the secreted tumor-associated antigen 90k is a potent immune

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOI. 269, No. 28, Issue of July 15, PP. 18401-18407, 1994 Printed in U.S.A.

The Secreted Tumor-associated Antigen 90K Is a Potent Immune Stimulator*

(Received for publication, December 6, 1993, and in revised form, March 17, 1994)

Axel UllrichSS, Irmi SuresS, Maurizia D’Egidion, Bahija JallalS, T. J. Powelll/, Ronald HerbstS, Andreas DrepsS, Mohammad AzamS, Menachem Rubinstein**, Clara Natolil, Laura K. Shawverll, Joseph SchlessingerSS, and Stefan0 Iacobellin From the $Department of Molecular Biology, Max-Planck-Znstitut fur Biochemie, A m Klopferspitz 18A, 82152 Martinsried, Federal Republic of Germany, Wattedra di Oncologia Medica, Universita G. D’Annunzio, Via dei Vestini, 6, 66100 Chieti, Italy, IISUGEN, Inc., Redwood City, California 94063, **Weizmann Institute of Science, 76100 Rehovot, Israel, and $$Department of Pharmacology, New York University Medical Center, New York, New York 10016

Immunization of mice with conditioned media from human breast cancer cells yielded the monoclonal antibody SP-2, which recognized an antigen of approximately 9&95 kDa. This protein, designated 90K, was found to be present in the serum of healthy individuals and at elevated levels in the serum of subpopulations of patients with various types of cancer and AIDS. Here we report the primary structure of the SP-2 antigen and demonstrate its relationship to a family of proteins which carry a scavenger receptor cysteine-rich domain. Northern blot analysis of normal tissues, primary tumors, and tumor-derived cell lines indicates a broad expression spectrum of the 9OK gene at widely varying levels. Functional characterization reveals stimulatory effects of 90K on host defense systems, such as natural killer cell and lymphokine-activated killer cell activity, and indicates that its immunostimulatory effects may be mediated through the induction of interleukin-2 and possibly other cytokines.

Monoclonal antibodies (mAbs)’ recognizing tumor-associated antigens (TAAs) for a number of cancer types have been reported, but the proportion of those that recognize molecules released in significant amounts into the bloodstream is small (for review, see Ref. 7). Moreover, the biological functions and possible roles of such TAAs in the context of tumor-host rela- tionships are largely unknown. Previous studies using mAb SP-2, derived from the fusion of spleen cells of mice repeatedly immunized with conditioned medium of human breast cancer cell cultures, led to the identification of the 90K glycoprotein, which was found in both healthy individuals and at elevated

Istituto Superiore di Sanita’-Project on AIDS, 1993 (Rome, Italy) (to * This work was supported by grants from Minister0 della Sanita’,

S. I.); Associazione Italiana per la Ricerca sul Cancro, 1993; Consiglio Nazionale delle Richerche Special Project “Applicazioni Cliniche della Ricerca Oncologica”; and SUGEN, Inc. (to A. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankm/EMBL Data Bank with accession numberls) X79089. The nucleotide sequence(s) reported in this paper has been submitted

2513; Fax: 49-89-857-7866. 1 To whom all correspondence should be addressed. Tel.: 49-89-8578-

mor-associated antigen; HW, human immunodeficiency virus; FCS, fe- The abbreviations used are: mAb, monoclonal antibody; TAA, tu-

tal calf serum; IL-2, interleukin-2; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; PBL, peripheral blood lymphocyte; NK, natural killer; LAK, lymphokine-activated killer; MR, maximal release; SR, spontaneous release; MTT, 3443- dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SRCR, scavenger receptor cysteine-rich domain; bp, base pair; kb, kilobase pair.

concentrations in the serum of patients with various types of cancer (1-4). Recently, 90K protein levels were found to be elevated in the serum of patients infected by the human immunodeficiency virus (HIV) (5, 6, 8-10), even in the apparent absence of neoplastic complications.

Because of the intriguing correlation between abnormal 90K serum levels, cancer, and HIV infection, we characterized the protein by cDNA cloning and sequence analysis in order to elucidate its molecular identity and approach the question of its biological function. Recently, after completion of the present study, Koths et al. (11) reported the cDNA structure of MAC- 2-BP, a binding protein for the soluble lactose/galactose-specific lectin Mac-2, which proved to be identical to the 90K cDNA sequence we have obtained. Mac-2, a 32-kDa protein, was first identified by Ho and Springer (12) as an antigen that is highly expressed by activated inflammatory macrophages. Subse- quent studies localized Mac-2 in a variety of cell types, including islets of Langerhans, dendritic cells, alveolar macrophages, Kupffer cells, and small intestine epithelial cells (13). Mac-2 represents the principal non-integrin lamin-binding protein of activated macrophages (14), and i t likely plays a role, by virtue of its laminin-binding property, in the interaction of these cells with the basement membrane, which may be of relevance for their ability to migrate into tissue spaces. In addition, Mac-2 has been implicated in events as diverse as immune response modulation, tumor progression, and metastasis (15). In this study we describe the expression of the 90K gene in normal and neoplastic tissues and provide evidence for a role of this glycoprotein in the stimulation of host defense systems against pathogens and cancer cells.

