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Loss of Methylthioadenosine Phosphorylase and Elevated Ornithine Decarboxylase Is Common in Pancreatic Cancer

¹ Division of Population Science and ² Division of Medical Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania

	ABSTRACT

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Purpose: Loss of the methylthioadenosine phosphorylase (MTAP) gene at 9p21 is observed frequently in a variety of human cancers. We have shown previously that MTAP can act as a tumor suppressor gene and that its tumor suppressor function is related to its effect on polyamine homeostasis. Ornithine decarboxylase is a key enzyme in the regulation of polyamine metabolism. The aim of this study is to analyze MTAP and ornithine decarboxylase (ODC) expression in primary pancreatic tumor specimens.

Experimental Design: We measured MTAP and ODC activity in protein extracts derived from 30 surgically resected tumor samples and eight normal pancreas samples. In a subset of six samples, we also examined MTAP DNA using interphase fluorescence in situ hybridization. In addition, we examined the effect of the ODC inhibitor difluoromethylornithine on two pancreatic adenocarcinoma-derived cell lines.

Result: MTAP activity was 2.8-fold reduced in adenocarcinomas and 6.3-fold reduced in neuroendocrine tumors compared with control pancreas. Conversely, ODC activity was 3.6-fold elevated in adenocarcinomas and 3.9-fold elevated in neuroendocrine tumors compared with control pancreas. Using interphase fluorescence in situ hybridization, we found in tumor samples that 43 to 75% of the nuclei had lost at least one copy of MTAP locus, indicating that loss of MTAP activity was at least partially because of deletion of the MTAP locus. We also show that inhibition of ODC by difluoromethylornithine caused decreased cell growth and increased apoptosis in two MTAP-deleted pancreatic adenocarcinoma-derived cell lines.

Conclusions: MTAP activity is frequently lost, and ODC activity is frequently elevated in both pancreatic adenocarcinoma and neuroendocrine tumors. Inhibition of ODC activity caused decreased cell growth and increased apoptosis in pancreatic tumor-derived cell lines. These findings suggest that MTAP and polyamine metabolism could be potential therapeutic targets in the treatment of pancreatic cancer.

	INTRODUCTION

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Pancreatic cancer is the fifth leading cause of cancer mortality in the United States, with >30,000 deaths per year (1) . The vast majority (85%) of pancreatic cancers are adenocarcinomas derived from the exocrine pancreas. At the time of diagnosis, most pancreatic adenocarcinomas have already metastasized to regional lymph nodes, liver, peritoneum, or lungs (2) . Pancreatic adenocarcinoma is among the most lethal of human cancers with a five-year survival rate of <5%. Most other pancreatic tumors arise from islets of the endocrine pancreas (3) . Unlike pancreatic adenocarcinomas, there is significantly greater survival for neuroendocrine tumors, ranging from 44 to 87% depending on the specific stage and tumor subtype (4) .

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the catabolism of methylthioadenosine, a byproduct of polyamine biosynthesis (Fig. 1) . Loss of MTAP expression has been observed in many different tumor-derived cell lines and primary tumors including gliomas, osteosarcoma, melanoma, non–small-cell lung cancers, and T-cell acute lymphocytic leukemia (5, 6, 7, 8, 9) . Loss of MTAP activity in tumors is primarily thought to be because of homozygous deletion of the MTAP gene that is located on human chromosome 9p21, approximately 100 Kb distal to the p16(INK4a)/p14(ARF) tumor suppressor gene (10) . Until recently, it was thought that loss of MTAP was simply because of its proximity to p16 (i.e., bystander effect), but more recent data suggest that MTAP may function as a tumor suppressor itself. In both non–small-cell lung cancer and gliomas, loss of MTAP has been shown to occur in the absence of loss of p16(INK4a) (8 , 11) . Furthermore, re-expression of MTAP in MCF-7 breast adenocarcinoma cells abolishes anchorage-independent growth in vitro, and inhibits tumor formation in SCID mice (12) . Finally, MTAP expression in the human melanoma cell line Mel Im resulted in a substantial reduction in invasive potential as measured by a Boydon Chamber (7) .

