CANCER RESEARCH55. 6040-6044, December 15, 1995]

Advances in Brief

Reduced Motility Related Protein-i (MRP-1/CD9) Gene Expression as a Factor ofPoor Prognosis in Non-Small Cell Lung Cancer1

Masahiko Higashiyama, Toshihiko TaM, Yoshiaki leki, Masashi Adachi, Cheng-long Huang, Takashi Koh,Ken Kodama, Osamu Doi, and Masayuki Miyake2Department of Surgery. The Center for Adult Diseases of Osaka, Osaka 537 (M. H., K. K., 0. DI: Department of Thoracic Surgery and Department V of Oncology, KitanoHospital, Tazuke Ko@fukaiMedical Research Institute, 13-3 Kamiyama-cho, Kita-ku, Osaka 530 fT. T., V. 1., M. A., C. H., M. M.]: and Department of Pathology and TumorBiology, Faculty of Medicine, Kvoto University, Kyoto 606 fT. K.], Japan

Abstract

Motility related protein-i (MRP-1) is a transmembrane glycoprotein

that is identical to the CD9 antigen. In previous studies, we showed thatvarious types of cultured tumor cells transfected with MRP-1/CD9 cDNAhave low motiilty and diminished metastatic potential to the lung. Morerecently we used immunohistochemical procedures, immunoblotting, andreverse transcription-PCR to demonstrate that the level of MRP-I/CD9expression was inversely related to the clinical stage of a given carcinomaof the breast. In addition, we found that the primary tumors of almost50% of the patients had higher MRP-1JCD9 levels than their respective

metastatic lymph nodes. In consideration of these findings, we have nowapplied reverse transcnption-PCR to determine MRP-1/CD9gene expression in lung cancer. We analyzed tumor tissues of 109 patients: 49 tumorswere stage I; 15 were stage H; and 45 were stage ifi. We found that 67patients had MRP-1/CD9-positive tumors, and that gene expression wasreduced in the tumors of the remaining 42 individuals. The overall rate ofsurvival was strikingly higher among patients with positive tumors than inthose whose tumors had reduced gene expression (62.3 versus 34.9%;P < 0.001). This also pertained to patients with adenocarcinomas of thelung (55.4 versus 26.0%; P < 0.001). Multivariate analysis with the Coxregression model indicated that MRP-1/CD9 positivity correlated betterwith overall survival rate than did other variables, except lymph nodestatus. Our data suggest that low MRP-1/CD9 expression by tumors of thelung may be associated with poor prognosis. It is conceivable that testingfor MRP-1/CD9 may identify node-negative lung cancer patients andpatients with adenocarcinomas who are at high risk for early diseaserecurrence.

Introduction

We have shown previously that MRP- 1@ is a transmembrane glycoprotein that is recognized by the murine MAb M31-l5, whichinhibits cell motility ( 1). We further established that the sequence ofMRP-l cDNA is identical to that of the CD9 antigen (1), a Mr24,000—27,000 glycoprotein widely expressed in hematopoietic tissues as well as in nonhematopoietic tissues (2—4).Although thephysiological functions of CD9 remain unknown, anti-CD9 MAbstrigger platelet activation mediated by the IgG Fc receptor type II.This activation involves phospholipase A2 and phospholipase C stimulation, which in turn induces an increase in cytoplasmic calcium andprotein tyrosine phosphorylation (5—7),thus pointing to the directparticipation of CD9 in signal transduction. In efforts to clarify the

Received 9/26/95; accepted 11/1/95.The costs of publication of this article were defrayed in part by the payment of page

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

I This work was supported in part by Grants-in-Aid from the Japanese Ministry of

Education (to M. M.) and from the Sagawa Foundation for Promotion of Cancer Research(to M. M.).

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: MRP-l, motility related protein-I ; MAt,, monoclonal

antibody; RT-PCR, reverse transcription-PCR; SCLC, small cell lung cancer; NSCLC,non-SCLC.

functions of MRP-l/CD9, we transfected various types of culturedcells with plasmid constructs containing MRP-1/CD9 cDNA (8).These experiments disclosed that motility and proliferation of MRP1/CD9-expressing cells were suppressed, and that the extent of theseeffects were directly related to the levels of MRP-1/CD9 expression.Moreover, transfected cells of the mouse melanoma line BL6 had alower metastatic potential than the parent BL6 cells (8). These findings would suggest that MRP-l/CD9 regulates cell motility, and thatthe glycoprotein is a receptor for negative signal ligands.

