mucin gene expression in normal, preneoplastic, and ... · and mvc4 gene expression. in contrast,...

[CANCER RESEARCH 55. 2681-2690, June 15, 1995]

Mucin Gene Expression in Normal, Preneoplastic, and Neoplastic HumanGastric Epithelium1

Samuel B. Ho,2 Laurie L. Shekels, Neil W. Toribara, Young S. Kim, Carolyn Lyftogt, David L. Cherwitz, and

Gloria A. NiehansDepartments of Medicine tinti Pathology, University' of Minnesota and Veterans Administration Medicai Center, Minneapolis, Minnesota 55417 Â¡S.B. H., L. L. S., C. L,

D. L. C., G. A. N.I, and Departments of Medicine and Pathology, University of California and Veterans Adntinistralitm Medical Center, San Francisco, California V4121[N. W. T., Y. S. K. l

ABSTRACT

Mucins synthesized by malignant cells may contribute (via decreasedcellular adhesion and immune recognition) to cancer invasion and mÃ©tastases. Human mucins are derived from a heterogeneous family of genes,labeled MUCI-6. Our aim was to determine the pattern of mucin gene

expression in normal, preneoplastic (intestinal metaplasia), and malignantgastric specimens. Probes and antibodies for specific mucin tandem repeatsequences were used for RNA and immunohistochemical analysis. Normalstomach mucosa was characterized by expression of Miti. MUC5, andMUC6 mRNA and immunoreactive protein, without MUC2, MUC3,and MVC4 gene expression. In contrast, high levels of MUC2 and MUC3mucin mRNA and immunoreactive protein were found in specimens withintestinal metaplasia. Gastric cancers exhibited markedly altered secretory mucin mRNA levels compared with adjacent normal mucosa, withdecreased levels of MVC5 and Aft/Co mRNA and increased levels ofMUC3 and MUC4 mRNA. Overall, immunoreactive MVC1 mucin wasdetected in 72% of 33 gastric cancers, and secretory mucin core peptideswere expressed in 34% (MVC2), 45% (MUC3), 19% (A/Ã•/C5), and 57%(MUC6) of these specimens. Coexpression of multiple (three or more)mucin core proteins occurred in 15 of 25 (60%) advanced (stages HI andIV) cancers compared with 1 of 8 (12.5%) early (stages I and II) cancersl /' < 0.048). We conclude that human gastric epithelium has a unique

mucin gene pattern, which becomes markedly altered in preneoplastic andneoplastic specimens. Increased mucin gene heterogeneity in gastric ade-

nocarcinomas is associated with advanced cancer stage.

INTRODUCTION

Gastric mucins are critical cytoprotective proteins synthesized bygastric epithelial cells. Numerous alterations of gastric mucins havebeen described in inflammatory, metaplastic, and malignant diseasesof the stomach (1). The transition from neutral mucins in normalstomach to sulfomucins in intestinal metaplasia has been shown torepresent a marker of increased risk for gastric cancer (1, 2). Thetransition from intestinal metaplasia to adenocarcinoma is characterized by further qualitative and quantitative alterations of mucin-related antigens (1, 3). Alterations of mucin-type glycoproteins may

contribute to changes in cancer cell growth regulation, immune recognition, and cellular adhesion, which in turn may influence theinvasive and metastatic capabilities of the cancer (4-8). Several

studies of cancers of the lung and colon have associated increasedexpression of mucin proteins (9), sialylated mucin antigens (10), andblood group H antigens (11) with increased cancer stage and diminished prognosis.

The identification of unique mucin genes has enabled us to begininvestigations of the molecular processes that underlie mucin alter-

Reccived 8/17/94; accepted 4/19/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore he hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a Veterans Administration Merit Review Award (S. B. H.,

N. W. T.), a National Research Service Award (L. L. S.), a Veterans Administration MedicalInvestigator Award (Y. S. K.), and the Research Service of the Veterans Administration.

2 To whom requests for reprints should be addressed, at Department of Gastroentcr-

ology (11l-D), Veterans Administration Medical Center, 1 Veterans Drive, Minneapolis,

MN 55417.

ations in disease states. To date, seven human mucin genes have beencloned and sequenced with the use of a variety of human organ-

specific cDNA libraries. Nucleotide sequence data indicate that thesemucin core proteins contain regions with a high content of O-glyco-

sylation sites arranged in tandem, repeated arrays (tandem repeats).However, each mucin is distinct due to differences in tandem repeatsequences, lengths, and unique nonrepctitive sequences (Table 1).One mucin (MUCI) is membrane associated and contains a mem-brane-spanning sequence at the COOH-terminal end (12-14). Secretory mucins are non-membrane bound and are expressed highly in the

gastrointestinal and respiratory tracks. Mucin genes isolated fromintestinal cDNA libraries include MUC2 and MUC3 (15, 16), andmucin genes isolated from bronchial tissue cDNA libraries includeMUC4 and MUC5 (17-19). The MUC5 and MUC6 mucins were

isolated from a human gastric cDNA library (20, 21). In addition, asecretory type mucin, termed MUC7, has been cloned recently withthe use of a salivary gland cDNA library; however, published data todate indicate that this mucin is not expressed in gastric epithelium(22). We have observed previously that expression of mucin genesMUCl-3 is relatively organ specific, and that extensive loss of normal

mucin gene regulation may occur after malignant transformation (23,24). To date, little is known concerning the expression of specificmucin genes in gastric neoplasms. To study the molecular mechanisms underlying mucin gene expression in normal and neoplastichuman stomach, we used specific mucin tandem repeat cDNA probesand antibodies to determine the relative expression of human mucingenes in normal stomach and preneoplastic (intestinal metaplasia) andmalignant gastric epithelium.

