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Int. J. Cancer: 77, 295–301 (1998)
r 1998 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
EXTRACELLULAR MATRIX MODULATES EXPRESSION OF GROWTH FACTORS
AND GROWTH-FACTOR RECEPTORS IN LIVER-COLONIZING COLON-CANCER
CELL LINES
Isabel ZVIBEL1,*, Zamir HALPERN1 and Moshe PAPA1,2
Metastasis Research Group, Gastroenterology Institute, Tel Aviv Medical Center, Tel Aviv, Israel
2Surgical Oncology Department, Shiba Medical Center, Tel Hashomer, Israel
1Liver
Site-specific metastasis is determined by the extracellular
matrix (ECM) of the colonized organ. We have shown that
hepatocyte-derived ECM stimulated proliferation of coloncancer cells via induction of autocrine growth factors and
their receptors. The ECM component responsible was heparin proteoglycan. We therefore investigated the effect of
exogenously added heparin on colon cell lines of varying
liver-colonizing potential. The cells were grown on typical
liver matrix components, such as fibronectin and collagens
type I and IV. We assessed the effect of these matrix
components on clonal growth, proliferation and expression of
autocrine growth factors and their receptors. The clonal
growth of the KM12 cells was not affected by heparin, while
the other cell lines were inhibited by heparin. Cell proliferation in weakly metastatic KM12, but not in strongly metastatic KM12SM, was inhibited by heparin on plastic. Weakly
metastatic LS174T, but not strongly metastatic LiM6, was
inhibited by heparin on fibronectin. Expression of erb-B2 was
also differently modulated by heparin in weakly metastatic vs.
highly metastatic cells. In weakly metastatic cells, heparin
reduced erb-B2 levels when cells were on plastic and fibronectin, while in strongly metastatic cells, erb-B2 was induced by
heparin. In all 4 cell lines, mRNA for cripto was induced by
heparin when the cells were grown on fibronectin. In KM12SM
cells, amphiregulin was induced by heparin in cells on fibronectin and collagen IV. We show that soluble heparin, similar in
its carbohydrate chemistry to liver heparin proteoglycan,
regulates the growth of colon-cancer cells. This effect depends on other matrix components found in the liver and is
mediated in part by EGF family members. Int. J. Cancer
77:295–301, 1998.
r 1998 Wiley-Liss, Inc.
Paget (1889), observing that different tumors metastasize preferentially to certain organs, suggested the ‘‘seed and soil’’ hypothesis,
to explain site-specific metastasis. He proposed that the newly
colonized organ must be a fertile soil for tumor cells. The local
environment of the colonized organ can dictate the establishment
and growth of tumor cells. The tumor cells need to recognize and
attach both to the local extracellular matrix (ECM) components and
to the cells of the target organ (Zvibel and Kraft, 1993). In addition,
each organ secretes paracrine growth stimulatory or inhibitory
factors that will affect the growth of the tumor cells (Radinsky,
1993).
In earlier studies with hepatomas and breast carcinomas, we
have shown that survival of cancer cells on organ-specific extracellular matrix (ECM) correlates with cell ability to metastasize to that
organ in vivo (Doerr et al., 1989). The ECM component responsible
for discriminating between strongly and weakly metastatic cells
was heparin, which was found to act via induction of autocrine
growth factors in the tumor cells (Zvibel et al., 1991).
We have also investigated the role of liver ECM on the growth of
colon-cancer cell lines of varying liver-colonizing potential (data
not shown). We found that hepatocyte-derived ECM selectively
enhances the clonal growth of strongly metastatic cells, but not of
weakly metastatic ones. The effect of liver ECM on cell proliferation however, was uniformly stimulatory of growth in all cell lines.
Hepatocyte ECM mediated the effect on cell proliferation by
increasing expression of 2 members of the EGF family, erb-B2 and
cripto, with no effect on the expression of EGF receptor or
amphiregulin. We performed various modifications of the ECM
produced by hepatocytes and showed that at least some of the ECM
effects were due to heparin proteoglycans (data not shown).
