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Hepatocyte Extracellular Matrix Modulates Expression of Growth Factors and Growth Factor Receptors in Human Colon Cancer Cells
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
245, 123–131 (1998)
EX984229
Hepatocyte Extracellular Matrix Modulates Expression of Growth Factors and Growth Factor Receptors in Human Colon Cancer Cells Isabel Zvibel,*,1 Shlomo Brill,* Zamir Halpern,* and Moshe Papa*,† *Liver Metastasis Research Group, Gastroenterology Institute, Tel Aviv Sourasky Medical Center, Weizman 6, Tel Aviv, Israel; and †Surgical Oncology Department, Shiba Medical Center, Tel Hashomer, Israel
We investigated the role of hepatocyte extracellular matrix (ECM) on the growth of human colon cancer cell lines. We cultured four cell lines with different liver-colonizing potential on ECM derived from primary rat hepatocyte cultures. We investigated the effect of ECM on cell proliferation, clonal growth, and expression of growth factors and growth factor receptors. The highly metastatic cells showed better clonal growth and produced larger colonies on ECM. The proliferation of all colon cancer cell lines was enhanced on hepatocyte ECM, yet inhibited on fibroblast ECM. Screening of autocrine growth factors and receptors showed that the cells expressed growth factors and receptors of the EGF family: EGF receptor, erb-B2, amphiregulin, and cripto. The expression of cripto mRNA, but not of amphiregulin, was induced in KM12SM cells grown on ECM. All colon cancer cell lines grown on ECM showed increased expression of erb-B2. The effect of ECM on erb-B2 expression was mediated by the heparin chains of heparin proteoglycan. ECM from hepatocytes grown in the presence of nitrophenyl-b-D-xylopyrannoside or sodium chlorate, which prevent formation of heparin proteoglycan, as well as ECM treated with heparinase, had no effect on erb-B2 expression. Our studies suggest a role for liver ECM as a determinant of colon cancer metastasis. Liver ECM acts, in part, via induction of members of the EGF family of growth factors and their receptors. © 1998 Academic Press
INTRODUCTION
The phenomenon of site-specific metastasis has been described as early as 1889, when Paget suggested the “seed and soil” hypothesis [1], proposing that the newly colonized organ must be a fertile soil for tumor cells. The local milieu of the colonized organ can dictate the establishment and growth of tumor cells. The metastatic cells need to recognize and attach to local extra1 To whom reprint requests should be addressed. Fax: 972-3-6974622.
cellular matrix (ECM) components and cells of the organ [2, 3]. Similarly, each organ can secrete paracrine growth stimulatory or inhibitory factors that will act on the tumor cells [4, 5]. We have previously shown that the ability of hepatomas and breast carcinomas to metastasize to a specific organ in vivo directly correlated with the ability of the cells to survive at clonal density on ECM derived from the organ [6]. In our present studies, we examined the role of liver ECM in colon cancer site-specific metastasis to the liver. The ECM of the normal liver, found in the space of Disse and secreted by endothelial cells, Kupffer cells, and hepatocytes, consists mainly of collagens type I and type VI, fibronectin, discrete aggregates of type IV collagen and filaments of type V and type III collagen [7], as well as the hepatocytespecific heparin proteoglycan (PG). The cell surface hepatocyte PG belongs to the syndecan family [8] and its glycosaminoglycan (GAG) chains are very similar to mast cell-produced heparin [9]. In our previous studies, the ECM component that affected the clonal growth efficiency in hepatomas and breast cancer cell lines was found to be heparin [6]. We found that heparin inhibited clonal growth of nonmetastatic and poorly metastatic cells but did not affect the highly metastatic cells [6]. Our studies suggested that organ-specific PGs and GAGs regulate the expression of autocrine growth factors in metastatic cells [10]. The autocrine growth factors can trigger either proliferation or differentiation signals [11], depending on the differentiated state of the tumor cells and the context of ECM components present. Colon carcinoma cells have been shown to secrete several autocrine growth factors and, in particular, members of the EGF family, both receptors and ligands, were found to be important in colon cancer cells [12–18]. The number of EGF receptors as well as c-met were found to correlate with colon cancer metastasis to the liver [15]. Most human colon cancers and cell lines express the EGF receptor ligands TGF a, amphiregulin, and cripto [16]. Erb-B2, found to be amplified in breast tumors and to correlate with poor prognosis, was not found in normal colon tissue, but its expression
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0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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was significantly increased in adenomas and adenocarcinomas of the colon [17, 18]. Both cripto and amphiregulin were found to be autocrine growth factors for colon carcinoma cell lines, and decreasing their expression or mode of action by antisense RNA or blocking with antibodies reduced tumor cell growth in nude mice and cell proliferation in vitro [13, 14]. The aim of our present studies was to investigate the role of primary rat hepatocyte-derived ECM on the growth of human colon cell lines with liver-colonizing potential. We tested the effect of hepatocyte ECM on the clonal growth, proliferation, and expression of autocrine growth factors and their receptors in particular members of the EGF family in these colon cancer cell lines. MATERIALS AND METHODS Cell lines. The cells used in our studies were the low metastatic cell line KM12 (Dr. Michael Sela, Weizmann Institute, Rehovot, Israel) and its liver colonizing derivate, KM12SM (Dr. J. Fidler, M.D. Anderson Cancer Center, Houston, TX). The other pair of low and high metastatic colon cells were LS174T (Dr. Betty Schwartz, Soroka Medical Center, Beer-Sheva, Israel) and its liver colonizing derivate, LiM6 (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% fetal calf serum (Biological Industries, Beit Haemek, Israel), 100 mg/ml penicillin and 100 mg/ml streptomycin. For the experimental studies, the cell lines were plated in SSM and then changed after 4 h to a medium described by Block [19], with slight modifications. The medium used was DMEM supplemented with 100 mg/ml penicillin/streptomycin, 2 mg/ml bovine serum albumin, 610 mg/ml nicotinamide, 740 ng/ml ZnSO4:7H2O, 20 ng/ml CuSO4:5H2O, 5 mM glutamine, 5 mg/ml insulin, 5 mg/ml iron-saturated transferrin, 5 ng/ml selenous acid, and 1027 M dexamethasone. This medium was termed hormonally defined medium (HDM). All the medium additives were obtained from Sigma (St. Louis, MO). Growth curves. Colon cell lines were plated in 24-well plates containing hepatocyte-derived ECM. After 4 h, the medium was changed to the HDM described above. The cells were trypsinized with 0.5% trypsin and 2 mM EDTA at various time points and counted using a hemocytometer. Cells were plated in duplicate and each experiment was repeated at least three times. Clonal growth. Colon cell lines were plated at a density of 200 cells per well in 6-well plates on TCP or hepatocyte-derived ECM. The cells were grown in HDM, with medium changes every 4 days. After 2 weeks, the colonies were stained with 0.25% crystal violet in methanol and counted under a binocular. Each cell line was plated in triplicate. Primary cultures of rat hepatocytes. Primary cultures of rat hepatocytes were established by perfusing the liver of adult Wistar rats using the Berry and Friend method [20]. The cells were plated on collagen I-coated plates and in the HDM of Block [19] and described above. Hepatocytes were plated at a density of 6 3 106 cells per 10-mm2 dishes, 2 3 106 cells per well in 6-well plates, and 2 3 105 cells per well in 24-well plates. In order to modify the heparin chains produced, hepatocytes were grown in the presence of 1 mM nitrophenyl b-D-xylopyrannoside or 30 mM sodium chlorate, with medium changes every 2 days. After 5 days, hepatocyte plates were treated for 10 min at room temperature with 0.5% Triton X-100 and then washed with PBS. Removal of heparin chains from proteoglycans
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was done by treating ECM plates with 0.05 U/ml heparinase for 22 h at 37°C. Primary cultures of fetal rat fibroblasts. Rat embryos from Fisher rats gestation day 13 were cut with a scalpel and the cells digested with 0.5% trypsin solution in PBS. After 20 min of digestion at 37°C, the cells were passed through a mesh washed and plated at a density of 3 3 105 in 24-well plates coated with collagen I. After 4 days, monolayers were treated with 0.5% Triton X-100 for 10 min at room temperature and then washed with PBS. Northern blot analysis. Plates were washed with cold phosphatebuffered saline (PBS) and total RNA was isolated by the method of Chomczynski and Sacchi [21]. RNA samples were resolved by electrophoresis through 1% agarose and denaturing formaldehyde gels in MOPS buffer and then transferred to Hybond N paper. RNA were fixed to the blots by UV crosslinking and then hybridized with cDNA probes radioactively labeled with [32P]dCTP by primer extension [22]. The cDNA probes used were: IGF I, IGF II (Dr. G. Bell), TGF b-1, amphiregulin, cripto, aFGF, TGF a, and IGF I receptor (American Tissue Culture Collection, ATCC), HGF (Dr. T. Nakamura), and c-met (Dr. Wande Woude). All probes were human, with the exception of c-met and TGFa (mouse) and HGF (rat). Western blots. Total protein was extracted by incubating the cells 30 min on ice in lysis buffer (250 mM sucrose, 5 mM MgCl2, 10 mM Tris, pH 8.0, 0.5% Triton X-100, and 1 mM PMSF). The extracts were normalized to total protein content, determined using Bradford Reagent (Sigma, St. Louis, MO). Protein (10 –20 mg per lane) were separated by SDS–PAGE (Bio-Rad Protean II minigel) and blotted onto Hybond C extra (Amersham, Arlington Heights, IL). The blots were blocked overnight in 5% milk. Blots were then incubated with antibodies to erb-B2, EGF receptor, IGF II, and TGFa (Santa Cruz Biotechnologies, Santa Cruz, CA), washed, and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, ME). The blots were then subjected to chemiluminescent detection and fluorography using X-ray film (New England Nuclear, Boston, MA).