MATERIALS AND METHODS

Tissue Specimens. Human tissue specimens were obtained from surgicalresections, fixed in buffered formalin, and embedded in paraffin with the useof standard methods. "Normal" tissue was obtained from histologically normal

specimens immediately adjacent to carcinomas (transitional mucosa) or histologically normal mucosa obtained from the resection margins of the surgicalspecimen. Specimens for RNA isolation were collected immediately aftersurgery, snap frozen in isopentane at â€”¿�6()Â°C,placed in sterile jars, and storedat â€”¿�70Â°C.Specimens used for formalin fixation were collected separately from

specimens used for RNA isolation. Tissue specimens were collected in accordance with human studies guidelines and approval.

Prior to RNA isolation the frozen tissue specimens were placed in a cryostat.Several 5-ju.m sections were taken and stained with hematoxylin and cosin to

verify the histology of the specimen (cardia, fundus, antrum, intestinal metaplasia, and type of cancer). If specimens of gastric cancer included segmentsof normal mucosa, these areas were trimmed from the tissue block prior toRNA isolation.

The use of histologically normal resection margins from patients undergoing gastric resection may not be the ideal normal control. We have comparedpreviously the immunohistochemical staining using mucin antibodies in normal surgical resection margins of cancer specimens to the staining observed innormal biopsy specimens from patients with normal stomachs determined byendoscopy (21). We found that the quality of staining for all antibodies was nodifferent in these two groups, with the exception that the intensity of stainingwas slightly less in the endoscopie biopsies compared with the surgical

2681

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MUCIN GENE EXPRESSION IN GASTRIC CANCER

Table 1 Human murin genes

DesignationMUClIUUC2MUC3MUC4MUC5MUC6SourceMammary

pancreaticIntestinalIntestinalTracheohronchialTracheobronchial,

gastricGastricChromosomal

locationIq21q24llplS73llplSllplSType"memsecsecsecsecsecNo.of a.a.

int.r.202317168169Tandem repeat a.a.sequenceGSTAPPAHGVTSAPDTRPAPPTTTPITTTTTVTPTPTPTGTQTHSTPSFTSSITTTETTS(T)SS(A)ST(GHA)T(P)L(P)VT(D)bTTSTTSAPSPFSSTGPMTATSFQTTTTYPTPSHPQTTLPTHVPPFSTSLVTPSTGTVIRet.121516181720

TPTHAQMATSASIHSTPTGTIPPPTTLKATGSTHTAPPMTPTTSGTSQAHSSFSTAKTSTSLHSHTSSTHHPEVTPTSTTTITPNPTSTGTSTPVAHTTSATSSRLPTPFTTHSPPTGS

" mem, membrane bound; sec, secreted; a.a., amino acid; t.r., tandem repeat.

Letters in parentheses, imperfectly conserved.

resection margins. These data support the use of resection margins as normal

tissue.Cancers were categorized histologically with the use of the classification

method by Lauren (25) and included intestinal type (well, moderately well, andpoorly differentiated) and diffuse-type adenocarcinomas. Cancer stage was

determined from surgical and pathological reports and classified according tocriteria published previously (26). Specimens included in this study consistedof: stage I (n = 3); stage II (n = 5); stage III (n = 20); and stage IV (n = 5).

Antibodies. Antibodies, type, and epitope specificity are listed in Table 2.The mAb 139H2 was kindly provided by John Hilkens (Netherlands CancerInstitute, Amsterdam). The antisera MRP was raised in rabbits against asynthetic MUC2 tandem repeat consensus sequence (KYPTTTPISTTTM-

VTPTPTPTGTQT) as described previously (15). The K and Y residues wereadded to allow glutaraldehyde conjugation and radioiodination for futurestudies. The antisera M3P was raised in rabbits against synthetic MUC3tandem repeat sequence (KTTSNSTPSFTSSITTTETTSHS) conjugated tokeyhole limpet hemocyanin (16) with the use of similar methods. AntibodyFHF was raised in rabbits against purified human gastric mucin, which hadbeen deglycosylated with hydrogen fluoride as described previously (20). Theantibody M5P-cl was raised in chickens, and antibody M5P-bl was raised inrabbits against a synthetic MUC5 tandem repeat peptide (KTTSAPTTSTT-SAPTTS), consisting of two 8-amino acid tandem repeat consensus sequence

units conjugated to keyhole limpet hemocyanin. Results with the use of thesetwo antibodies were similar and grouped under the heading M5P. The antibodyM6P also was raised in chickens against a 23-amino acid peptide (KPPFTTH-

SPPTGSSPFSSTGPMT) conjugated to keyhole limpet hemocyanin. This peptide corresponds to a portion of the 169-amino acid tandem repeat unit of the

MUC6 mucin. Antibodies against synthetic MUC5 and MUC6 tandem repeatsare specific and do not cross-react with other mucin tandem repeats. The

detailed production and characterization of these antibodies are describedseparately (21).

