Expression of the Tumor Suppressor GeneMaspin in HumanPancreatic Cancers1

Nicolai Maass,2 Takashi Hojo, Michael Ueding,Jutta Luttges, Gunter Kloppel, Walter Jonat, andKoichi NagasakiDepartments of Gynecologic Oncology [N. M., M. U., W. J., K. N.],and Pathology [J. L., G. K.], University of Kiel, 24105 Kiel,Germany, and Growth Factor Division, National Cancer CenterResearch Institute, Tokyo 104-0045, Japan [T. H.]

ABSTRACTThe tumor suppressor genemaspin, a unique member

of the serpin superfamily, inhibits cell motility, invasion, andmetastasis in breast and prostate cancers. Maspin is ex-pressed in normal human mammary and prostate epithelialcells but down-regulated during cancer progression. In thisstudy, we analyzed the expression ofmaspin in varioushuman cancer cells by means of Northern blot and immu-nohistochemistry.Maspin gene expression proved to be up-regulated in pancreatic cancer. Maspin expression was notdetected in any of 6 gastric cancers, 4 melanomas, or 6 of 7breast cancer cell lines examined. In contrast, 5 of 9 pan-creatic cancer cell lines showed maspin expression, althoughmaspin expression was not detected in normal pancreatictissue. Furthermore, maspin was expressed in 23 of 24 tu-mor specimens obtained from pancreatic cancer patients aswell as all high-grade precancerous lesions (PanIN3 andintraductal carcinoma extension). In contrast, no expressionwas observed in normal and low-grade precancerous lesions.Our results show that maspin is a new factor associated withpancreatic cancer. In addition, the detection of maspin inpancreatic tumor tissues and its lack of expression in allnormal pancreatic tissues suggests that maspin may be auseful marker of primary human pancreatic cancer.

INTRODUCTIONMaspin (mammary serpin) is a serine protease inhibitor

related to the serpin family (1). Themaspingene was originally

identified in normal mammary epithelium by subtractive hybrid-ization on the basis of its expression at the mRNA level (1). Itwas shown to have tumor suppressive activity attributable toinhibition of breast cancer cell motility, invasion, and metastasis(2–4). Maspin is aMr 42,000 protein with sequence homologyto other inhibitory serpins (2, 5). Maspin, which is located at thecell membrane and the extracellular matrix, does not act as aclassical inhibitory serpin with antiprotease activity againsttrypsin-like serine proteases (6–8).

Maspin is expressed in normal human mammary and pros-tate epithelial cells but down-regulated during cancer progres-sion. The loss ofmaspingene expression with increasing ma-lignancy is regulated at the transcriptional level (9). Recentpublications have discussed the participation of cytosine meth-ylation and chromatin condensation in the down-regulation ofmaspin expression during neoplastic progression (10).

Although at present the molecular and biological mecha-nisms of the function(s) of maspin remain unknown there isevidence that maspin interacts with the p53 tumor suppressorpathway and may function as an inhibitor of angiogenesisinvitro andin vivo (11, 12). Using Northern blot analysis, reversetranscription PCR and immunohistochemistry, we found furtherevidence of decreasing maspin expression with increasing ma-lignancy in human breast cancer tissues (13). Pembertonet al.(14) demonstrated the presence of maspin in the epithelium ofseveral normal human organs (such as prostate, thymus, testis,small intestine, and colon) and particularly in the myoepitheliumof the breast, where it is localized and probably functions bothintra- and extracellularly. Because themaspingene is expressedin the epithelium of other glands, it is conceivable that it mayplay a similar role in the pancreas as well. For this reason, wewere interested in determining whether the tumor suppressorfunction described for maspin in mammary carcinomas can alsobe detected in pancreatic cancers. Interestingly, our data re-vealed a different pattern ofmaspingene expression from that inbreast cancer cells. Maspin was not expressed in normal humanpancreatic cells but showed strong expression in pancreaticcancer cells as well as a weaker but detectable expression inprecancerous pancreatic lesions. These results suggest thatmaspin is a new factor associated with pancreatic cancer.