Our current studies investigated the role of exogenously added
heparin, in the context of various matrix components found in liver
ECM, such as fibronectin and collagens type I and IV, on the
growth of 4 human colon-cancer cell lines of varying livercolonizing capability. We found that the effect of heparin on
colon-cancer cells was dictated both by the metastatic potential of
the cell and by the type of matrix component on which the cells
were plated.
MATERIAL AND METHODS
Cell lines
The cells used in our studies were the weakly metastatic cell line
KM12 (obtained from Dr. M. Sela, Weizmann Institute, Rehovot,
Israel) and its liver-colonizing derivate, KM12SM (kindly provided by Dr. J. Fidler, M.D. Anderson Cancer Center, Houston,
TX). The other pair of weakly and strongly metastatic colon cells
were LS174T (from Dr. B. Schwartz, Soroka Medical Center,
Beer-Sheva, Israel) and its liver-colonizing derivate, LiM6 (kindly
provided by Dr. R. Bresalier, Henry Ford Hospital, Detroit, MI).
Cell culture
Cell lines were grown in serum-supplemented medium (SSM)
composed of DMEM supplemented with 10% FCS (Biological
Industries, Beit Haemek, Israel), 100 µg/ml penicillin and 100
µg/ml streptomycin. For the experimental studies, the cell lines
were plated overnight for 4 hr in SSM, then in a medium composed
of DMEM supplemented with penicillin/streptomycin 100 µg/ml,
0.1% BSA, 50 µg/ml ascorbic acid, 2 mM glutamine, 10 µg/ml
insulin, 10 µg/ml transferrin (iron-saturated), 0.2 ng/ml triiodotyronine and 15 mM HEPES. All the medium additives were obtained
from Sigma (St. Louis, MO).
The cells were plated on uncoated plates (TCP), fibronectincoated plates (1.7 µg/cm2 ), collagen-IV-coated plates (3.4 µg/cm2 )
and collagen-I-coated plates (16.6 mg/cm2 ) in the presence or
absence of 100 µg/ml porcine intestine heparin (Sigma). The
coating of the plates was done by incubation at 37°C for 30 min.
Growth curves
Colon cell lines were plated in 24-well plates, coated as
described above; the cells were trypsinized with 0.5% trypsin and 2
mM EDTA, and counted using a hemocytometer. Cells were plated
in duplicates, and each experiment was repeated at least 3 times.
Clonal growth
Colon cell lines were plated at a density of 200 cells per well in
6-well plates coated as described above. The cells were grown in
colon HDM, with or without 10 µg/ml porcine intestine heparin,
with medium changes every 4 days. After 2 weeks, the colonies
*Correspondence to: Liver Metastasis Research Group, Gastroenterology Institute, Tel Aviv Medical Center, Weizman 6, Tel Aviv, Israel. Fax:
972-3-697 4622. E-mail: [email protected]
Received 11 November 1997; Revised 15 January 1988
ZVIBEL ET AL.
296
were stained with 0.25% crystal violet in methanol and counted
under a binocular. Each cell line was plated in triplicates.
Northern-blot analysis
Plates were washed with cold PBS and total RNA was isolated
from the cells by the method of Chomczynski and Sacchi (1987).
RNA samples were resolved by electrophoresis through 1%
agarose, submerged-slab, de-naturing formaldehyde gels in MOPS
buffer, then transferred to Hybond-N paper (Sambrook et al.,
1989). RNA was fixed to the blots by UV cross-linking, then
hybridized with cDNA probes radioactively labeled with 32PdCTP
by primer extension (Feinberg and Vogelstein, 1984). Blots were
hybridized at 42°C for 48 hr, washed and subjected to autoradiography using X-ray film and enhancing screens. The cDNA probes
used were human cripto and human amphiregulin (ATCC, Rockville, MD).
