Pigment Cell Research Suppl. 2 219-233 (1992)

The Use of Novel Antineoplastic Agents to Inhibit the Growth and Metastasis of Malignant Melanoma and other CancemKENNETH OLDEN', SHEILA A. NEWTON', TOSHIHMO NAGAI', YOSHIAKI YASUDA', KRZYSZTOF GRZEGORZEWSIU', PASCAL BRETON', OLADIPO OREDIPE' AND SANDRA L. WHITE'72 'Howard University Cancer Center and 'Department of Microbiology, Howard University College of Medicine, Washington, DC 20060

INTRODUCTION Since researchers have begun to elucidate the biochemical mechanisms involved in the progression of malignant disease, efforts to develop therapeutic agents to block tumor growth and metastasis are now progressing rapidly. In fact, several promising antimetastatic agents may soon be ready for use in human trials. In the case of hematogeneous metastasis, tumor cells escape from the primary tumor mass and enter the circulatory system either by direct shedding of cells into vascular clefts, as in certain sarcomas; or more frequently, intravasation is preceded by invasion of cells into the stromal tissue surrounding the primary tumor. It is likely that this process involves both controlled secretion of degradative enzymes and directed migration toward capillaries and lymphatics (1-5). Once in the circulation, tumor cell arrest can potentially occur by a number of different mechanisms including indirect trapping through formation of tumor cellplatelet-endothelial call complexes, direct adhesion to recognition proteins on the luminal surface of endothelial cells, and

by adhesion to extracellular matrix proteins in the subendothelium (6-9). Homo- and heterotypic aggregation of tumor cells and platelets appears to increase the implantability of malignant cells by increasing the size of tumor cell emboli (10-13). A number of tumor cell lines have been reported to aggregate pl ate1 ets spontaneously in vitro and to induce thrombocytopenia when injected intravenously into mice (14-16). It is also likely that trauma to endothelial cells in the microvasculature of target organs, caused by passage of tumor cells and exacerbated by chemotherapeutic agents, triggers the binding of platelets to exposed endothelial basement membrane (13). This in turn may lead to the disturbances in clotting time, fibrinolysis, and platelet function that are all characteristic of patients with malignant disease. Subsequent to arrest, in a series of steps that may mirror the release phase of metastasis, tumor cells pass through the endothelial cell layer, reinvade the underlying basement membrane and proliferate .in subendothelial connective tissue. Potentially, any step of the

K. Olden et al.

220 m e t a s t a t i c cascade o r above sequence o f

f i b r i n o g e n , type I V and V collagen, and

events can be t a r g e t e d f o r i n t e r r u p t i o n .

heparan s u l f a t e proteoglycans.

However, t h e most common a n t i m e t a s t a t i c

m a j o r i t y o f research on these p r o t e i n s has

regimens are aimed a t ( i ) i n h i b i t i o n o f tumor

been concerned w i t h e l u c i d a t i o n o f t h e

The

v a s c u l a r i z a t i o n ; ( i i ) i n h i b i t i o n o f the

biochemical mechanisms by which they

formation o f homo- and h e t e r o t y p i c c e l l

promote c e l l attachment i n v i t r o , and

aggregates d u r i n g i n t r a v a s c u l a r t r a n s p o r t ;

subsequent development o f antiadhesive

( i i i ) i n h i b i t i o n o f adhesion t o and invasion

agents t h a t could be used i n probing the

o f basement membrane/extracellular m a t r i x ,

f u n c t i o n s o f the i n d i v i d u a l f a c t o r s

and ( i v ) augmentation o f h o s t immune e f f e c t o r mechanisms (17).

m.

I n t h i s a r t i c l e , the

approaches used i n our l a b o r a t o r y t o i n h i b i t

Following t h e i n v i t r o i d e n t i f i c a t i o n

tumor growth and metastasis w i l l be summa-

o f the RGD t r i p e p t i d e as an important

r i z e d i n t h r e e sections:

r e c o g n i t i o n determinant i n t h e c e n t r a l

f i r s t , t h e evidence

t h a t metastasis o f 616-F10 murine melanoma

c e l l - b i n d i n g domain o f f i b r o n e c t i n (18-

can be i n h i b i t e d by c o - i n j e c t i o n o f tumor

20), s y n t h e t i c peptides c o n t a i n i n g t h i s

c e l l s w i t h t h e pentapeptide GRGDS (Gly-Arg-

sequence have been found t o e x h i b i t

Gly-Asp-Ser);

p o t e n t i n h i b i t o r y e f f e c t s on v a r i e t y o f

second, the evidence t h a t

a l t e r a t i o n s i n c e l l surface carbohydrate

b i o l o g i c a l processes i n v i v o .