RESULTS
The Effect of Hepatocyte ECM on the Clonal Growth of Colon Cancer Cell Lines We assessed the effect of hepatocyte-derived extracellular matrix on the clonal growth of four human colon cancer cell lines with low and high liver-colonizing potential. Primary rat liver cultures were established as described under Materials and Methods, the cells were removed after 4 days with Triton X-100 treatment, and 200 colon cells were plated on the ECMcovered 60-mm2 dish. After 2 weeks, the colonies formed were stained and counted and the results are shown in Fig. 1. Both low-colonizing liver cell lines KM12 and LS174T formed the same number of colonies on tissue culture plastic (TCP) and on hepatocyte-derived ECM. The colon cell lines with high liver metastasizing potential, KM12SM and LiM6, formed more colonies on hepatocyte-derived ECM than on plastic (Fig. 1). Moreover, in the case of LiM6, the colonies growing on hepatocyte ECM were larger than those on plastic, as shown in Fig. 2.
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON CELLS
FIG. 1. Clonal growth of colon cancer cell lines on plastic (TCP) versus hepatocyte ECM. Two hundred cells from each cell line were plated per 60-mm2 dish, uncoated or coated with ECM deposited by primary cultures of rat hepatocytes. The cells were cultured in HDM as described under Materials and Methods. The number of colonies formed was counted in triplicates after 2 weeks of culture.
The Effect of ECM on the Proliferation of Colon Cancer Cell Lines We next investigated the effect of hepatocyte ECM on the proliferation of the four colon cancer cell lines. As shown in Fig. 3, hepatocyte ECM stimulated proliferation of all four cell lines compared to cell proliferation on plastic. The experiments were repeated six times and the ECM effect on cell proliferation was statistically significant (P , 0.005). In order to test whether the ECM component responsible for the stimulatory effect was heparin proteoglycan, we modified the ECM produced by hepatocytes by growing them for 4 days in the presence of 1 mM nitrophenyl-b-D-xylopyrannoside, which prevents the addition of heparin chains to proteoglycans [23], or 30 mM sodium chlorate, which at this dose completely prevents the sulfation of GAGs [24]. Another treatment used to remove heparin chains from heparin proteoglycans in the ECM was to incubate the hepatocyte ECM with 0.05 U/ml
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heparinase for 2 h at 37°C prior to seeding the colon cancer cell lines. Hepatocyte ECM, after nitrophenyl-b-D-xylopyrannoside, but not after heparinase treatment, reduced the stimulatory ECM effect on the proliferation of all four cell lines (Fig. 3). Chlorate-treated ECM also reduced the proliferation of all the cell lines, with the exception of KM12 (Fig. 3). Reduced cell proliferation on the modified ECM was not statistically significant. However, we observed in all six experiments the same trend, namely, that cell proliferation on nitrophenyl-b-D-xylopyrannoside or chlorate-treated ECM was higher than cell proliferation on TCP. This result suggests that highly sulfated heparin proteoglycan chains are only part of the stimulatory component in hepatocyte ECM. In order to show that the effect of hepatocyte ECM was specific, we also grew the colon cell lines on ECM from primary cultures of fetal rat fibroblasts. The results are presented in Fig. 4. Fibroblast ECM had an inhibitory effect on the proliferation of all four colon cell lines. The inhibitory effect was less marked for LiM6 cells (Fig. 4). The Effect of Hepatocyte-Derived ECM on the Expression of Growth Factors and their Receptors in Colon Cancer Cell Lines One mechanism though which hepatocyte ECM could affect growth is by modulating the expression of autocrine growth factors and their receptors in the colon cancer cell lines. In order to test this hypothesis, we first screened the autocrine growth factors and receptors expressed by the colon cancer cell lines. The screening was done using molecular probes or specific antibodies and results are summarized in Table 1 and Table 2. The growth factors detected belong mainly to the EGF and EGF receptor family: all four cell lines expressed EGF receptor and erb-B2, as well as amphiregulin, an EGF receptor ligand. Cripto, another member of the EGF family, was present in all four cell lines.
FIG. 2. Photomicrograph of a representative colony of LiM6 on plastic (A) and on hepatocyte ECM (B). The bar represents 140 mm. The magnification in A and B is the same.
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FIG. 3. Growth curves of the four colon cancer cell lines cultured on tissue culture plastic (TCP), hepatocyte-deposited ECM (H.matrix), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xylose), ECM deposited by hepatocytes treated with 30 mM chlorate (chlorate), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (heparinase). Twenty thousand cells were plated on day 0, changed to HDM after 4 h, and counted in duplicates after 1, 3, and 4 days. The plots are means of three different experiments.
The other growth factor weakly detected in the cells was aFGF. We next investigated the effect of hepatocyte ECM on the expression of EGF receptor and erb-B2 in the colon cancer cells, as detected in Western blots with receptor-specific antibodies. The expression of EGF receptor was not modified in cells grown on hepatocyte ECM (data not shown). However, the expression of erb-B2 was greatly increased in all four cell lines cultured on hepatocyte ECM versus plastic (Fig. 5). The hepatocyte ECM component responsible for this induction of erb-B2 is heparin proteoglycan, since this induction was strongly reduced when the cells were grown on ECM from nitrophenyl-b-D-xylopyrannoside, chlorate, or heparinase-treated hepatocytes (Fig. 5). The effect of hepatocyte ECM on the level of mRNA for cripto and amphiregulin was assessed using molecular probes. Both cripto and amphiregulin were expressed by all four cell lines, but amphiregulin showed very low expression in KM12 cells. Three different cripto mRNA species, of 1.7 (the main transcript), 2.5,
and 4.5 Kb, were present in the four cell lines. Amphiregulin mRNA was present as a single mRNA transcript of 1.4 Kb. In KM12, LS174T, and LiM6 cells, cripto mRNA expression was not affected when the cells were grown on hepatocyte ECM (Figs. 6A and 6B). In KM12SM cells, hepatocyte ECM increased the expression of cripto, and this effect was slightly reduced on ECM of nitrophenyl-b-D-xylopyrannoside-treated hepatocytes and less so on chlorate- and heparinasetreated ECM (Fig. 6A), implying that heparin proteoglycans could be partially mediating this effect. Amphiregulin mRNA expression was not affected by hepatocyte ECM in KM12SM, the cell line that expressed the highest abundance of this growth factor mRNA (Fig. 7), or in the other cells lines (data not shown). DISCUSSION
The aim of our studies was to investigate some of the parameters involved in determining site-specific me-
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EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON CELLS
FIG. 4. Growth curves of colon cancer cell lines on plastic (TCP) versus ECM deposited by fetal rat fibroblasts (fibr.ECM). Twenty thousand cells were plated on day 0, changed to HDM after 4 h, and counted in duplicates on day 1, 3, and 4. The plots are means of three different experiments.