MATERIALS AND METHODSCell Culture and Clinical Specimens. The following

human cancer cell lines were studied: breast cancer (MCF-7,ZR-75–1, SK-BR-3, BT-20, T47D, MDA-MB-231, MDA-MB-468), pancreatic cancer (BxPC-3, AsPC-1, MIAPaCa-2, CA-PAN-1, CAPAN-2, PANC-1, PSN-1, KP2, FA-6), gastric can-cer (MKN-1, MKN-7, MKN-28, MKN-45, MKN-74, KATOIII), and melanoma (SEKI, G361, A375, MeWo). All of thecancer cell lines were cultured in RPMI 1640 supplementedwith 10% FCS. HMECs were purchased from Clonetics (SanDiego, CA), maintained according to supplier’s instructions, and

Received 10/2/00; revised 1/12/01; accepted 1/19/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by Dr. Mildred Scheel Stiftung, Deutsche Kreb-shilfe, and by the grant-in-aid from the Ministry of Health and Welfare,Japan, for the 2nd Term Comprehensive 10-Year Strategy for CancerControl and for Cancer Research (9-32 and 10-28).2 To whom requests for reprints should be addressed, at Department ofGynecologic Oncology, University of Kiel, Michaelisstrasse 16, 24105Kiel, Germany. Phone: 49-431-597-2100; Fax: 49-431-86200; E-mail:nmaass@email.uni-kiel.de.3 The abbreviations used are: HMEC, human mammary epithelial cell;PanIN, pancreatic intraductal neoplasia.

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assessed at early passages. Total RNA was extracted from cellswhen cultures reached 80% confluence, as described previously(15, 16).

Whipple resection specimens were obtained from 24 pa-tients [14 female and 10 male; mean age, 69.6 years (range46–76)] with ductal adenocarcinoma of the pancreas head froma series of 70 pancreatic resections performed in the years1996–1999 in the Department of Surgery, University of Kiel(Kiel, Germany). Histological classification and grading wereperformed according to the criteria of WHO 1996 and Luttgesetal. (17, 18). Of the carcinomas, one was classified as grade 1, 15as grade 2, and 8 as grade 3. One tumor was staged as stage Idisease, 6 as stage II, 14 as stage III, and 3 as stage IV. Ductallesions were classified according to the recently proposed PanINclassification (19–21). These samples where obtained from dif-ferent areas of the same surgical specimens as the carcinomas.In addition, three surgical specimens of normal pancreas (from1 male and 2 female patients) were also investigated and servedas control tissues. The staining of the carcinomas, normal pan-creatic tissues, intraductal tumor extensions, and hyperplasticduct epithelia was evaluated. The cytoplasmic staining intensitywas scored as follows: 0, no staining; 1, faint; 2, moderate; and3, strong. The cytospin specimens were scored in the samemanner.

RNA Isolation and Northern Blot. MaspincDNA waskindly provided by Dr. Ming Zhang (Baylor College of Medi-cine, Houston, TX). Total cell RNA was isolated from thecancer cell lines using the RNeasy Mini kit (QIAGEN, Hilden,Germany). A 2.5-kbEcoRI/XhoI fragment from themaspincDNA plasmid (pMZ-32) was labeled with32P using a Re-diprime DNA labeling system (Amersham Life Science, Arling-ton Heights, IL) and used in Northern analyses of total RNA asdescribed previously (15, 16). For standardization, membraneswere stripped and reprobed with the probe 36B4 under similarconditions to assess RNA loading and transfer efficiency (22,

23). The human multiple tissue Northern (MTN) blots (Clon-tech, Palo Alto, CA) were used to determine the tissue distri-bution.

