Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential

JOURNAL OF CELLULAR PHYSIOLOGY 160:275286 (1994)

Binding and Degradation of Hyaluronan by Human Breast Cancer Cell Lines Expressing Different Forms of CD44: Correlation With

lnvasive Potential MARTINE CULTY,* MEHRAN SHIZARI, ERIK W. THOMPSON, AND

CHARLES B. UNDERHILL Department of Cell Biology and The Vincent T. Lombard; Cancer Research Center,

Georgetown University Medical Center, Washington, D.C. 20007

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1 ). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < O.OOOl), as well as with the invasive potential of the cells. Scatchard analysis of the [3H] hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions i s to degrade hyaluronan. o 1994 WiIey-Liss, Inc.

CD44 defines a family of surface glycoproteins involved in cell migration and adhesion to extracellular matrices, which have been implicated in a number of phenomena, including morphogenesis, inflammation, lymphocyte homing, and tumor metastasis (Haynes et al., 1991; Underhill, 1992). CD44 comes in a wide variety of different molecular weight forms, ranging from 80 to well over 200 kDa. This diversity has been attributed to alternative splicing (Brown et al., 1991; Cooper et al., 1992; Screaton et al., 1992) and to variations in the degree of glycosylation of the proteins (Carter and Wayner, 1988; Jalkanen et al., 1988).

The ability of CD44 to bind hyaluronan was initially suggested by the existence of sequence homology between the N-terminal domain of CD44 and that of the cartilage link protein (Goldstein et al., 1989; Stamenk- ovic et al., 1989), and further confirmed by studies re- porting the identity of CD44 with the hyaluronan receptor which binds specifically to hyaluronan (Culty et al., 1990; Aruffo et al., 1990). However, some contro- versy exists as to whether all of the different forms of CD44 can bind hyaluronan. Studies by Stamenkovic et al. (1991) presented evidence that cells transfected with a high molecular weight epithelial form of CD44 (CD44E) lack the capacity to bind hyaluronan. In contrast, studies by He et al. (1992) have indicated that high molecular weight forms of CD44 containing addi- 0 1994 WIILEY-LISS. INC.

tional sequences in the membrane proximal domain can bind hyaluronan.

Besides its role in cell adhesion, CD44 appears to be critically involved in the degradation of hyaluronan by both macrophages and SV40 transformed 3T3 cells, since antibodies to CD44 prevented both the binding of hyaluronan to the surfaces of these cells and its subse- quent uptake and degradation (Culty et al., 1992). In addition, agents such as chloroquine and NH,Cl, which block the activity of lysosomal enzymes, also prevented the breakdown of hyaluronan. These results indicate that CD44 mediates the attachment of hyaluronan to the cell surface so that it can be internalized and degraded by lysosomal enzymes.

Several lines of evidence suggest that CD44 is involved in tumor metastasis. First, high levels of CD44 were associated with certain types of carcinomas, high grade gliomas, and many non-Hodgkin’s lymphomas (Stamenkovic et al., 1989; Horst et al., 1990; Kuppner et al., 1992). More direct evidence that the expression of CD44 is related to the metastatic behavior of tumor cells comes from the work of Gunthert et al. (1991), who

Received November 16,1993; accepted March 1,1994. *To whom reprint requestsicorrespondence should be addressed.

276 CULTY ET AL.

TABLE! 1. Comparison of invasive characteristics of human breast cancer cell lines and CD44 expression'

Cell Estrogen line receptor Vimentin Invasion Chemotaxis CD44

ZR-75-1 SK-Br-3 MCF-7 T47D ADR MDA-436 MDA-435 MDA-231 BT549 MDA-468 Hs578T

+ +

++ +

+++ +++ +++

+++++ +++++

+ +++++

+ +

++ +

+++ ++++

++ +++++ +++++

+++ ++++

-

+ + + +

+++ +++ +++

++++ ++++

+++++ 'The various characteristics of the human breast cancer cell lines described above are from Thompson et al. (1992). The presence or absence of the estrogen receptor and vimentin are indicated hy + or -. The invasion and chemotaxis assays were determined by quantifying the migration of cells in Boyden chamber assays using fibroblast conditioned medium as chemoattractant. To study the chemoinvasion, the polyearbonate filters were coated with a uniform layer of matrigel, constituting a barrier that the cells had to degrade in order to reach the filters and migrate through them. To determine the chemotactic behavior of cells, the filters were coated with a thin layer ofcollagen IV that promotes cell attachment and allows the free migration of the cells toward the gradient ofconditioned medium. Both invation and chemotaxis assays were graded as 7% of the MDA-231 activities (+, &20%; + +, 20405; + + +, 4-08; + + + +, 6 M 0 6 ; + + + + +, >808). The relative amounts of CD44 in the cell lines are taken from Figure 1.

showed that nonmetastatic rat pancreas cell lines could be converted into a metastatic phenotype by transfect- ing them with a cDNA coding for a high molecular weight variant of CD44 (termed CD44v), expressed only by metastatic cell lines. Sy et al. (1991) have also shown that there was a marked increase in tumor formation and metastatic behavior of lymphoma cells transfected with the cDNA for an 85 kDa CD44 (CD44H). Consequently, Matsumura and Tarin (1992) proposed that the expression of CD44 variants may be a useful marker for the assessment of the metastatic potential of tumors. However, the mechanism by which CD44 participates in tumor progression is still a mys- tery.

