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 REFERENCES BEERLI, R.R. and HYNES, N.E., Epidermal-growth factor-related peptides activate distinct sub-sets of erbB receptors and differ in their biological activities. J. biol. Chem., 271, 6071–6076 (1996). CHOMCZYNSKI, P. and SACCHI, N., Single-step method of RNA isolation by acid-guanidinium-thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 56–159 (1987). 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., Inhibition of cripto expression and tumorigenicity in human colon-cancer cells by anti-sense RNA and oligodeoxynucleotides. Oncogene, 9, 291–298 (1994). DOERR, R., ZVIBEL, I., CHIUTEN, D., D’OLIMPIO, J. and REID, L.M., Clonal growth of tumors on tissue-specific biomatrices and correlation with organ-site specificity of metastases. Cancer Res., 49, 384–392 (1989). FEDARKO, N.S. and CONRAD, H.E., A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells. J. Cell Biol., 102, 587–599 (1986). FEINBERG, A.P. and VOGELSTEIN, B., A technique for radiolabeling DNA restriction-endonuclease fragments to high specific activity. Anal. Biochem., 137, 266–267 (1984). JOHNSON, G.R., SAEKI, T., GORDON, A.W., SHOYAB, M., SALOMON, D.S. and STROMBERG, K., Autocrine action of amphiregulin in a colon-carcinoma cell line and immunochemical localization of amphiregulin in human colon. J. Cell Biol., 118, 741–751 (1992). JOHNSON, G.R. and WONG, L., Heparan sulfate is essential to amphiregulininduced mitogenic signaling by the epidermal-growth-factor receptor. J. biol. Chem., 269, 27149–27154 (1994). KAPITANOVIC, S., RADOSEVIC, S., KAPITANOVIC, M., ANDELINOVIC, S., FERENCIC, Z., TAVASSOLI, M., PRIMORAC, D., SONICKI, Z., SPAVENTI, S., PAVELIC, K. and SPAVENTI, R., The expression of p185 (HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterology, 112, 1103–1113 (1997). LONARDO, F., DI MARCO, E., KING, C.R., PIERCE, J.H., SEGATTO, O., AARONSON, S.A. and DI FIORE, P.P., The normal erb-b2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol., 2, 992–1003 (1990). LONN, U., LONN, S., NILSSON, B. and STENKVIST, B., Prognostic value of erb-B2 and myc amplification in breast-cancer imprints. Cancer, 75, 2681–2687 (1995). LYON, M., DEAKIN, J.A. and GALLAGHER, J.T., Liver heparan sulfate structure. J. biol. Chem., 269, 11208–11215 (1994). MARTINEZ-HERNANDEZ, A. and AMENTA, P.S., Morphology, localization, and cellular origin of hepatic extracellular matrix. In: M.A. Zern and L.M. Reid (eds.), Extracellular matrix: its chemistry, biology and pathobiology, Vol. 22, pp. 201–255, Marcel Dekker, New York (1993). PAGET, S., Lancet, 1, 571 (1889). RADINSKY, R., Paracrine growth regulation of colon-carcinoma organspecific metastasis. Cancer Metastasis Rev., 12, 345–361 (1995a). RADINSKY, R., Molecular mechanisms for organ-specific colon-carcinoma metastasis. Europ. J. Cancer, 31A, 1091–1095 (1995b). REILLY, C.F., FRITZE, M.S. and ROSENBERG, R.D., Anti-proliferative effects of heparin on vascular smooth-muscle cells are reversed by epidermal growth factor. J. cell. Physiol., 121, 149–157 (1987). SAEKI, T., SALOMON, D.S., JOHNSON, G.R., GULLICK, W.J., MANDAI, K., YAMAGAMI, K., MORIWAKI, S., TANADA, M., TAKASHIMA, S. and TAHARA, E., Association of epidermal-growth-factor-related peptides and type-I receptortyrosine-kinase receptors with prognosis of human colorectal carcinomas. Jap. J. clin. Oncol., 25, 240–249 (1995). SAMBROOK, J., FRITSCH, E.F. and MANIATIS, T., Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989). ZVIBEL, I., BRILL, S. and REID, L.M., Insulin-like growth-factor regulation of gene expression in rat and human hepatomas. J. cell. Physiol., 162, 36–43 (1995). ZVIBEL, I., HALAY, E. and REID, L.M., 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 (1991). ZVIBEL, I. and KRAFT, A., Extracellular matrix and metastasis. In: M.A. Zern and L.M. Reid (eds.), Extracellular matrix: its chemistry, biology, and pathobiology, Vol. 22, pp. 559–580, Marcel Dekker, New York (1993).