laminin and fibronectin increase the steady state level of the 67 kd high affinity...

Vol. 168, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 16, 1990 Pages 1110-1117

Laminin and fibronectin increase the steady state

level of the 67 kD high affinity metaatask-associated

laminin receptor UIRNA in human cancer cells

Vincent Castronovo*and Mark E. Sobel

Tumor Invasion and Metastasis Section, Laboratory of Pathology,

National Cancer Institute, Bethesda , Maryland 20892

Received March 9, 1990

Among the various known laminin binding proteins, the 67 kD

high affinity laminin receptor (LR) is intimately involved

during tumor invasion and metastasis. In this study, we report

that laminin and fibronectin, two attachment glycoproteins,

significantly increased the total cellular level of 67 kD LR mRNA

in two human cancer cell lines, T47D breast carcinoma cells and

A2058 melanoma cells. Neither GRGDS nor YIGSR synthetic peptides

induced such a stimulatory effect. Since the steady state level

of LR mRNA has been shown to control the number of receptors

expressed at the cell surface, these results suggest that

contact of the cancer cells with laminin and fibronectin in the

host matrix may be an important regulatory mechanism by which

cancer cells maintain a high number of LR at their cell surface

as they progress through the several steps of tumoral invasion

and metastasis. 01990 Academic Press, Inc.

Tumor invasion and metastasis are the result of complex

pathological interactions between malignant cells and host stroma

(l-3). During their progression, the invading tumor cells have to penetrate and traverse a number of tissue compartments separated

from each other by two types of extracellular matrix:

interstitial matrix and basement membranes (1,2). The latter are

specialized thin extracellular sheets which constitute barriers

that cancer cells must cross (1,4). A major event during tumoral

*To whom correspondence and reprint requests should he addressed at Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2A33, Bethesda, MD 20892.

0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 1110


invasion and metastasis is the attachment of cancer cells to

both types of extracellular matrix, mediated by cell surface

receptors to attachment factors such as fibronectin and laminin

(l-3,5) .

Laminin is the major component of basement membrane (6). Interactions between laminin and cancer celis are important at

several steps of the metastatic cascade (1,2,7) and are mediated

through several laminin binding proteins (8-12). Among these, a

67 kD high affinity laminin receptor appears to be intimately

involved during tumor invasion and metastases (8,13,14). A strong

direct correlation has been observed between the number of 67 kD

laminin receptors expressed at the cell surface and the

metastatic capabilities of malignant cells, particularly in

breast and colon cancer (1,15,16). Estrogen and progesterone,

known to modulate the invasive and metastatic potential of

steroid receptor positive breast cancer cells, significantly increased the expression of laminin receptor both at the protein

and the mRNA level in human breast carcinoma T47D cells (17).

cDNA clones encoding the 67 kD laminin receptor have been

identified both in human and mouse (16, 18-20). RNA blot

analysis showed that the steady state level of 67 kD laminin

receptor mRNA correlated directly with the number of cell surface

laminin receptors in a variety of human cancer cells (18). The

synthesis of the receptor in most cells thus appears to be

controlled by the amount of mRNA available for translation.

During invasion, cancer cells actively influence the

surrounding extracellular matrix (1). In return, the host extracellular matrix components exert biochemical and

mechanical influences on the cancer cell via plasma membrane receptors (21). Fibronectin and laminin are known to stimulate

attachment, spreading, migration and proliferation of normal and

cancer cells (1,5). In addition, laminin has been shown to

stimulate the production of type IV collagenase by malignant

melanoma cells (21). Thus, attachment of the tumor cell to the

basement membrane via laminin receptor(s) may induce a cascade

of events, one of which is production of proteolytic enzymes to

degrade other components of the basement membrane such as type

IV collagen.

The three-step hypothesis suggested by Liotta et al. (1) proposed that continued invasion of the matrix may take place by

cyclic repetition of tumor cell attachment, local degradation of

the matrix and cell locomotion into the altered matrix. This

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theory implies a high turnover in the synthesis of several gene

products involved in each of these steps. Expression of the 67 Kd

laminin receptor is probably involved in the attachment step.

Since the mRNA level of this receptor controls the number of

receptors expressed at the cell surface (18), we examined in this

study the possible modulation of laminin receptor mRNA levels in cancer cells by laminin or fibronectin.

MATERIAL AND METHODS

Cell Lines and Culture Conditions The human breast carcinoma T47D cell line was obtained

from the American Tissue Type Collection. Human melanoma A2058 cells were previously described (23). The cells were routinely grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10 % heat inactivated fetal bovine serum, penicillin (100 pg/ml) and streptomycin (100 ug/ml) obtained from Gibco, in an atmosphere of 5% CO2/95% air under saturating concentrations of humidity at 37OC.