To address this question, we examined the expression of CD44 and its interaction with hyaluronan in a panel of human breast cancer cell lines. These cells have been previously characterized by Thompson et al. (1992) with regard to their expression of estrogen receptor, vimentin, and invasive activity based on in vitro and in vivo assays, and represent a convenient model system for tumor invasion, since they cover a vast range of invasive potentials. Here, we found that the most invasive cells as judged by in vitro assays were those that expressed the most CD44. Moreover, we showed that all these CD44 positive cell lines were able to bind hyaluronan, although they expressed variants of CD44 that differed in their affinity for hyaluronan, which may reflect differences in the progression of these cells toward the acquisition of an invasive phenotype. More importantly, all these cells lines were able to degrade hyaluronan. Thus, one of the main functions of CD44 in invasive tumor cells may be to mediate the degradation of hyaluronan.

MATERIALS AND METHODS Sources and preparation of biochemicals

The two monoclonal antibodies (mAb) against human CD44 were obtained from the following sources: BU52 from The Binding Site (San Diego, CA) and the Hermes-1 was kindly donated by Dr. Butcher (Depart- ment of Pathology, Stanford, CA). In the case of Her- mes-1, which also partially blocks [3H]hyaluronan

binding to CD44 (Culty et al., 19901, the antibody was isolated from ascites fluid of nude mice (Bioproducts, Indianapolis, IN) by chromatography on DEAE Migel blue columns as recommended by the manufacturer (BioRad, Richmond, CAI. Nonspecific rat and mouse IgG were purchased from Sigma (St. Louis, MO).

The [3Hlhyaluronan was prepared using a protocol previously described (Underhill et al., 1983). Rat fibro- sarcoma cells were grown in 98% Dulbecco's modified Eagles medium (DMEM), 2% fetal calf serum (FCS) supplemented with L3H1acetate for 1 day. The conditioned medium was collected, digested with pronase, and dialyzed extensively against distilled water. The [3H]hyaluronan was purified by precipitation with cetylpyridinium chloride and then redissolved in a saline solution. The amount of hyaluronan was determined by a uronic acid assay, as described by Bitter and Muir (1962). The three different preparations of [3Hlhy- aluronan used in this study had the following specific activities: preparation 1,6.0 x lo4 cpm/bg Na hyaluronan; preparation 2, 1.7 x lo5 cpm/Fg Na hyaluronan; and preparation 3, 8.9 x lo4 c p d k g Na hyaluronan. The same preparation of labeled hyaluronan was used within each experiment.

Cell culture The human breast cancer cell lines used in this study

are listed in Table 1, along with several characteristics such as the presence of estrogen receptor, vimentin, and their in vitro invasive behavior, which were previously determined by Thompson et al. (1992). These in vitro experiments showed that the acquisition of vimentin correlated well with the invasive behavior of the cells. Similar results were obtained when the tissue invasiveness of the cell lines was assessed following injection into the mammary pads of N Cr nulnu nude mice. How- ever, some cells lines did not grow in nude mice, proba- bly due to the existence of tumor-host interactions such as residual immune responses. In spite of these limita- tions, these experiments suggested that the acquisition of vimentin is required, although not sufficient, for breast cancer cells to become invasive. The following abbreviations have been used in Table 1: MDA-231 for

HYALURONAN DEGRADATION BY CD44' TUMOR CELLS 277

MDA-MB-231; MDA-435 for MDA-MB-435; MDA-436 for MDA-MB-436, MDA-468 for MDA-MB-468; and ADR for MCF-7ADR. The cells were grown in 90% DMEM (GIBCO, Grand Island, NY), 10% FCS containing 100 U/ml penicillin, and 100 pg/ml streptomycin (Sigma). In addition, the human bladder carcinoma cell line, HCV-29T, obtained from Dr. Knudson (Rush Med- ical College, Chicago, IL), which was used as a standard for comparing the amount of CD44, was also cultured in the same media. In some experiments, we included the MCF-1OA cell line, a spontaneously immortalized human breast epithelial cell line, as a model for normal breast tissue. This cell line was provided by Dr. Soule (Michigan Cancer Foundation, Detroit, MI) and was grown in DMEMIF12 containing low levels of calcium as described by Soule et al. (1990).