s t r u c t u r e by treatment o f tumor c e l l s w i t h

example, i n j e c t i o n o f GRGDS o r i t s

For

swainsonine o r castanospermine i n h i b i t the

d e r i v a t i v e s i n t o amphibian and i n s e c t

metastasis o f B16-FlO Relanoma; and t h i r d ,

embryos d u r i n g g a s t r u l a t i o n was found

the evidence t h a t swainsonine i n h i b i t s tumor

t o completely h a l t t h i s key e a r l y migra-

growth and metastasis when administered

t o r y process, presumably by b l o c k i n g t h e

s y s t e m i c a l l y v i a mechanisms i n v o l v i n g immune

interaction o f migrating c e l l s with

modulation.

f i b r o n e c t i n - l i k e molecules (21-23). S i m i l a r l y , when a decapeptide c o n t a i n i n g RGDS was i n j e c t e d i n t o avian embryos,

ANTIADHESIVE APPROACH INVOLVING FIBRONECTIN

neural c r e s t c e l l s f a i l e d t o leave t h e i r

From an examination o f the steps i n v o l v e d i n

neural tube, and i n s t e a d formed a b l o c k o f

t h e m e t a s t a t i c cascade, i t i s c l e a r t h a t t h e r e

stationary tissue (24).

s i t e o f o r i g i n on t h e dorsal face o f t h e

are m u l t i p l e i n t e r a c t i o n s o f tumor c e l l s w i t h e x t r a c e l l u l a r matrices, and since t h e blockage

A d d i t i o n a l l y , the homing o f hemopoie-

o f o n l y one c r u c i a l step i s t h e o r e t i c a l l y

t i c precursor c e l l s t o t h e embryonic

enough t o abrogate metastasis, i t i s t h e r e f o r e

thymus gland, a process important i n

conceivable t h a t s p e c i f i c i n h i b i t o r s o f

e s t a b l i s h i n g t h e immune system, a l s o

adhesion p r o t e i n f u n c t i o n might be able t o

appears t o depend on c e l l i n t e r a c t i o n s

block t h e c o l o n i z a t i o n process.

w i t h f ibronectin; t h e GRGDS p e p t i de was

Over the past

15 years, a number o f prominent connective

found t o i n h i b i t t h i s chemotactic homing

t i s s u e and basement membrane p r o t e i n s have

event ( 2 5 ) , as w e l l as i n v a s i o n across

been i d e n t i f i e d whose l o c a l i z a t i o n i n v i v o

human amnion basement membranes.

would be c o n s i s t e n t w i t h a r o l e i n tumor c e l l

l a r l y , human tumor c e l l i n v a s i o n across

adhesion.

These in c l ude f ib r o n e c t i n ,

laminin, v i t r o n e c t i n / s e r u m spreading f a c t o r ,

basement membranes was blocked by RGD-containing peptides.

Simi-

K. Olden et al. As a functional extension of its effects on cellular adhesion, fibronectin is also able to stimulate fibroblastic cell migration in a variety of in vitro assays (26-30). Furthermore, when introduced into one well of a Boyden chamber in a standard chemotaxis assay, fibronectin can promote the directed migration of cells across a nitrocellulose filter (31-34). High molecular weight cell-binding fragments of fibronectin containing the GRGDS pentapeptide also possesses this activity (31-34), implying that either this signal itself, or a site close to it, mediates the migratory response. As an alternative explanation for the activity of fibronectin in Boyden chamber experiments, it has been proposed that migratory cells can recognize gradients of substrate-absorbed adhesion proteins, such as fibronectin, and translocate from a region of low density to one of high density ( 3 5 ) . This phenomenon, termed haptotaxis, may occur in chemotaxis assays when only one chamber is filled with fibronectin solution and the protein sticks to the filter that separates the chambers. In keeping with its proposed role in cellular migration in vivo, fibronectin has been found both in the embryo and adult at sites of tissue remodeling Synthetic peptides based on the RGD (36-42). sequence have been reported to completely block neural crest cell migration in vitro and in vivo (21), as have antifibronectin antibody Fab' fragments directed against the central cell-binding domain, and a monoclonal antibody that binds close to the GRGDS site (30,42,43).