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. MTAP-related metabolic pathways. Metabolites are shown in regular type, whereas enzymes are shown in italics. MTA, methylthioadenosine; SAM-S, adenosylmethionine; dSAM, decarboxy-S-adenosylmethionine. Dotted line shows repression of ODC activity by 2-keto-methylthiobutyrate (MTOB) as shown in Subhi et al. (13) .

In this report, we analyzed MTAP and ODC status in surgically resected pancreatic tumors. We found that loss of MTAP activity was common in both pancreatic adenocarcinomas and neuroendocrine tumors and that this loss was associated with elevated ODC activity in both tumor types.

	MATERIALS AND METHODS

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Patient Samples.
Tumors and normal pancreatic tissue were obtained through the tumor bank at Fox Chase Cancer Center. Fresh surgical material was flash frozen and stored at –70°C. Before cryopreservation, a sample was obtained for diagnostic purposes. These H&E-stained tumor sections were reviewed by a pathologist (H. C.) and assessed for percentage of tumor cells. For the neuroendocrine tumors >95% of the material consisted of tumor. In contrast, adenocarcinoma samples were typically comprised of 25 to 50% cancer cells, with the remainder consisting mostly of fibrous extracellular stroma. Two of the normal pancreas samples were obtained from fresh (<4 h) autopsies. These were obtained from the Cooperative Human Tissue Network-Eastern Division. The specimens were obtained in accordance with a protocol approved by the Fox Chase Cancer Center Institutional Review Board.

MTAP and ODC Activity.
Protein extracts were prepared by thawing a small portion of tissue on ice in the presence of a buffer containing 10 mmol/L Tris-HCL and a protease inhibitor mixture (Roche Biochemical, Indianapolis, IN). The sample was homogenized with an OMNI 1000 homogenizer, centrifuged at 10,000 x g for 15 minutes, and the supernatant collected. A Slide-A-Lyzer dialysis cassette with M_r = 10kDal (Pierce Biotechnology Inc., Rockford, IL) was used to dialyze the extract against 40 mmol/L potassium phosphate (pH 7.4) for 90 minutes. Ornithine decarboxylase activity was assayed by measuring the ¹⁴CO₂ formed by decarboxylation of (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) C-labeled L-ornithine (5 mCi/mmol, Maravek Biochemical) in 30 minute at 37°C as described previously (13) . MTAP activity was determined with a photometric assay to measure adenine production as described previously (12) . Reactions generally used 20 µg of cell extract. One unit of MTAP catalyzes the formations of 1 µmol of adenine/minute, whereas 1 unit of ODC catalyzes the formation of 1 nmol of CO₂/hour.

Fluorescence In situ Hybridization.
Fluorescence in situ hybridization (FISH) and detection of immunofluorescence was carried out as described previously with some modifications (16) . Briefly, a human BAC clone for the MTAP gene (RP11–70L8) was biotinylated by random priming in a reaction containing 600 ng DNA, in a total volume of 100 µL with the Bioprime DNA-labeling system (Invitrogen). Spectrum orange-labeled satellite (CEP9) DNA probe (Vysis, Downers Grove, IL) was used for identification of human chromosome 9 centromere. Frozen pancreatic tumors were minced, treated with hypotonic solution (0.075M KCl for 15–20 minutes), and fixed in methanol acetic acid (3:1). Isolated nuclei were then dropped on slides and used for interphase FISH. Probes were denatured and hybridized at 37°C overnight. Biotinylated probe was detected with FITC-labeled avidin (Vector, Burlingame, CA) and amplified by the addition of antiavidin antibody (Vector) and a second layer of FITC-labeled avidin. The slides were counterstained with 4',6-diamidino-2-phenylindole and observed with a Zeiss Axiophot epiflourescence microscope equipped with a cooled charge-coupled device camera (Photometrics, Tucson, AZ) operated by a Macintosh 4',6-diamidino-2-phenylindole, FITC, and Spectrum Orange signals. A total of 11 samples were prepared, but in only six cases (see Table 1 ) were there sufficient nuclei to perform quantitation.

