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Clinical Cancer Research Vol. 6, 566-571, February 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Neurotensin Receptor-1 mRNA Analysis in Normal Pancreas and Pancreatic Disease

Li Wang, Helmut Friess1, Zhaowen Zhu, Hans Graber, Arthur Zimmermann, Murray Korc, Jean-Claude Reubi and Markus W. Büchler

Department of Visceral and Transplantation Surgery [L. W., H. F., Z. Z., H. G., M. W. B.], Institute of Pathology [A. Z.], and Division of Cell Biology and Experimental Cancer Research [J-C. R.], Institute of Pathology, University of Bern, Inselspital, CH-3010 Bern, Switzerland, and Departments of Medicine, Biological Chemistry, and Pharmacology, University of California, Irvine, California 92697 [M. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By autoradiography, neurotensin (NT) binding is specifically detectable in pancreatic cancer, but not in the normal pancreas, chronic pancreatitis (CP), or other pancreatic disorders. In the present study, we investigated whether this is due to NT receptor-1 (NTR-1) mRNA up-regulation and whether NTR-1 mRNA could also be used as a specific diagnostic marker and treatment target in pancreatic cancer.

Fifteen normal pancreas tissue samples, 20 CP samples, and 30 pancreatic cancer samples were studied. Expression and localization of NTR-1 mRNA was investigated by Northern blot analysis and in situ hybridization. Furthermore, consecutive tissue sections were analyzed for NTR-1 mRNA expression and NT binding.

By Northern blot analysis, NTR-1 mRNA expression was 4.4-fold (P < 0.01) and 3.0-fold (P < 0.01) higher in pancreatic cancer and CP tissue samples, respectively, compared with normal controls. There was no difference in NTR-1 mRNA levels between CP and cancer samples (P > 0.05). In pancreatic cancer, the NTR-1 mRNA levels were higher in advanced tumor stage (stages III and IV) than early tumor stage (stages I and II; P < 0.05), but no difference was found between well/moderately differentiated (grades 1 and 2) and poorly differentiated/undifferentiated cancers (grades 3 and 4; P > 0.05). By in situ hybridization, NTR-1 mRNA signals were weakly present in the cytoplasm of acinar and ductal cells of the normal pancreas. Moderate to intense NTR-1 mRNA signals were present in the cytoplasm of acinar cells dedifferentiating into tubular complexes and degenerating acinar cells of CP samples. In the cancer samples, NTR-1 mRNA was moderately to intensely expressed in the cytoplasm of cancer cells. When on consecutive tissue sections NTR-1 mRNA expression was compared with the presence of NTR-1, measured by receptor autoradiography, a correlation was found in carcinomas but not in CP samples, in which no receptors were detectable by autoradiography.

The enhanced expression of NTR-1 mRNA in pancreatic cancer cells further suggests that neuroendocrine hormones might modulate pancreas cancer cell behavior. However, its relatively high levels in CP excludes NTR-1 mRNA as a specific parameter for pancreatic cancer and for the differentiation of pancreatic cancer from CP.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic cancer is the fourth or fifth leading cause of cancer-related death in most Western countries (1) . Although there has been some improvement recently in the strategies used to diagnose and treat pancreatic cancer, its prognosis is still poor, due to the low rate of early detection, the difficulty of clearly differentiating pancreatic cancer from benign pancreatic lesions, and its aggressive growth behavior (2) . To achieve better insight into the disease mechanisms, it is important to better understand the molecular characteristics of pancreatic cancer. It has already been demonstrated that classical growth factors and their receptors (such as epidermal growth factor, TGF-{alpha},2 epidermal growth factor receptor family, TGF-ßs, and TGF-ß receptors) have an influence on the process of pancreatic cancer progression (3, 4, 5, 6) . Although there is experimental evidence that gastrointestinal hormones can stimulate pancreatic cancer cell growth, little is known about their potential functions in pancreatic cancer growth in humans in vivo.

NT, a tridecapeptide, was originally isolated from bovine hypothalamus (7) . It is mainly present in the central nervous system and functions as a neurotransmitter. However, NT is also found in specialized enteroendocrine cells (N cells) and in neurons within the myenteric plexus, indicating that this peptide also has a wide spectrum of physiological actions in the gastrointestinal system (8) . This includes the stimulation of pancreatic (9) and biliary secretion (10) , stimulation of colonic motility (11) , and inhibition of small bowel and gastric motility (12) . However, NT also stimulates cell growth of prostate cancer cells (13) , small-cell lung cancer (14) , and cultured human pancreatic cancer cells (15) . These observations indicate that this neuroendocrine hormone might also possess an important role in the regulation of cancer cell growth. Recent studies have found that the undifferentiated pancreatic cancer cell line MIA PaCa-2 is growth stimulated by NT, that it expresses functional NTRs, and that the NT-stimulated cancer cell growth can be strongly inhibited by a nonpeptide NTR antagonist (16 , 17) .