MATERIALS AND METHODS Cell Lines, Tissue Specimens, and Antibodies-Human tumor speci-

mens and normal tissues were obtained from surgical biopsies. Breast cancer and other tumor-derived cell lines were obtained from the ATCC. HT-2 cells, an IL-2-dependent murine cell line, were passaged every 2 days in RPMI + 10% FCS supplemented with 40 unitshl recombinant murine IL-2 (Genzyme). The SP2 mAb has been described previously (1). The 19B5 mAb was isolated after immunization of mice with recombinant 90K protein isolated from conditioned media of NIH 3T3 cells transfected with a 90K cDNA expression plasmid.’

Molecular Cloning of9OK-A cDNA library from MCF7 poly(A+) RNA in hgtX0 (16) was screened with a 32P end-labeled (5 x lo6 cpm/pM) 66-nucleotide long, single-stranded synthetic DNA probe (5‘-GTG AAT GAT GGC GAC ATG TCC CTG GCT GAT GGC GGC GCC ACC AAC CAG GGC CGG GTG GAGATC TTC-3‘) based on the NHZ-terminal90K amino acid sequence VNDGDM(C)LADGGATNQGRVEIF (17). Hybrid- ization was at relaxed stringency in 30% formamide, 5 x SSC, washing at 42 “C in 0.2 x SSC, 0.1% SDS. cDNA inserts were subcloned sepa-

’ B. Jallal, in preparation.

18401

18402 Tumor-associated Antigen 9OK

rately or as EcoRI partial into Bluescript KSII (Stratagene) and se- quenced by the dideoxy chain termination (18) or Maxam and Gilbert (19) sequencing method.

Dansient Expression in 293 Fibroblasts-The expression plasmid was constructed by introducing a 2147-bp ClaI (position 726 in Blue- script I1 KS)-XhoI (position 2118 in Fig. 1) restriction fragment into the eukaryotic, cytomegalovirus promoter-based expression vector p C W . Human embryonic kidney fibroblasts (293; ATCC CRL 1573) were grown in DMEM containing 10% FCS and antibiotics. One day prior to transfection, 2 x lo6 cells were seeded into each well of a six-well dish. Transfections were carried out according to the protocol of Chen and Okayama (20) with a total of 4 pg of CsCl gradient-purified plasmid DNNwell. 16 h after addition of precipitates, cells were washed once with DMEM and fresh growth medium was added.

For metabolic labeling, cells were grown overnight with [35S]methionine (50 pCi/ml) in methionine-free DMEM (0.5 muwell) containing 1% dialyzed FCS. The formation of N-glycosidic linkages was blocked by addition of tunicamycin to a final concentration of 0.1 or 1 pg/ml for 16 h. Cells were lysed on ice with 0.3 ml of lysis buffer containing 50 mM HEPES, pH 7.5,150 mM NaCl,1.5 mM MgCl,, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 200 unitdml aprotinin, 10 mM sodium pyrophosphate, 10 pg/ml leupeptin. Lysates were transferred to microcentrifuge tubes, vortexed for 10 s, and pre- cleared by centrifugation at 12,500 rpm for 15 min at 4 "C.

For immunoprecipitations, 10 pl of protein A-Sepharose (swollen and prewashed in 20 mM HEPES, pH 7.5) and 1 pg of mAb SP-2 was added to the cleared lysate and incubated at 4 "C for 3 h. The conditioned medium was used for immunoprecipitations after adding aprotinin (200 unitdml) and phenylmethylsulfdnyl fluoride (2 mM final) and preclear- ing by centrifugation. Precipitates were washed three times with 1 ml of washing buffer (lysis buffer with 0.1% Triton X-100). SDS sample buffer was added, and the samples were boiled and loaded on SDS- PAGE for separation of precipitated proteins.

Purification of 9OK-Pooled ascitic fluid from patients with ovarian carcinoma or serum-free conditioned medium of NIH 3T3 cells that were transfected with an expression plasmid containing the 90K cDNA were precipitated with ammonium sulfate at 43% saturation. The re- mainder of the purification procedure was carried out as previously described (17). Protein concentration was determined according to Bradford (21), and purity was confirmed by SDS-PAGE.

Northern Blot Analysis-For Northern blots, total RNA was prepared by the guanidine thiocyanate method (22). The RNA was resolved on a 1.2% agarose, formaldehyde gel, transferred to nitrocellulose, UV cross- linked, and hybridized in a formamide-based hybridization solution at 42 "C with 32P-labeled cDNA inserts. The filters were washed twice at room temperature in 2 x SSC, 0.1% SDS for 20 min and twice at 55 "C in 0.1 x SSC, 0.1% SDS, and exposed to x-ray film. Full-length cDNA probes of 90K and the HER2lneu receptor oncogene (23) were used as hybridization probes. Intactness and quantity of the analyzed RNA samples was monitored by ethidium bromide staining and reprobing of the filters with a GAPDH probe.