Determination of CD44 content of the cell lines The various cell lines were first rinsed with calcium

and magnesium-free-phosphate-buffered saline (CMF- PBS) and then extracted in 0.1% Na deoxycholate, 0.5 M NaCl, 0.02 M Tris, pH 8.0 (DOC buffer). The protein contents were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Samples were serially diluted in DOC buffer, and 50-100 pl samples of each dilution were applied to duplicate wells of a dot blot apparatus (BioRad) containing a premoistened sheet of nitrocellulose. For comparison, serial dilutions of a 250 pg/ml extract of HCV-29T cells, which express significant amounts of CD44 (Culty et al., 1990; Nemec et al., 19871, were applied to each sheet. After allowing the samples to adsorb to the sheets for 20 min, a vacuum was applied and the wells were washed with water. The sheet was then placed in a 5% solution of nonfat milk for 1 hr to block residual protein binding sites. The sheet was then incubated for 1 hr in a solution of the BU52 mAb (ascites) diluted 1 to 10,000 in 10% calf serum, 90% CMF-PBS containing 0.05% (v/v) Tween 20. This was followed by a solution of peroxidase labeled anti-mouse antibody (Kirkegaard and Perry, Gathersburg, MD) diluted 1 to 500 in the same buffer. Finally, the blot was incubated in 0.03% H202, 0.2 mg/ml3-amino-9-ethylcarbazole in 0.05 M Na acetate, pH 5.0, for 5-20 min according to Graham et al. (1965). After drying, the optical density of each dot was determined as previously described by Wang and Underhill (1992). When the optical density of the standard curve for the HCV-29T cell extract was plotted on semi-log paper, the curve was linear over the range of 0.5-15 pg/ml protein. The amount of CD44 in each cell line extract was determined relative to that of the HCV-29T cells, with each unit corresponding to the amount of immunoreactivity present in 1.0 pg of protein from HCV-29T cells. Control experiments showed that below 1 mglml of protein, the adsorption of CD44 to nitrocellulose was directly proportional to its concentration. The background level of immunoreactivity was determined by processing the blot under identical conditions except that nonspecific mouse IgG was substituted for the BU52 mAb.

Degradation of C3H]hyaluronan The assay for hyaluronan degradation was similar to

that previously described (Culty et al., 1992). Cells

were plated at 100,000-150,000 cells/ml in 24 multi- well plates andpown in medium containing 5% FCS and 2 pg/ml of [ Hlhyaluronan. After 40 hr, the media were collected and 200 pl protease E (Sigma; 20 mg/ml, predialyzed against distilled water and filtered) was added on the cell layers. The cells were digested over- night, combined back with their media and incubated for an additional 4 hr. The mixtures were then centrifuged in Centricon 30 Microconcentrators (Amicon, Danvers, MA). High molecular weight hyaluronan re- mained in the upper chamber, while the fragments which passed through the membrane were collected and processed for scintillation counting. The data are expressed in terms of cpm of hyaluronan fragments produced per microgram of protein in the cell layer. This latter value was determined by extracting representative wells with DOC buffer, after washing them with CMF-FBS, at the end of the incubation period and assaying them for protein. The background level of degradation was measured in each experiment and subtracted from each value. It was determined by incubating [3H]hyaluronan in medium without cells for an equivalent length of time, treating it with pronase, and passing it through centricon microconcentrators. This background varied depending of the preparation of [3Hlhyaluronan used (batch 1: 169 & 32; batch 2: 481 ? 27; batch 3: 163 2 36). In some experiments, antibodies (Hermes-1 and nonspecific rat IgG) were included in the medium along with the [3H]hyaluronan.

[3Hlhyaluronan binding assays Hyaluronan binding activity was determined on de-

tergent extracts of cells as previously described (Culty et al., 1990). For this, the cells were washed with CMF- PBS, extracted in DOC buffer, and 200 pl aliquots were mixed with 1 pg of [3Hlhyaluronan (final volume was 250 pl). After shaking for 20 min, 250 p1 of saturated (NH,),SO, was added, followed by 25 pl of nonfat milk, and the samples were centrifuged at 9,OOOg for 5 min. The tubes were rinsed twice with 50% saturated (NH,),SO,, the pellets were dissolved in water, and processed for scintillation counting. The background level of binding was determined by including an excess of nonlabeled hyaluronan (60 pg) in some samples, and varied depending upon the amount of protein present. In general, this background corresponded to approximately 4 cpm per microgram of protein in the cell extract. The results are expressed in terms of specific binding (i-e., background subtracted) of [3Hlhyaluro- nan normalized to protein, which was measured for each cell extract as described above.

In some experiments, the cell extracts were incubated with antibodies (Hermes-1 or nonspecific rat IgG) for 20 min prior to the addition of [3Hlhyaluronan.

Western blotting Cell monolayers were washed with CMF-PBS and

further solubilized in Laemmli sample buffer (Laem- mli, 1970) lacking P-mercaptoethanol and the protein content was determined as described above. Samples from each cell line containing 10 pg of protein were electrophoresed on an 8% sodium dodecyl sulfate (SDS) polyacrylamide gel and then transferred to a sheet of nitrocellulose (Immobilon-NC, Millipore, Bedford, MA)

278

4.00

CULTY ET AL.