tastasis. Our model comprised four colon cancer cell lines of high and low liver-colonizing potential and we assessed the role of hepatocyte-derived ECM in the growth of these cell lines. We found that hepatocyte ECM selectively enhances the clonal growth of the highly metastatic cells, but not of the low metastatic ones. The effect of liver ECM on the cell proliferation was, however, uniformly stimulatory of growth in all cell lines. We showed that hepatocyte ECM also in-
creased the expression of two members of the EGF family, erb-B2 and cripto, but did not affect the expression of EGF receptor or amphiregulin. By modifying the ECM produced by hepatocytes, we showed that some of its effects were due to heparin proteoglycans. Site-specific metastasis is determined by properties of both tumor cells and host: the ability of the tumor cells to detach from the primary tumor, invade the lymphatics or blood circulation, and survive, extrava-
TABLE 1 Expression of Autocrine Growth Factors in Human Colon Cell Lines Cell line
Met.Pot
IGF I
IGF II
TGFa
TGFb
cripto
amphir.
aFGF
HGF
KM12 KM12S LS174T LiM6
low high low high
– – – ND
– – – –
– – – ND
V V ND ND
V V V V
V V V V
V V V ND
– – ND ND
Note. All of the above growth factors were detected using molecular probes. Expression of IGF II and TGF a was also assessed using specific antibodies. V: denotes that the growth factor was expressed. –: denotes that the growth factor was not detected.
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TABLE 2 Expression of Growth Factor Receptors in Human Colon Cell Lines Cell line
Met.Pot
IGF I R
EGF R
erb-b2
c-met
KM12 K12SM LS174T LiM6
low high low high
– – – ND
V V V V
V V V V
– – – ND
Note. The expression of IGF I receptor and c-met was assessed using molecular probes, and the expression of EGF receptor and erb B2 was assessed using specific antibodies. V: denotes that the receptor was detected. –: denotes that the receptor was not detected.
sate, and attach in the newly colonized organ, attract blood vessels by producing angiogenic factors and proliferating in the target organ [2, 3]. The target organ can enhance or inhibit the growth of the tumor cells either by soluble growth factors produced by the cells of the organ or embedded in the ECM [4, 5], or the target organ ECM can affect the proliferation of the tumor cells [2]. We have previously shown that tumor cells’ ability to metastasize in vivo correlates with the ability of the cells to survive at clonal densities on ECM derived from that organ [6]. The ECM components responsible for this effect were heparin proteoglycans, which acted via induction of autocrine growth factors in the tumor cells [10]. In the present studies, we tested whether liver ECM could be a determinant in colon cancer propensity to metastasize to this organ. We established a model in which we grew colon cancer cell lines of varying livercolonizing ability on ECM produced by cultures of primary rat hepatocytes. As in previous studies with hepatomas and breast cancers, the effect of hepatocyte ECM was different on high and low metastatic cells when they were plated at clonal densities, but was stimulatory for all cell lines, reardless of their metastatic potential, when the cells were plated at high densities. This effect of hepatocyte ECM on cell proliferation was quite specific, since ECM derived from fibroblasts inhibited the proliferation of the colon cancer cell lines. The ECM deposited by fibroblasts is very different than that secreted by epithelial cells, consisting of collagen type I, fibronectin, and chondroictin or dermatan sulfate proteoglycans. The stimulatory effect of hepatocyte ECM on colon cancer cells was accompanied by an induction in the expression of a member of the EGF family, namely erb-B2. In KM12SM highly metastatic cells, hepatocyte ECM also increased cripto mRNA expression. The EGF family, both ligands and receptors, play a major role in the growth of epithelial tumors, and their expression was increased in these tumors [17, 18]. The
EGF receptor family comprises four known receptors: EGF receptor, or erb-B1, erb-B2, erb-B3, and erb-B4. Of those, EGF receptors levels were found to be increased in metastatic colon cancers, while amplified expression of erb-B2 in breast tumors correlates with a bad prognosis. The members of this family can form either homo- or heterodimers with each other and can bind and respond to different ligands, according to the receptors in the complex. The known growth factors of the EGF family are EGF, transforming growth factor a (TGF a), heparin-binding EGF-like growth factor (HBEGF), betacellulin, amphiregulin, and cripto [25]. The heregulins are the ligands for erb-B3 and erb-B4 [25]. Amphiregulin, HB-EGF, and heregulins are heparinbinding growth factors, and their effect on a given cell can be modified by the presence of heparin [26 –28]. Both amphiregulin and HB-EGF require cell surface heparin proteoglycans for signaling through the EGF receptor [27, 28]. Both cripto and amphiregulin, expressed in the cell lines that we worked with, were shown to be autocrine growth factors for colon cancer cells, and reduction of their expression resulted in decreased cell proliferation and decreased tumor growth [13, 14]. Erb-B2 is not expressed in normal colon mucosa; yet it is expressed in a large number of colon cancers [17, 18]. Its role in colon cancer is yet to be determined; however, it was shown that when overexpressed, this receptor undergoes autophosphorylation in the absence of a ligand [29]. Erb-B2, but not EGF receptor
FIG. 5. Western blots showing expression of erb-B2 in the four colon cell lines. Total protein was extracted after 4 days from cells cultured on tissue culture plastic (TCP), hepatocyte-deposited ECM (ECM), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xyl), ECM deposited by hepatocytes treated with 30 mM chlorate (chlor), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (hep). Five micrograms of total protein was loaded on SDS–PAGE gels, blotted on Hybond C extra (Amersham), and incubated with anti human erb-B2 antibodies (Santa Cruz, CA), followed by the appropriate second antibody and detected by ECL.
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FIG. 6. Cripto mRNA expression in Northern blots of total RNA extracted after 4 days from the colon cancer cell lines grown on tissue culture plastic (TCP), hepatocyte-deposited ECM (ECM), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xyl), ECM deposited by hepatocytes treated with 30 mM chlorate (chlor), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (hep). Ten micrograms of total RNA were 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. The ethidium bromide staining shows the amount of RNA in each lane.
and amphiregulin expression, were increased by hepatocyte ECM. This effect was observed in all four cell lines. In addition, cripto mRNA was increased in KM12SM cells. Increased expression of a growth factor does not always result in enhanced proliferation. The possibility exists that the state of differentiation of a cell determines its response to specific growth factors. In the normal intestine, TGF a or TGF b have different effects as well as different distributions in the stem cells at the base of the cript or in the more differentiated cells at the tip of the villus [30, 31]. As they differentiate, both goblet cells and enterocytes lose their ability to respond to TGF a with a proliferative response [12].
FIG. 7. Amphiregulin mRNA expression in a Northern blot of total RNA extracted after 4 days from KM12SM cells grown on tissue culture plastic (TCP) and hepatocyte-deposited ECM (ECM). The blot was hybridized with a human cDNA probe to amphiregulin. The ethidium bromide staining shows the amount of RNA in each lane.
In order to identify the active ECM component we performed various modifications of heparin proteoglycans composition or chemistry in the hepatocyte ECM. Nitrophenyl-b-D-xylopyranoside is an acceptor for nascent heparin chains and therefore prevents the formation of cell surface heparin proteoglycans [23]. However, it was shown that in various cells, this substance can also decrease the amount of fibronectin in the ECM deposited [32]. Treatment of the hepatocytes with this chemical resulted in an ECM that lost its ability to stimulate cell proliferation or induce erb-B2, but did not affect cripto induction. Heparinase treatment, which degrades heparin chains, had a weaker effect than nitrophenyl-b-D-xylopyranoside treatment on both erb-B2 induction and cell proliferation. This could be due to limited accessibility of the chains to the enzyme. Sodium chlorate treatment of hepatocytes, an inhibitor of the sulfation of GAGs [24], also resulted in slightly reduced cell proliferation and erb-B2 expression, indicating that sulfated heparin chains are necessary for both of these effects. Hepatocyte ECM also increased the mRNA levels for cripto, but only in KM12SM cells. On ECM from hepatocytes treated with nitrophenyl-b-D-xylopyranoside, cripto mRNA was reduced, suggesting that heparin proteoglycan in the hepatocyte ECM also regulates the expression of this growth factor. The exact mechanism by which heparin affects cell growth is yet to be determined. Highly sulfated species of heparin were produced by hepatocytes and inhibited their growth by being internalized to the nucleus [33]. Heparin can modify the number and affinity of cell surface receptors [34] or modify the effect of a given growth factor on specific cells [27, 28, 35]. Many of the
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heparin binding growth factors absolutely require heparin for signal transduction [27, 28, 35]. Inside the nucleus, heparin can bind transcription factors, such as jun and fos [36], therefore affecting gene transcription. Our studies show an effect of hepatocyte ECM on the growth of colon cancer cell lines and imply that some of these effects are mediated via members of the EGF family, namely erb-B2 and cripto. Further studies are required to investigate the role of erb-B2, cripto, and amphiregulin, in conjunction with hepatocyte ECM, for each cell line.
14.
15. 16.
17. We thank Meirav Varon for excellent technical assistance. These studies were supported by generous gifts from the Lewkowicz family and from Shlomo Agami.
REFERENCES 1. 2.
3.
4.
5.
6.
7.
8.
9. 10.
11.
12.
13.
Paget, S. (1889) Lancet 1, 571. Zvibel, I., and Kraft, A. (1993). Extracellular matrix and metastasis. In “Extracellular Matrix: Its Chemistry, Biology, and Pathobiology” (M. A. Zern, and L. M. Reid, Eds.), vol. 22, Dekker, New York. Nicolson, G. L. (1989). Metastatic tumor cell interactions with endothelium, basement membrane and tissue. Curr. Opin. Cell Biol. 1, 1009 –1019. Radinsky, R. (1993). Paracrine growth regulation of human colon carcinoma organ-specific metastasis. Cancer Metastasis Rev. 12, 345–361. Nicolson, G. L., and Dulski, K. M. (1986). Organ specificity of metastatic tumor colonization is related to organ-selective growth properties of malignant cells. Int. J. Cancer 38, 289 – 294. Doerr, R., Zvibel, I., Chiuten, D., D’Olimpio, J., and Reid, L. M. (1989). Clonal growth of tumors on tissue-specific biomatrices and correlation with organ-site specificity of metastases. Cancer Res. 49, 384 –392. Martinez-Hernandez, A., Amenta, P. S. (1993). Morphology, localization, and cellular origin of hepatic extracellular matrix. In “Extracellular Matrix: Its Chemistry, Biology, and Pathobiology” (M. A. Zern, and L. M. Reid, Eds.), vol. 22, Dekker, New York. Pierce, A., Lyon, M., Hampson, I. N., Cowling, G. J., and Gallagher, J. T. (1992). Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver. J. Biol. Chem. 267, 3894 –3900. Lyon, M., Deakin, J. A., and Gallagher, J. T. (1994). Liver heparan sulfate structure. J. Biol. Chem. 269, 11208 –11215. Zvibel, I., Halay, E., and Reid, L. M. (1991). Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: Relevance to clonal growth of tumors. Mol. Cell. Biol. 11, 108 –116. Zvibel, I., Brill, S., and Reid, L. M. (1995). Insulin-like growth factor II regulation of gene expression in rat and human hepatomas. J. Cell. Physiol. 162, 36 – 43. Huang, F., Sauma, S., Yan, Z., and Friedman, E. (1995). Colon absorptive cells lose proliferative response to TGF a as they differentiate. Exp. Cell Res. 219, 8 –14. Ciardello, F., Tortora, G., Bianco, C., Selvam, M. P., Basolo, F., Fontanini, G., Pacifico, F., Normanno, N., Brandt, R., Persico, G. M., Salomon, D. S., and Bianco, A. R. (1994). Inhibition of
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Cripto expression and tumorigenicity in human colon cancer cells by antisense RNA and oligodeoxynucleotides. Oncogene 9, 291–298. Johnson, G. R., Saeki, T., Gordon, A. W., Shoyab, M., Salomon, D. S., and Stromberg, K. (1992). Autocrine action of amphiregulin in a colon carcinoma cell line and immunochemical localization of amphiregulin in human colon. J. Cell Biol. 118, 741–751. Radinsky, R. (1995). Molecular mechanisms for organ-specific colon carcinoma metastasis. Eur. J. Cancer 31A, 1091–1095. Ciardello, F., Kim, N., Saeki, T., Dono, R., Persico, M. G., Plowman, G. D., Garrigues, J., Radke, S., Todaro, G. J., and Salomon, D. S. (1991). Differential expression of epidermal growth factor-related proteins in human colorectal tumors. Proc. Natl. Acad. Sci. USA 88, 7792–7796. Kapitanovic, S., Radosevic, S., Kapitanovic, M., Andelinovic, S., Ferencic, Z., Tavassoli, M., Primorac, D., Sonicki, Z., Spaventi, S., Pavelic, K., and Spaventi, R. (1997). The expression of p185 (HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterology 112, 1103–1113. Saeki, T., Salomon, D. S., Johnson, G. R., Gullick, W. J., Mandai, K., Yamagami, K., Moriwaki, S., Tanada, M., Takashima, S., and Tahara, E. (1995). Association of epidermal growth factor-related peptides and type I receptor tyrosine kinase receptors with prognosis of human colorectal carcinomas. Jpn. J. Clin. Oncol. 25, 240 –249. Block, G. D., Locke, J., Bowen, W. C., Petersen, B. E., Katyal, S., Strom, S. C., Riley, T., Howard, T. A., and Michalopoulos, G. K. (1996). Population expansion, clonal growth and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF a in a chemically defined (HGM) medium. J. Cell Biol. 132, 1133–1149. Berry, M. N., and Friend, D. J. (1969). High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structure study. J. Cell Biol. 43, 506 –520. Chomcznyski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 56 –159. Feinberg, A. P., and Vogelstein, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266 –267. Kanwa, Y. S., Rosenzweig, L. J., and Jakubovski, M. L. (1986). Xylosylated proteoglycan-induced Golgi alterations. Proc. Natl. Acad. Sci. USA 83, 6499 – 6503. Bame, K. J., Reddy, R. V., and Esko, J. D. (1991). Coupling of N-deacetylation and N-sulfation in a chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Cell Biol. 266, 12461–12468. Beerli, R. R., and Hynes, N. E. (1996). Epidermal growth factorrelated peptides activate distinct subsets of erbB receptors and differ in their biological activities. J. Biol. Chem. 271, 6071– 6076. Johnson, G. R., and Wong, L. (1994). Heparan sulfate is essential to amphiregulin-induced mitogenic signaling by the epidermal growth factor receptor. J. Biol. Chem. 269, 27149 –27154. Aviezer, D., and Yayon, A. (1994). Heparin-dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF. Proc. Natl. Acad. Sci. USA 91, 12173–12177. Schuger, L., Johnson, G. R., Gilbride, K., Plowman, G. D., and Mandel, R. (1996). Amphiregulin in lung branching morphogenesis: Interaction with heparan sulfate proteoglycan modulates cell proliferation. Development 122, 1759 –1767. Lonardo, F., Di Marco, E., King, C. R., Pierce, J. H., Segatto, O., Aaronson, S. A., and Di Fiore, P. P. (1990). The normal erb-b2
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON CELLS product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol. 2, 992–1003. 30. Thomas, D. M., Nasim, M. M., Gullick, W. J., and Alison, M. R. (1992). Immunoreactivity of transforming growth factor alpha in the normal adult gastrointestinal tract. Gut 33, 628 – 631. 31. Barnard, J. A., Beauchamp, R. D., Coffey, R. J., and Moses, H. L. (1989). Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc. Natl. Acad. Sci. USA 86, 1578 –1582. 32. Canfield, A. E., Sutton, A. B., Hiscock, D. R., Gallagher, J. T., and Schor, A. M. (1994). Alpha and beta-xylosides modulate the synthesis of fibronectin and thrombospondin 1 by endothelial cells. Biochem. Biophys. Acta 1200, 249 –258. Received June 8, 1998 Revised version received July 30, 1998
33.
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Fedarko, N. S., and Conrad, H. E. (1986). A unique heparan sulfate in the nuclei of hepatocytes: Structural changes with the growth state of the cells. J. Cell Biol. 102, 587–599. 34. Reilly, C. F., Fritze, M. S., and Rosenberg, R. D. (1987). Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J. Cell. Physiol. 121, 149–157. 35. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841– 848. 36. Busch, S. J., Jackson, R. L., Sassone-Corsi, P. (1990). Heparin inhibits transcriptional activation by Fos and Jun oncoproteins. Trans. Assoc. Am. Physicians 103, 21–27.
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EX984229
Hepatocyte Extracellular Matrix Modulates Expression of Growth Factors and Growth Factor Receptors in Human Colon Cancer Cells Isabel Zvibel,*,1 Shlomo Brill,* Zamir Halpern,* and Moshe Papa*,† *Liver Metastasis Research Group, Gastroenterology Institute, Tel Aviv Sourasky Medical Center, Weizman 6, Tel Aviv, Israel; and †Surgical Oncology Department, Shiba Medical Center, Tel Hashomer, Israel
We investigated the role of hepatocyte extracellular matrix (ECM) on the growth of human colon cancer cell lines. We cultured four cell lines with different liver-colonizing potential on ECM derived from primary rat hepatocyte cultures. We investigated the effect of ECM on cell proliferation, clonal growth, and expression of growth factors and growth factor receptors. The highly metastatic cells showed better clonal growth and produced larger colonies on ECM. The proliferation of all colon cancer cell lines was enhanced on hepatocyte ECM, yet inhibited on fibroblast ECM. Screening of autocrine growth factors and receptors showed that the cells expressed growth factors and receptors of the EGF family: EGF receptor, erb-B2, amphiregulin, and cripto. The expression of cripto mRNA, but not of amphiregulin, was induced in KM12SM cells grown on ECM. All colon cancer cell lines grown on ECM showed increased expression of erb-B2. The effect of ECM on erb-B2 expression was mediated by the heparin chains of heparin proteoglycan. ECM from hepatocytes grown in the presence of nitrophenyl-b-D-xylopyrannoside or sodium chlorate, which prevent formation of heparin proteoglycan, as well as ECM treated with heparinase, had no effect on erb-B2 expression. Our studies suggest a role for liver ECM as a determinant of colon cancer metastasis. Liver ECM acts, in part, via induction of members of the EGF family of growth factors and their receptors. © 1998 Academic Press
INTRODUCTION
The phenomenon of site-specific metastasis has been described as early as 1889, when Paget suggested the “seed and soil” hypothesis [1], proposing that the newly colonized organ must be a fertile soil for tumor cells. The local milieu of the colonized organ can dictate the establishment and growth of tumor cells. The metastatic cells need to recognize and attach to local extra1 To whom reprint requests should be addressed. Fax: 972-3-6974622.
cellular matrix (ECM) components and cells of the organ [2, 3]. Similarly, each organ can secrete paracrine growth stimulatory or inhibitory factors that will act on the tumor cells [4, 5]. We have previously shown that the ability of hepatomas and breast carcinomas to metastasize to a specific organ in vivo directly correlated with the ability of the cells to survive at clonal density on ECM derived from the organ [6]. In our present studies, we examined the role of liver ECM in colon cancer site-specific metastasis to the liver. The ECM of the normal liver, found in the space of Disse and secreted by endothelial cells, Kupffer cells, and hepatocytes, consists mainly of collagens type I and type VI, fibronectin, discrete aggregates of type IV collagen and filaments of type V and type III collagen [7], as well as the hepatocytespecific heparin proteoglycan (PG). The cell surface hepatocyte PG belongs to the syndecan family [8] and its glycosaminoglycan (GAG) chains are very similar to mast cell-produced heparin [9]. In our previous studies, the ECM component that affected the clonal growth efficiency in hepatomas and breast cancer cell lines was found to be heparin [6]. We found that heparin inhibited clonal growth of nonmetastatic and poorly metastatic cells but did not affect the highly metastatic cells [6]. Our studies suggested that organ-specific PGs and GAGs regulate the expression of autocrine growth factors in metastatic cells [10]. The autocrine growth factors can trigger either proliferation or differentiation signals [11], depending on the differentiated state of the tumor cells and the context of ECM components present. Colon carcinoma cells have been shown to secrete several autocrine growth factors and, in particular, members of the EGF family, both receptors and ligands, were found to be important in colon cancer cells [12–18]. The number of EGF receptors as well as c-met were found to correlate with colon cancer metastasis to the liver [15]. Most human colon cancers and cell lines express the EGF receptor ligands TGF a, amphiregulin, and cripto [16]. Erb-B2, found to be amplified in breast tumors and to correlate with poor prognosis, was not found in normal colon tissue, but its expression
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was significantly increased in adenomas and adenocarcinomas of the colon [17, 18]. Both cripto and amphiregulin were found to be autocrine growth factors for colon carcinoma cell lines, and decreasing their expression or mode of action by antisense RNA or blocking with antibodies reduced tumor cell growth in nude mice and cell proliferation in vitro [13, 14]. The aim of our present studies was to investigate the role of primary rat hepatocyte-derived ECM on the growth of human colon cell lines with liver-colonizing potential. We tested the effect of hepatocyte ECM on the clonal growth, proliferation, and expression of autocrine growth factors and their receptors in particular members of the EGF family in these colon cancer cell lines. MATERIALS AND METHODS Cell lines. The cells used in our studies were the low metastatic cell line KM12 (Dr. Michael Sela, Weizmann Institute, Rehovot, Israel) and its liver colonizing derivate, KM12SM (Dr. J. Fidler, M.D. Anderson Cancer Center, Houston, TX). The other pair of low and high metastatic colon cells were LS174T (Dr. Betty Schwartz, Soroka Medical Center, Beer-Sheva, Israel) and its liver colonizing derivate, LiM6 (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% fetal calf serum (Biological Industries, Beit Haemek, Israel), 100 mg/ml penicillin and 100 mg/ml streptomycin. For the experimental studies, the cell lines were plated in SSM and then changed after 4 h to a medium described by Block [19], with slight modifications. The medium used was DMEM supplemented with 100 mg/ml penicillin/streptomycin, 2 mg/ml bovine serum albumin, 610 mg/ml nicotinamide, 740 ng/ml ZnSO4:7H2O, 20 ng/ml CuSO4:5H2O, 5 mM glutamine, 5 mg/ml insulin, 5 mg/ml iron-saturated transferrin, 5 ng/ml selenous acid, and 1027 M dexamethasone. This medium was termed hormonally defined medium (HDM). All the medium additives were obtained from Sigma (St. Louis, MO). Growth curves. Colon cell lines were plated in 24-well plates containing hepatocyte-derived ECM. After 4 h, the medium was changed to the HDM described above. The cells were trypsinized with 0.5% trypsin and 2 mM EDTA at various time points and counted using a hemocytometer. Cells were plated in duplicate and each experiment was repeated at least three times. Clonal growth. Colon cell lines were plated at a density of 200 cells per well in 6-well plates on TCP or hepatocyte-derived ECM. The cells were grown in HDM, with medium changes every 4 days. After 2 weeks, the colonies were stained with 0.25% crystal violet in methanol and counted under a binocular. Each cell line was plated in triplicate. Primary cultures of rat hepatocytes. Primary cultures of rat hepatocytes were established by perfusing the liver of adult Wistar rats using the Berry and Friend method [20]. The cells were plated on collagen I-coated plates and in the HDM of Block [19] and described above. Hepatocytes were plated at a density of 6 3 106 cells per 10-mm2 dishes, 2 3 106 cells per well in 6-well plates, and 2 3 105 cells per well in 24-well plates. In order to modify the heparin chains produced, hepatocytes were grown in the presence of 1 mM nitrophenyl b-D-xylopyrannoside or 30 mM sodium chlorate, with medium changes every 2 days. After 5 days, hepatocyte plates were treated for 10 min at room temperature with 0.5% Triton X-100 and then washed with PBS. Removal of heparin chains from proteoglycans
1
was done by treating ECM plates with 0.05 U/ml heparinase for 22 h at 37°C. Primary cultures of fetal rat fibroblasts. Rat embryos from Fisher rats gestation day 13 were cut with a scalpel and the cells digested with 0.5% trypsin solution in PBS. After 20 min of digestion at 37°C, the cells were passed through a mesh washed and plated at a density of 3 3 105 in 24-well plates coated with collagen I. After 4 days, monolayers were treated with 0.5% Triton X-100 for 10 min at room temperature and then washed with PBS. Northern blot analysis. Plates were washed with cold phosphatebuffered saline (PBS) and total RNA was isolated by the method of Chomczynski and Sacchi [21]. RNA samples were resolved by electrophoresis through 1% agarose and denaturing formaldehyde gels in MOPS buffer and then transferred to Hybond N paper. RNA were fixed to the blots by UV crosslinking and then hybridized with cDNA probes radioactively labeled with [32P]dCTP by primer extension [22]. The cDNA probes used were: IGF I, IGF II (Dr. G. Bell), TGF b-1, amphiregulin, cripto, aFGF, TGF a, and IGF I receptor (American Tissue Culture Collection, ATCC), HGF (Dr. T. Nakamura), and c-met (Dr. Wande Woude). All probes were human, with the exception of c-met and TGFa (mouse) and HGF (rat). Western blots. Total protein was extracted by incubating the cells 30 min on ice in lysis buffer (250 mM sucrose, 5 mM MgCl2, 10 mM Tris, pH 8.0, 0.5% Triton X-100, and 1 mM PMSF). The extracts were normalized to total protein content, determined using Bradford Reagent (Sigma, St. Louis, MO). Protein (10 –20 mg per lane) were separated by SDS–PAGE (Bio-Rad Protean II minigel) and blotted onto Hybond C extra (Amersham, Arlington Heights, IL). The blots were blocked overnight in 5% milk. Blots were then incubated with antibodies to erb-B2, EGF receptor, IGF II, and TGFa (Santa Cruz Biotechnologies, Santa Cruz, CA), washed, and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, ME). The blots were then subjected to chemiluminescent detection and fluorography using X-ray film (New England Nuclear, Boston, MA).
RESULTS
The Effect of Hepatocyte ECM on the Clonal Growth of Colon Cancer Cell Lines We assessed the effect of hepatocyte-derived extracellular matrix on the clonal growth of four human colon cancer cell lines with low and high liver-colonizing potential. Primary rat liver cultures were established as described under Materials and Methods, the cells were removed after 4 days with Triton X-100 treatment, and 200 colon cells were plated on the ECMcovered 60-mm2 dish. After 2 weeks, the colonies formed were stained and counted and the results are shown in Fig. 1. Both low-colonizing liver cell lines KM12 and LS174T formed the same number of colonies on tissue culture plastic (TCP) and on hepatocyte-derived ECM. The colon cell lines with high liver metastasizing potential, KM12SM and LiM6, formed more colonies on hepatocyte-derived ECM than on plastic (Fig. 1). Moreover, in the case of LiM6, the colonies growing on hepatocyte ECM were larger than those on plastic, as shown in Fig. 2.
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON CELLS
FIG. 1. Clonal growth of colon cancer cell lines on plastic (TCP) versus hepatocyte ECM. Two hundred cells from each cell line were plated per 60-mm2 dish, uncoated or coated with ECM deposited by primary cultures of rat hepatocytes. The cells were cultured in HDM as described under Materials and Methods. The number of colonies formed was counted in triplicates after 2 weeks of culture.
The Effect of ECM on the Proliferation of Colon Cancer Cell Lines We next investigated the effect of hepatocyte ECM on the proliferation of the four colon cancer cell lines. As shown in Fig. 3, hepatocyte ECM stimulated proliferation of all four cell lines compared to cell proliferation on plastic. The experiments were repeated six times and the ECM effect on cell proliferation was statistically significant (P , 0.005). In order to test whether the ECM component responsible for the stimulatory effect was heparin proteoglycan, we modified the ECM produced by hepatocytes by growing them for 4 days in the presence of 1 mM nitrophenyl-b-D-xylopyrannoside, which prevents the addition of heparin chains to proteoglycans [23], or 30 mM sodium chlorate, which at this dose completely prevents the sulfation of GAGs [24]. Another treatment used to remove heparin chains from heparin proteoglycans in the ECM was to incubate the hepatocyte ECM with 0.05 U/ml
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heparinase for 2 h at 37°C prior to seeding the colon cancer cell lines. Hepatocyte ECM, after nitrophenyl-b-D-xylopyrannoside, but not after heparinase treatment, reduced the stimulatory ECM effect on the proliferation of all four cell lines (Fig. 3). Chlorate-treated ECM also reduced the proliferation of all the cell lines, with the exception of KM12 (Fig. 3). Reduced cell proliferation on the modified ECM was not statistically significant. However, we observed in all six experiments the same trend, namely, that cell proliferation on nitrophenyl-b-D-xylopyrannoside or chlorate-treated ECM was higher than cell proliferation on TCP. This result suggests that highly sulfated heparin proteoglycan chains are only part of the stimulatory component in hepatocyte ECM. In order to show that the effect of hepatocyte ECM was specific, we also grew the colon cell lines on ECM from primary cultures of fetal rat fibroblasts. The results are presented in Fig. 4. Fibroblast ECM had an inhibitory effect on the proliferation of all four colon cell lines. The inhibitory effect was less marked for LiM6 cells (Fig. 4). The Effect of Hepatocyte-Derived ECM on the Expression of Growth Factors and their Receptors in Colon Cancer Cell Lines One mechanism though which hepatocyte ECM could affect growth is by modulating the expression of autocrine growth factors and their receptors in the colon cancer cell lines. In order to test this hypothesis, we first screened the autocrine growth factors and receptors expressed by the colon cancer cell lines. The screening was done using molecular probes or specific antibodies and results are summarized in Table 1 and Table 2. The growth factors detected belong mainly to the EGF and EGF receptor family: all four cell lines expressed EGF receptor and erb-B2, as well as amphiregulin, an EGF receptor ligand. Cripto, another member of the EGF family, was present in all four cell lines.
FIG. 2. Photomicrograph of a representative colony of LiM6 on plastic (A) and on hepatocyte ECM (B). The bar represents 140 mm. The magnification in A and B is the same.
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FIG. 3. Growth curves of the four colon cancer cell lines cultured on tissue culture plastic (TCP), hepatocyte-deposited ECM (H.matrix), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xylose), ECM deposited by hepatocytes treated with 30 mM chlorate (chlorate), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (heparinase). Twenty thousand cells were plated on day 0, changed to HDM after 4 h, and counted in duplicates after 1, 3, and 4 days. The plots are means of three different experiments.
The other growth factor weakly detected in the cells was aFGF. We next investigated the effect of hepatocyte ECM on the expression of EGF receptor and erb-B2 in the colon cancer cells, as detected in Western blots with receptor-specific antibodies. The expression of EGF receptor was not modified in cells grown on hepatocyte ECM (data not shown). However, the expression of erb-B2 was greatly increased in all four cell lines cultured on hepatocyte ECM versus plastic (Fig. 5). The hepatocyte ECM component responsible for this induction of erb-B2 is heparin proteoglycan, since this induction was strongly reduced when the cells were grown on ECM from nitrophenyl-b-D-xylopyrannoside, chlorate, or heparinase-treated hepatocytes (Fig. 5). The effect of hepatocyte ECM on the level of mRNA for cripto and amphiregulin was assessed using molecular probes. Both cripto and amphiregulin were expressed by all four cell lines, but amphiregulin showed very low expression in KM12 cells. Three different cripto mRNA species, of 1.7 (the main transcript), 2.5,
and 4.5 Kb, were present in the four cell lines. Amphiregulin mRNA was present as a single mRNA transcript of 1.4 Kb. In KM12, LS174T, and LiM6 cells, cripto mRNA expression was not affected when the cells were grown on hepatocyte ECM (Figs. 6A and 6B). In KM12SM cells, hepatocyte ECM increased the expression of cripto, and this effect was slightly reduced on ECM of nitrophenyl-b-D-xylopyrannoside-treated hepatocytes and less so on chlorate- and heparinasetreated ECM (Fig. 6A), implying that heparin proteoglycans could be partially mediating this effect. Amphiregulin mRNA expression was not affected by hepatocyte ECM in KM12SM, the cell line that expressed the highest abundance of this growth factor mRNA (Fig. 7), or in the other cells lines (data not shown). DISCUSSION
The aim of our studies was to investigate some of the parameters involved in determining site-specific me-
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FIG. 4. Growth curves of colon cancer cell lines on plastic (TCP) versus ECM deposited by fetal rat fibroblasts (fibr.ECM). Twenty thousand cells were plated on day 0, changed to HDM after 4 h, and counted in duplicates on day 1, 3, and 4. The plots are means of three different experiments.
tastasis. Our model comprised four colon cancer cell lines of high and low liver-colonizing potential and we assessed the role of hepatocyte-derived ECM in the growth of these cell lines. We found that hepatocyte ECM selectively enhances the clonal growth of the highly metastatic cells, but not of the low metastatic ones. The effect of liver ECM on the cell proliferation was, however, uniformly stimulatory of growth in all cell lines. We showed that hepatocyte ECM also in-
creased the expression of two members of the EGF family, erb-B2 and cripto, but did not affect the expression of EGF receptor or amphiregulin. By modifying the ECM produced by hepatocytes, we showed that some of its effects were due to heparin proteoglycans. Site-specific metastasis is determined by properties of both tumor cells and host: the ability of the tumor cells to detach from the primary tumor, invade the lymphatics or blood circulation, and survive, extrava-
TABLE 1 Expression of Autocrine Growth Factors in Human Colon Cell Lines Cell line
Met.Pot
IGF I
IGF II
TGFa
TGFb
cripto
amphir.
aFGF
HGF
KM12 KM12S LS174T LiM6
low high low high
– – – ND
– – – –
– – – ND
V V ND ND
V V V V
V V V V
V V V ND
– – ND ND
Note. All of the above growth factors were detected using molecular probes. Expression of IGF II and TGF a was also assessed using specific antibodies. V: denotes that the growth factor was expressed. –: denotes that the growth factor was not detected.
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TABLE 2 Expression of Growth Factor Receptors in Human Colon Cell Lines Cell line
Met.Pot
IGF I R
EGF R
erb-b2
c-met
KM12 K12SM LS174T LiM6
low high low high
– – – ND
V V V V
V V V V
– – – ND
Note. The expression of IGF I receptor and c-met was assessed using molecular probes, and the expression of EGF receptor and erb B2 was assessed using specific antibodies. V: denotes that the receptor was detected. –: denotes that the receptor was not detected.
sate, and attach in the newly colonized organ, attract blood vessels by producing angiogenic factors and proliferating in the target organ [2, 3]. The target organ can enhance or inhibit the growth of the tumor cells either by soluble growth factors produced by the cells of the organ or embedded in the ECM [4, 5], or the target organ ECM can affect the proliferation of the tumor cells [2]. We have previously shown that tumor cells’ ability to metastasize in vivo correlates with the ability of the cells to survive at clonal densities on ECM derived from that organ [6]. The ECM components responsible for this effect were heparin proteoglycans, which acted via induction of autocrine growth factors in the tumor cells [10]. In the present studies, we tested whether liver ECM could be a determinant in colon cancer propensity to metastasize to this organ. We established a model in which we grew colon cancer cell lines of varying livercolonizing ability on ECM produced by cultures of primary rat hepatocytes. As in previous studies with hepatomas and breast cancers, the effect of hepatocyte ECM was different on high and low metastatic cells when they were plated at clonal densities, but was stimulatory for all cell lines, reardless of their metastatic potential, when the cells were plated at high densities. This effect of hepatocyte ECM on cell proliferation was quite specific, since ECM derived from fibroblasts inhibited the proliferation of the colon cancer cell lines. The ECM deposited by fibroblasts is very different than that secreted by epithelial cells, consisting of collagen type I, fibronectin, and chondroictin or dermatan sulfate proteoglycans. The stimulatory effect of hepatocyte ECM on colon cancer cells was accompanied by an induction in the expression of a member of the EGF family, namely erb-B2. In KM12SM highly metastatic cells, hepatocyte ECM also increased cripto mRNA expression. The EGF family, both ligands and receptors, play a major role in the growth of epithelial tumors, and their expression was increased in these tumors [17, 18]. The
EGF receptor family comprises four known receptors: EGF receptor, or erb-B1, erb-B2, erb-B3, and erb-B4. Of those, EGF receptors levels were found to be increased in metastatic colon cancers, while amplified expression of erb-B2 in breast tumors correlates with a bad prognosis. The members of this family can form either homo- or heterodimers with each other and can bind and respond to different ligands, according to the receptors in the complex. The known growth factors of the EGF family are EGF, transforming growth factor a (TGF a), heparin-binding EGF-like growth factor (HBEGF), betacellulin, amphiregulin, and cripto [25]. The heregulins are the ligands for erb-B3 and erb-B4 [25]. Amphiregulin, HB-EGF, and heregulins are heparinbinding growth factors, and their effect on a given cell can be modified by the presence of heparin [26 –28]. Both amphiregulin and HB-EGF require cell surface heparin proteoglycans for signaling through the EGF receptor [27, 28]. Both cripto and amphiregulin, expressed in the cell lines that we worked with, were shown to be autocrine growth factors for colon cancer cells, and reduction of their expression resulted in decreased cell proliferation and decreased tumor growth [13, 14]. Erb-B2 is not expressed in normal colon mucosa; yet it is expressed in a large number of colon cancers [17, 18]. Its role in colon cancer is yet to be determined; however, it was shown that when overexpressed, this receptor undergoes autophosphorylation in the absence of a ligand [29]. Erb-B2, but not EGF receptor
FIG. 5. Western blots showing expression of erb-B2 in the four colon cell lines. Total protein was extracted after 4 days from cells cultured on tissue culture plastic (TCP), hepatocyte-deposited ECM (ECM), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xyl), ECM deposited by hepatocytes treated with 30 mM chlorate (chlor), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (hep). Five micrograms of total protein was loaded on SDS–PAGE gels, blotted on Hybond C extra (Amersham), and incubated with anti human erb-B2 antibodies (Santa Cruz, CA), followed by the appropriate second antibody and detected by ECL.
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FIG. 6. Cripto mRNA expression in Northern blots of total RNA extracted after 4 days from the colon cancer cell lines grown on tissue culture plastic (TCP), hepatocyte-deposited ECM (ECM), ECM deposited by hepatocytes treated with 1 mM nitrophenyl-b-D-xylopyranoside (xyl), ECM deposited by hepatocytes treated with 30 mM chlorate (chlor), and hepatocyte-deposited ECM treated for 2 h with 0.05 m/ml heparinase (hep). Ten micrograms of total RNA were 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. The ethidium bromide staining shows the amount of RNA in each lane.
and amphiregulin expression, were increased by hepatocyte ECM. This effect was observed in all four cell lines. In addition, cripto mRNA was increased in KM12SM cells. Increased expression of a growth factor does not always result in enhanced proliferation. The possibility exists that the state of differentiation of a cell determines its response to specific growth factors. In the normal intestine, TGF a or TGF b have different effects as well as different distributions in the stem cells at the base of the cript or in the more differentiated cells at the tip of the villus [30, 31]. As they differentiate, both goblet cells and enterocytes lose their ability to respond to TGF a with a proliferative response [12].
FIG. 7. Amphiregulin mRNA expression in a Northern blot of total RNA extracted after 4 days from KM12SM cells grown on tissue culture plastic (TCP) and hepatocyte-deposited ECM (ECM). The blot was hybridized with a human cDNA probe to amphiregulin. The ethidium bromide staining shows the amount of RNA in each lane.
In order to identify the active ECM component we performed various modifications of heparin proteoglycans composition or chemistry in the hepatocyte ECM. Nitrophenyl-b-D-xylopyranoside is an acceptor for nascent heparin chains and therefore prevents the formation of cell surface heparin proteoglycans [23]. However, it was shown that in various cells, this substance can also decrease the amount of fibronectin in the ECM deposited [32]. Treatment of the hepatocytes with this chemical resulted in an ECM that lost its ability to stimulate cell proliferation or induce erb-B2, but did not affect cripto induction. Heparinase treatment, which degrades heparin chains, had a weaker effect than nitrophenyl-b-D-xylopyranoside treatment on both erb-B2 induction and cell proliferation. This could be due to limited accessibility of the chains to the enzyme. Sodium chlorate treatment of hepatocytes, an inhibitor of the sulfation of GAGs [24], also resulted in slightly reduced cell proliferation and erb-B2 expression, indicating that sulfated heparin chains are necessary for both of these effects. Hepatocyte ECM also increased the mRNA levels for cripto, but only in KM12SM cells. On ECM from hepatocytes treated with nitrophenyl-b-D-xylopyranoside, cripto mRNA was reduced, suggesting that heparin proteoglycan in the hepatocyte ECM also regulates the expression of this growth factor. The exact mechanism by which heparin affects cell growth is yet to be determined. Highly sulfated species of heparin were produced by hepatocytes and inhibited their growth by being internalized to the nucleus [33]. Heparin can modify the number and affinity of cell surface receptors [34] or modify the effect of a given growth factor on specific cells [27, 28, 35]. Many of the
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heparin binding growth factors absolutely require heparin for signal transduction [27, 28, 35]. Inside the nucleus, heparin can bind transcription factors, such as jun and fos [36], therefore affecting gene transcription. Our studies show an effect of hepatocyte ECM on the growth of colon cancer cell lines and imply that some of these effects are mediated via members of the EGF family, namely erb-B2 and cripto. Further studies are required to investigate the role of erb-B2, cripto, and amphiregulin, in conjunction with hepatocyte ECM, for each cell line.