If, as postulated, adhesion to specific extracellular matrix molecules is a prerequisite for successful organ colonization by metastatic cells, then interference

i r i t h i J adhcaivG intorastion w i t h a n t i adhesive agents, such as GRGDS, should represent a potential strategy for

preventing metastasis. To investigate the possible role of the major cell interaction site of fibronectin in tumor metastasis, the synthetic peptide GRGDS ( 3 mg/mouse) was coinjected with 70,000 816F10 murine melanoma cells into the 1 ateral tai 1 vein of syngenei c C57B1/6 mice as previously described ( 4 4 ) . When the melanotic tumor nodules present in the lungs were enumerated two weeks later, we observed an 85% decrease in the number of colonies in the lungs of the peptide treated mice ( 4 5 ) . Similar results were obtained when cells and peptide were injected simultaneously or sequentially into separate tail veins, indicating that inhibition of metastasis is not an artifact of premixing. Control experiments were done to demonstrate that the antimetastatic activity of GRGDS was concentration dependent and that the peptide did not merely retard growth nor impair cellular tumorigenicity. For example, growth in soft agar and at subcutaneous sites was not affected by the presence of the peptide. Also, peptide treatment did not increase the frequency of extrapulmonary metastases. Since there are potentially many fibronectin-dependent adhesive steps involved in metastasis and invasion, experiments were performed to determine more precisely the particular site of action o f GRGDS. To investigate this matter, two types of experiments were performed. First, we examined the activity of the peptide in Natural Killer (NK) cell-deficient or platelet-depleted (thrombocytopenic) mice because it is we1 1 established that both NK cells (46,47) and platelets (8,48) play prominent roles in metactacls. Also. i t has been rcported t h a t GRGDS can inhibit platelet aggregation (49). Second, we monitored the effect o f peptide on

221

222

K. Olden et a].

the kinetics of retention of radiolabeled tumor cells in the lungs of a syngeneic host. The number of platelets in circulating mouse blood was decreased by injection of a specific antiplatelet serum prior to the intravenous injection of B16-FlO cells with or without peptides ( 5 0 ) . We observed that depletion of platelet count by as much as 55% had no demonstrable effect on the antimetastatic activity of GRGDS; that is, the peptide was able to inhibit metastasis in both normal and platelet-depleted mice to approximately the same extent (2 77%). Similar results were obtained with mice depleted of NK cell activity by systemic administration of acetylsalicylic acid (50). While these studies strongly support the notion that the antimetastatic activity of GRGDS is not dependent on a platelet- or NK cell-mediated type mechanism, the results are not conclusive. This matter is presently being further explored in our laboratory using light and electron microscopy to visualize the melanin stained tumor cells from time of release into circulation until micrometastases are evident in the lung. Also, the extent of platelet aggregate formation induced by B16-FlO cells in vitro i s being examined in the presence and absence of graded concentrations of GRGDS. When the time course of pulmonary retention of [1251]-IUdR-labeled cells was examined, we observed that approximately 96% of the cells, administered in the absence of GRGDS, were recovered in the lung within 2 minutes after injection into the lateral tail vein; however, a significantly lower number of cells (approximately 71%) was recovered during this same period when the tumor cells were injected with GRGDS. Additionally, the rate of loss of cells from the lung of mice treated with GRGDS

was substantially higher than from control animals; for example, half of the cells were cleared from the lungs of the GRGDS-treated animals in 90 minutes, whereas it took 160 minutes for a comparable number of cells to be cleared from the lungs of control animals. The more rapid loss of radiolabeled cells from the lungs of the GRGDS-treated animals continued over the next few hours to such an extent as to account for the peptide-mediated inhibition of pulmonary colonization. These results strongly suggest that the primary effect of GRGDS is to inhibit arrest and promote clearance in the lung, consistent with the putative antiadhesive function of this peptide. Since GRGDS has a circulation half-life of approximately 8 minutes (50), it is unlikely that inhibition of later steps of invasion is the explanation for the antimetastatic effect that we have reported (46). To further test the hypothesis that GRGDS decreased the capacity of tumor cells to form strong adhesive interactions necessary for retention in the microvasculature of the lung, standard vitro assays were performed to measure the peptide inhibition of cell attachment and spreading on immobilized fibronectin (50). Consistent with the proposed mechanism of action, GRGDS markedly inhibited the adhesion of B16-Fl0 cells at a concentration of 500 ug/ml. Peptide-mediated inhibition of spreading was completely reversed by washing and resuspension of the cells in normal growth medium, indicating that the antiadhesive activity of GRGDS was not due to toxicity. Finally, the specificity o f GRGDS was tested in the lung colonization assay using a