Immunohistochemistry. Previous studies have demonstrated that uniquemucin tandem repeats can be identified with the use of polyclonal and mAbsin pathological specimens with the use of immunohistochemical techniques

Table 2 Mucin core peptide antibodies and mucin cDNAprobes

MucingeneMUCl

MUC2MUC3MUC5

+ MUC6MUC5MUC6Mucin

geneMUCl

MUC2MUC3MUC4MUC5MUC6Name139H2

MRPM3PFHFM5P

M6PTypeMouse

mAbRabbit pAb"

Rabbit pAbRabbitpAbChicken

pAbChickenpAbcDNA

(length)*pum

24p (280)SMUC 41 (836)SIB 139 (260)M4oligo (48)4F (494)3F (540)SpecificityRef.MUCl

core protein 70Synthetic MUC2 tandem repeat peptide 15Synthetic MUC3 tandem repeat peptide 16Deglycosylated gastric mucin 20

(MUC5 + 6 core proteins)Synthetic MUC5 tandem repeat peptide 21Synthetic MUC6 tandem repeat peptide21Sequence

specificityMUCl

tandem repeatsMUC2 tandem repeatsMUC3 tandem repeatsMUC4 tandem repeatsMUC5 tandem repeatsMUC6 tandem repeatsRef.12

151618

17,2120

" pAb, polyclonal antibody.'' Length in bp.

(24, 27-29). Furthermore, we have shown that MUC2 and MUC3 tandem

repeat immunoreactivity in normal colon and colon cancers correlates withMUC2 and MUC3 mucin gene expression determined by in vitro hybridization

(30). Formalin fixation has been shown to be adequate for reactivity with mAb139H2 and antibodies MRP, FHF, M3P, M5P, and M6P (20, 21, 31). Thestreptavidin-peroxidase technique was used as described previously (24).

Briefly, tissue sections were deparaffinized, rehydrated, incubated with fresh3% hydrogen peroxide in methanol for 10 min, and then washed with buffer(PBS). Normal goat or rabbit serum (5%) was applied for 20 min and removedby blotting. Next the sections were incubated with primary antisera for 90 minat room temperature, washed three times in buffer, and incubated with thebiotinylated secondary antibody (1:75 dilution in PBS) for 20 min. Afterwashing, the sections were incubated with streptavidin-peroxidase conjugate

(10 ng/ml) for 30 min, followed by repeated washing. Next the sections wereincubated with diaminobenzidine in 0.03% hydrogen peroxide for 10 min,washed, counterstained with methyl green, rinsed in tap water, and mounted.Immunoreactivity was graded as â€”¿�(negative), Â±(trace positive), + (positive),

or ++ (strongly positive). A specimen was considered focally positive ifimmunoreactive cells were found in at least 5% (objective, X10) to <50% oflow power fields. A specimen was considered to have widespread reactivity ifpositive cells were present in a50% of low power fields. Biotinylated goatanti-rabbit IgG; biotinylated rabbit anti-mouse IgG, IgA, and IgM; biotinylatedrabbit anti-chicken IgY; streptavidin-peroxidase conjugate, and normal rabbit

and goat serum were obtained from Zymed Laboratories (San Francisco, CA).Diaminobenzidine was obtained from Sigma Chemical Co. (St. Louis, MO).Statistical analysis was performed with the use of ANOVA and x2 contingency

table analysis with the use of the Statview SE+Graphics program (AbucusConcepts, Inc., Berkeley, CA).

Immunohistochemical Controls. Negative controls included substitutingsimilar dilutions of preimmune rabbit, mouse, and chicken antibodies for theprimary antibodies, which resulted in negative staining. In addition, workingdilutions of anti-MRP, anti-M3P, anti-M5P, and anti-M6P antibodies were

preabsorbed with the use of 10-20 /ig/ml of synthetic MUC2, MUC3, MUC5,

and MUC6 peptides, respectively, which resulted in negative staining whenused on positive specimens (21, 24).

RNA Analysis. Total RNA was isolated from tissue specimens with the useof guanidinium thiocyanate-phenol-chloroform extraction (32). After verifica

tion of specimen histology by cryostat sectioning, the frozen tissue specimens(100-500 mg) were placed in guanidinium thiocyanate solution and homogenized on ice with the use of a Polytron PT-10~35 (Brinkman Co., Westbury,

NY). RNA was extracted with the use of phenol, chloroform, and isoamylalcohol, as described (32). The RNA was washed twice with 70% ethanol, oncewith absolute ethanol, resuspended in water treated with diethylpyrocarbonate,and quantitated spectrophotometrically. RNA samples (10 jag) were separatedon 1.2% agarose gels in the presence of formaldehyde. The integrity andamount of RNA loaded on each gel was determined by staining with ethidiumbromide (1 fÂ¿g/mlin 100 mM ammonium acetate). Samples with evidence ofrRNA degradation were discarded. The RNA was transferred to Nytran nylonmembranes (Schleicher & Schuell, Keene, NH) by capillary action and cross-

linked with the use of UV radiation. Slot blot analysis was also performed byapplying 0.4-4.0 fig of total RNA to Â£-probenylon membranes with the useof a slot blot apparatus (Bio-Rad Laboratories, Richmond, CA). The mem-