Western blots
Cells were collected from the plates with 2 mM EDTA and total
protein was extracted by incubating the cells 30 min on ice in lysis
buffer (250 mM sucrose, 5 mM Mg2Cl, 10 mM Tris, pH 8.0, 0.5%
Triton X-100 and 1 mM PMSF); insoluble material was pelleted at
17,000 g. The extracts were normalized to total protein content,
determined using Bradford reagent (Sigma) and boiled for 5 min in
sample buffer containing SDS and b-mercaptoethanol; 5 to 10 µg
protein per lane was separated by SDS-PAGE (BioRad Protean II
Minigel, Hercules, CA) and blotted onto Hybond-C extra (Amersham Life Science, Arlington Heights, IL). The blots were blocked
overnight in 5% milk, incubated with antibodies to erb-B2 and
EGF receptor (Santa Cruz Biotechnologies, Santa Cruz, CA),
washed, and incubated for 1 hr with the appropriate horseradishperoxidase-conjugated secondary antibody (Jackson Laboratories,
Bar Harbor, ME). The blots were then subjected to chemiluminescent detection (ECL, Amersham Life Science) and fluorography
using X-ray film (NEN Life Science Products, Boston, MA).
RESULTS
Effect of heparin and matrix components on the clonal growth
of colon-cancer cell lines
The 4 cell lines were plated at a density of 200 cells/60 mm2 dish
on uncoated dishes or plates coated with fibronectin, collagen type
IV and collagen type I. The medium was changed to the hormonally defined medium (HDM) described in ‘‘Material and Methods’’
with or without 10 µg/ml heparin. The cells were grown for 2
weeks, after which the colonies formed were fixed and counted.
Representative results are shown in Figure 1. The clonal growth of
the weakly metastatic KM12 cell line was not affected by heparin,
with the exception of colonies growing on collagen I, where
heparin increased the number of colonies formed (Fig. 1). KM12
cells attached and spread well to plastic, but KM12SM, LS174T
and LiM6 grew in separate colonies, forming rounded, 3H dimensional clusters. When these cells were plated on matrix components, they tended to attach and spread. KM12SM formed significantly more colonies on fibronectin and collagen type IV than on
plastic. Both LS174T and LiM6 formed more colonies on collagens
type IV and type I than on plastic. Heparin strongly inhibited clonal
growth of the 3 cell lines, particularly on fibronectin and collagen
type IV (Fig. 1).
Effect of heparin and matrix components on the proliferation
of colon-cancer cell lines
Heparin inhibited the proliferation of KM12 mainly when the
cells were growing on plastic (TCP) and on collagen I (Fig. 2a).
The growth of the strongly metastatic cell line KM12SM was not
inhibited by heparin when the cells were on plastic, but there was
strong inhibition of cells cultured on fibronectin and collagen IV
(Fig. 2a).
The weakly metastatic LS174T was inhibited by heparin when
the cells were on plastic and fibronectin, but not on collagen IV and
FIGURE 1 – Clonal growth of colon-cancer cell lines on tissueculture plates (TCP), and plates coated with fibronectin (fibr), collagen
IV (coll IV) and collagen I (coll I). The cells were plated and changed
after a day to HDM with or without 100 µg/ml porcine intestine
heparin. The number of colonies formed was counted in triplicates after
2 weeks.
FIGURE 2 – Growth curves of colon-cancer cell lines plated on
tissue-culture plates (TCP), and plates coated with fibronectin (fibr),
collagen IV (col IV) and collagen I (col I). (a) KM12 and KM12SM;
(b) LS174T and LiM6 cells. The cells were plated and changed after 4
hr to HDM with or without 100 µg/ml porcine intestine heparin. Cells
(20,000) were plated and counted in duplicates after 1, 3 and 4 days.
The plots are means of 3 different experiments.
FIGURE 2
298
ZVIBEL ET AL.
collagen I (Fig. 2b). The proliferation of the strongly metastatic
LiM6 was decreased by heparin when the cells were plated on
collagen IV and collagen I, but not on fibronectin, thus showing
the opposite pattern of LS174T (Fig. 2b). Indeed, LiM6 appeared
to be stimulated by heparin when the cells grew on plastic and
fibronectin.