To determine whether these experimental results are relevant withrespect to actual human tumors, we investigated MRP-1/CD9 expression in breast cancer (9). That study revealed that MRP-l/CD9 expression was inversely related to the clinical stage of a given breastcancer, and that in almost 50% of patients, the protein levels werelower in the metastatic lymph nodes than in the respective primarytumors (9). These findings, obtained with immunohistochemicalmethods and immunoblotting, were corroborated by determiningMRP-1/CD9 gene expression with a quantitative RT-PCR-based assay. Overexpression of the MRP-J/CD9 gene by mammary tumorswas not detected (9). We have now investigated whether the levels ofMRP-1/CD9 gene expression in tumor tissues are of value for predicting the clinical behavior of lung cancer. In this report, we presentthe results of the RT-PCR-based analysis of tumor specimens from109 lung cancer patients for whom adequate clinicopathological andpost-surgical follow-up data were available.

Materials and Methods

Clinical Characteristics of the Patients and Surgical Specimens. Thisstudy was carried out on 109 patients with lung cancer (up to stage HIB

tumors) who underwent surgery at the Department of Surgery of the Center forAdult Diseases ofOsaka and the Department ofThoracic Surgery ofthe KitanoHospital, Medical Research Institute of Osaka, between January 1991 andDecember 1992. Clinical records for these patients were available, and thehistopathological diagnoses were fully documented. The postsurgical pathological stage of each tumor was classified according to the tumor-nodemetastasis system (10). The salient clinical characteristics of the patients arepresented in Table 1. This report includes follow-up data as of August 1, 1995.The median follow-up for all patients was 43.7 months, with a range of 31—55months. Five patients who had two or more forms of cancers and four lungcancer patients who died as a consequence of accidents were not included in

the study. In addition, seven patients with advanced stage lung cancer (stage

IV) were also excluded because, in such individuals, the effect of a singlefactor such as the MRP-1/CD9 status could be considerably diluted, andbecause they usually have cancer cells at metastatic sites, even if their primary

tumors are resected.One-half of each freshly resected tumor tissue was immediately embedded

in optimum cutting temperature compound (Miles, Kankakee, IL) and frozen

at —80°C.Frozen sections were cut on the cryostat to a thickness of 6 @mandimmediately stained with hematoxylin and eosin to verify the presence ofcancer cells. One-half of each tumor specimen containing only cancer cells wasused for RT-PCR.

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----@11

23456789101112a

Table I Relationship of MRP-1/CD9 gene expression and various prognosticfactorsMRP-

l/CD9statusTotalCharacteristics(n)(+)

(—)PvalueAge

at Surgery(yrs)60432617>606641

25Ns'2SexFemale

Male29 801712

5030NSTumor

statusT12924512

T350 223317

9 130001‘1481

7Nodal

statusN0613922N1

N218 23144

13100029N3716Pathological

stageStageI493514Stagell

Stage lIla15 31132

18 130001‘Stage

IIlb14113HistologyAdenocarcinoma7344

29Squamouscell carcinoma322210NSLarge

cell carcinoma413Total

number of patients1096742a

NS, not significant.

MRP-1/CD9 EXPRESSION IN LUNG CANCER

A1353 (ba)

1O78@\@

8726033107281

B

Fig. I . A, agarose gel electrophoresis of RTPCR-amplified 800-bp MRP-l/CD9 DNA. Lane 1.size marker; Lane 2, human lung adenocarcinomacell line MACIO (positive control); Lanes 3, 5, and7, primary adenocarcinomasof the lung with positive MRP-1/CD9 gene expression; Lanes 4, 6, and8, primary adenocarcinomas of the lung with reduced MRP-1/CD9 gene expression; Lanes 9 and11, MRP- //CD9-positive primary squamous cellcarcinomas of the lung; Lanes 10 and 12, primarysquamous cell carcinomas of the lung with reducedMRP-1/CD9 gene expression. B, agarose gel electrophoresis of amplified @-actinDNA (internalPCR control) of the same specimens.