2682




branes were prehybridized in 50% formamide, 5X SSPE,3 0.3% SDS, 5XDenhardt's solution, and 200 /j,g salmon sperm DNA and incubated at 42Â°C

overnight. The nylon membranes were then hybridized to radiolabeled cDNAprobes as described (15). cDNA probes were labeled with [32P]dCTP (3000

Ci/mmol dCTP triethyl ammonium salt; DuPont New England Nuclear, Boston, MA) with the use of random hexamer primers (GIBCO-BRL, GrandIsland, NY; Ref. 33). Radiolabeled probes were purified with the use of a G-50

Sephadex column (Boehringer Mannheim, Indianapolis, IN) according tomanufacturer's directions. After hybridization, the membranes were washed

with the use of high stringency conditions: two washes with 2X SSC-0.1%SDS for 30 min at room temperature, one wash with 0.1% SSC-0.1% SDS forl h at room temperature, followed by one wash with 0.1% SSC-0.1% SDS at55-65Â°C for 30 min. Autoradiography was performed with the use of Kodak

X-OMAT AR film (Eastman Kodak Co., Rochester, NY) with a Cronexintensifier screen (DuPont) at -70Â°C for 24 h. After autoradiography, each

membrane was stripped and reprobed. The characteristics of the cDNA probesused in this study are listed in Table 2. The probe for MUC1 (pum24p) waskindly provided by Dr. Dallas Swallow (MRC Human Biochemical GeneticsUnit, London, England). The MUC2 (probe SMUC41), MUC3 (probe sibl39),MUC5 (probe 4F) and MUC6 (probe 3F) sequences have been describedpreviously (15, 16, 20).

Three 48-mer oligonucleotide probes were constructed with the use of

MUC4 tandem repeat sequences as published previously by Porche! et al. (18).The MUC4 sequence differs from other secretory mucins in that the sequenceof the tandem repeats is not well conserved among the different tandemrepeats. Three different oligonucleotide probes were constructed to correspondwith the three most common tandem repeat sequences in the published MUC4cDNA. An oligonucleotide probe 5'-AACGATCAGAGTAGTGGTATT-TCACC-3' corresponding to sequence 4011-4036 of human 28S rRNA was

used to verify equal loading (34). These oligonucleotides were purified withthe use of an OPC cartridge (Applied Biosystems, Foster City, CA) and labeledwith [32P]dCTP with the use of terminal deoxynucleotidyl transferase

(GIBCO-BRL; Ref. 35). Radiolabeled nucleotides were purified with the useof a G-25 Sephadex column (Boehringer Mannheim) according to manufacturer's directions. Prehybridization and hybridization conditions were as listed

earlier. After hybridization the membranes were washed three times at roomtemperature (15 min each) in 6X SSPE-0.1% SDS and once at 37Â°C(15 min)

in 2X SSPE-0.1% SDS. Autoradiograms were prepared as above. Positive

controls were used on each blot and consisted of RNA isolated from humansurgical resection specimens of colon, lung, breast, and small intestine. Hybridization signals were quantitated with the use of a Bio-Rad densitometer

and computer software. Densitometric units were calculated for each sampleafter normalization of the readings with corresponding densitometric readingsobtained with the use of the 28S rRNA probe.

RESULTS

Mucin Gene Expression in Normal Tissues. Northern blot analysis revealed a unique distribution of secretory mucin mRNAs in thegastrointestinal tract. In contrast with other secretory-type mucins,

normal stomach contained high levels of MUC5 and MUC6 mRNA,with lesser amounts found in small intestine and ileum (Fig. 1). Inaddition, normal duodenum was found to contain high levels ofMUC6 mRNA. MUC2, MUC3, and MUC4 gene expression wereabsent in normal gastric specimens, whereas MUC2 and MUC3 mucinmRNAs (Figs. 1 and 2) and immunoreactivity (Figs. 3 and 4) wereexpressed uniformly at high levels in gastric specimens with intestinalmetaplasia and in duodenum, small intestine, ileum, and colon. MUC4mRNA was limited to colonie tissue (Fig. 1) and bronchial tissue (notshown). Lack of detection of MUC4 mRNA in intestinal metplasiaspecimens may be due to the limited number of samples available forthis assay.

MUC1 mRNA demonstrated a polymorphic pattern of discretebands between 4.4 and 9.49 kb, with no evidence for RNA degrada-

3 The abbreviation used is: SSPE, 3.6 M sodium chloride-0.2 M sodium phosphate

dibasic-0.02 M EDTA.

tion. In contrast, secretory mucins displayed a polydisperse patternwhen the same blots were used for Northern blot analysis, which istypical for secretory mucins (see "Discussion" and Ref. 36).

M5P immunoreactivity was localized to the supra- and perinuclear

areas of mucinous foveolar cells lining the surface of the gastricepithelium in over 90% of cardia, fundus, and antrum specimensexamined (Fig. 3). In contrast, M6P immunoreactivity was localizedprimarily to the peri- and supranuclear area of mucous neck cells of

the fundus and the glandular cells of the cardia, antrum glands, andduodenal Brunner's glands (Fig. 4C; Ref. 21).

The intestinal metaplasia in gastric specimens used for RNA isolation was focal and interspersed heavily with areas of normal, non-

metaplastic gastric epithelium. Therefore, specimens of intestinalmetaplasia processed for RNA analysis contained MUC5 and MUC6mRNA levels comparable with normal gastric tissue, as indicated inFig. 2. For the immunohistochemical analysis only the intestinalmetaplasia glands were graded (Fig. 3). Intestinal metaplasia wasprimarily nonreactive with M5P and M6P antibodies, with the exception of rare mixed glands that appeared to contain both gastric andintestinal-like goblet cells. Therefore, MUC5 and MUC6 mRNA were

detected in every tissue sample of intestinal metaplasia; however,immunoreactivity corresponding to MUC5 and MUC6 was detectedrarely within the intestinal metaplasia glands. In contrast, intestinalmetaplasia was characterized by prominent MUC2 immunoreactivityin the supranuclear area of goblet cells and strong MUC3 reactivity inprimarily columnar-type cells (Fig. 4, A and B). The perinuclear and

supranuclear staining of MRP appears to be due to compression of thecytoplasm by mature mucous granules in goblet cells.

Antibody FHF was raised against purified human gastric mucin thathad been deglycosylated with hydrogen fluoride. FHF reactivity included the distribution of both M5P and M6P and also reacted to alesser extent with MUC2-expressing goblet cells in intestinal meta

plasia (Fig. 3). Presumably, the purified mucin used as the antigen forFHF antisera included a mixture of MUC5, MUC6, and possiblyMUC2 proteins.

MUC1 mRNA was present in normal gastric tissues (Fig. 2) and inother normal gastrointestinal tissues examined, with the exception ofminimal MUC1 mRNA in one sample of small intestine (Fig. 2). Innormal stomach, MUC1 immunoreactivity was faint and located in theapical membranes and cytoplasm of parietal cells located in fundicglands. In addition, trace 139H2 reactivity was present in rare antralglands and antral surface mucous cells.

Mucin Gene Expression in Gastric Cancers. Striking differencesin both the quality and quantity of mucin gene expression wereobserved in gastric cancers. With the exception of the MUC1 trans-

membrane mucin, expression of normal gastric mucins was decreasedin gastric cancers. MUC5 and MUC6 mRNA levels (Figs. 1 and 2)and immunoreactive protein (Fig. 3) were decreased markedly in mostcancers when compared with normal gastric specimens. M5P andM6P reactivity were observed in only 19 and 57% of specimens,respectively (Fig. 5, B and C). In contrast, aberrant expression ofmucins MUC2, MUC3, and MUC4 was observed frequently in gastriccancers (Figs. 1-3). MUC2 immunoreactivity was observed in 34% of

33 gastric cancers, and MUC2 mRNA was detected at low levels in31% of 13 cancer RNA specimens. MUC3 immunoreactivity waspresent in 45% of 33 cancer specimens, and varying levels of MUC3mRNA was detected in all 13 cancer RNA specimens. Gastric cancersand normal stomach contained similar levels of MUC1 mRNA; however, strong 139H2 immunoreactivity was observed more often ingastric cancers compared to normal gastric epithelial cells (Fig. 5A).

The cellular localization of mucin peptides in gastric cancers wasaltered frequently. For example, the transmembrane MUC1 proteinoften was observed to be expressed diffusely in the cytoplasm of

2683



MUCIN CiENE EXPRESSION IN GASTRIC CANCER

abcdefghijk l m n abcdefghijk Imn

Fig. 1. Northern blot analysis of mucins in gastrointestinal tissues. Blots were probed and erased serially with the use of cDNA or oligonucleotide probes corresponding tomembrane-bound MUC1 mucin (A) and secretory mucins MUC2 (B), MUCJ (C), MUC4 (D), MUCS (Â£),and MUC6 (F). Leading was verified with the use of a 28S rRNA probe

(C). Lanes: normal esophagus (<;), intestinal metaplasia (b, c), normal stomach (d, e), normal pancreas (/). normal duodenum (g), normal small intestine (/i), normal ileum (i), normalproximal colon (j), normal distal colon (k), and gastric adenocarcinoma (/-/<). Ordinine, size markers in thousands.

malignant cells in addition to the expected apical membrane localization. Similarly, secretory mucin proteins often were expressed diffusely rather than localized to the perinuclear or supranuclear areas ofmucin-synthesizing cells (Fig. 5, B and C). The intensity of cancer

staining, when present, was also often increased (+ or + +) compared

to the reactivity present in normal gastric epithelium (Â± or +).Intense mucin core peptide immunoreactivity is observed frequently in malignant cells and may reflect diminished tandemrepeat glycosylation (24, 37).

Expression of mucin core peptides is related to the histological2684



MUC1

T

NL IM CA

n=11 n-3 n-13


MUC2

NL IM CA

MUC3 MUC4 MUC5 MUC6

NL IM CA NL IM CA NL IM CA NL IM CA

Fig. 2. Slot blot analysis of mucin mRNA in surgical resection specimens of normal gastric epithelium (/VÂ¿),intestinal metaplasia (IM), and gastric adenocarcinoma (CA). Blotswere probed, erased, and reprobed with the use of specific mucin cDNAs as indicated. Results are normalized for RNA loading with the use of a 28S rRNA signal. Columns, mean;bars, Â±SEM.

;90-80-70-;80-50-40-30-20-10-

0-1â€¢3Â¡ a 3 Â¡ a 1Â¡lilis

lilis liliin

^ll

iliÂ¡-<lll-<cÂ¡iHE.

Fig. 3. Immunohistochemical analysis of normal gastric samples, intestinal metaplasia(/.A/.) and gastric cancers (CA). Serial sections from each specimen were analyzed withthe antibodies listed on top of the graph (see "Materials and Methods"). Numbers of

specimens analyzed are listed once and are the same for each antibody.

differentiation of the tumor. As illustrated in Fig. 6, mucin corepeptides were expressed more frequently by well- and moderatelywell-differentiated intestinal cancers compared with poorly differentiated intestinal or diffuse-type cancers. The distribution of positive

reactivity was quite diverse, with both focal and widespread distributions noted in most histological subtypes of cancers.

Coexpression of Mucin Core Peptides. Each cancer was categorized by degree of differentiation, surgical stage, and the presence ofspecific mucin tandem repeat peptides determined by immunohisto-

chemistry (Fig. 7). The numerous combinations of mucin peptidesobserved indicate that considerable heterogeneity exists in mucin geneexpression in gastric cancers. Overall, 23 of 33 (66.6%) cancersexpressed 2 or more different mucin peptides. These cancers oftencontained discrete cell populations that synthesized different mucins,as exemplified by Fig. 8. The most common mucin core peptideexpressed in gastric cancers was derived from the transmembraneMUC1 gene and was detected in 25 of 33 (75.8%) of cancers. MUC6was the most common secretory mucin, found in 19 of 33 (57.6%),followed by MUC3 mucin found in 15 of 33 (45.4%) of cancers.Immunoreactivity for one or more secretory mucin core peptides waspresent in 75.8% of cancers. As illustrated in Table 3 and Fig. 7A,

A

X

v

Fig. 4. Immunohistochemical staining of intestinal metaplasia. A, antibody MRP. Note the supranuclear and perinuclear reactivity in goblet cells only. 8, antibody M3P. Reactivityis present in columnar and rare goblet cells. C, antibody M6P. Reactivity is present in antral glands, an area of intestinal metaplasia is negative. Bars, 100 /xm; countcrstain is methylgreen.

2685




B

Fig. 5. Immunohistochemical staining of gastric cancers. A, expression of MUC1 core peptide detected by antibody 139H2. Note the strong apical membrane reactivity. B, expressionof MUC5 core peptide detected by antibody M5P. C, MUC6 core peptide detected by antibody M6P. Note the diffuse, nonpolarized cytoplasmic staining. Bars, 100 /xm; counterstainis methyl green.

multiple mucin core peptides were expressed more frequently by well-and moderately well-differentiated intestinal cancers compared withpoorly differentiated intestinal or diffuse-type cancers. In addition,

expression of multiple mucin core peptides correlated with increasedsurgical stage (Fig. IB). When specimens are grouped according tothe total number of immunoreactive mucin core peptides expressed,early (stage I and II) cancers were significantly more likely to expresssi mucin compared with advanced (stages III and IV) cancers (Fig.9). In contrast, coexpression of 3 or more mucin core proteins occurred in 15 of 25 (60%) advanced cancers compared with 1 of 8(12.5%) early cancers (P = 0.048).

DISCUSSION

These data demonstrate that human gastric epithelium has a uniqueorgan- and cell-specific mucin gene pattern characterized by high

*â€¢ g PIIntestinal-Well/Moderats5-C1o."

3-o

8t2~C1-

n.H

Intestinal-Poor-11â€¢s-n-1

Diffuse-i

i-ipni

iMUC1 - + -MUC2 - - tMUC3 - - -MUC5 - - -MUC6 - - -

100 139H2 MRP M3P FHF MSP M6P

Ã¤

â€”¿�70-

2 60-

I 50-

o 40-:

20 -ÃŒ

10-ÃŒ

0

localreactivity

widespreadreactivity

5 o. o i o. D EO.Q EO.O

Fig. 6. Immunohistochemical analysis of gastric cancers categorized according tohistology. W/M, well- or moderately well-differentiated intestinal cancers; P, poorlydifferentiated intestinal cancers; D, diffuse-type cancers. Serial sections from each specimen were analyzed with the antibodies listed on top of the graph (see "Materials andMethods"). Numbers of specimens analyzed are listed once and are the same for each

antibody.

B.

I *

I"o

ae

1-

II 1nin

D Stage I

D Stage II

Stage III

Stage IV

MUC1 -MUC2 -MUC3 -MUC5 -MUC6 -

Fig. 1. Coexpression of mucin core peptides in gastric cancers. Cancers are categorizedaccording to histology (A) and stage (B). Abscissa, positive (+) or negative (â€”)reactivity

for each mucin core peptide.

2686




Fig. 8. Immunohistochemical staining of gastriccancers. A, moderately well-differentiated cancerstained with antibody M6P. B, serial section of thesame specimen as in A stained with antibody MRP.Note that the arrows point to the same area on eachspecimen. This cancer contains two different populations of mucin-synthesizing cells. Bars, 100 urn;

counterstain is methyl green.

$*m

C"â€¢¿�Ã¯- *rÂ£:<

Table 3 Relationship of number of mucin core peptides expressed in gastricadenocarcinomas with histolÃ³gica! differentiation and surgical stage

No. ofmucin core peptides

expressed^

1 mucin2 mucins

a3 mucinsDiffuse"

(%)3/5

(60)fc

1/5 (20)1/5 (20)Poor

%5/11(45)

4/11(36)2/11 (18)IntestinalWell/moderate

(%)2/17

(12)2/17(12)

13/17 (76f" Number of specimens with positive immunoreactivity/total number of specimens.

P - 0.0486 compared with poor and well/moderate histologies.c P = 0.0041 compared with diffuse and poor histologies.

levels of MUC1, MUC5, and MUC6. The process of neoplastictransformation in the stomach is associated with a decrease in thesemucins and the additional expression of mucin genes normally expressed by the intestine (MUC2, MUC3, and MUC4). Advanced stagegastric cancers and cancers with greater differentiation are significantly more likely to express multiple mucin core peptides comparedwith early stage and less differentiated cancers. These results supportour previous observation of extensive loss of normal mucin generegulation in other gastrointestinal and nongastrointestinal adenocarcinomas. Using antibodies and cDNA probes that recognize the tandem repeats of membrane-bound (MUC1 ) and secreted (MUC2 and

MUC3) mucins, we observed that cancers frequently demonstratethree types of alterations: (a) loss of normal mucin gene expression;(b) increased mucin core peptide immunoreactivity; and (c) expression of mucin core peptides and mRNA not found in correspondingnormal epithelium (23, 24, 38, 39). Similar alterations in gastricmucins were observed in the gastric cancers studied in this paper.

Secretory mucins typically demonstrate a polydisperse mRNA pattern on Northern blot analysis. The reason for this polydispersity isunknown but may be due to either rapid turnover of mucin message orincreased susceptibility of very long mucin mRNAs to degradation(36). Analysis of the same samples and blots with the MUC1 cDNAprobe (Fig. 1) or with a housekeeping probe, glyceraldehyde 3-phos-

phate dehydrogenase (not shown), demonstrates single discrete bands.In addition, ethidium bromide staining of the RNA gels demonstratescomparable size distribution, as well as intact 28S and 18S bands (not

shown). This rules out excessive RNA degradation as a cause of theappearance of secretory mucin mRNAs in our samples. The cDNAand oligonucleotide probes recognize portions of mucin tandem repeats; hence, the intensity of slot blot hybridization may be considereda quantitative measurement of the amount of tandem repeat RNApresent. Some caution should be taken in interpreting the intensity asa direct measure of the whole mucin message, since the cause of thepolydispersity is not known. In this regard we have observed thattissue specimens with strong mucin RNA signals obtained on slot blotusually demonstrate strong intensity of staining as evidenced byimmunohistochemistry, indicating that mRNA levels determined byslot blots may be a semiquantitative reflection of translated mucinprotein.

Adenocarcinomas of the stomach can be classified into two majortypes (25). The "intestinal" type is composed of distinct glands with

Stage l+ll (n=8)

Stage III+IV (n=25)

< 1 MUCIN 2 MUCINS > 3 MUCINS

â€¢¿�p=0.008vs.stage III+IV

"p=0.048 vs. stage l+ll

Fig. 9. Expression of mucin core peptides in relation to surgical stage, n = 8, stage Iand II patients; n = 25, stage III and IV patients.

2687




polarized cells resembling colon carcinoma. The "diffuse" type rarely

demonstrates glandular morphology and is composed of solitary cellsor small clusters of cells. The intestinal type of gastric cancer is thepredominant type in elderly populations at high risk and is precededby well-defined precancerous lesions, such as intestinal metaplasia

and atrophie gastritis. The diffuse type is relatively more frequent inlow risk populations and is not as often preceeded by intestinalmetaplasia (1). Marked differences in mucin gene expression werealso noted in gastric cancers categorized by histological type. Intestinal-type cancers were more likely to express mucins compared withdiffuse-type cancers. These data suggest that the ability of gastric

cancers to synthesize mucins is largely a function of their ability todifferentiate. We have observed previously that the aberrant expression of the mature products of a variety of cell types or lineages is acharacteristic feature of gastrointestinal cancers and may represent anembryonic or stem cell-like derepression of dormant synthetic path

ways (40, 41). Expression of MUC5 and MUC6 mucin genes ischaracteristic of the gastric mucous cells, whereas MUC2 and MUC3mucin genes may be considered markers for intestinal or coloniedifferentiation. Therefore, expression of these mucin core peptides ina cancer may reflect the original cell type involved in malignanttransformation. In the present study the majority of intestinal-typecancers express MUC2 and/or MUC3-related peptides, supporting the

theory that these cancers result from transformation of intestinalmetaplasia.

The transition from MUC5 and MUC6 mucin gene expression innormal gastric mucosa to MUC2 and MUC3 mucin genes in intestinalmetaplasia is associated with the appearance of new carbohydrateantigens. Torrado et al. (42) observed that anomalous expression ofLe1'antigen in Le(a~b+) subjects was present in 70% of 122 intestinal

metaplasia specimens. Murata et al. (43) reported that intestinalmetaplasia was associated with frequent deletions of ABH, Lex, and

Ley antigens expressed by normal gastric mucosa. In addition, expression of sialylated Le" and Le" antigens was observed in 71-100% of

intestinal metaplasia specimens, depending on the secretor status ofthe patients. These data suggest that expression of specific glycosyl-transferase and mucin genes may be coregulated in cell- or tissue-

specific distributions. Studies by Paulson et al. (44) have demonstrated that sialyltransferase enzymes are expressed in a tissue-

specific distribution. Protein sequences may provide recognitionsequences for certain O- and AMinked glycosyltransferases (45, 46).

Additional experiments are needed to determine the exact relationshipof specific mucin tandem repeat units, carbohydrate antigens, andglycosyltransferases in gastric mucosa.

Previous studies have also described the findings of increasedmucin heterogeneity in gastric cancers (both intra- and intertumor).Well-differentiated cancers typically contain more sulfomucin (char

acteristic of mature surface mucin cells), whereas sialomucins(characteristic of intestinal goblet cells) predominate in moderatelyand poorly differentiated cancers (1, 47). Biochemical characteristicsof glycoproteins purified from gastric secretions of patients withgastric cancer contain increased sialic acid and sulfated blood groupantigens compared with normals (48). In addition, studies using mAbshave indicated that mucin carbohydrates are frequently shortened oraltered in gastric cancers. Aberrant antigens such as blood groupsincompatible with the normal blood group of the individual, sialylatedLea (antigen 19-9), sialylated Le", and sialyl-Tn antigens also appear

(43, 49-55). We have observed a marked heterogeneity or mosaicism

in mucin core peptide expression that is similar to the heterogeneity ofcarbohydrate antigen expression noted previously in gastric cancers(3). These data suggest that differences in carbohydrate antigen expression may be associated with differences in mucin gene expression.In addition, previous studies have identified a number of poorly

defined mucin antigens expressed by gastric cancers and normal smallintestine or colon but not by normal gastric epithelium. These includethe SIMA, LIMA (56), BD-5 (57), and M3 antigens (58). Our datasuggest that these antigens may represent MUC2-, MUC3-, and/orMt/C4-related mucins; however, further comparative studies are

needed.Cancer mucins may be exploited as possible therapeutic and diag

nostic targets. Cytotoxic T cells derived from patients with breast,pancreatic, or ovarian cancer recognize cell surface MUCl-type coretandem repeat sequences (59-61). Cytotoxic T cells readily recognizetarget cells that are transfected with cDNA encoding only two 20-

amino acid tandem repeat sequences (62). Stimulation of an antitumorimmune response can also be accomplished by immunization withspecific carbohydrate antigens, such as T, Tn, and sialyl-Tn, which are

located in the core carbohydrate region of mucin molecules (63, 64).Several mucin-related antigens have also demonstrated utility as se-

rological assays and for targeting radiolabeled antibodies (65). Mucincore peptide expression in gastric cancers was observed most commonly with antibody 139H2 (MUC1 mucin gene), with positiveimmunoreactivity in 75.8% of specimens. The addition of one secretory mucin antibody (MUC6) to the MUC1 antibody increases thepositive rate to 81.8%. Overall, 87.9% of gastric cancers displayedÂ¡mmunoreactivity to at least one mucin. These observations indicatethat diagnostic or therapeutic uses of mucin core peptide antibodiesfor gastric cancers would be most effective with the use of a combination of transmembrane MUC1 mucin and at least one secretorymucin.

Expression of multiple mucin core peptides in gastric cancerswas associated strongly with increased tumor stage. Two interpretations of this observation are possible: (a) expression of multiplemucins may only secondarily reflect the increased dedifferentia-

tion and genetic alterations found in advanced cancers; and (b)increased mucin gene expression per se may contribute to tumorcell growth and metastatic abilities. An increasing number ofstudies support a role for mucins in altering the biological behaviorof cancers. Highly mucinous human colon cancer cell lines demonstrate greater tumorigenicity and metastatic behavior in nudemouse models, which can be reduced by treatment of the cells withan inhibitor of glycoprotein glycosylation (4, 5, 66). In addition,increased sialylated cell surface glycoproteins have been associated with enhanced metastatic potential in a variety of otherexperimental animal models (67, 68). Increased sialylated mucinantigens (10, 69) and MUCl-core protein (9) have been detected in

metastatic and advanced stage human colon cancers compared withthe primary and early stage colon cancers. These data indicate thatfurther research regarding mucin gene expression and the biological behavior of gastric cancers is warranted.

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1995;55:2681-2690. Cancer Res Samuel B. Ho, Laurie L. Shekels, Neil W. Toribara, et al. Neoplastic Human Gastric EpitheliumMucin Gene Expression in Normal, Preneoplastic, and

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