NT can bind two subtypes of NTRs, NTR-1 and NTR-2 (18 , 19) . The human NTR-1 consists of 418 amino acids and belongs to a large superfamily of receptors coupled to G proteins (20) . NTR-1 is present in different organs, and its biological function has been studied in vitro and in vivo, whereas the exact function of NTR-2 is presently not known (18, 19, 20, 21) . Recently, we reported that by autoradiography, NT binding sites in human pancreatic cancer tissues are strongly present, whereas NT binding is minor or absent in CP and in normal controls (22) . These findings suggested that NTR binding might be a specific marker for differentiation of CP from pancreatic cancer and that it might be useful as a selective factor for genetic targeting.

Therefore, the purpose of the present study was to analyze NTR-1 mRNA expression and localization in normal pancreas, CP, and human pancreatic cancer tissues by Northern blot analysis and in situ hybridization to further evaluate whether increased NT binding by autoradiography is caused by NTR up-regulation on the mRNA level. If this were true, this gene would qualify for NTR promotor-regulated prodrug activation. In addition, we studied whether NTR-1 mRNA analysis might be a reliable marker to differentiate pancreatic cancer from CP.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tissue Collection.
Normal human pancreatic tissue samples were obtained from 15 individuals (9 female and 6 male) who were free of pancreatic disease, through an organ donor program in which no candidates for pancreatic transplantation were present. The median age of the organ donors was 55 years, with a range of 28–67 years. All normal tissue samples were obtained from the head of the pancreas to ensure comparability with the CP and cancer samples.

CP tissues were obtained from 3 female and 17 male patients undergoing a pancreatic head resection due to CP. The median age of the CP patients was 48 years, with a range of 37–64 years.

Pancreatic cancer tissues were obtained from 30 patients (17 female and 13 male) undergoing a partial duodenopancreatectomy (Whipple resection) for pancreatic cancer. The median age of the pancreatic cancer patients was 62 years, with a range of 53–73 years. According to the tumor-node-metastasis classification and histopathological grading of the Union International Contre Cancer (23) , there were 4 stage I, 5 stage II, 18 stage III, and 3 stage IV tumors. Tumor grading showed 9 well-differentiated (grade 1), 7 moderately differentiated (grade 2), 12 poorly differentiated (grade 3), and 2 undifferentiated (grade 4) tumors.

Freshly removed tissue samples were snap frozen in liquid nitrogen in the operating room upon surgical removal and maintained at -80°C until use for RNA extraction and in situ hybridization. The studies were approved by the Human Subject Committee of the University of Bern.

Northern Blot Analysis.
Total RNA was extracted using the single-step guanidinium isothiocyanate method (24) , followed by electrophoresis under denaturing conditions in a 1.2% agarose/1.8 M formaldehyde gel, as previously reported (25, 26, 27) . The gels were stained with ethidium bromide for verification of RNA integrity and loading equivalency. The RNA was electrotransferred onto nylon membranes (Gene Screen, DuPont, Boston, MA) and cross-linked by UV irradiation. The filters were then prehybridized, hybridized, and washed under highly stringent conditions. The blots were prehybridized overnight at 42°C in a buffer containing 50% formamide, 1% SDS, 0.75 M NaCl, 5 mM EDTA, 5x Denhardt’s solution, 100 µg/ml salmon sperm DNA, 10% dextran sulfate, and 50 mM sodium phosphate (pH 7.4). Hybridization followed under the same conditions, with incubation of 1 x 106 cpm/ml of the32P-labeled NTR-1 cDNA probe and then washing twice at 50°C in 2x SSC and three times at 55°C in 0.2x SSC and 2% SDS.

To assess equivalent RNA loading and transfer, all blots were rehybridized with a mouse 32P-labeled 7 S cDNA probe, which cross-hybridizes with human 7 S RNA (25, 26, 27) . Hybridization was done as for NTR-1, with the exception that only 1 x 105 cpm/ml 32P-labeled 7 S cDNA probe was used in the hybridization solution.