Cytotoxicity Assay-PBLs were isolated from heparinized venous blood from apparently healthy individuals after centrifugation through Ficoll. PBL at 2 x lo6 celldml were incubated in the presence of 90K or conditioned medium from SK-BR-3 human breast cancer cells for 16 h. At the end of the incubation period, PBLs were washed twice with warm Hank's balanced salt solution. Target cells for the assays of lymphocyte natural killer (NK) cytotoxic activity were the human erythroleukemia cell line K562, an NK-sensitive cell line, and for the lymphokine-activated killer cell (LAK) activity the Burkitt's lymphoma cell line Daudi, an NK-resistant cell line. Cells were labeled by incubating with 100 pCi 51Cr/107 cells for 1 h at 37 "C, then washed (twice) and incubated for 30 min a t 37 "C in RPMI culture medium. Subsequently, the labeled cells were washed two more times, and 2 x lo5 cells were added to the wells of a round-bottom tissue culture microplate. Effector cells were added to produce final effectortarget cell ratios as described in the legend to Fig. 4. Plates were briefly centrifuged and then incubated at 37 "C for 4 h. Supernatants were removed using a Skatron harvesting apparatus (Skatron, Lier, Norway), and radioactivity was determined with a y counter. A solution of 0.1 N HCl and culture medium were used instead of effector cells to determine maximal (MR) and spontaneous (SR) release, respectively, of radioactivity from target cells. The percentage of specific tumor cell lysis was calculated by dividing (sample cpm - cpm SR) by (cpm MR - cpm SR) and multiplying by 100 to yield the percent specific lysis value.

IL-2 Measurements-PBLs were isolated by standard procedures from blood of apparently healthy donors. Cells were suspended a t 4 x 106/ml in RPMI + FCS and plated in 24-well plates (0.5 mVwel1). C o d

and 90K in RPMI + FCS were added to a final volume of 1 ml. After incubation for 48 h at 37 "C, cell supernatants were collected, clarified by centrifugation, subjected to one cycle of freeze-thawing, and tested for IL-2 activity employing IL-2-dependent murine HT-2 cells. Samples and recombinant controls were serially diluted in 96-well flat bottom plates in a volume of 50 puwell. lo4 HT-2 indicator cells were then added. Following 18 h of incubation at 37 "C, the plates were pulsed with 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) for 4 h, and the resulting formazan crystals were solubi- lized with acidified isopropanol. Optical density was measured on a dual wavelength microplate reader (sample wavelength = 570 nm, ref- erence wavelength = 630 nm). IL-2 concentrations in the samples were determined by regression analysis and comparison to a standard curve, obtained with a recombinant cytokine control.

RESULTS

9OK cDNA Sequence-The cloned 2206-nucleotide long 90K cDNA was derived from an mRNA with an open reading frame of 1886 nucleotides, which was defined by the presence of the 22-amino acid 90K amino-terminal peptide sequence (17) (Fig. 1). The open reading frame of 185 nucleotides upstream from the amino-terminal valine residue contained only one methionine codon, which preceded a 17-amino acid long sequence with the characteristics of a signal peptide. This putative initiation codon was flanked by nucleotides that were in agreement with Kozak's rule for a translation initiation consensus sequence (24). The protein coding region was terminated by an in-frame stop codon at position 1887 and followed by 320 untranslated nucleotides with a potential ATTAAApolyadenylation signal 18 bp upstream from the polyadenylated 3' end of the mRNA. The predicted sequence of the 585-amino acid long mature protein included seven potential NH,-linked glycosylation sites (Fig. 1).

Comparison of the derived sequence with the GenBankTM database did not reveal any significant homology to known DNA sequences. However, the cloned DNA sequence is identical to the recently reported sequence of a binding protein for a macrophage lectin specific for galactose (11). Examination of the deduced amino acid sequence of 90K revealed the presence of an approximately 120-amino acid residue long structural motif, which was first identified in the type 1 macrophage scavenger receptor (25) and defines the SRCR motif gene family. In several of the members of this family, which includes exclusively cell surface and secreted proteins, SRCR motif re- peats form a major part of their extracellular portion, suggesting a role for this structural element in interactions with ho- mologous or distinct protein targets. Interestingly, all currently characterized mammalian members of this family appear to be involved in critical functions of the cellular host defense system (reviewed in Ref. 26).