T -

1.50

1 .oo

0.50

0.00 v-

li,

f N

Human Breast Cancer Cell Lines

Fig. 1. The relative amounts of CD44 immunoreactivity associated with the different human breast cancer cell lines. Confluent cultures of the cell lines were extracted in DOC buffer, serially diluted, and applied to nitrocellulose using a dot blot apparatus. The sheets of nitrocellulose were stained for CD44 using the BU52 mAb and the optical density was determined as described in Materials and Meth-

at 0.9 amp for 30 min, using a transblot cell (Idea, Corvalis, OR). The nitrocellulose sheet was blocked in a solution of 5% nonfat milk and then stained with the BU52 mAb as described above. The background stain- ing was determined by substituting nonspecific mouse IgG for the BU52 mAb and was negative.

Gel filtration chromatography The cell monolayers were rinsed with CMF-PBS and

extracted in DOC buffer, and then centrifuged at low speed to remove most of the insoluble material. A por- tion of this sample was analyzed directly for f3H1hyalu- ronan binding activity and the remaining 2 or 3 ml was applied to the top of a 112 x 1.5 cm column of Sephacryl S-300 superfine (Pharmacia, Uppsala, Sweden). The column was eluted with DOC buffer and 2 ml fractions were collected. The eluted fractions were then analyzed for protein, CD44 immunoreactivity by Western and dot blotting, and [3H]hyaluronan binding. The protein content was determined on 100 pl aliquots of each fraction using the bicinchoninic acid assay. The determination of CD44 content by dot blot analysis was done as described above on 100 p1 aliquots of the fractions. The [3Hlhyaluronan binding was measured as described above, except that 480 p1 aliquots of fractions were used in a final volume of 500 r~.l containing 2 pg of [3H]hyaluronan. For Western blot analysis, 500 pl aliquots of each fraction were precipitated with 75% ethanol over- night at -20°C. The samples were centrifuged, the pel- let dried and then solubilized in 60 ~1 Laemmli buffer

ods. Each unit represents the amount of immunoreactivity present in 1 pg of protein from HCV-29T cells. The cell lines have been arranged in order of increasing amounts of CD44. Each of the values shown is derived from three to five serial dilutions of a single sample, and is representative of that obtained from multiple samples. The error bars represent the mean & standard deviation.

lacking 6-mercaptoethanol. Aliquots of 30 p1 were analyzed by Western blot as described above.

RESULTS Expression of CD44 by different human breast

cancer cell lines The amount of CD44 immunoreactivity present in

each cell line was quantified by dot blot analysis with the BU52 mAb (see Materials and Methods section). As shown in Figure 1, the amounts of CD44 varied significantly between the different cell lines. In general, the cell lines that expressed the most CD44 were also the most invasive, as judged by in vitro chemoinvasion and chemotaxis assays (see Table 1). The chemoinvasive behavior of cells was determined by measuring cell migration through a matrigel coated filter in a Boyden chamber toward a gradient of fibroblast conditioned medium. With the exception of the MDA-468 cell line, the cell migration paralleled the levels of CD44 present in the cells. The measurement of chemotactic activity, which was carried out in similar conditions except that the filter was only coated with a thin layer of collagen, also revealed a very good correlation with the levels of CD44 in the cells. At the extremes, the Hs578T cell line that expressed the greatest amount of CD44 was invasive, as judged by migration through matrigel and chemotaxis in Boyden chamber assays, while the ZR- 75-1 cell line, which did not express detectable levels of CD44, was judged to be noninvasive in both of these


TABLE 2. The effect of the Hermes-1 mAb on the deeradation of 1'Hlhvaluronan'

Experiment Cell Antibody Amount [3H]hyaluronan degraded Inhibition no. line added Der samole ( C D ~ L L P orotein) (%)

1 MDA-468 None 0 5.2 -c 0.3 0 Control IgG 100 Pg 5.1 * 0.1 2 Hermes-1 (pure) 100 kg 0.6 * 0.2 88 Hermes-1 (ascited 20 &1 1.3 f 0.2 75

2 Hs578T None 0 11.2 f 1.8 0 Control IgG 20 Pg 13.3 2 2.1 -18 Hermes-1 (pure) 20 Pg 5.4 2 0.6 52

'The cells were cultured in the presence of2 pgiml r3H1hyaluronan (experiment 1, perparation 3; experiment 2, preparation 2). To some ofthe cultures were added either purified antibodies or ascites fluid of Hermes-1 (approximately 5 mg/ml antibody). ARer 40 hr, the extent of degradation was determined using Centricons. The amount of degradation is normalized to the protein in the cell layer at the end of the incubation period, which waa 123 pgiwell for experiment 1 (MDA-468) and 142 pgiwell for experiment 2 (Hs578T). The mean and range of duplicate determinations are given. Note: the same set of cells were used in experiment 1 of Tables 2 and 3.