All blots were exposed at -80°C to Fuji X-ray films with Kodak intensifying screens for 1–10 days. The intensity of the radiographic bands was quantified by a computerized video imaging system and the Image-pro-plus 3.0 software (Media Cybernetics, Silver Spring, MD). The ratios of the optical densities of the RNA levels (NTR-1/7 S) were calculated for each sample and used for statistical analysis.

The human NTR-1 (hNTR-1) cDNA probe used for Northern blot analysis was a 2.8 kb BamHI/HindIII fragment (containing full sequence of the coding region) of hNTR-1 plasmid, which was kindly provided by Dr. Pascale Chalon (Unite Biologie Molecularire du Gene, Sanofi Elf Biorecherches, France; Refs. 28 and 29 ). A 190-bp BamHI/BamHI fragment of mouse 7 S cDNA was subcloned into the pGEM 7ZF(+) (Promega Biotechnology, Madison, WI). Both the hNTR-1 and the 7 S cDNA probes were labeled with [{alpha}-32 P]dCTP (Du Pont, International, Regensdorf, Switzerland) by using a random primer labeling system (Roche Diagnostics, Rotkreuz, Switzerland).

In Situ Hybridization.
In situ hybridization was performed as reported previously (27 , 30) . Briefly, 10-µm frozen tissue sections were postfixed with 4% paraformaldehyde in PBS for 5 min and incubated with 0.2 M HCl for 20 min. The samples were prehybridized at 50°C for at least 1 h in 50% formamide (v/v), 4x SSC, 2x Denhardt’s solution, and 250 µg of RNA/ml. Hybridization was performed overnight at 50°C in 50% (v/v) formamide, 4x SSC, 2x Denhardt’s solution, 500 µg of RNA/ml, and 10% dextran sulfate (w/v). The final concentration of the digoxigenin-labeled NTR-1 probes (antisense and sense) was approximately 0.5ng/µl. After hybridization, the sections were washed and treated with RNase (Roche Diagnostics). The samples were then incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics; dilution, 1:500). For color reaction, 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma, Buchs, Switzerland) were used. For control experiments, the slides were incubated with RNase or with the corresponding sense probes. Pretreatment of the slides with RNase abolished the hybridization signal produced by the antisense probe. Furthermore, incubation with the sense probe failed to produce in situ hybridization signals.

For in situ hybridization, antisense and sense hNTR-1 cRNA probes were prepared from a 220-bp KspI/PstI fragment of the human NTR-1 cDNA, which was generated by reverse transcription-PCR and was subcloned into the pGEM-T Easy vector (Promega Biotechnology). This vector carries promoters for the DNA-dependent SP6 and T7 RNA polymerases. The authenticity of the subcloned DNA fragment was confirmed by sequencing using the dye terminator method (ABI 373A, Perkin-Elmer, Rotkreuz, Switzerland). The sense and antisense cRNA probes were labeled with digoxigenin (25, 26, 27) . After linearization, the cDNAs were transcribed using the Ribomax system (Promega Biotechnology). The transcription resulted in digoxigenin-labeled antisense riboprobes specific for the NTR-1 mRNA. The corresponding sense probes were prepared in an analogous manner. The digoxigenin-labeled antisense and sense probes were stored in diethylpyrocarbonate-treated water at -70°C until further use.

NTR-1 Autoradiography.
NTR-1 autoradiography was performed on 20-µm frozen sections as previously reported (22) . 125I-[Tyr3]-NT was used as radioligand. Slides were incubated at 4°C for 1 h. For control experiments, consecutive tissue sections were incubated with nonradioactive NT. Following four washings, the slides were exposed to X-ray films and exposed for 7 days at +4°C.

In all, tissue sections from five patients with normal pancreas, four patients with CP, and four patients with pancreatic cancer were processed. Consecutive slides from these patients were used for in situ hybridization to evaluate whether NTR-1 mRNA expression and NTR-1 binding are simultaneously present.

Statistical Analysis.
Results are expressed as median and range or mean ± SE. For statistical analysis, the Mann-Whitney U test was used. Significance was defined as P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Northern Blot Analysis.
Total RNA was extracted from all normal, CP, and cancer samples and analyzed by Northern blot analysis. Much lower levels of NTR-1 mRNA were visible in normal pancreas samples. In comparison with the normal pancreas, 14 of 20 (70%) CP samples and 25 of 30 (83%) pancreatic cancer samples exhibited higher NTR-1 mRNA levels (Fig. 1)Citation . Densitometric analysis revealed that NTR-1 mRNA levels were 3.0-fold increased (P < 0.01) in CP samples and 4.4-fold increased (P < 0.01) in pancreatic cancer samples compared with normal controls. Comparison of CP with pancreatic cancer revealed that the levels of NTR-1 mRNA expression were not different (P > 0.05), although the mean value of absorbance of pancreatic cancer samples was higher than in CP samples (Fig. 2)Citation . Further analysis in pancreatic cancer samples revealed that NTR-1 mRNA levels were higher in advanced tumor stages (stages III and IV) than earlier stages (stages I and II; P < 0.05) and that the tumor differentiation (grades 1 and 2 versus grades 3 and 4) had no influence on NTR-1 mRNA expression levels (P > 0.05; Table 1Citation ).