Apoptosis Assay.
The MiaPaca-2 cells undergoing apoptosis were determined with Guava Nexin kit in its Guava Personal Cell Analysis System (Guava Technologies, Hayward, CA) according to the manufacturer’s instruction. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and incubated with various concentration of DFMO for different time periods. Cells were trypsinized and collected by centrifuging at 1,000 rpm for 5 minutes at 4°C. After washing with ice-cold 1x Nexin buffer, cells were resuspended in the same buffer and then labeled with Annexin V-PE and 7-amino-actinomycin D on ice and in a dark place for 20 minutes. The proportion of apoptotic cells was detected by Guava Personal Cytometer.

Statistics.
All statistics were done with Quick Statistica 4.1 (StatSoft, Inc). All mean comparisons were evaluated for significance with the non-parametric Mann-Whitney U test unless otherwise stated. Correlations used the Pearson product-moment correlation R.

	RESULTS

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Analysis of MTAP Activity.
Initially, we attempted to measure MTAP levels by Western blotting with polyclonal antiserum (12) . We found that the antiserum cross-reacted with a non-MTAP-related protein that migrated similarly to MTAP in SDS-PAGE, making it impossible to quantitate MTAP levels (data not shown). To circumvent this problem, we measured MTAP enzyme activity directly in whole cell extracts from a total of 38 surgical specimens (Table 2 ; Fig. 2A ). The breakdown of these specimens was as follows: 19 adenocarcinomas, 8 neuroendocrine tumors, 8 normal pancreas, 1 acinar carcinoma, 1 ampullary carcinoma, and 1 invading leiomyosarcoma. The ductal carcinomas (n = 19) had on average 0.18 units of MTAP, whereas the neuroendocrine tumors (n = 8) had 0.08 units. Both tumor types had significantly reduced activity relative to the control samples (0.18 versus 0.51, P = 0.0005; 0.08 versus 0.51, P = 0.0007). Eleven of 19 adenocarcinomas (58%) and eight of eight neuroendocrine samples (100%) had activities outside the range of the normal pancreas samples (0.232–1.079 units). The acinar carcinoma, the ampullary carcinoma and the sarcoma sample all had MTAP levels within the control range (0.332, 0.380, and 0.364 units, respectively). Because about half of all of the patients with pancreatic adenocarcinoma were pretreated with chemotherapy and radiation, we were concerned that the necrosis caused by the pretreatments might be associated with artificially low MTAP levels. Comparing the MTAP activity in samples from pretreated versus untreated adenocarcinoma patients, we found that activity was not significantly altered (0.16 versus 0.20 units, P = 0.74), suggesting that this was not the case. Our results indicate that loss of MTAP activity is common in both pancreatic adenocarcinoma and neuroendocrine tumors.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. MTAP and ODC activity in pancreatic tumor tissue. A, scatter diagram of MTAP activity in different tumor types. NE, neuroendocrine; AC, adenocarcinoma; Normal, normal pancreas; other, leiomyosarcoma, acinar, ampullary carcinoma. Long horizontal bar show mean. Vertical bars indicates 95% CI interval for mean (1.96*SE). B, identical to A except for ODC activity.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Interphase FISH for MTAP. A, nuclei from normal pancreas (N3) probed with MTAP Bac (green) and a probe for chromosome 9 centromere (red). B, nuclei from adenocarcinoma (A5). C, nuclei from neuroendocrine tumor (NE3).

We also found a statistically significant inverse correlation between MTAP and ODC activity in analysis of all 38 samples (r = –0.38, P = 0.017). However, much of this correlation was driven by the normal pancreas samples, all of which had high MTAP and low ODC status. Confining our examination to only the tumor samples the correlation is somewhat weaker and loses its statistical significance (r = –0.22, P = 0.239). However, we did find that tumors with ODC levels of >10 units/mg (n = 9) had significantly reduced MTAP levels compared with tumors with ODC levels of <10 units/mg (n = 21; 0.09 versus 0.20, P = 0.02). This result indicates that high levels of ODC activation is associated with reduced levels of MTAP.

P16 Expression.
Because MTAP is located adjacent to the p16(INK4a) gene, we examined p16 protein levels in our samples by Western blot analysis of protein extracts. We found detectable levels of p16 in only three neuroendocrine tumor samples, the single acinar tumor, the leiomyosarcoma, and one of the adenocarcinoma samples (see Table 2 ). We did not find detectable levels of p16 in any of the normal pancreas extracts, confirming previous observations that only a small subset of cells express p16 in normal pancreas (18) . Of the six tumor samples with detectable p16 levels, three had MTAP levels significantly reduced (NE3, NE5, and NE6), suggesting that they had mutations that inactivated MTAP but not p16(INK4a). NE3 was one of the samples in which interphase FISH was done, and it was shown to have a large percentage of nuclei that have lost either one or both copies of MTAP. This suggests that MTAP deletion can occur in the absence of p16(INK4a) deletion.

Effect of ODC Inhibitors on an Adenocarcinoma-Derived Cell Lines.
We next tested the effect of ODC inhibition on growth of MiaPaca-2 and Panc-1 cells, two human pancreatic adenocarcinoma-derived cell lines. It has been shown previously that both MiaPaca-2 and Panc-1 cells are deleted for MTAP and have high levels of ODC activity. We treated these cells with various concentrations of DFMO, an ornithine analog that binds irreversibly to the ODC active site and inhibits enzyme function (19) . We found that DFMO effectively inhibited the growth of both cell lines (Fig. 4A) in a concentration-dependent manner. This decrease in growth was also associated with an increase in apoptosis. Treatment with increasing concentrations of DFMO resulted in increasing levels of cleaved poly(ADP-ribose) polymerase, a marker for apoptosis (Fig. 4B) . We also assayed apoptosis by staining with annexin V and 7-aminoactinomycin D. The addition of 400 µmol/L DFMO resulted in a 2.5-fold increase in apoptotic cells in the Panc-1 cells and a 3-fold increase in MiaPaca-2 cells. These experiments show that inhibition of ODC by DFMO can kill pancreatic tumor cells.

View larger version (61K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. DFMO inhibits MiaPaca-2 and Panc-1 cell growth and induces apoptosis. A, one-thousand MiaPaca-2 or Panc-1 cells were plated in 96-well tissue culture plates in the presence of 0–500 µmol/L DFMO. After 48 and 72 hours, cell growth was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Each data point was done in triplicate (B) Ten-thousand MiaPaca-2 or Panc-1 cells were plated in 6-well tissue culture plates and exposed to the indicated amount of DFMO. After 72 hours, the cells were harvested, and total protein was isolated and evaluated for poly(ADP-ribose) polymerase p85 expression by Western blot. The lower band shows cleaved poly(ADP-ribose) polymerase, whereas the upper band is an unknown cross-reacting protein that serves as a convenient internal control. C identical to B except that after 72 hours, cells were analyzed for Annexin V and 7-aminoactinomycin D staining as described in Materials and Methods.

	DISCUSSION

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Previously, we showed that re-expression of MTAP in MTAP-deficient MCF-7 breast adenocarcinoma cells resulted in a loss of anchorage-dependent growth and tumorigenicity in SCID mice, indicating that MTAP acted as a tumor suppressor gene (12) . We also showed that expression of MTAP resulted in significantly reduced levels of ODC activity and that this effect explains at least part of MTAP’s tumor suppressor qualities (12 , 13) . In this report, we show for the first time that ODC levels are significantly elevated in pancreatic adenocarcinoma and neuroendocrine tumor specimens. We also show that there is an inverse correlation between MTAP activity and ODC activity in the tissue samples we analyzed, consistent with the hypothesis that MTAP inactivation results in elevated ODC. However, we did notice that ODC levels tended to be higher in tumor samples relative to nontumor samples with similar MTAP levels. This suggests that although loss of MTAP can elevate ODC activity, there may also be additional alterations responsible for the elevated ODC levels observed in tumor cells. However, the tumor samples with the highest ODC levels tended to have extremely low MTAP levels, suggesting that to achieve extremely high levels of ODC it is necessary to inactivate MTAP.

A possible limitation of our study concerns the use of total tumor specimens. We can make no conclusions about the homogeneity of MTAP and ODC expression within the cells of the tumor mass. Our FISH data suggest that there is considerable heterogeneity within the tumor, as different nuclei had differing amounts of MTAP loss. In addition to tumor cells, there is also reactive nonmalignant stroma that is a substantial component of the tumor mass. This is especially true in the adenocarcinoma specimens, which are characterized by having a high degree of desmoplastic stroma (21) . We did not observe a correlation between the amount of stroma in a sample and MTAP levels. This is evident in our comparison of pretreated versus nonpretreated patients. In general, the amount of stroma was higher in the pretreated patients, but we did not observe a statistically significant difference in MTAP or ODC levels. This suggests that either a large percentage of stroma is extracellular material or that the stromal-producing cells express very little MTAP and ODC. Clearly, for future studies it would be useful to have high quality monoclonal antibodies to be able to do immunohistochemistry, allowing for determination of MTAP and ODC levels in individual tumor and stromal cells.

Our finding that pancreatic tumors have a high frequency of MTAP loss and ODC overexpression suggests possible new therapeutic approaches to pancreatic cancer. MTAP is a key enzyme in the methionine salvage pathway (Fig. 4) . The absence of MTAP causes three potential metabolic stresses on a cell. First, because MTAP normally salvages adenine from MTA, the absence of MTAP makes a cell more dependent on de novo purine synthesis inhibitors. In fact, pancreatic adenocarcinoma cell lines deleted for MTAP have been shown to have increased sensitivity for drugs that interfere with de novo purine synthesis, such as azaserine and methotrexate (17 , 22) . Absence of MTAP would also be expected to reduce production of methionine and thus have a higher requirement for methionine then normal cells. Our lab has found that MCF-7 cells expressing MTAP have reduced dependence on methionine compared with isogenic cells lacking MTAP (unpublished data). We also observed that there is an association between MTAP status and the ability of cells to use homocysteine instead of methionine as a source of methionine (23) . Finally, in the work presented here, we show that inhibition of ODC by DFMO can inhibit cell growth and induce apoptosis in pancreatic adenocarcinoma-derived cell lines. The concentration of DFMO used in the experiments shown here are achievable in the clinic (24) . Taken together, these lines of evidence suggest that it might be possible to target MTAP-deleted pancreatic tumors with a combination of purine synthesis inhibitors, treatments that lower plasma methionine (methioninase; ref 25 ), and ODC inhibitors. Should our findings be confirmed in additional studies, it would seem worthwhile to explore these potential drug combinations in animals models of pancreatic cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. Maureen Murphy for use of the Guava Personal Cell Analysis System and the Fox Chase Cancer Center Tumor Bank Facility for their assistance.

	FOOTNOTES

 
Grant support: This work was supported by American Cancer Society Grant RSG0315701, a Translational Research Grant from Fox Chase Cancer Center, core Grant CA06927 and R01-CA77429 from the NIH, and by an appropriation from the Commonwealth of Pennsylvania.

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.

Note: A. Subhi and B. Tang contributed equally to this work.

Requests for reprints: Warren D. Kruger, Division of Population Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-3030; Fax 215-214-1623; E-mail: warren.kruger{at}fccc.edu

Received 5/18/04; revised 7/29/04; accepted 8/16/04.

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