20

15

10

5

0

0 1 2 3 4

CD44, unitslpg


Fig. 2. The degradation of [3Hlhyaluronan by the human breast can- label passing through the membrane. The background level of degra- cer cell lines. Each of the cell lines was cultured for 40 hr in 1 d of dation was determined by incubating ['Hlhyaluronan in medium medium containing 2 p,g [3Hlhyaluronan. At the end of the incubation without cells. This background has been subtracted from each value. period, the cell layer and medium were digested with protease, and The insert shows the degradation of hyaluronan relative to CD44 then applied to Centricon 30 microconcentrators. The amount of concentration (correlation coefficient, r = 0.951; P < 0.0001). [3Hlhyaluronan broken down was determined from the amount of Mean -t standard deviation.

assays. Similarly, the cell lines expressing high amounts of CD44 were generally associated with the absence of estrogen receptors and the acquisition of the intermediate filament protein vimentin, both of which have been shown to indicate a poor prognosis in human breast cancer (Sommers et al., 1989). This trend is consistent with other studies indicating that the expression of CD44 is correlated with the metastatic behavior of tumor cells (Gunthert et al., 1991; Sy et al., 1991).

Degradation of [3H]hyaluronan To examine the degradation of hyaluronan, the cells

were cultured in the presence of [3Hlhyaluronan and after 40 hr, the resulting fragments were detected us-

ing Centricon 30 microconcentrators (see Materials and Methods). As shown in the insert of Figure 2, the degradation of hyaluronan was closely correlated with the amount of CD44 (coefficient of correlation, r = 0.951; P < 0.0001). In general, the cell lines that expressed the most CD44 could also degrade the most hyaluronan. This correlation was excellent, despite the fact that other factors are clearly involved in the degradation process, such as the rate of endocytosis and the amount of lysosomal hyaluronidase.

The involvement of CD44 in hyaluronan degradation was further supported by the observation that Her- mes-1 mAb, which is directed against an epitope close to the hyaluronan binding domain of CD44, blocked the

280 CULTY ET AL.

TABLE 3. The effect of the Hermes-1 mAb on the binding of [3H]hyaluronan to detergent extracts of cells'

Experiment Cell Antibody no. line added

1 MDA-468 None Control IgG Hermes-1 (pure) Hermes-1 (ascites)

Hermes-1 (ascites)

Hermes-1 (ascites)

2 MDA-468 None

Bt549 None

Amount per sample

20 Pg 20 Pg 50 ~1 0

20 pl 0

20 &l

0

[3Hlhyaluronan bound (cpmlpg protein)

139 t 18 152 + 19 36 + 4 3 + 1

82 t- 2 3 + 1

105 t 6 5 t l

Inhibition (%)

0 -9 74 98 0

97 0

95

'The cells were extracted in DOC buffer and assayed for ['Hlhyaluronan binding activity (experiment 1, preparation 3; experiment 2, preparation 2). Either purified antibody or ascites fluid (approximately 5 mglml antibody) was added to some of the samples. The results are normalized to the amountof protein per sample, which in experiment 1 was 20.6 pg and in experiment 2 was 292 pg for MDA-468 and 131 pg for BT549. The mean and range of duplicate determinations are given.

C .- CI

2 70 n * O t

C

m a 10 I

Y

-- I

4 1 I

0-

0 1 2 3 4

CD44, unitslpg


Fig. 3. Binding of [3H]hyaluronan by detergent extracts of the human breast cancer cell lines. The cells were dissolved in DOC buffer, and 250 p,l of this extract was incubated with 1 kg of I3H1hyaluronan. The CD44 and any bound [3H]hyaluronan were then precipitated by the addition of an equal volume of saturated (NH,),SO,. The amounts of [3H]hyaluronan in the precipitates were determined. In each case, the background level of binding was determined by including a large

excess of nonlabeled hyaluronan in some samples and this was subtracted from each value. Each determination was done in duplicate or triplicate in two to nine independent experiments for each cell line. The insert shows the correlation between the amounts of [3HJhyaluro- nan binding and CD44 content for each cell line (correlation coefficient, r = 0.667; P = 0.025). Mean ? standard deviation.

degradation of hyaluronan by both MDA-468 and Hs578T cells (see Table 2).

Binding of [3Hlhyaluronan To determine the extent of hyaluronan binding, the

cells were extracted in DOC buffer, mixed with l3H1hy- aluronan, and CD44 and the bound [3Hlhyaluronan were precipitated with (NH,),S04. As shown in the insert of Figure 3, the correlation between [3Hlhyaluro- nan binding and CD44 content was not as good as that of [3Hlhyaluronan degradation and CD44 content (coefficient of correlation, r = 0.667; P = 0.025). However, it should be noticed that the binding and degradation assays were performed under different conditions; the

degradation was carried out over a period of 2 days in living cells, whereas the binding was done on a detergent extract of cell proteins. The greatest deviation between CD44 expression and hyaluronan binding was observed in the Hs578T and MDA-435 cell lines, which bound less [3Hlhyaluronan than anticipated from the amount of CD44 immunoreactivity. We then examined a number of factors which could account for this dis- crepancy.