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  		Fig. 1. Northern blot analysis of NTR-1 mRNA in normal, CP, and pancreatic cancer tissues. Twenty µg of total RNA were subsequently size fractionated, blotted, and hybridized with [{alpha}-32P]dCTP-labeled hNTR-1 cDNA and 7 S cDNA probes.

 


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  		Fig. 2. Densitometric analysis of Northern blot hybridization signals. The ratio of absorbances of NTR-1 with the corresponding 7 S signals was calculated. Columns, mean of calculated ratios; bar, SE. *, P < 0.01 CP versus normal pancreas; **, P < 0.01 pancreatic cancer versus normal pancreas; ***, P > 0.05 CP versus pancreatic cancer.

 

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  		Table 1 Relationship of NTR-1 mRNA expression levels to tumor stage and tumor differentiation in pancreatic cancer

 
In Situ Hybridization.
To localize the exact site of NTR-1 mRNA expression, in situ hybridization was performed in normal, CP, and pancreatic cancer samples. In the normal pancreas, NTR-1 mRNA was weakly present in the cytoplasm of some acinar cells and ductal cells (Fig. 3A)Citation . In CP, moderate to strong NTR-1 mRNA signals were found in the cytoplasm of acinar cells dedifferentiating into tubular complexes and in degenerating acinar cells (Fig. 3B)Citation . In pancreatic cancer samples, moderate to strong NTR-1 mRNA signals were present in the cytoplasm of cancer cells (Fig. 3C)Citation . In the control experiments, no NTR-1 mRNA signals were obtained by in situ hybridization using the NTR-1 sense probe (Fig. 3D)Citation .



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  		Fig. 3. In situ hybridization. In the normal pancreas, a weak NTR-1 mRNA signal was present in acinar and ductal cells (A; x 200). NTR-1 mRNA signal was moderately to strongly present in acinar cells dedifferentiating into tubular complexes and the remaining degenerating acinar cells of CP (B; x 200) and cancer cells (C; x 200). Hybridization with a sense probe showed negative staining (D; x 200).

 
Comparison of NTR-1 mRNA Expression and NTR-1 Autoradiography.
On consecutive tissue sections of five patients with normal pancreas, four patients with CP, and four patients with pancreatic cancer, NTR-1 mRNA expression and NTR-1 ligand binding were evaluated. In situ hybridization revealed weak NTR-1 mRNA expression in the normal pancreas, moderate NTR-1 mRNA expression in three CP samples, and strong expression in one CP sample. In contrast, all CP samples were negative by NTR-1 receptor autoradiography (Table 2)Citation .


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  		Table 2 Comparison of NTR-1 mRNA expression and NT binding by NTR autoradiography in normal pancreas, chronic pancreatitis, and pancreatic cancer. -, negative; +, weak signal; ++, moderate signal; +++, strong signal. CA, pancreatic cancer.

 
In pancreatic cancer, two samples exhibited moderate NTR-1 mRNA expression and two samples exhibited strong expression. NTR-1 autoradiography was weakly positive in one cancer sample, moderately positive in one cancer sample, and strongly positive in two cancer samples (Table 2)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pancreatic cancer is an extremely aggressive malignant disorder with a definitely poor prognosis. We have learned from molecular studies in the past that the clinical characteristics of pancreatic cancer are much influenced by growth factors, growth factor receptors, apoptosis-inhibiting genes, and so forth (3 , 4 , 25 , 31) . NT is a neurotransmitter that also stimulates MIA PaCa-2 cell growth, and subsequent studies have revealed that NT-induced growth stimulation is mediated via NTR, which is present on the cell surface of these cells (14) . Recent autoradiographic binding analysis in resected pancreatic cancer revealed the presence of high levels of NTR-1 binding sites in 75% of pancreatic cancers, suggesting that gastrointestinal hormones and neurotransmitters might also be involved in the regulation of pancreatic cancer cell behavior (22) . Furthermore, the presence of NTR has also been observed in other malignancies, suggesting that NT/NTR signaling might be of importance in cancer cell growth (13 , 14) .