Effect of heparin and matrix components on the expression
of autocrine growth factors in colon-cancer cell lines
The 4 cell lines express various receptors and ligands of the
EGF-receptor family, such as the EGF receptor, erb-B2, cripto and
FIGURE 3 – Western blots showing EGF-receptor expression in the
colon-cancer cell lines. Total protein was extracted after 2 days of
culture on tissue-culture plates (TCP), and plates were coated with
fibronectin (fibr), collagen IV (col IV) and collagen I (col I). The cells
were plated and changed after 4 hr to HDM with or without 100 µg/ml
porcine intestine heparin. Total protein (5 µg) was loaded on SDS
PAGE gels, blotted to Hybond-C extra and incubated with anti-human
EGF receptor. Detection was performed by ECL.
amphiregulin (data not shown). We studied the expression of these
receptors and growth factors when the colon-cancer cells were
growing on various matrix components, in the presence or absence
of heparin. The expression of EGF receptor was not affected by
heparin in the various culture conditions in either the weakly
metastatic KM12 or the strongly metastatic KM12SM cells
(Fig. 3).
The levels of erb-B2 were affected differently in the weakly
metastatic KM12 and in the strongly metastatic KM12SM. Heparin
induced erb-B2 expression in KM12SM cells grown on plastic,
fibronectin and collagen I, while it reduced erb-B2 in KM12 cells
cultured on plastic (Fig. 4a).
In the weakly metastatic LS174T, heparin reduced erb-B2 in
cells on plastic and fibronectin, while in the strongly metastatic
LiM6 cells, heparin induced higher expression of erb-B2 when the
cells were grown on plastic and fibronectin (Fig. 4b). Erb-B2 was
reduced by heparin in LiM6 cells plated on collagen I.
Cripto, a member of the EGF family, is expressed by all 4 cell
lines, as 3 mRNA transcripts of 1.7 kb, 2.5 kb and 4.5 kb. Its mRNA
levels were reduced by heparin in KM12 cells grown on plastic,
collagen IV and collagen I, and increased in cells grown on
fibronectin (Fig. 5a). In the strongly metastatic KM12SM, cripto
mRNA was induced by heparin in cells cultured on plastic and
fibronectin, and reduced on cells grown on collagens type I and
type IV (Fig. 5a). In both LS174T and LiM6, heparin increases the
levels for cripto mRNA in cells plated on fibronectin (Fig. 5b).
Amphiregulin mRNA is expressed, albeit at in low abundance, in
all the 4 cell lines, as one mRNA species of 1.5 kb. Its highest
expression was found in KM12SM cells. Amphiregulin mRNA
expression was induced by heparin in KM12SM plated on fibronectin and on collagen IV (Fig. 6).
DISCUSSION
Our current studies have focused on the role of exogenously
added heparin on the growth of colon-cancer cell lines of low and
high liver-colonizing capabilities. We found that heparin affected
cell proliferation differently, depending on the matrix component
on which the cells grew, and on the metastatic potential of the cell.
FIGURE 4 – Western blots showing erb-B2 expression in the colon-cancer cell lines. (a) KM12 and KM12SM; (b) LS174T and LiM6. Total
protein was extracted after 2 days of culture on tissue-culture plates (TCP) and plates coated with fibronectin (fibr), collagen IV (col IV) and
collagen I (col I). The cells were plated and changed after 4 hr to HDM with or without 100 µg/ml porcine intestine heparin. Total protein (5 µg)
was loaded on SDS PAGE gels, blotted to Hybond-C extra and incubated with anti-human erb-B2 antibodies. Detection was performed by ECL.
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON-CANCER CELL LINES
299
A
B
FIGURE 5 – Cripto mRNA expression in Northern blots of total RNA from colon-cancer cells cultured on tissue-culture plates (TCP) and plates
coated with fibronectin (fibr), collagen IV (col IV) and collagen I (col I). The cells were plated and changed after 4 hr to HDM with or without 100
µg/ml porcine intestine heparin, and total RNA was extracted after 2 days. Total RNA (10 µg) was run in a formaldehyde gel, blotted onto
Hybond-N (Amersham) and hybridized with a human cDNA probe for cripto labeled with 32P-d CTP by oligolabeling using random primers. (a)
KM12 and KM12SM; (b) LS174T and LiM6. Ethidium-bromide staining shows amount of RNA in each lane.