One possible factor was that other hyaluronan binding proteins were present in the detergent extracts. To test this possibility, the effects of the Hermes-1 mAb on [3Hlhyaluronan binding was examined. As shown in Table 3, this antibody blocked most of the binding of


[3Hlhyaluronan to detergent extracts of MDA-468 and BT549 and between MDA-468 and BT 549. This suggests that CD44 is the major, if not the only, protein responsible for binding hyaluronan under the assay conditions used. It should be noted, however, that the assay was carried out under conditions of high ionic strength, which enhances the binding of hyaluronan to CD44, but may inhibit the binding to other proteins (Hardwick et al., 1992; Yannariello-Brown et al., 1992).

Characterization of hyaluronan binding to CD44 from different cell lines

Another factor that could influence the binding of hyaluronan is the presence of inhibitors. Indeed, most of the cell lines expressing high levels of CD44 also synthesized hyaluronan, which could competitively inhibit the binding of [3Hlhyaluronan used in the assay (unpublished observations). To circumvent this prob- lem, the CD44 was partially purified from four representative cell lines (MDA-468, MDA-231, Hs578T, and MDA-435) by gel filtration chromatography on Sephacryl S-300 (representative profile shown in the insert of Fig. 4). The purification of CD44 from these cell lines did lead to two to fourfold increase in the total amount of [3H]hyaluronan binding, suggesting that inhibitors such as endogenous hyaluronan had been re- moved.

The active fractions were pooled (see example in Fig. 4 insert) and the binding of [3H]hyaluronan was analyzed by Scatchard plot. As shown in Figure 4, there were two types of binding curves corresponding to two pairs of cell lines, which differed by fourfold in both the Kd and the B,, (see Table 4). Interestingly, the cell lines presenting high Kd and low B,, (MDA-435 and Hs578T) were those for which CD44 levels and hyaluronan binding did not correlate. The difference in the K d

suggests a structural alteration in the CD44 that af- fects the binding affinity for hyaluronan. In addition, the difference in the B,,,/CD44 suggests that some of the CD44 present in the MDA-435 and Hs578T cell lines lack the capacity to bind hyaluronan.

Binding of [3Hlhyaluronan by different molecular weight forms of CD44

One possible explanation for the difference in hyaluronan binding characteristics is the particular molecular weight forms of CD44 expressed by the tumor cells. Western blotting of the tumor cell lines revealed that they expressed varying amounts of a low molecular weight form of 85 kDa and some high molecular weight forms of 110, 150, and 180 kDa (see Fig. 5). Interest- ingly, the cell lines which expressed the greatest amounts of CD44 tended to have a larger proportion of the 85 kDa band (see ADR through Hs578T). In contrast, the cell lines that expressed small amount of CD44 (SK-Br-3, MCF-7, and T47D) possessed mainly higher molecular weight forms similar to those of the MCF-1OA cell line, which is derived from normal tissue. Given the diversity of CD44 molecules expressed by these cells, it is clearly possible that only some were capable of binding hyaluronan and this could account for the variation between the cell lines.

To examine this possibility, four of the cell lines (MDA-468, MDA-231, Hs578T, and MDA-435) were ex-

5

4

N 0

x 3 Q)

r

!! k ICI

0 g 2 rn

1

0

M D A-46 8/\\/ rn

A-2 '

M D A-435

$&.-4s578T , \\ 0 1 2 3 4 5 6 7 8

Bound, cpm x

Fig. 4. Scatchard plot analysis of [3HIhyaluronan binding by CD44 from four different cell lines. The CD44 was partially purified by molecular-sieve chromatography on Sephacryl 5-300. The insert shows an example of the profile obtained for the MDA-468 cell line. The cells were extracted in DOC buffer and applied to the column, which was eluted with DOC bdfer. Two milliliter fractions were collected and analyzed for protein (*) and for CD44 immunoreactivity (0). The positions of the void and total volumes are indicated. For each cell line, alternate fractions of the area indicated by the bracket (CD44 immunoreactive zone) were pooled and analyzed for [3Hlhyaluronan binding (preparation 3) by Scatchard plot. The different cell lines examined are: MDA-231 (0); MDA-468 (m); MDA-435 (0); and Hs578T (a).