In the present study, we found overexpression of NTR-1 mRNA in 83% of pancreatic cancer samples. Although NTR-1 protein could not be directly investigated due to the unavailability of specific antibodies, in conjunction with the previous report on NT binding studies, our data suggest that increased NTR-1 mRNA synthesis is also translated into protein in pancreatic cancer cells. Although peripheral NT serum levels in healthy controls and pancreatic cancer patients are not different, the presence of higher NTR-1 density in pancreatic cancer cells might enhance NT functions in pancreatic cancer patients (32) . Furthermore, NT seems to have completely different functions in the normal pancreas and in pancreatic cancer in vivo. In the normal pancreas, NT stimulates exocrine pancreatic secretion (9) , and in pancreatic cancer cells, it influences cell proliferation (15) .

Our study covers two new interesting aspects of NTR-1. One is the distribution of NTR-1 in the normal pancreas, and the other is the suggestion that neuroendocrine hormones can influence the growth behavior of pancreatic cancer cells. The distribution of NTR-1 mRNA expression has not been studied before in the normal pancreas. We know that exogenous NT stimulates exocrine pancreatic secretion in rats and dogs, indicating either direct or indirect stimulatory effects on pancreatic acinar and/or ductal cells. We found the expression of low levels of NTR-1 mRNA in pancreatic acinar and ductal cells, which strongly indicates that NT released from enteroendocrine cells has direct effects on pancreatic cells and functions in humans.

At present, it is not known whether the expression of NT mRNA is increased in pancreatic cancers. However, the treatment of MIA PaCa-2 with the nonpeptide NTR antagonist SR 48692 inhibits NT-induced cancer cell growth, indicating that by blocking the NT/NTR pathway, it might be possible to negatively influence cancer cell proliferation (17) .

Pancreatic tissue samples obtained from patients with CP also exhibited increased NTR-1 mRNA expression compared with normal controls. In comparison with pancreatic cancer samples, no statistical difference was found, although the mean NTR-1 mRNA expression level was lower in CP samples. However, significantly higher NT serum levels were measured in CP patients in comparison with normal controls and pancreatic cancer patients (32) , suggesting that in CP, NT signaling via NTR-1 might be markedly increased. We postulate two potential functions of NT signaling in CP. Reduction of adequate exocrine pancreatic secretion due to the loss of exocrine parenchyma might lead to an up-regulation of secretion-stimulating pathways, including neuroendocrine hormones. On the other side, NTR-1 signaling might also be involved in remodeling and dedifferentiation of acinar cells into duct-like structures, because by in situ hybridization, the strongest NTR-1 mRNA signals were always present in these cellular structures.

It is surprising that by NTR-1 autoradiography, only pancreatic cancer samples and not CP samples exhibited increased NT binding (22) . It is probable that in CP NTR-1 mRNA is not translated into protein, which may result in low NTR-1 binding capacity. Similar observations have been made for somatostatin receptors, in which a discrepancy between a high receptor mRNA expression and the absence of the receptor protein has also been reported in pancreatic cancer (33) . Alternatively, it is possible that in CP, most of the NTRs are occupied and thereby masked by endogenous NT, which is increased in the serum, and that this does not happen in pancreatic cancer, in which the serum levels of NT are lower (32) .

In conclusion, neuropeptides such as NT may play a role in growth regulation and progression of pancreatic cancer. It remains questionable whether it has a functional role, i.e., tissue remodeling, in CP. Additional studies are needed to clarify whether modulation of neuroendocrine hormones can therapeutically influence cancer growth in human pancreatic cancer in vivo.


   ACKNOWLEDGMENTS
 
The hNTR-1 plasmid used for preparation of the probe used in Northern blot analysis was kindly provided by Dr. Pascale Chalon (Unite Biologie du Gene, Sanofi Elf Biorecherches, BP 137, 31676 Labege Cedex, France).


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Department of Visceral and Transplantation Surgery, University of Bern, Inselspital, CH-3010 Bern, Switzerland. Phone: 41-31-632-9578; Fax: 41-31-632-9732; E-mail: helmut.friess{at}Insel.ch Back

2 The abbreviations used are: TGF, transforming growth factor; NT, neurotensin; NTR, NT receptor; NTR-1, NTR type 1; NTR-2, NTR type 2; CP, chronic pancreatitis. Back

Received 7/ 8/99; accepted 10/26/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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