The same differential effect of heparin was observed on the
modulation of erb-B2 and cripto expression in the 4 cell lines.
In order to complete the process of metastasis, tumor cells
require certain properties: the ability to detach from the primary
tumor, to invade the lymphatics or blood circulation and survive, to
extravasate and attach in the newly colonized organ, to attract
blood vessels by producing angiogenic factors, and to proliferate in
the target organ. The growth of tumor cells in the newly colonized
organ can be modulated either by the ECM of the target organ
(Doerr et al., 1989) or by soluble growth factors produced by the
cells of the organ (Radinsky, 1995a).
We found that tumor-cell propensity to metastasize in vivo to a
certain organ correlates with the ability of the cells to survive at
clonal densities on ECM derived from that organ (Doerr et al.,
1989). The ECM components responsible for this effect were
heparin proteoglycans. One of their mechanisms of action was via
induction of autocrine growth factors in the tumor cells (Zvibel et
al., 1991). The growth factors induced resulted in the differentiation of weakly metastatic cells (Zvibel et al., 1995).
The ECM of the normal liver is found in the space of Disse. The
ECM is secreted mainly by endothelial cells and hepatocytes and
consists primarily of collagens type I and type VI, fibronectin,
300
ZVIBEL ET AL.
FIGURE 6 – Amphiregulin mRNA expression in a Northern blot of
total RNA from KM12SM cells cultured on tissue-culture plates (TCP)
and plates coated with fibronectin (fibr), collagen IV (col IV) and
collagen I (col I). The cells were plated and changed after 4 hr to HDM
with or without 100 µg/ml porcine intestine heparin and total RNA was
extracted after 2 days. The blot was hybridized with a human cDNA
probe to amphiregulin. Ethidium-bromide staining shows amount of
RNA in each lane.
discrete aggregates of type-IV collagen and filaments of type-V and
type-III collagen (Martinez-Hernandez et al., 1993). One of the
unique components of liver ECM is the hepatocyte-specific heparin
proteoglycan (PG). This PG is anchored on the cell surface of
hepatocytes and belongs to the syndecan family. Its glycosaminoglycan chains are very similar in carbohydrate sequences and sulfation
to mast-cell-produced heparin (Lyon et al., 1994).
We have found that ECM deposited by primary cultures of rat
hepatocytes was stimulatory for the proliferation of colon-cancer
cell lines and preferentially enhanced the clonal growth of colon
cells with high liver-colonizing ability (data not shown).
The present studies investigated the effect of ECM components
known to be abundant in the liver on the growth of colon-cancer
cells. We used commercially available heparin to assess its effect
on the clonal growth and proliferation of colon-cancer cells plated
on fibronectin and collagens type I and IV.
Soluble heparin proved to be inhibitory both to clonal growth
and to cell proliferation of colon-cancer cells. However, this effect
was observed only when the cells were plated on certain matrices.
KM12, but not KM12SM, was inhibited by heparin when the cells
were plated on plastic, and LS174T, but not LiM6, was inhibited by
heparin when the cells grew on fibronectin.
We decided to investigate some of the mechanisms through
which heparin could regulate colon-cell proliferation. We focused
on expression of members of the EGF family, since growth factors
and their receptors belonging to this family play an important role
in the growth of epithelial tumors and cell lines, and show
increased expression in epithelial tumors (Kapitanovic et al., 1997;
Saeki et al., 1995). The EGF-receptor family consists of 4 known
receptors: EGF receptor, or erb-B1, erb-B2, erb-B3 and erb-B4.
erb-B2-amplified expression in breast tumors correlates with a bad
prognosis (Lonn et al., 1995). Although its role in colon cancer is
yet to be determined, erb-B2, absent in normal colon mucosa, is
expressed in a large number of colon cancers (Kapitanovic et al.,
1997; Saeki et al., 1995). Over-expression of erb-B2 results in
receptor autophosphorylation in the absence of a ligand (Lonardo
et al., 1990).
The members of the EGF-receptor family can form homodimers
or heterodimers with each other. The homo- or heterodimers can
bind and respond to different ligands, according to the receptors in
the complex. The known ligands are EGF, transforming growth
factor alpha (TGFa), heparin-binding EGF-like growth factor
(HB-EGF), betacellulin, amphiregulin (Beerli and Hynes, 1996).
Another member of the EGF family with an unknown receptor is
cripto. The neu differentiation factors (NDFs)/heregulins are the
ligands for erb-B3 and erb-B4.
In earlier studies (data not shown), the stimulatory effect of
hepatocyte ECM was accompanied by increased expression of
erb-B2. In LS174T and LiM6 cells, regulation of erb-B2 by heparin
correlated with the effect of heparin on cell proliferation. Heparin
inhibited LS174T and stimulated LiM6 when the cells were plated
on fibronectin, and in those conditions, erb-B2 was reduced in
LS174T and increased in LiM6 cells. We observed that heparin
reduced erb-B2 expression in weakly metastatic cells on plastic and
fibronectin, and increased erb-B2 expression in strongly metastatic
cells in the same conditions.
Heparin inhibited the growth of vascular smooth-muscle cells by
decreasing the number of EGF receptors (Reilly et al., 1987).
EGF-receptor levels were increased in metastatic colon cancers,
and cells selected on the basis of their increased expression of EGF
receptors showed increased ability to form liver metastases (Radinsky, 1995). However, we did not observe any effect of heparin on
expression of the EGF receptor in the colon cell lines.
The inhibitory effects of soluble heparin could be explained as a
direct effect or by its acting in concert with the autocrine growth
factors secreted by the colon-cancer cells. Heparin has been shown
to inhibit the growth of hepatocytes directly by becoming internalized to the nucleus (Fedarko and Conrad, 1986), and, when it acts
with certain growth factors, such as amphiregulin, heparin can have
either a stimulatory or an inhibitory effect on a certain cell.
Both cripto and amphiregulin, expressed in the 4 colon-cancer
cell lines we worked with, were shown to be autocrine growth
factors for colon-cancer cells. Reduction of their expression by
transfection of anti-sense oligonucleotides or by the use of
antibodies led to decreased cell proliferation and slower tumor
growth (Ciardello et al., 1994; Johnson et al., 1992). The different
cripto mRNA transcripts observed in our cells differ from those
reported by others, raising the possibility that these cells regulate
cripto mRNA in a manner different from that of other colon cell
lines.
Amphiregulin, HB-EGF and heregulins are heparin-binding
growth factors, and their effect on a given cell can be modulated by
the presence of heparin (Johnson et al., 1992). Moreover, neither
amphiregulin nor HB-EGF are able to signal through the EGF
receptor in the absence of cell-surface heparin proteoglycans
(Johnson and Wong, 1994).
In KM12SM cells, amphiregulin mRNA expression was induced
by heparin in cells cultured on fibronectin and on collagen IV,
suggesting an inhibitory role for amphiregulin in KM12SM cells.
We also found that KM12, the cell line expressing the least amount
of amphiregulin, was also the least sensitive to heparin inhibition.
We are currently investigating the effect of amphiregulin, with or
without heparin, on the colon-cancer cell lines.
Our studies show that soluble heparin, similar in its chemistry to
liver heparin proteoglycan, regulates growth of colon-cancer cells.
This effect depends on other matrix components found in the liver.
Some of the molecules responsible for this growth regulation are
members of the EGF family, including receptors and ligands.
ACKNOWLEDGEMENTS
We thank Miss M. Varon for excellent technical assistance.
These studies were supported by generous gifts from the Lewkowicz family and from Mr. S. Agami.
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON-CANCER CELL LINES
301
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