Although the 90K cDNA predicts a mature polypeptide of M , 63,000, its apparent molecular weight in SDS gels varies de- pending on the tissue source from 90-98 kDa in SDS-PAGE (not shown). To verify that the observed size difference was in fact due to N-linked glycosylation and that the mature protein was secreted into the culture medium, we overexpressed the protein in human 293 cells. Cells were metabolically labeled with [35S]methionine in the presence or absence of tunicamycin, and immunoprecipitates from cell extracts as well as supernatants were analyzed by gel electrophoresis and autoradiography. As shown in Fig. 2 (lune 81, the SP-2 antibody recognized a single protein of apparent M, 95,000 in conditioned media of control cells that had been transfected with the plasmid vector only, indicating that 293 cells normally produce this protein. A severalfold increase of the SP-2 antigen band was found in the conditioned media of cells transfected with the expression plasmid containing the cDNA insert (lune 2). When cells were grown in the presence of 0.1 or 1.0 pg/ml tunicamycin (lanes 4 and 6, respectively), N-linked glycosylation was inhibited, which in turn resulted in a marked decrease of secreted pro-

Tumor-associated Antigen 9OK 18403

~CCAGTCMCAGAOOCATCCTGTGGCTG~GAGG-TCCACACGGCCGTTGCAGCTACCGCAGCC~TCTG-TCCA-C~C 131 CACGCTCCATACTGGGAGAQGCl'TC~CA 32

M T P P R L P W V W L L V A G T Q G V N D G D M R A M A m CCT CCG AGG CTC TTC TGG GTG TGG CTG CTG GTT OCA GGA ACC CAA ODC 206 AT0 C W GTG AAC GAT GGT GAC

L T A N L E A Q A L W K E P G S V T M S V D A E a00 CTG ACT GCC AAC CTG GAG GCC CAD GCC CTG TGG AAG GAG CCD GGC AGCQT GTC ACC ATG AGT GTG GAT GCT QAG 680

175 CGG GGC TGC GAC CTG TCC ATC AGC GTG AAT GTG CAG GGC GAG GAC GCC CTG GGC TTC TOT GGC CAC ACG GTC ATC 656 R G C D L S I S V N V Q G E D A L G F C G H T V I

GAA ACC AGG AGC ACC CAC ACC CTG GAC CTC TCC AGG GAG CTC TCG GAG GCC CTT GGC CAG ATC TTT GAC AGC CAG 581 E T R S T H T L D L S R E L S E A L G Q I P D S Q 150

125 506 DCc GAC TGC AAG TCC CTG GGC TGG CTG AAG AM: AAC TGC AGG CAC GAG ADA GAC GCT GGT 6 0 GTC Toc ACCQT

A D C X S L G W L K S N C R H E R D A G V V C T

100 431 ADA OCT GCC TTC GGG CAA GGA TCA GGC CCC hTC ATG CTG GAC GAG GTC CAG TGC ACG GGA ACC DAD GCC TCA C M

R A A F G Q G S G P I M L D E V Q C T G T E A S L

75 A T Q A L G 356 AAC CTG Tffi GAC CTG ACT GAT K C AGC GTC GTC TGC C W GCC CTG GGC TTC GAG% GCC ACC CAQ GCT CTG GQC

N L W D L T D A S V V C R A L G F E

50 281 CTG CCC GAT Coo GGC GCC ACC AAC CAD GGC CGC GTG GAG ATC TTC TAC AGA ODC CAG TGG GGC ACT OTG W GAC

L A D G G A T N Q G R V E I P Y R G Q W G T V C D

25

C V P M V R D L L R Y P Y S R R I D I T L ~ ~ V X 295 TGT GTG CCC ATG GTC AGG GAC CTT CTC AGG TAC TTC TAC TCC CGA ADD ATT GAC ATC ACC CTG TCG TCA GTC AAG 806

C P H K L A S A Y G A R Q L Q G Y C A S L F A I L 250 TGC TTC CAC AAG CTG GCC TCT GCC TAT OOO GCC AGG CAG CTG CAG GGC TAC TGC GCA AGC CTC TTT GCC ATC CTC 881

L P Q D P S F Q M P L D L Y A Y A V A T G D A L L 275 CTC CCC CAG GAC CCC TCG 'PTC CAD ATG CCC CTG GAC CTG TAT GCC TAT GCA GTG GCC ACA GSG GAC GCC CTG CTG 956

E K L C L Q P L A W N F E A L T Q A E A W P S V P GAG AAG CTC TGC CTA CAD TTC CTG GCC TGG AAC TTC GAG GCC TTG ACG CAG GCC GAG GCC TGG CCC AGT GTC CCC 1031

300

T D L L Q L L L P R S D L A V P S E L A L L K A V 325 ACA GAC CTG CTC CAA CTG CTG CTG CCC AGG AGC GAC CTG GCG GTG CCC AGC GAG CTG GCC CTA CTG AAG K C GTG 1106

D T W S W G E R A S H E E V E G L V E K I R F P M GAC ACC TGG AGC TGG GGG GAG CGT GCC TCC CAT GAG GAG GTG GAG GGC TTG GTG GAG AAG ATC CGC TTC CCC ATG 1181

350

M L P E E L F E L Q P ATG CTC CCT GAG GAG CTC TTT GAG CTG CAG TTC % CTG TCC CTG TAC TGG AGC CAC GAG GCC CTG TTC CAG AAG 1256

L S L Y W S H E A L P Q K 375

K T L Q A L E F H T V P P Q L L A R Y X G L AAG ACT CTG CAD GCC CTG GAA TTC CAC ACT 0% CCC TTC CAD TTG CTG GCC CGG TAC AAA GGC CTG QC CTC ACC 1331

L T 400

E D T Y K P R I Y T S P T W S A F V T D S S W S A 425 GAG GAT ACC TAC AAG CCC CGG ATT TAC ACC TCG CCC ACC TGG AGT GCC TTT GTG ACA GAC AGT TCC TGG AGT GCA 1406

R K S Q L V Y Q S R R G P L V K Y S S D Y F Q A P CDO AAQ TCA CAA CTG GTC TAT CAD TCC AGA C W GGG CCT TTQ GTC AAA TAT TCT TCT GAT TAC TTC CAA OCC CCC 1481

450

TCT GAC TAC AGA TAC TAC CCC TAC CAG TCC TTC CAG ACT CCA CAA CAC CCC AGC TTC CTC TTC CAG GAC AAG ADD 1556 S D Y R Y Y P Y Q S F Q T P Q H P S F L F Q D K R 475

V S W S L V Y L P T I Q S C W N Y G F S C S S D E GTG TCC TGG TCC CTG GTC TAC CTC CCC ACC ATC CAD AGC TGC TGG AAC TAC GGC TTC TCC TGC TCC TCG GAC GAG 1631

500

L P V L G L T K S G G S D R T I A Y E N K A L M L 525 CTC CCT GTC CTG GGC CTC ACC AAG TCT GGC GGC TCA GAT CGC ACC ATT GCC TAC GAA AAC AAA GCC CTG AT0 CTC 1706

C E G L F V A D V T D P E G W K A A I P S A L D T TGC GAA GGG CTC TTC GTG GCA GAC GTC ACC GAT TTC GAG GGC TGG AAG GCT GCG ATT CCC AGT GCC CTG GAC ACC 1781

550

S S K S T S S P P C P A G H F N G F R T V I R P % AGC TCG AAG AGC ACC TCC TCC TTC CCC TGC CCG GCA GGG CAC TTC AAC GGC TTC CGC ACG GTC ATC CGC CCC 1856 575

P Y L T S S G V D TTC TAC CTG ACC% TCC TCA GGT GTG GAC TAGACGCGTGGCCAADD6GGTGACCGGAGAACCCCAGGACGCCCTCACMC- 1945

585

TCCCCTCCTCGGCTTCCTTCTCTCTGCAATGACCTTCAACMCCGGCCACCAGATGTCGCCCTACTCACCTGAGGCTCAGCTT~TTAC~ 2044 AWCTTCCACTAOOOTCCACCAGGAGTTCTCCCACCACCTCACCAGTTTCC-GGT~CCA~AGGCCCTCGAGGTTGCTC-TCCCCC~ 2143 A G C C C C T f f i T C ~ T C T G C C C ~ G T C A C T O G T C T G A C 6 C 2206

FIG. 1. Nucleotide and predicted amino acid sequence of SOK. The putative signal peptide is boxed; the SRCR homology region is shaded. Potential asparagine-linked glycosylation sites are circled.

tein. SP-2 immunoprecipitates from the corresponding cell lysates (lanes 1 , 3, and 5) contained only trace amounts of the 95-kDa protein but showed several protein bands of M, 69,000- 77,000, likely representing glycosylation intermediates of 90K. The virtual absence of full-length 95-kDa protein in all cell lysate immunoprecipitates is likely due to association of this form with the membrane fraction or its rapid secretion.

Expression of 9OK mRNA in Normal and %mor Tissues-To approach functional aspects of this novel polypeptide, we examined by Northern blot analysis a variety of normal tissues, tumor cell lines, and primary tumor samples. All examined normal human tissues, with the exception of peripheral blood lymphocytes, contained high levels of 2.2-kb 9OK-specific mRNA. Generally, the highest levels of hybridizing RNA were found in normal gastrointestinal tissues, such as colon, duodenum, stomach, and small intestine (Table I). Because of the previously reported increase in 90K levels in the serum of cancer patients (2), we analyzed mRNA levels in human tumor-

derived cell lines and primary tumors (Table I1 and Fig. 3). While 21 lines of different cancer cell types expressed the 2.2-kb mRNA to varying extents, this mRNA could not be detected at all in the MDA-MB-453 mammary carcinoma cell line (Fig. 3 A ) and the histiocytic lymphoma-derived line, U937 (not shown). RNA analysis of primary cancer tumors of various types showed that in comparison with normal tissue, the examined samples frequently exhibited in some cases dramati- cally increased levels of 90K mRNA (Fig. 3B). Rather dramatic differences in 90K mRNA expression levels in tumor-derived clonal cell lines (Fig. 3 A ) , in conjunction with previous immu- nohistochemical analyses with SP-2 antibody (1) and in situ hybridization experiments (not shown), indicated that this was not simply due to the enrichment of epithelial cells producing normal levels of 90K mRNA in tumor tissue compared with normal tissue controls or other tumors with higher stromal cell content. Moreover, some tumors, such as the majority of the kidney carcinomas analyzed, actually expressed lower levels of

18404 %mor-associated Antigen 9OK

Fraction: L M L M L M L M

c

FIG. 2. Transient expression of 90K in 293 fibroblasts. 293 cells were either transfected with the pCMV-9OK expression plasmid (lanes 1-6) or with the insertless plasmid pCMV as control (lanes 7 and 8 ) as described under “Materials and Methods.” 16 h prior to lysis, cells were treated with tunicamycin (lanes 3-6) a t 0.1 pg/ml (lanes 3 and 4 ) or 1.0 pg/ml (lanes 5 and 6 ) and metabolically labeled with [3sSlmethionine. Cells were lysed, and both the cleared lysate (L) as well as the conditioned media (M) were subjected to immunoprecipitation with mAb SP-2. Immunoprecipitated proteins were separated by 8.0% SDS-PAGE and detected by autoradiography.

TABLE I Expression of 9OK-specific mRNA in normal tissues

Tissue mRNA“

Adrenal gland + Bladder ++ Bone marrow + Brain + Colon +++ Duodenum +++ Small intestine ++ Kidney + Liver + Fetal liver ++ Lung +++ Mammary gland + Muscle (striated) + Myometrium + Ovary ++ Pancreas + Placenta + Spleen ++ Stomach +++

a Relative levels of mRNA are qualitatively assessed as high (+++), intermediate (++), or low (+).

2.2-kb mRNA than normal kidney tissue from the same individual (not shown), and approximately 30% of all mammary carcinomas tested did not show any, or a subnormally low 2.2-kb mRNA hybridization signal (Fig. 3, C and D). Remark- ably, about 60% of these 90K mRNA-negative tumors exhibited overexpression of the oncogenic HER2 lneu receptor tyrosine kinase gene (23) (Fig. 30) and therefore represented a category that was characterized by aggressive growth and poor prognosis for the patient (27). Other mammary carcinomas and cell lines, such as SK-BR-3, T47-D, and MDA-MB175 expressed both 90K and HER2heu mRNAs at high levels, suggesting that suppression of 90K expression was not caused by a constitutive HER2heu signal but by other mechanisms, including somatic gene d e l e t i ~ n . ~

Biological Function of 9OK-The structural similarity to several polypeptides with established roles in the immune system, in combination with the sometimes enhanced and in some cases subnormal expression in cancer tissues and corresponding abnormal serum antigen levels in significant fractions of patient populations with cancer (21, virus infections such as HIV (51, or autoimmune disease such as rheumatoid arthritis and sys- temic lupus erythematosus,4 suggested a role for the SP-2 an-

P. Knyazev and A. Ullrich, manuscript in preparation. S. Iacobelli, manuscript in preparation.

TABLE I1 Expression of 9OK-specific mRNA in primary tumors

Tumor type” No. mRNAh

Mammary carcinoma 14 +++ 25 8

17 - ++ +

Colon carcinoma 3 +++ 6 ++ 6 +

Stomach carcinoma 3 ++ 2 + 1 -

Leukemias Acute myoblastic 1 +++

Acute lympho- 1 +++ 2 ++

Chronic myelocytic 1 +++ 1 ++

Squamous carcinoma 1 +++ 4 ++ 6 + 4 -

APUDoma 1 - Ewing 1 +++ Hepatoblastoma 1 +++ Liposarcoma 1 ++ Neuroblastoma 1 - Ovarian thecoma 1 +++ Pheochromocytoma 1 - Rhabdomyosarcoma 1 + VIPoma 1 +++ Wilms tumor 1 + APUD, amine precursor uptake and decarboxylation; VIP, vasoin-

* Relative levels of mRNA are qualitatively assessed as high (+++),

1 +

blastidymphocytic

testinal peptide.

intermediate (++), low (+I, or undetectable (-1.

tigen in the transmission of stimulatory signals to the cellular components of the immune system. To test this hypothesis, we examined its effects on NK cell activity and spontaneous LAK cell activity on freshly prepared PBLs from normal individuals. As shown in Fig. 4A, exposure of freshly prepared PBLs from healthy individuals to different concentrations of immunoafinity-purified 90K from pooled sera of cancer patients (17) for 16 h markedly stimulated lysis of K562 and Daudi target cells. These activities, which were optimal between 0.5 and 1 pglml, were confirmed with conditioned media from SK-BR-3 mammary carcinoma cells, but failed to be induced with media after passage over an SP-2 mAb immunoafinity column (Fig. 4, B and C), which depleted more than 95% of 90K as evaluated by immunoradiometric assay (6) (not shown). Recombinant 90K, which was immunopurified from serum-free conditioned media of NIH 3T3 cells that were transfected with an expression plasmid containing the cDNA under the cytomegalovirus early promoter control, had similar stimulatory effects on the cyto- lytic activity of PBLs (Fig. 4, B and C), which was abolished by coincubation with the 9OK-specific mAb 19B5 (Fig. 4, B and C), but not with control mAb 4D5 against an irrelevant antigen (not shown).

To investigate the mechanism by which 90K exerts its im- mumostimulatory activity, we examined its effect on the secretion of IL-2, an important lymphokine in the generation of cytotoxic effector cells such as NK and M. Costimulation of PBL with the T-cell mitogen ConA and purified recombinant 90K protein resulted in significantly higher secretion of IL-2, as compared to stimulation with ConA alone (Fig. 5). At the highest doses of reagents tested (10 pg/ml ConA and 20 pg/ml90K)

Tumor-associated Antigen 9OK 18405

A

B N T N T N T

2.2 kb+

c l I 1 - 2.2 kb

N T

1 , y M -2.2kb

I 1 MCF7

YLr

MCF7 -1

D 4.8 k b + r 1 I

l Z J 4 3 O I t 3 Y I U 1 1

FIG. 3. Northern blot analysis of primary mammary carcinoma tumor RNAs. "P-Labeled 90K (2206 bp) and HER2heu (4810 bp) cDNA probes were used to characterize mRNA expression in mammary carcinoma cell lines and normal breast tissues in comparison with tumors of the same individual and randomly collected primary mammary carcinoma tissues by Northern blot analysis of poly(A+) RNA(3 pgflane). A, mammary carcinoma-derived cell lines; 90K cDNA probe. B , four primary mammary carcinoma tumors (2 ' ) in comparison with matched normal mammary tissue ( N ) ; 90K cDNA probe. C, 22 randomly collected mammary carcinoma tumors with MCF7 RNA controls; 90K cDNA probe. Negative samples contained intact RNA as determined by staining with ethidium bromide and hybridization with a GAPDH probe

2-6) tumor RNAs were hybridized with both 90K (2.2-kb band) and (not shown). D, selected 90K positive (lanes 7-11) and negative (lanes

HER2heu (4.8 kb band) cDNAprobes. Lane 1 contains an MCF7 mRNA control.

this augmentation was approximately 6-fold. Similar results were obtained in separate experiments using PBL from random donors, various batches of purified 90K, and conditioned media from cells which produce either natural or recombinant 90K (data not shown).

DISCUSSION We have cloned a full-length cDNA for the 90K tumor-asso-

ciated protein and established functions for this protein that strongly suggest a role in the immune defense against cancer cells and possibly other pathogens such as viruses. Analysis of the deduced amino acid sequence revealed interesting struc-

200 400 600 800 1000 1200 1400 1600 1800 2000

90K (nglrnl)

B loo 90 3

1 10 30 60 90

c 100, E/T ratio

$. 70 .- v) 60

- 40 a,

..

- % 50 - 0 30

20 10 0

1 10 30 60 90

E/T ratio FIG. 4.90K stimulates NK and w( activity of peripheral blood

lymphocytes. A, 90K was purified from pooled sera of cancer patients as previously described (4) and tested for its dose-dependent activity in NK (A) and LAK (A) assays with K562 and Daudi cells as targets, respectively. Analogous experiments ( B , w ( , C, NK) included stimulation of PBLs with conditioned media from SK-BR-3 human mammary carcinoma cells (darkly hatched bars), immunopurified recombinant 90K at 200 ng/ml (gray bars), SP-2 mAb immunoafinity-depleted SK- BR-3 conditioned media (lightly hatched bars), and SK-BR-3 conditioned media in the presence of anti-9OK mAb 19B5 (open bars; 1 pgl ml). Because of the quantitative variability of the assays due to differences in donor PBLs, representative examples of at least five independent experiments are shown.

tural features and homologies with previously characterized proteins. The primary structure of the 90K TAA is identical to the recently reported amino acid sequence of the Mac-2-BP (11, 28) and to the partially characterized L3 lung carcinoma associated antigen (29,30). Like 90K, an increase in the circulating levels of L3 antigen has been reported to be associated with various malignant and nonmalignant diseases (29). From this we conclude that the 90K, Mac-BBP, and L3 antigens are identical, independently isolated proteins.

Comparison of the deduced 90K amino acid sequence the SwissProt, PIR, and Mips X data bases, using the search algo- rithm FASTA, revealed the presence of a structural motif that is approximately 120-amino acid residues long and had been first identified in the type I macrophage scavenger receptor (25). This scavenger receptor cysteine-rich domain is highly conserved between evolutionally distant species, such as sea

18406 %mor-associated Antigen 9OK

'E 80 7

q - , 0

0 2.5 5 10

ConA (pg/ml)

FIG. 5. 90K augments the secretion of IL-2 by mitogen-stimulated PBL. PBL were cultured with the indicated doses of ConA and/or

hatched bars, 20 pg/ml) for 48 h. Supernatants were assayed for IL-2 purified recombinant 90K (black bars, none; open bars, 10 pg/ml;

bioactivity by their ability to support the growth of IL-2-dependent HT-2 cells.

urchin and man. It occurs once in 90K, type I scavenger receptor, and complement factor I, three times in the T-cell surface glycoproteins CD5Ly-1 (31-34) and CD6 (351, and in four cop- ies in the sea urchin Strongylocentrotus purpuratus speract receptor, which is the binding site for oocyte peptides that activate sperm functions (36). All of the previously characterized SRCR-containing proteins are transmembrane polypeptides which carry the structural motif in their extracellular domains. In contrast, 90K has all the structural features of a secreted polypeptide, which is confirmed by its occurrence in cell conditioned medium and serum.

The 90K gene appears to be expressed by most human normal tissues, and frequently at elevated levels in tumor cell lines and primary tumors as estimated by Northern blot analysis (Fig. 3) and immunoprecipitations from conditioned cell culture media (not shown). This is in contrast to the report by Koths et al. (111, who found immunoreactive Mac-2-BP by Western analysis in human fluids such as breast milk, saliva, tears, and semen, but not in other human tissue samples and cell lines. Intriguingly, we found an inverse correlation between the expression of 90K and HER2/neu mRNA in human mammary carcinomas. These data show that of about one-third of the tumors which express either low levels or no 90K mRNA, about 60% overexpress HER2/neu, a known marker of aggressive progression of breast tumors and poor prognosis for the patient (27). While we do not yet know whether constitutive HER2heu signaling directly leads to suppression of 90K gene expression or whether silencing of this gene represents one step in the development of more aggressive tumors, our observation sug- gests that production of elevated levels of 90K by the tumor may correlate with a more favorable clinical outcome. This is in agreement with preliminary data showing that high 90K levels in mammary tumors are associated with a more differentiated phenotype and by the observation that tumor cell lines express- ing high levels of 90K frequently do not grow in nude mice, while those with low 90K expression are more likely to form solid tumors (not shown). A possible connection between the recent finding of autoimmune responses in subsets of breast cancer patients with HER2heu-overexpressing tumors (37) and differential 90K expression characteristics is a matter of speculation and remains to be experimentally substantiated.

These observations in conjunction with our findings of elevated expression levels in tissues containing cavity lining se- cretory epithelia are especially intriguing in light of the stimulatory activities of 90K on NK and LAK cytotoxic effector cells, which are critical elements in the body's immune response. A connection between 90K and stimulation of host defense systems is further substantiated by our demonstration of in-

creased IL-2 secretion by Cod-treated PBLs after exposure to 90K. Production of this lymphokine, which plays a key role in cell-mediated immune responses, is likely a secondary effect that may be primarily the result of cytokine secretion by 9OK- activated accessory cells such as macrophages. Ageneral role in the immune defense system of organisms is further supported by the recent finding that in its aggregated form 90K binds the Mac-2 lectin, which is expressed by activated macrophages and appears to play a role in the mediation of a cellular immune response (121, possibly by stabilizing the adhesion of T cells to accessory cells. Moreover, its expression appears to be induced by interferon (38, 391, and serum levels correlate with those of endogenous interferon, &-microglobulin, and neopterin (9), suggesting that it may act on cells of the monocyte-macrophage lineage, which are particularly interferon-responsive. Finally, the elevated serum levels that have been found in pathological conditions associated with a progressive immune deficit, such as cancer (2-4) and HIV infection (5, 6, 8-10), suggest that under certain circumstances where the immune stimulation signalling cascade is disrupted downstream, overproduction of 90K may be the result of disease progression due to subversion of the normal cellular immune defense system.

Taken together, these data suggest that 90K could be a broadly active immune stimulator molecule whose production by normal cells is enhanced by pathogenic events such as oncogenic transformation or virus infection of cells, or tissue in- vasion by other types of pathogens. Locally increased levels of this factor may, presumably through the induction of cytokines, alert specific cellular elements of the host defense and activate their target-specific cytocidal functions. Mechanisms that sup- press 90K production or interfere with its signalling function may be required for the escape of pathogenically transformed cells from these immune defense systems. 90K may therefore represent a suppressor gene product not only for tumors but for pathogenic events in general.

Acknowledgments-We thank Dr. Peter Hirth for discussions and advice, Randy Schreck for expert technical assistance in the ConA costimulation assay, and Jeanne Arch for expert preparation of this manuscript.

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