TABLE 4. Binding constants for CD44 from four different cell lines'

Cell K d B,JCD44 line HaHA, pg/ml NaHA, ng/Unit CD44

MDA-468 4.0 3.49 MDA-231 3.7 3.68 Hs578T 14.1 0.81 MDA-435 22.5 0.69

~

'The Kd and the B, were calculated from the slope and intercept of the Scatchard plots shown in Figure 4. The amount of hyaluronan bound at saturation was normalized to CD44 immunoreactivity, which was: 53 U/ml for MDA-468; 54 U/ml for MDA-231: 102 U/ml for Hs578T and 61 Urn1 for MDA-435. Note: the Kd is calculated in terms of the concentration of Na hyaluronan in the assay solution prior to the addition of the (NHJzS04.

tracted in detergent and then applied to gel filtration columns to separate out the various forms of CD44 (see example of MDA-468 in Fig. 4 insert). As shown in Figure 6a, the different molecular weight forms of CD44 from the MDA-468 cell line were partially separated from each other. The same fractions were also

282 CULTY ET AL.

Fig. 5. Western blot analysis of extracts from the various human breast cancer cell lines. The cells were extracted in Laemmli sample buffer lacking p-mercaptoethanol, and 10 kg of protein were electrophoresed on an 8% SDS-polyacrylamide gel. The proteins were electro- phoretically transferred to a sheet of nitrocellulose, which was then

blocked in nonfat milk and stained for CD44 using the BU52 mAb. The positions of the prestained high and low molecular weight standards (BioRad) are shown on the left, while the positions of the different forms of CD44 are indicated on the right.

assayed for [3H Jhyaluronan binding activity (expressed as cpdml) and CD44 immunoreactivity (Fig. 6b). Fig- ure 6c shows that the binding of [3H]hyaluronan relative to CD44 immunoreactivity was constant across the different fractions. This suggested that all of the different molecular weight forms of CD44 expressed by the MDA-468 cells have the ability to bind hyaluronan. Similar results were obtained with the MDA-231 cell line.

In contrast to the above, when the Hs578T cell line was analyzed in a similar fashion, several differences became apparent (see Fig. 7). First, the specific activity varied across the different fractions, the highest values being associated with the fractions containing the 85 kDa form (Fig. 7c). This suggests that, in this cell line, only the 85 kDa form of CD44 can bind hyaluronan, while the high molecular weight forms are inactive. In addition, the amount of [3H]hyaluronan bound by the 85 kDa CD44 from this cell line was only 3050% of that from the MDA-468 and MDA-231 cell lines. Anal- ysis of the MDA-435 cell line gave results similar to that of the Hs578T cell line. These results indicate that

CD44 from the various cell lines is heterogeneous with regard to [3Hlhyaluronan binding.

DISCUSSION The present study demonstrates that the expression

of CD44 and degradation of hyaluronan correlate with the invasive potential of human breast cancer cell lines as measured by in vitro assays. The overexpression of CD44 was associated with various markers of tumor progression, including the loss of estrogen receptor, acquisition of vimentin, and increased invasive and chemotactic activities. Interestingly, the MDA-468 cell line, which expressed high levels of CD44 but scored differently for the two invasion assays, has been re- cently found to be heterogeneous with respect to vimentin expression (Azzam et al., 1993) and may represent a transitional form between the noninvasive and invasive cell types. More importantly, there was an excellent correlation between the expression of CD44 by the various cell lines and their ability to degrade hyaluronan. The role of CD44 in the degradation of hyaluronan

HYALURONAN DEGRADATION BY CD44+ TUMOR CELLS 283

a

25

20

15

1 0

5

n

I I '

- 6 8 10 12 14 16 18 20 22 24 26

0 ' 6 8 10 12 14 16 18 20 22 24 26

Fraction

Fig. 6. Analysis of Sephacryl S-300 fractions ofMDA-468 cell extract by Western blotting, CD44 immunoreactivity, and L3H]hyaluronan binding activity (preparation 2). The fractions indicated in the insert of Figure 4 were further analyzed with respect to the following. a: Western blot analysis of each fraction shows the distribution of the different forms of CD44. The arrows on the right indicate the positions

of the different molecular weight forms of CD44. b The amount of [3H]hyaluronan binding activity (*) and CD44 immunoreactivity (0) for each fraction are shown. c: The specific activity of [3Hlhyaluronan binding relative to CD44 immunoreactivity shows only minor variations across the fractions.

284 CULTY ET AL.

a

b

P /

I /- / '. \

8 10 12 14 16 18 20 22 24 26 28

C g 1.00 r X U P n 0,

0.50 0

& -

Y ri n 0.00 0

v)

1, /v' +-m-m-

8 10 12 14 16 18 20 22 24 26 28

Fraction

1.50

1 .oo

0.50

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Fig. The blot

7. Analysis of Sephacryl S-300 fractions of Hs578T cell extract. fractions were analyzed with respect to the following. a: Western analysis of each fraction shows the distribution of the different

forms of CD44. The arrows on the right indicate the positions of the different molecular weight forms of CD44. b The amount of ["Hlhy- aluronan binding activity (0) and CD44 immunoreactivity (0) for each

fraction are shown. Note that the scale for binding activity is different from that in Figure 6b. c: The specific activity of [3Hlhyaluronan binding relative to CD44 immunoreactivity shows that most of the activity is associated with fractions containing the 85 kDa form and not with those containing the higher molecular weight forms.


was demonstrated by the fact that this process was blocked by the Hermes-1 mAb against CD44.