K. Olden et al. series of closely related peptides or free amino acids. The most instructive peptides were the pentapeptides GRGES and GRDGS, in which aspartic acid was substituted by glutamic acid and the central Gly and Asp were transposed, respectively. Neither of these peptides caused significant inhibition of pulmonary colonization. In contrast, the tetrapeptide RGDS was almost as effective in blocking pulmonary colonization as the pentapeptide GRGDS, and the inverted peptide SDGR was about half as effective. Tripeptides, dipeptides and free amino acids had no effect on metastasis. Following comparisons of the inhibitory profile of a library of homologous peptides on both experimental metastasis in vivo and cellular adhesion in vitro, the antiadhesive activity of these peptides appears to correlate closely with antimetastatic activity (46,50). However, the particular adhesive events that are both crucial for metastatic colonization and sensitive to GRGDS inhibition await identification. It will be important, therefore, to define whether GRGDS affects tumor cell adhesion to the vessel wall, to endothelial basement membrane/ extracellular matrix, invasion through these structures, or even the directed migration of cells through underlying connective tissue. Also, it is becoming increasingly apparent that adhesion proteins contain multiple cell interaction sites. For fibronectin, four sites that interact directly with cells are now known. Once specific peptide probes are developed for each of these different sites, it will be instructive to compare their effects on adhesion, migration, and metastasis. Furthermore, although the relative activit y 69 GRGDS h 6 M l b J e i n 8 V p 8 V l M m t a l

metastasis assays most closely parallels their ability to block fibronectin-

223

mediated adhesim in vitro, as opposed to adhesion supported by other matrix molecules containing the RGDX signal (collagens, vitronectin, fibrinogen, von Willebrand factor, and thrombospondin), the role of fibronectin has not been demonstrated unequivocally. The development of peptide probes that possess absolute specificity for different adhesive ligands should also yield important information about their roles in metastasis. To test further the potential useful-

ness of GRGOS as an antimetastatic agent, studies were done to determine whether GRGDS-mediated inhibition of metastasis translated into prolongation of survival for the host (50). In studies employing intravenous injection of 30,000 B16-Fl0 melanoma cells with GRGDS (3 mg/mouse), no deaths among the C5781/6 host mice were recorded. In a typical experiment, mice that received tumor cells alone died (8/8) between 32 and 46 days after injection of tumor cells (mean survival time of 35.8 days); whereas, mice (0/8) that received both tumor cells and peptide survived over a period of 15 months. Replication of this experiment with a larger tumor burden (50,000cells) resulted in no significant difference in th'e mean survival time (28.9 f 1.1 versus 27.3 5 1.9) between control and peptide-treated animals. Thus, peptide therapy was completely effective in preventing metastasis formation provided the tumor burden was not too high. Therefore, if the circulation half-life of GRGDS could be improved, or if an appropriate continuous infusion protocol could be developed, then it is highly likely that GRGDS could be used to limit metastatic disease. However, since malignant cells are able t o use several adhesion proteins during t a r g e t organ colonization, it is conceivable that the anti-

I(. Olden et al.

224 metastic e f f e c t s o f anti-adhesive peptides

1 0 0 - f o l d l e s s than t h a t o f t h e 75KDa c e l l

s p e c i f i c f o r each o f these f a c t o r s might be

b i n d i n g fragment o r i n t a c t f i b r o n e c t i n ,

a d d i t i v e ; thus a d m i n i s t r a t i o n o f a c o c k t a i l

t h i s suggested t h a t a d d i t i o n a l i n f o r m a t i o n

o f such i n h i b i t o r s may prove t o be most

was r e q u i r e d f o r n a t i v e a c t i v i t y .

e f f i c a c i o u s i n preventing metastasis.