Attachment glycoproteins and synthetic peptides Laminin was purified from the mouse Engelbreth-Holm-Swarm

sarcoma as described (6). Human fibronectin was purchased from Collaborative Research, Inc. The purity of both laminin and fibronectin was verified by gel electrophoresis. Synthetic peptides (YIGSR, YIGSE, GRDGS and GRGES) were synthesized on a Biosearch 9600 peptide synthesizer.

Attachment factor effect assays Plastic culture dishes (diameter 13.5 cm, Falcon 3025) were

coated with laminin or fibronectin by incubation overnight at 370 with 15 ml of a solution containing 10 fig/ml laminin or fibronectin in phosphate buffered saline (PBS), 2% bovine serum albumin (BSA), pH 7.4. After the coating, the dishes were washed twice with PBS containing 2% BSA. A2058 or T47D cells were harvested at a preconfluent stage of growth by treatment with 0.1% trypsin/SmM ethylene-diaminetetraacetic acid. The cells were washed twice in DMEM without serum and added in the same medium to the plastic culture dishes coated with laminin or fibronectin. Uncoated plastic dishes were used as control. To investigate the possible effect of the synthetic peptides YIGSR or GRGDS on the level of laminin receptor mRNA , plastic culture dishes were coated with these peptides as well as with their corresponding controls (YIGSE and GRGES, respectively), following the same protocol described for laminin and fibronectin. In some experiments, the peptides were added at a concentration of 50 pg/ml to the culture medium of A2058 or T47D cells which were seeded on control plastic dishes.

RNA blot analysis Total cellular RNA was isolated from cells grown in

the absence of serum on laminin , fibronectin or plastic for 24 hours using the guanidium thiocyanate method as described (24). After electrophoresis on 1.0% gels containing methyl- mercuric hydroxide as the denaturing agent, the fractionated RNA was transferred to activated diazobenzyloxymethyl cellulose paper (Schleicher & Schuell) .as described (25) and hybridized to

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nick translated 32P-labeled laminin receptor cDNA insert (18) for 16-24 h at 420 C. After hybridization, blots were stringently washed as described (25), and exposed to X-ray film using Cronex Hi plus intensifying screens (DuPont). Blots were stripped free of probe by incubating in 99% formamide and then water, both at 70° C for 5 min, followed by exposure to X-ray film to ensure complete removal of probe. Blots were subsequently hybridized with beta-actin cDNA (26) as a control to rule out differences in the amount of RNA loaded on the gel. Hybridization signals were quantified by densitometric scanning of multiple autoradiograms of various timed exposures. In some experiments, RNA slot blots (Schleicher & Schuell) containing 1, 5 and 10 pg total cellular RNA were hybridized to the cDNA probes.

RESULTS

Levels of laminin receptor mRNA in A2058 and T47D cells which

had been cultured for 24 hours on plastic, laminin or fibronectin

were determined by Northern and slot blot hybridizations. As

shown in figure 1, the laminin receptor cDNA probe hybridized to

a unique size transcript of approximately 1,400 bases. In both

melanoma and breast cancer cell lines, attachment to laminin and

fibronectin increased the level of laminin receptor mRNA. The

effect of fibronectin was more pronounced than that of laminin.

P L F P L F fj 300, ,

1,400 E - 4 B E IOO- 0 z K E 2

0 1 A2058 T47D A2058 T47D

Figure 1. Nothern blot hybridization. Cells were cultured for 24 hours on plastic (P), laminin (L) or fibronectin (F). Total cellular RNA extracted from A2058 or T47D cells was hybridized with a human laminin receptor cDNA probe as described in Methods. The length of the hybridized mRNA (1400 bases) was dete.rmined by comparison with the known size of rRNA and with the sizes of lambda DNA digested with Hind III.

Figure 2. Modulation of laminin receptor mRNA levels in A2058 and T4lD cells by laminin and fibronectin. Each column represents the laminin receptor mRNA level (+ SD) quantified bv densitometrv and expressed relative to the amount of a&in mRNA, as described-in Methods. The cells were grown on plastic ( n ), laminin ( q ) or fibronectin ( q ). Each experiment was repeated at least twice.

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Indeed, the relative steady state level of the laminin receptor

mRNA was 1.96 times higher in A2058 cells grown on laminin than

on plastic (p< 0.001) and 2.8 times higher in A2058 cells grown

on fibronectin than on plastic (pl 0.001) (fig. 2). A comparable

stimulatory effect was observed in T47D cells. In the latter, the

steady state level of laminin receptor mRNA was 1.4 fold and

1.94 fold higher in cells grown on laminin or fibreonectin,

respectively, than in cells grown on plastic (ps 0.001) (fig. 2).

The more pronounced effect of fibronectin over laminin was

significant in both A2058 and T47D cells (p< 0.008).

It has been reported that YIGSR and GRGDS are amino acid

sequences which can mimic some of the biological activities of

laminin or fibronectin respectively (27-29). We investigated the

possibility that the stimulatory effect of laminin and

fibronectin on the steady state level of laminin receptor mRNA

could be produced by exposure of cancer cells to these

synthetic peptides. Thus, the melanoma and breast cancer cells

were grown for 24 hours on YIGSR- or GRGDS- coated plastic

dishes. As a control, cells were grown on plastic dishes coated

with the corresponding inactive peptide YIGER or GRGES,

respectively. The analysis of the RNA extracted from these

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Figure 3. Modulation of laminin receptor mRNA levels in A2058 and T47D cells by YIGSR ( H) and its control peptide YIGSE (H). Each column represents the laminin receptor mRNA level (+ SD) quantified by densitometry as described in figure 2. Figure 4. Modulation of laminin receptor mRNA level in A2058 and T47D cells by GRGDS ( n ) and its control peptide GRGES (H,. Each column represents the laminin receptor mRNA level (5 SD) quantified by densitometry as described in figure 2.

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cells after 24 hours showed that neither YIGSR (fig. 3) nor

GRGDS (fig. 4) induced a signficant increase in the steady

state level of laminin receptor mRNA.

DISCUSSION

Tumor invasion and metastasis are the result of complex

pathological interactions between malignant tumor cells and the

host stroma. These interactions affect both the host tissues and

the cancer cells. Malignant cells degrade the matrix in

actively invaded areas , induce production of matrix components

by host cells in response to the local presence of the tumor,

or may themselves synthesize and deposit matrix components such

as collagen (1,2). Conversely, the host extracellular matrix

regulates certain activities of cancer cells such as proteolytic

enzyme secretion or motility (1,2).

Attachment of cancer cells to extracellular matrix components,

especially to laminin and fibronectin, is an important event

occurring many times during tumoral invasion and metastasis

(1,3,5). This attachment is mediated through a variety of cell

surface receptors involving a variety of adhesion mechanisms.

Interaction between cancer cells and laminin has been the

subject of intensive studies over the past decade, since it is

considered to be a key step of the metastatic cascade (1). Among

several recently identified laminin binding cell surface

proteins (8-12), the 67 kD laminin receptor seems to be actively

used by cancer cells during the progression of tumor invasion and

metastasis. Indeed, increased cell surface expression of this

receptor has been correlated with the invasive and metastatic

capabilities of cancer cells (15,16). In highly invasive cancer

cells, the number of laminin receptors appears to be dramatically

increased and not saturated with laminin. In this study, we

showed that laminin significantly increased the level of mRNA in

two human cancer cell lines, A2058 and T47D. YIGSR, which has

been reported to inhibit cell attachment to laminin (27), was

unable to substitute for laminin in increasing the level of

laminin receptor mRNA. This suggests that the YIGSR sequence is

not sufficient to induce the laminin stimulatory effect.

Possibly, YIGSR is not the sequence of laminin responsible for

the interaction between the 67 kD laminin receptor and

laminin; it may bind bind to another cell surface laminin binding

protein to mediate its effect on cell attachment.

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Unexpectedly, fibronectin dramatically increased the level of

67 kD laminin receptor mRNA in both human melanoma and breast

cancer cell lines. The ability of cells to bind fibronectin and

related adhesion proteins appears to depend on a short,

hydrophilic amino acid sequence, RGDS, located in the cell

binding domain of the fibronectin molecule (28,29).

It has recently been reported that laminin also contains,

within its A chain, the sequence RGDS (30). We therefore tested

the hypothesis that the increase of laminin receptor mRNA levels

by fibronectin and by laminin was due to interaction of the cells

with this specific sequence. We showed that the peptide RGDS,

whether coated on plastic culture dishes or added in solution to

the culture medium, did not affect the level of laminin receptor

mRNA in A2058 and T47D cell lines. These results indicate that

the sequence RGDS is either not sufficient or is not involved at

all in the stimulatory effect observed.

During tumor progression, cancer cells synthesize and deposit

laminin. We showed previously an accumulation of laminin at the

invasive edge in both in vitro and in viva models for tumor

invasion (31). Fibronectin is a widely distributed extracellular

matrix protein (32). The induction of laminin receptor mRNA

following the interaction of cancer cells with laminin and/or

fibronectin may therefore be an important regulatory mechanism

which provides cancer cells with a continuous supply of laminin

receptors necessary for the continuation of tumor invasion and

metastasis.

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laminin and fibronectin increase the steady state level of the 67 kd high affinity...

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