There are several potential ways in which the CD44- mediated degradation of hyaluronan could influence the formation of primary and secondary tumors. For example, the fragments of hyaluronan could act as in- ductive agents for endothelial cells, resulting in the formation of new blood vessels. Indeed, West et al. (1985) have shown that fragments of hyaluronan (4-25 disaccharides) have a marked effect on angiogenesis. Another possibility is that the degradation of hyaluronan surrounding blood vessels leads to a weakening of their wall. This, in turn, could allow tumor cells to more easily penetrate the blood vessels, enter the circulation, and metastasize to different locations. Thus, CD44 would play a role equivalent and/or complementary to that of the collagenases in tumor invasion (Moscatelli and Rifkin, 1988).

Another conclusion of the present study is that human breast cancer cell lines express various forms of CD44, differing in both size and ability to bind hyaluronan. The existence of these variants was first indicated by the fact that the binding of [3Hlhyaluronan relative to CD44 immunoreactivity was significantly different between some of the cell lines. Further analysis of hyaluronan binding revealed that in two of the cell lines (MDA-468 and MDA-231), all of the different molecular weight forms of CD44 could bind hyaluronan with high affinity. In contrast, in two other cell lines (Hs578T and MDA-435), the high molecular weight forms of CD44 lacked the ability to bind hyaluronan, while the 85 kDa form bound with a low affinity. The data presented above suggest that the 85 kDa CD44 is mediating the degradation of hyaluronan by invasive tumor cells, since it was the only form of CD44 consistently present in the active fractions from these cell lines. This molecule could be homologous to that described by Sy et al. (1991)) which also binds hyaluronan, and promotes the tumor growth and metastatic behavior of lymphoma cells. Furthermore, our data showing that CD44 heterogeneity is also found within individual cell lines, suggest that the expression of CD44 by tumor cells does not follow a simple pattern (such as the production of one isoform related to the acquisition of an invasive phenotype), but rather it can lead to the expression of variants of CD44 with different functions. This is consistent with the fact that tumor cells are, by nature, deregu- lated cells presenting the abnormal expression of proteins which may be altered structurally and function- ally, and that it is the conjunction of some of these alterations which may result in the acquisition of an invasive phenotype.

At present, the structural basis for these variations in hyaluronan binding is unclear. Such differences may be due to alternative RNA splicing as well as to different glycosylation and/or phosphorylation patterns. Along these lines, the gene for CD44 is very complex, comprising a t least 19 exons (Screaton et al., 1992). It is possible that subtle differences in the splicing of the exons, which do not significantly alter the molecular weight of the protein, could cause differences in the binding affinity for hyaluronan. Clearly, this is a topic that requires further investigation.

The results of this study may account for some of the

divergent observations concerning the hyaluronan binding capacity of different molecules of CD44. Sta- menkovic et al. (1991) have shown that a cDNA isolated from epithelial cell lines encoded for a 130 kDa CD44 form that lacked the capacity to bind hyaluronan. On the other hand, He et al. (1992) have found that some high molecular weight isoforms of CD44 can bind hyaluronan and that structural variations occurring in the middle domain of the molecule do not necessarily affect its ability to bind hyaluronan. Indeed, in the present study, we found that both the 85 and 150 kDa forms of CD44 could bind hyaluronan with high affinity in some of the breast cancer cell lines (MDA-468 and MDA-231). Thus, depending upon the particular cell line being examined, different results can be obtained with regard to the binding of hyaluronan by CD44.

Despite the variation in hyaluronan binding activity between the different cell lines, there was a remarkable correlation between the amount of CD44 present and the ability of the cells to degrade hyaluronan. It is prob- able that there is some degree of compensation for the low affinity form of CD44 under physiological conditions. Indeed, the Hs578T cell line, which expressed the low affinity form of CD44, was the most active in de- grading hyaluronan. There is evidence that under physiological conditions, several molecules of CD44 can cooperatively bind hyaluronan (Underhill, 1992). Thus, an increase in the number of molecules of CD44 or their distribution on the cell surface could compensate for the lower affinity of individual molecules. Similarly, an increase in lysosomal hyaluronidase activity could also result in enhanced degradation.

The present study illustrates the structural and func- tional heterogeneity of CD44 in tumor cells. Moreover, the use of a panel of tumor cells presenting various invasive potentials allowed us to determine whether there was any relation between the interaction of CD44 with hyaluronan and the in vitro invasive behavior of the cells. Indeed, we found that CD44 was overex- pressed in all invasive cell lines and that there was an excellent correlation between the expression of CD44 by the cells and their ability to degrade hyaluronan. These results suggest that the CD44 molecules expressed by invasive tumor cells may mediate the adhesion to hyaluronan as well as its degradation. The rela- tionship between the degradation of hyaluronan and tumor invasion is an area that deserves further study.

ACKNOWLEDGMENTS We thank V. Papadopoulos and P. Pavasant for their

constructive criticism of this work. This work was supported by United States Public Health Service grant CA35592 awarded to Charles Underhill by the Na- tional Cancer Institute, Department of Health and Hu- man Services.

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Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential