shown in all (100%) of the investigated PanINs with more than 90% of cells staining positive. To our knowledge, this is the first investigation of 5-LOX in PanIN lesions. The 5-LOX metabolites 5(S)-HETE and LTB₄ stimulate pancreatic cancer cell proliferation, whereas 5-LOX inhibitors and LTB₄ receptor antagonists inhibit pancreatic cancer cell growth in vitro and in vivo by inducing apoptosis through the mitochondrial pathway (19, 21). If these data are considered in the light of marked activation of 5-LOX in PanIN lesions, it is reasonable to speculate that targeting 5-LOX in these very early ductal lesions could delay progression towards pancreatic adenocarcinoma. Recently, 5-LOX inhibitors have been introduced into the clinic for the treatment of chronic inflammatory diseases and are well tolerated (22). However, as Maitra et al. pointed out in their study, the effectiveness of such an approach needs to be shown in clinical trials in people at high risk for pancreatic cancer (13). It is notable that 5-LOX is up-regulated in PanIN lesions from patients with primary chronic pancreatitis as well as in the malignant environment of pancreatic adenocarcinoma. Chronic pancreatitis patients are at increased risk of pancreatic cancer and could also benefit from chemopreventive therapy. It is also tempting to speculate that 5-LOX inhibitor therapy would prevent pancreatic cancer development in patients with hereditary pancreatitis. Interestingly, in normal pancreatic tissues from multiorgan donors, a few scattered cells show strong nuclear staining for 5-LOX, particularly in the larger pancreatic ducts, raising the question of whether these cells could have the potential for development of PanINs and pancreatic adenocarcinomas.

Because lipoxygenase metabolites influence insulin secretion, the observation that 5-LOX is up-regulated in islets adjacent to pancreatic cancers may have importance in the understanding of the relationship between pancreatic cancer and diabetes, as well as the importance of islets for the development of pancreatic adenocarcinoma (16). Therefore, observing the effects of 5-LOX inhibitors on pancreatic cancer-associated diabetes would be of particular interest.

5-Lipoxygenase promotes cell proliferation and increases growth-inhibitory response to the 5-lipoxygenase inhibitor Rev5901 in NIH3T3 cells. 5-LOX protein was not detected in the empty vector–transfected NIH3T3 cells, whereas prominent expression of 5-LOX was visualized by Western blotting in stable 5-LOX–expressing cells (Fig. 2A). 5-LOX overexpression significantly increased the proliferation of NIH3T3 cells, as measured by both thymidine incorporation and cell counting (Fig. 2B and C), compared with control cells. To confirm that the increased proliferation in 5-LOX–expressing cells was mediated by overexpressed 5-LOX, a 5-LOX inhibitor, Rev5901, was employed. Compared to control cells, 5-LOX–expressing NIH3T3 cells were more sensitive to Rev5901-induced growth inhibition (Fig. 2C). The 5-LOX inhibitor also significantly inhibited cell proliferation in control cells, possibly because of low level background expression of 5-LOX or because of other growth inhibitory effects of this chemical inhibitor. Indeed, this compound is known to act as a LTB₄ receptor antagonist and we have previously shown that LTB₄ antagonists can inhibit cellular proliferation as discussed above (19).

View larger version (28K): [in this window] [in a new window]   Figure 2. A, up-regulation of 5-LOX protein (Western blotting) in NIH3T3 cells after stable transfection with 5-LOX. B and C, 5-LOX promotes cell proliferation and increases growth-inhibitory response to the 5-LOX inhibitor Rev5901 in transfected NIH3T3 cells, shown by [3H]thymidine incorporation (B) and cell counting (C).

	Acknowledgments

 
Grant support: The National Cancer Institute Specialized Program of Research Excellence program (CA72712), the American Institute for Cancer Research (00B065), and the Michael Rolfe Foundation. R. Hennig received a fellowship award from the Deutsche Forschungsgemeinschaft.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/15/04. Revised 3/ 7/05. Accepted 4/27/05.

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