RT-PCR Analysis. Total cellularRNA was purifiedfrom freshor frozentumor tissues by the acid guanidinium thiocyanate procedure (11).Preparationsof the MRP-l/CD9-positive human lung adenocarcinoma cell line MAC10were used as positive controls. The first-strand cDNA was prepared from 5 @.tgof total RNA using Moloney murine leukemia virus reverse transcriptase(Pharmacia, Piscataway, NJ). After heating the RNA solution for 10 mm at65°Cand chilling on ice, the reaction mixture was incubated for 1 h at 37°C.For PCR amplification, we used 1-s.d aliquots of the reaction mixture. Inpreliminary experiments, we titrated the initial amount of cDNA and thenumber of amplification cycles required to obtain reproducible quantitativeRI-PCR data on MRP-1/CD9 gene expression as described previously (datanot shown; Ref. 12). All subsequent assays were carried out under conditions

that yielded amplification of both MRP-l/CD9 and (3-actin (used as theinternal standard) DNA within a linear range. On the basis of the nucleotide sequence of MRP-l/CD9 cDNA (1-3), 5'-TGCATCTGTATC

CAGCGCCA-3' and 5'-CTCAGGGATGTAAGCTGACT-3' were used assense and antisense primers, respectively. This primer pair amplifies an 800-bpfragment (nucleotides 19—818)that includes the full-length MRP-l/CD9cDNA (9). The reaction mixture was overlaid with 50 pA of mineral oil and

subjected to 30 PCR amplification cycles of 40 s at 94°C, 40 s at 60°C, and 90

S at 72°C. The sense and antisense primers for (3-actin cDNA amplification

(14) were 5'-CTGTCTGGCGGCACCACCAT-3' and 5'-GCAACT AAGTCATAGTCCGC-3', respectively; the same temperature profile was used for30 cycles. Tubes containing all ingredients except templates, included in all

runs, served as negative reaction controls. The amplified DNA samples were

electrophoresed on I % agarose gels, the bands, visualized with ethidium

bromide, and photographed with a Polaroid camera. Densitometric analysis of

the photographic negatives was used for band quantification (14).SpecimenClassificationBased on RT-PCR ReSUlts.The densitometric

value obtained for MRP-l/CD9 of a given tumor sample was divided by thecorresponding f3-actin value and referred to as MRP-l/CD9 expression ratio (9,

12). The rate of reduction in MRP-I/cD9 gene expression in a tumor was

computed by dividing the respective expression ratio by that of the human lungadenocarcinoma cells MACb. Tumors with values of more than 0.7 wereconsidered as having no reduction (positive) in gene expression, and those with

values of @0.7as having reduced MRP-I/CD9 gene expression.

Statistical Analysis. The x@test was used to compare the associationbetween the incidence of MRP-1/CD9 reduction and several clinical andpathological parameters. The Kaplan-Meier method was utilized to estimate

the probability of the overall survival as a function of time (15). Comparisons

were made with the log-rank test (16). The Cox proportional hazards modelwas used with the SAS statistical package (SAS Institute, Cary, NC); five

factors (sex, age at surgery, T stage, N stage, and tumor MRP-I/CD9 status)

were studied (17); scores were assigned to each variable for the regressionanalysis. All P values are based on two-tailed statistical analysis; a P value of

<0.05 was considered to indicate statistical significance.

Results

Relationship between MRP-1/CD9 Gene Expression andKnown Prognostic Factors. Of the 109 primary lung cancers studied, 67 (61.5%) were evaluated as MRP-1/CD9 gene positive (+) and

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u@—@ MRP—1/CD9

,I@— 13 actin

1 2 3 4 5 6 7 8 9 101112

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Table 2 Overallsurvival rate of 109 patients with NSCLC according to clinicopathological characteristics and MRP-1/CD9statusMRP-

1/CD9status(no.of patients)Median survival (days)Overall survival rate(%)MRP-l

MRP-lMRP-l MRP-lMRP-IMRP-lCharacteristics(+)(—)(i-) (—)(+) (—)PvalueAge

at Surgery (yr)6026 17NR― 42955.917.7<0.001>604

1 25NR 99380.4 47.10.006SexFemale17

12NR 91866.747.6<0.001Male5030NR 54265.030.0<0.001Nodal

statusN0

N1N@

N339

2214 413 10

I 6NR

NR1512 927406 379

1288 24692.3

58.742.9 25.025.6 10.00.0 0.00.001

0.0310.250

0.126Tumor

statusT1

T,T31424

533 179 131 7NR

NRNR 9181102 3511288 62855.6

60.084.7 39.741.7 23.1

0.0 28.60.339

0.0010.155

0.696Pathological

stagestage Istage IIstage lilastageIlIb35

1413 218 13I 13NR

NR1512 927880 4401288 33994.3

63.542.3 0.041.7 30.80.0 15.40.005

0.0370.349

0.446Histology

Adenocarcinoma44 29NR 44055.426.0<0.001Squamouscell carcinoma

Large cell carcinoma2210

I 3NRNR

500 91880.260.0

0.0 33.30.1850.515Total

number of patients67 42NR 62862.334.9<0.001a

NR.notreached.

MRP-I/CD9 EXPRESSION IN LUNG CANCER

particularly evident in patients with N0 and N, nodal status (Table 2).In addition, there was a significant difference in the overall survivalrate between patients whose adenocarcinomas were MRP-1/CD9 positive and those that were not (55.4 versus 26.0%; P < 0.001) (Fig. 3A).

A

0 10 20 30 40 50 60(M)

Months after Surgery

Fig. 3. A, overall survival of 73 patients with adenocarcinoma of the lung in relationto tumor MRP-1/CD9 status. B, overall survival of 32 patients with squamous cellcarcinoma of the lung in relation to tumor MRP-I/CD9 status.

(‘5

Ce>

C,)

Ce

>0

a,Ce

Ce>

:@C,)

Cea,>0

20

Months after Surgery

Fig. 2. Overall survival of 109 lung cancer patients in relation to tumor MRP-I/CD9status. The P value was determined with the log-rank test.

42 (38.5%) as having reduced (—)gene expression (Fig. 1). Therewere no statistically significant relationships (f) between gene expression and the age of the patients at surgery, gender, or tumorhistology (Table 1). In contrast, MRP-1/CD9 expression was associated with lymph node status (P = 0.029), as well as with tumor status(P = 0.001); 36.1% of the N0 stage patients had reduced gene expression, as compared to 22.2% of those with N1 stage and 43.5% ofpatients with N2 stage. Among the seven patients with N3 stagedisease, only one was MRP-1/CD9 gene positive. With respect totumor status, the proportion of patients whose tumors had reducedgene expression increased from 17.2% of those with T1 stage to87.5% of patients with T4 stage.

Association of Tumor MRP-1/CD9 Gene Expression and Overall Survival. Comparing the survival among all 109 patients revealedthat the overall survival rate for the 42 patients whose tumors hadreduced expression of the MRP-1/CD9 gene was strikingly lower thanthat of the 67 with MRP-1/CD9-positive tumors (Table 2; Fig. 2). Therespective rates were 34.9 and 62.3% (P = 0.001). This difference was

Months after Surgery

B

(%)100

a, 90

@8o-i@ 70

.2 60@ 50

‘@40@ 30@ 20

0 10

MRP—1/CD9 positive (n=22)

MRP-1/CD9 reduced (n=10)

P=0,l 85

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95%VariablesAssigned

score@3SEWald x2HazardratioconfidenceintervalPvalueMRP-

l/CD9Negative0—1.19750.339612.43510.3020.155-0.5870.0004Positive1Age

(yr)@6000.46830.39411.4121.5970.738—3.4580.2347>601SexFemale0—0.69880.42932.64940.4970.214—1.1530.1036MaleIT

statusT1I0.17570.17860.96751.1920.840—1.6920.3253T,2T33T44N

statusN000.85660.170125.36362.3551.687—3.2870.0001N1IN22N33

MRP-1/CD9 EXPRESSION IN LUNG CANCER

Table 3 Value offive variables in predicting overall survival of 109 patients withNSCLC

minants by tumor tissues may reflect the clinical aggressiveness ofNSCLC (30, 31).

As our results indicate, the reduction in MRP-I/CD9 gene expression by tumors of the lung was associated with poor prognosis. Thelink between reduced MRP-1/CD9 expression and poor prognosis wasindependent of tumor stage, nodal stage, age at surgery, and sex. TheMRP-1/CD9 reduction was strongly associated with an increased riskof recurrence, especially among patients with N0 or@ stage diseaseand with stage I or stage II disease. On the other hand, becausepatients with N2 or N3 stage already have systemic disease, MRP-1/CD9 does not seem to be a prognostic predictor for such individuals.However, the overall survival rate of adenocarcinoma patients whosetumors were MRP-1/CD9 positive was significantly better than that ofpatients whose adenocarcinomas had reduced gene expression. Thesefindings would suggest that the status of the MRP-1/CD9 gene in atumor may have prognostic implications for lung cancer patients withearly stage disease and with adenocarcinomas.

MRP-l/CD9 belongs to a structurally distinct family of cell membrane glycoproteins, the so-called transmembrane 4 superfamily,which includes CD37 (32), CD53 (33), ME491/CD63 (34), TAPA-l/CD81 (35), R2/CD82 (36), CO-029 (37), Sm23 (38), and others.These proteins, variously expressed on leukocytes and a variety oftissues, have four highly conserved hydrophobic domains that areassumed to span the lipid bilayer and a few extracellular N-glycosylated domains (4, 39). Although the precise biochemical functions ofthe transmembrane of superfamily remain unknown, alterations in theexpression of ME491/CD63, R2/CD82, and MRP-l/CD9 appear to becorrelated with tumor metastases. Thus, ME491/CD63 is expressed inthe early stages of tumor progression, and its decrease is associatedwith an increased metastatic potential of malignant human melanomas(34). R2/CD82 is identical to the predicted product encoded by KAJ-1,a metastasis suppressor gene of prostate cancer (40). It is believed thatdecreased KAI-I expression may be involved in the malignant progression of prostate cancer (40). As demonstrated before, MRP-l/CD9gene expression is reduced in the advanced stages of breast cancer (9)and, as shown here, in lung cancer as well.

Even though it has not been shown that adjuvant therapy increasesdisease-free and overall survival of early-stage lung cancer patients, itis possible that this treatment modality may be important for patientswhose tumors have diminished expression of the MRP-1/CD9 gene.On the other hand, patients with MRP-1/CD9-positive tumors may notrequire adjuvant therapy after surgery. Although its actual functions insolid tumors have not been established, our results do indicate thatdiminished MRP-1/CD9 expression appears to be associated withmetastases and suggest that this reduction may represent a potentiallyuseful prognostic marker in lung cancer. It is thus conceivable thattesting for MRP-l/CD9 status in conjunction with other molecular andbiochemical assays may improve the prognostic evaluation of lungcancer patients and enhance the ability of the clinician to prospectively identify patients who are likely to have early disease recurrenceand who may benefit from adjuvant therapy. In the near future, wewant to carry out the large prospective study of correlations of othermarkers, K-ras mutations, p53 abnormalities, and blood group analyses with MRP-1/CD9 status.

Acknowledgments

We thank Dr. Masato Yagita for critical editorial help with the manuscript.

References

6043

In contrast, no significant differences in survival were detected amongthe 32 patients with squamous cell carcinoma (Fig. 3B).

Prognostic Value of MRP-1/CD9 Status. The variables used inthe Cox regression analysis are shown in Table 3. The estimatedprognostic value of each variable in relation to overall survival amongthe 109 patients studied is expressed as a P value. Only two variables(tumor MRP-1/CD9 status and N status) were found to be significantprognostic factors of survival. The other variables listed in Table 3(age at surgery, sex, T status) had no relation with the MRP-1/CD9status or its prognostic value.

Discussion

It has been suggested that lung cancer may become, in the nearfuture, the most common cause of death due to cancer in Japan ( 18).From a clinical and therapeutic standpoint, all histological types oflung cancer are divided into two major groups: SCLC and NSCLC(19). Whereas SCLC is best treated with chemotherapy because of itscharacteristics of a systematic disease, the most effective form oftreatment for NSCLC is resection when the tumor is localized to thepulmonary parenchyma (19—21).However, the survival rates of patients with NSCLC have remained constant over the last decades,suggesting that the management of the disease has improved verylittle over the past 20 years. Even in resected stage I lung cancer,primary tumor relapses still occur in 20—30%of the patients (19—21).In view of the poor prognosis of lung cancer, it seems justified toevaluate the effectiveness of surgery plus adjuvant chemotherapy orradiotherapy (22—24).However, it is not clear which clinical characteristics determine the need for adjuvant therapy for a given patient.Hence, the search for reliable and sensitive prognostic tests is criticalbecause, if available, they would help identify patients for whomintensive adjuvant therapy is warranted (25). Recently, some molecular and glycolipid markers have been found to be associated withpoor prognosis, and their presence may be an indication for adjuvanttherapy in lung cancer patients. For example, a mutated K-ras oncogene is a potentially useful prognostic indicator because its presencehas been found to be associated with shorter survival, particularly ofpatients with an adenocarcinoma (26—28),and mutations of the tumorsuppressor gene p.53 may also be of prognostic importance, especiallyfor patients with advanced disease (18, 29). Moreover, changes in theexpression of ABH blood group antigens and of Lewis-related deter

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1995;55:6040-6044. Cancer Res   Masahiko Higashiyama, Toshihiko Taki, Yoshiaki Ieki, et al.   Lung CancerExpression as a Factor of Poor Prognosis in Non-Small Cell

) GeneMRP-1/CD9Reduced Motility Related Protein-1 (

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