Immunohistochemistry. A mouse antihuman maspinmonoclonal antibody was purchased from PharMingen Interna-tional (San Diego, CA). For the staining of HMECs and cancer celllines, cells were cultured in chamber slides for 24 h to 60–70%confluency, fixed with 4% paraformaldehyde in PBS and perme-ablized with methanol/3% H2O2 before blocking with 10% fetalbovine serum for 30 min. Cells were incubated with antihumanmaspin antibody (diluted 1:75) according to the manufacturer’sinstructions. Peroxidase-conjugated sheep antimouse IgG was usedas secondary antibody at a dilution of 1:75 and was color-devel-oped using diaminobenzidine. Cells were then counterstained withhematoxylin, dehydrated, and mounted. In addition, 5-mm sectionsof formalin-fixed, paraffin-embedded tissue samples from pancre-atic cancers, normal pancreatic tissues, and precancerous pancreaticlesions were analyzed. After microwave-based antigen retrievalwith 0.05 M Tris buffer (pH 9.0) for 15 min, the sections wereincubated with the antihuman maspin monoclonal antibody (dilut-ed 1:75) for 12 h. Bound antibodies were detected using theavidin-biotin complex technique. New Fuchsin/Naphtol AS-Biphosphate was used as a substrate and hematoxylin was used forcounterstaining.

RESULTSExpression of Maspin in Human Pancreatic Cancer

Cell Lines. To evaluate the expression of maspin in cancercells several human cancer cell lines and normal tissues wereinvestigated by means of Northern blot.Maspingene expressionwas not detected in any of the six gastric cancer, four melanomaand seven breast cancer cell lines with the exception of theMDA-MB-468 breast cancer cell line (Fig. 1,A, C, andD). Incontrast, maspin mRNA expression was observed in five of nine

Fig. 1 Northern blot analysisof maspin in cancer cell linesand human tissues.A, HMECsand seven human breast cancercell lines. B, nine pancreaticcancer cell lines.C, six gastriccancer cell lines.D, four mela-noma cell lines. Each lane con-tains 10 mg of total RNA. E,expression of maspin in normaltissues. The blot containing 2mg poly(A) RNA per lane waspurchased from Clontech. Theblots were hybridized with 2.5kb maspincDNA probe. 36B4was used as loading and trans-fer control.

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human pancreatic cancer cell lines (Fig. 1B). Maspin was highlyexpressed in BxPC-3 and AsPC-1, whereas low expression wasfound in CAPAN-2, KP2, and FA-6. In the normal tissues, highexpression of maspin mRNA was observed in mammary epi-thelial cells (Fig. 1A), whereas none of eight other normaltissues (heart, brain, placenta, lung, liver, skeletal muscle, kid-ney, and pancreas) showed expression of maspin mRNA (Fig.1E). It should be noted that, although a variety of pancreaticcancer cell lines exhibited maspin mRNA expression, maspinwas not detected in normal pancreatic tissues on Northern blotanalysis.

The expression of maspin in pancreatic cancer cells wasconfirmed by immunohistochemistry. Representative stainingresults in mammary epithelial cells and pancreatic cancer celllines are shown in Fig. 2. Positive staining for maspin wasobserved in HMECs (Fig. 2D), but no staining was seen in theMCF-7 breast cancer cell line (data not shown). Of the pancre-atic cancer cell lines, BxPC-3 and AsPC-1 showed strongmaspin staining (Fig. 2,A and B), and a weak signal wasobserved in KP2 (data not shown). As shown in Fig. 2C, thepancreatic cell line PANC-1 lacked maspin expression. Thesedata are consistent with Northern blot analysis.

Expression of Maspin in Surgical Specimens. Acinarcells and ductal epithelia from the tumor-associated pancreatictissues as well as the normal pancreas (control cases) stainednegatively (Fig. 3A; Table 1). A strong cytoplasmic reaction ofall tumor cells was observed in 23 of 24 ductal adenocarcinomas

(Fig. 3B; Table 1),which was diffusely distributed throughoutthe tumors. The staining intensity was generally strong andvaried only a little, except that cells with a broad clear cyto-plasm showed a faint positivity (Fig. 3C; Table 1). The onlycase that did not stain positively was a rare type of clear-cellductal adenocarcinoma (24). Intraductal non-clear-cell areas ofthis case, however, showed faint nuclear and cytoplasmic stain-ing. Some cells also exhibited nuclear staining that was alwaysaccompanied by strong cytoplasmic staining. Intraductal exten-sions of the carcinomas (17/17) and lesions of PanIN grade 3(7/7) stained positive also but with lower intensity. In contrast,ductal hyperplasia without dysplasia and low-grade dysplasia(Table 1, 8/8; PanIN 1A, 1B, and 2) such as mucinous cellhypertrophy or papillary hyperplasia stained negative (Fig. 3D;Table 1). In addition, foci of squamous intraductal metaplasia(6/6) showed cytoplasmic staining (Table 1). There was nocorrelation between the staining intensity and the histologicalgrade or stage of the tumors.

DISCUSSIONMaspin was originally described as a tumor suppressor

gene that affects cell motility and invasion (1). Recent findingssuggest that maspin is part of the p53 tumor suppressor pathway(12). Maspin expression is high in normal human mammary andprostate epithelial cells but is decreased in breast and prostatecancers and lost in metastatic cells. We examined the expression

Fig. 2 Maspin staining in HMECs and cancer cell lines.A, BxPC-3;B, AsPC-1;C, PANC-1;D, HMECs (3100). Cells were probed with antihumanmaspin antibody and counterstained with hematoxylin. In pancreatic cancer cell lines, maspin staining was moderate to strong in the cell lines BxPC-3and AsPC-1 and in HMECs. PANC-1 showed no staining.

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of maspin in various cancer cells using Northern blot analysisand immunohistochemistry.

Only a few reports of maspin expression in cancer cellshave been published (12, 25, 26). However, our Northern blotanalysis revealed that more than one-half of the pancreatic

cancer cell lines examined expressed maspin mRNA. Immuno-histochemical staining using a monoclonal maspin antibodyyielded identical expression patterns, indicating that up-regu-lated maspin mRNA expression is translated into protein inpancreatic cancer cells. Previously presented data of maspin

Fig. 3 Maspin staining in pancreatic cancer tissues.A, normal pancreatic tissue of the control cases with negatively stained acinar and ductal cells.B, invasive ductal adenocarcinoma of the pancreas with strong cytoplasmic and nuclear staining of the tumor cells and negative hyperplastic ductepithelium.C, invasive ductal adenocarcinoma showing strong cytoplasmic staining of tumor cells with dense cytoplasm and faint staining of thosewith clear and vacuolated cytoplasm.D, ductal papillary hyperplasia without nuclear atypia (PanIN1b,bottom half) without expression of maspin andPanIN3 lesion (strong nuclear abnormalities,top half) showing moderate cytoplasmic and nuclear staining.

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expression in normal pancreatic tissue showed conflicting re-sults: Pembertonet al. (14) could not detected maspin mRNAexpression by Northern blot but did detect maspin-like proteinexpression in glandular epithelia of the pancreas by immuno-staining using a polyclonal antibody. The discrepancy may beattributable to different characteristics of the antimaspin anti-bodies used, such as reaction with distinct epitopes or differentspecificity. A monoclonal antimaspin antibody was used in ourstudy. On the other hand, reduced or lacking mRNA expressionhas been reported in breast and prostate tumor cells, whereascorresponding normal cells exhibited high expression (1–4, 20).Although this difference between the pancreas and other organsneeds further analysis, maspin appears be an interesting factorassociated with pancreatic cancer.