14.
15. 16.
17. We thank Meirav Varon for excellent technical assistance. These studies were supported by generous gifts from the Lewkowicz family and from Shlomo Agami.
REFERENCES 1. 2.
3.
4.
5.
6.
7.
8.
9. 10.
11.
12.
13.
Paget, S. (1889) Lancet 1, 571. Zvibel, I., and Kraft, A. (1993). Extracellular matrix and metastasis. In “Extracellular Matrix: Its Chemistry, Biology, and Pathobiology” (M. A. Zern, and L. M. Reid, Eds.), vol. 22, Dekker, New York. Nicolson, G. L. (1989). Metastatic tumor cell interactions with endothelium, basement membrane and tissue. Curr. Opin. Cell Biol. 1, 1009 –1019. Radinsky, R. (1993). Paracrine growth regulation of human colon carcinoma organ-specific metastasis. Cancer Metastasis Rev. 12, 345–361. Nicolson, G. L., and Dulski, K. M. (1986). Organ specificity of metastatic tumor colonization is related to organ-selective growth properties of malignant cells. Int. J. Cancer 38, 289 – 294. Doerr, R., Zvibel, I., Chiuten, D., D’Olimpio, J., and Reid, L. M. (1989). Clonal growth of tumors on tissue-specific biomatrices and correlation with organ-site specificity of metastases. Cancer Res. 49, 384 –392. Martinez-Hernandez, A., Amenta, P. S. (1993). Morphology, localization, and cellular origin of hepatic extracellular matrix. In “Extracellular Matrix: Its Chemistry, Biology, and Pathobiology” (M. A. Zern, and L. M. Reid, Eds.), vol. 22, Dekker, New York. Pierce, A., Lyon, M., Hampson, I. N., Cowling, G. J., and Gallagher, J. T. (1992). Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver. J. Biol. Chem. 267, 3894 –3900. Lyon, M., Deakin, J. A., and Gallagher, J. T. (1994). Liver heparan sulfate structure. J. Biol. Chem. 269, 11208 –11215. Zvibel, I., Halay, E., and Reid, L. M. (1991). Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: Relevance to clonal growth of tumors. Mol. Cell. Biol. 11, 108 –116. Zvibel, I., Brill, S., and Reid, L. M. (1995). Insulin-like growth factor II regulation of gene expression in rat and human hepatomas. J. Cell. Physiol. 162, 36 – 43. Huang, F., Sauma, S., Yan, Z., and Friedman, E. (1995). Colon absorptive cells lose proliferative response to TGF a as they differentiate. Exp. Cell Res. 219, 8 –14. Ciardello, F., Tortora, G., Bianco, C., Selvam, M. P., Basolo, F., Fontanini, G., Pacifico, F., Normanno, N., Brandt, R., Persico, G. M., Salomon, D. S., and Bianco, A. R. (1994). Inhibition of
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Cripto expression and tumorigenicity in human colon cancer cells by antisense RNA and oligodeoxynucleotides. Oncogene 9, 291–298. Johnson, G. R., Saeki, T., Gordon, A. W., Shoyab, M., Salomon, D. S., and Stromberg, K. (1992). Autocrine action of amphiregulin in a colon carcinoma cell line and immunochemical localization of amphiregulin in human colon. J. Cell Biol. 118, 741–751. Radinsky, R. (1995). Molecular mechanisms for organ-specific colon carcinoma metastasis. Eur. J. Cancer 31A, 1091–1095. Ciardello, F., Kim, N., Saeki, T., Dono, R., Persico, M. G., Plowman, G. D., Garrigues, J., Radke, S., Todaro, G. J., and Salomon, D. S. (1991). Differential expression of epidermal growth factor-related proteins in human colorectal tumors. Proc. Natl. Acad. Sci. USA 88, 7792–7796. Kapitanovic, S., Radosevic, S., Kapitanovic, M., Andelinovic, S., Ferencic, Z., Tavassoli, M., Primorac, D., Sonicki, Z., Spaventi, S., Pavelic, K., and Spaventi, R. (1997). The expression of p185 (HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterology 112, 1103–1113. Saeki, T., Salomon, D. S., Johnson, G. R., Gullick, W. J., Mandai, K., Yamagami, K., Moriwaki, S., Tanada, M., Takashima, S., and Tahara, E. (1995). Association of epidermal growth factor-related peptides and type I receptor tyrosine kinase receptors with prognosis of human colorectal carcinomas. Jpn. J. Clin. Oncol. 25, 240 –249. Block, G. D., Locke, J., Bowen, W. C., Petersen, B. E., Katyal, S., Strom, S. C., Riley, T., Howard, T. A., and Michalopoulos, G. K. (1996). Population expansion, clonal growth and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF a in a chemically defined (HGM) medium. J. Cell Biol. 132, 1133–1149. Berry, M. N., and Friend, D. J. (1969). High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structure study. J. Cell Biol. 43, 506 –520. Chomcznyski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 56 –159. Feinberg, A. P., and Vogelstein, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266 –267. Kanwa, Y. S., Rosenzweig, L. J., and Jakubovski, M. L. (1986). Xylosylated proteoglycan-induced Golgi alterations. Proc. Natl. Acad. Sci. USA 83, 6499 – 6503. Bame, K. J., Reddy, R. V., and Esko, J. D. (1991). Coupling of N-deacetylation and N-sulfation in a chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Cell Biol. 266, 12461–12468. Beerli, R. R., and Hynes, N. E. (1996). Epidermal growth factorrelated peptides activate distinct subsets of erbB receptors and differ in their biological activities. J. Biol. Chem. 271, 6071– 6076. Johnson, G. R., and Wong, L. (1994). Heparan sulfate is essential to amphiregulin-induced mitogenic signaling by the epidermal growth factor receptor. J. Biol. Chem. 269, 27149 –27154. Aviezer, D., and Yayon, A. (1994). Heparin-dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF. Proc. Natl. Acad. Sci. USA 91, 12173–12177. Schuger, L., Johnson, G. R., Gilbride, K., Plowman, G. D., and Mandel, R. (1996). Amphiregulin in lung branching morphogenesis: Interaction with heparan sulfate proteoglycan modulates cell proliferation. Development 122, 1759 –1767. Lonardo, F., Di Marco, E., King, C. R., Pierce, J. H., Segatto, O., Aaronson, S. A., and Di Fiore, P. P. (1990). The normal erb-b2
EXTRACELLULAR MATRIX REGULATES GROWTH OF COLON CELLS product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol. 2, 992–1003. 30. Thomas, D. M., Nasim, M. M., Gullick, W. J., and Alison, M. R. (1992). Immunoreactivity of transforming growth factor alpha in the normal adult gastrointestinal tract. Gut 33, 628 – 631. 31. Barnard, J. A., Beauchamp, R. D., Coffey, R. J., and Moses, H. L. (1989). Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc. Natl. Acad. Sci. USA 86, 1578 –1582. 32. Canfield, A. E., Sutton, A. B., Hiscock, D. R., Gallagher, J. T., and Schor, A. M. (1994). Alpha and beta-xylosides modulate the synthesis of fibronectin and thrombospondin 1 by endothelial cells. Biochem. Biophys. Acta 1200, 249 –258. Received June 8, 1998 Revised version received July 30, 1998
33.
131
Fedarko, N. S., and Conrad, H. E. (1986). A unique heparan sulfate in the nuclei of hepatocytes: Structural changes with the growth state of the cells. J. Cell Biol. 102, 587–599. 34. Reilly, C. F., Fritze, M. S., and Rosenberg, R. D. (1987). Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J. Cell. Physiol. 121, 149–157. 35. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841– 848. 36. Busch, S. J., Jackson, R. L., Sassone-Corsi, P. (1990). Heparin inhibits transcriptional activation by Fos and Jun oncoproteins. Trans. Assoc. Am. Physicians 103, 21–27.