I t has

now been confirmed t h a t a t l e a s t one s y n e r g i s t i c s i t e e x i s t s i n t h e 75KDa

I t i s apparent from t h e above discussion

fragment t h a t i s e s s e n t i a l f o r f u l l c e l l

t h a t t h r e e s i g n i f i c a n t problems w i l l have t o

adhesion-promoting a c t i v i t y (51,52).

be resolved before the e f f e c t i v e n e s s o f GRGDS

fragment i n c l u d i n g both b i n d i n g s i t e s

as an adjuvant a n t i m e t a s t a t i c agent can be

might be more e f f e c t i v e as an antimeta-

improved.

F i r s t , i t w i l l be necessary t o

s t a t i c agent as the receptor a f f i n i t y

generate peptide probes t h a t e x h i b i t b i o l o g i -

might approximate t h a t o f n a t i v e

cal a c t i v i t y and receptor b i n d i n g a f f i n i t y

fibronectin.

comparable t o i n t a c t f i b r o n e c t i n .

A

Despite

the obvious importance o f the GRGDS sequence

I n summary, t h e s t u d i e s described

i n fibronectin-mediated adhesion, t h e drop i n

above are important t o e l u c i d a t e t h e

a c t i v i t y and a f f i n i t y t h a t takes place upon

mechanism(s) o f tumor c e l l adhesion t o

t r u n c a t i o n o f the 75KDa c e l l - b i n d i n g fragment

f i b r o n e c t i n and t h e r o l e o f these

t o the 11.5KDa fragment suggests t h a t

i n t e r a c t i o n s i n metastasis and invasion.

additional information i s required f o r

Since these i n t e r a c t i o n s appear t o be

optimal f u n c t i o n i n g o f t h i s domain.

e s s e n t i a l f o r metastasizing tumor c e l l s

Second,

i t may be important t o generate peptides t h a t

d u r i n g hematogenous dissemination, a

are l i k e l y t o e x h i b i t g r e a t e r o r absolute

r a t i o n a l b a s i s e x i s t s f o r the c u r r e n t

s p e c i f i c i t y f o r f i b r o n e c t i n.

A1 t e r n a t i v e l y ,

s t u d i e s t o develop such anti-adhesive

i t i s p o s s i b l e t h a t increased s p e c i f i c i t y

agents f o r use i n management o f human

might r e s u l t i n l o s s o f a n t i m e t a s t a t i c

malignancies.

a c t i v i t y since adhesion t o other basement

metastasis can be achieved, and t h i s

membrane/extracellular m a t r i x adhesion-

i n h i b i t i o n translated i n t o prolongation o f

promoting macromolecules would n o t be

s u r v i v a l , t h e u l t i m a t e t e s t o f any a n t i -

inhibited.

n e o p l a s t i c agent.

I n d i s c r i m i n a n t r e c o g n i t i o n by

Complete i n h i b i t i o n o f

receptors f o r other t i s s u e g l y c o p r o t e i n s c o n t a i n i n g RGD sequence may be an advantage. T h i r d , the c i r c u l a t i o n h a l f - l i f e o f the

INTERRUPTION OF METASTASIS BY ALTERATION

antiadhesive peptide GRGDS must be improved

OF SURFACE OLIGOSACCHARIDES

before i t can be evaluated i n the more c l i n i c a l l y r e l e v a n t spontaneous metastasis

Recently, several s t u d i e s have s t r o n g l y

experiments.

i m p l i c a t e d surface oligosaccharides i n c e l l - c e l l r e c o g n i t i o n and adhesion ( 5 3 ) .

To address these concerns, we have

F o r example, l e s s adhesive malignant c e l l s

i n i t i a t e d experiments t o i s o l a t e l a r g e r

f r e q u e n t l y possess more h i g h l y branched and

peptide fragments o f f i b r o n e c t i n contain-

s i a l y l a t e d N-linked oligosaccharides than

ing additional structural information

corresponding normal c e l l s .

crucial f o r f u l l biological activity.

t r e a t e d w i t h tunicamycin, an i n h i b i t o r o f

Since t h e a c t i v i t y and a f f i n i t y o f the RGD

p r o t e i n g l y c o s y l a t i o n , acquire a more

c o n t a i n i n g pentapeptide i s a p p r o x i a t e l y

spherical morphology and are l e s s adhesive t o

Also, c e l l s

I