Although cell lines often develop artificial gene changesduring long-term culturing, our studies showed that maspinexpression occurs not only in pancreatic cancer cell lines butalso in clinical pancreatic cancer tissues. Interestingly, we ob-served maspin staining in 23 of 24 pancreatic cancer tissues,which suggests that maspin expression is a common event inpancreatic cancer cells. Unlike the cancer tissues, no or faintexpression was observed in corresponding normal pancreatictissues and low-grade precancerous lesions. Furthermore,maspin expression seems to increase with increasing malig-nancy from normal pancreas tissue via precancerous lesions toinvasive carcinomas. These findings indicate that maspin ex-pression is of biological relevancein vivo for the developmentof pancreatic cancers. Although at present, the molecular andbiological mechanisms of maspin’s function are unknown, sev-eral authors adhere to the hypothesis that maspin functions at thelevel of invasion and metastasis by blocking tumor cell migra-tion and proliferation (2–4). Our findings, which show theup-regulation of maspin in pancreatic cancer, provide new in-formation about factors that regulate tumor cell development.

It has been shown repeatedly that distinct genes such as theK-rasoncogene and the tumor suppressor genesp53,p16,DCC,and DPC4/SMAD4are frequently altered in pancreatic cancerand may be essential for its genesis (27, 28). Interestingly, themaspingene is mapped on chromosome 18q21.3 in close prox-imity to theDCC andDPC4/SMAD4genes. Losses of chromo-some 18q including the loci for the genesDCC and DPC4/SMAD4are the most frequently identified genetic alterations inpancreatic cancer (29). Although the regulatory mechanism ofmaspin expression remains unknown, our study has added the

maspingene to the list of possible genes involved in pancreaticcarcinogenesis. The biological role of maspin expression inpancreatic cancer should be determined by further investigation.

Carcinoma of the pancreas is the fourth highest cause ofcancer-related death and shows the highest mortality rate of allcancers in most Western countries (30). Several tumor-associ-ated antigens, such as CEA, CA125, and CA19–9, are used tomonitor pancreatic cancer patients (31). However, they are nottumor specific and are commonly expressed in normal andbenign conditions (32, 33). The fact that maspin was detected inpancreatic cancer but was not expressed in normal pancreastissues suggests that maspin could serve as a useful marker forprimary human pancreatic cancer.

In conclusion, we have demonstrated that maspin may playan important role in the carcinogenesis of pancreatic cancer, inaddition to its tumor suppressor activity in breast and prostatecancer. We have shown thatmaspin gene expression is up-regulated in pancreatic cancer at the RNA and protein level, incontrast to its down-regulation in breast and prostate cancers.The function ofmaspinas a tumor suppressor gene involved intumor invasion, metastasis, and angiogenesis may not be limitedto breast and prostate cancer. Its relationship to carcinoma of thepancreas opens a new angle to the discussion on its function incancer.

ACKNOWLEDGMENTSWe thank Dr. Ming Zhang for providing the maspin cDNA

plasmid.

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Table 1 Immunohistochemically assessed expression of maspin in pancreatic ductal adenocarcinomas, associated duct lesions, and normalpancreatic tissue

No. of cases Staining intensity (% of cases)

Negative (0) Faint (1) Moderate (2) Strong (3)

Invasive ductal pancreatic adenocarcinoma (n5 24) 1a/24 (4) 3/24 (12) 4/24 (17) 16/24 (67)Intraductal carcinoma extension (n5 17)b 0/17 10/17 (59) 6/17 (35) 1/17 (6)PanIN3 (n5 5)b 0/7 6/7 (86) 1/7 (14) 0/7PanIN 1A, 1B, 2 (n5 8)b 8/8 (100) 0/8 0/8 0/8Squamous metaplasia, (n5 6)b 0/6 5/6 (83) 1/6 (17) 0/6Acinar cells and duct epithelium associated with carcinoma (n5 24) 24/24 (100) 0/24 0/24 0/24Acinar cells and duct epithelium of the control cases (n5 3) 3/3 (100) 0/3 0/3 0/3

a Clear cell carcinoma.b Number of cases that exhibited intraductal carcinoma extension or the various lesions.

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2001;7:812-817. Clin Cancer Res   Nicolai Maass, Takashi Hojo, Michael Ueding, et al.   Pancreatic Cancers

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